Genetic diversity within leukemia-associated immunophenotype-defined subclones in AML.

Acute myeloid leukemia (AML) is a highly heterogeneous disease showing dynamic clonal evolution patterns over time. Various subclones may be present simultaneously and subclones may show a different expansion pattern and respond differently to applied therapies. It is already clear that immunophenotyping and genetic analyses may yield overlapping, but also complementary information. Detailed information on the genetic make-up of immunophenotypically defined subclones is however scarce. We performed error-corrected sequencing for 27 myeloid leukemia driver genes in 86, FACS-sorted immunophenotypically characterized normal and aberrant subfractions in 10 AML patients. We identified three main scenarios. In the first group of patients, the two techniques were equally well characterizing the malignancy. In the second group, most of the isolated populations did not express aberrant immunophenotypes but still harbored several genetic aberrancies, indicating that the information obtained only by immunophenotyping would be incomplete. Vice versa, one patient was identified in which genetic mutations were found only in a small fraction of the immunophenotypically defined malignant populations, indicating that the genetic analysis gave an incomplete picture of the disease. We conclude that currently, characterization of leukemic cells in AML by molecular and immunophenotypic techniques is complementary, and infer that both techniques should be used in parallel in order to obtain the most complete view on the disease.

Establishment of harmonization in immunophenotyping: A comparative study of a standardized one-tube lymphocyte-screening panel.

BACKGROUND: Multiparameter flow cytometry has been increasingly used in the identification and characterization of leukemia and lymphoma. However, due to technical complexity, this method still presents some level of variation between laboratories. In an attempt to yield more reproducible results, restrictive, highly standardized procedures have been proposed. The objective of this work was to compare this standardized protocol to a more open and flexible procedure. METHODS: The levels of expression of markers from the Euroflow lymphoid screening tube (LST) panel were evaluated on a population of both healthy and diseased patients using the recommended monoclonal antibody (MoAb) combinations or an alternative combination of either different MoAb clones or different dyes. Results were expressed as the percentages of positive target cells for each marker. RESULTS: Our study shows excellent correlation between the two methods demonstrating that comparable results can be achieved through harmonization of the procedures rather than through the constraints of standardization. CONCLUSION: Our results demonstrate that the harmonization approach is feasible. This frees scientists from the restrictions imposed by a standardization approach.

Adult metachromatic leukodystrophy treated by allo-SCT and a review of the literature.

Five patients with adult-onset metachromatic leukodystrophy (MLD) underwent allo-SCT. Conditioning was reduced in intensity and grafts were obtained from voluntary unrelated donors. All but one graft were depleted of T-lymphocytes. Patient age at transplantation varied from 18 to 29 (median, 27) years. Two patients rejected their graft and MLD progressed. The recipient of the unmanipulated graft converted to complete donor chimerism with normalization of arylsulphatase A (ARSA) levels. Despite ARSA normalization, he deteriorated. Another patient was a mixed chimera. Following escalated doses of donor lymphocyte infusions he converted to complete donor chimerism. His levels of ARSA correlated positively with the percentage of donor cells and MLD was not progressive. The fifth patient died after 35 days from complications associated with GVHD. We conclude that results of allo-SCT in symptomatic MLD patients are poor. However, allo-SCT may stop progression of MLD in selected patients.

A pilot study on the immunogenicity of dendritic cell vaccination during adjuvant oxaliplatin/capecitabine chemotherapy in colon cancer patients.

BACKGROUND: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses in cancer patients, but so far its clinical efficacy is limited. Recent evidence supports an immunogenic effect of cytotoxic chemotherapy. Pre-clinical data indicate that the combination of chemotherapy and immunotherapy may result in an enhanced anti-cancer activity. Most studies have focused on the immunogenic aspect of chemotherapy-induced cell death, but only few studies have investigated the effect of chemotherapeutic agents on the effector lymphocytes of the immune system. METHODS: Here we investigated the effect of treatment with oxaliplatin and capecitabine on non-specific and specific DC vaccine-induced adaptive immune responses. Stage III colon cancer patients receiving standard adjuvant oxaliplatin/capecitabine chemotherapy were vaccinated at the same time with keyhole limpet haemocyanin (KLH) and carcinoembryonic antigen (CEA)-peptide pulsed DCs. RESULTS: In 4 out of 7 patients, functional CEA-specific T-cell responses were found at delayed type hypersensitivity (DTH) skin testing. In addition, we observed an enhanced non-specific T-cell reactivity upon oxaliplatin administration. KLH-specific T-cell responses remained unaffected by the chemotherapy, whereas B-cell responses were diminished. CONCLUSION: The results strongly support further testing of the combined use of specific anti-tumour vaccination with oxaliplatin-based chemotherapy.

Rapamycin and MPA, but not CsA, impair human NK cell cytotoxicity due to differential effects on NK cell phenotype.

Cyclosporin A (CsA), rapamycin (Rapa) and mycophenolic acid (MPA) are frequently used for GVHD prophylaxis and treatment after allogeneic stem cell transplantation (SCT). As NK cells have received great interest for immunotherapeutic applications in SCT, we analyzed the effects of these drugs on human cytokine-stimulated NK cells in vitro. Growth-kinetics of CsA-treated cultures were marginally affected, whereas MPA and Rapa severely prevented the outgrowth of CD56(bright) NK cells. Single-cell analysis of NK cell receptors using 10-color flow cytometry, revealed that CsA-treated NK cells gained a similar expression profile as cytokine-stimulated control NK cells, mostly representing NKG2A(+) KIR(-) NCR(+) cells. In contrast, MPA and Rapa inhibited the acquisition of NKG2A and NCR expression and NK cells maintained an overall NKG2A(-) KIR(+) NCR(+/-) phenotype. This was reflected in the cytolytic activity, as MPA- and Rapa-treated NK cells, in contrast to CsA-treated NK cells, lost their cytotoxicity against K562 target cells. Upon target encounter, IFN-γ production was not only impaired by MPA and Rapa, but also by CsA. Overall, these results demonstrate that CsA, MPA and Rapa each have distinct effects on NK cell phenotype and function, which may have important implications for NK cell function in vivo after transplantation.

CD3+/CD19+-depleted grafts in HLA-matched allogeneic peripheral blood stem cell transplantation lead to early NK cell cytolytic responses and reduced inhibitory activity of NKG2A.

Natural killer (NK) cells have an important function in the anti-tumor response early after stem cell transplantation (SCT). As part of a prospective randomized phase III study, directly comparing the use of CD3(+)/CD19(+)-depleted peripheral blood stem cell (PBSC) harvests with CD34(+)-selected PBSC harvests in allogeneic human leukocyte antigen-matched SCT, we here show that the use of CD3(+)/CD19(+)-depleted PBSC grafts leads to early NK cell repopulation and reconstitution of the CD56(dim) and CD56(bright) NK cell subsets, with concomitant high cytolytic capacity. In the CD34 group, this process took significantly longer. Moreover, in the CD3/19 group after reconstitution, a higher percentage of killer immunoglobulin-like receptor-positive NK cells was found. Although similar percentages of CD94-positive NK cells were found in both groups, in the CD34 group, almost all expressed the inhibitory CD94:NKG2A complex, whereas in the CD3/19 group, the inhibitory CD94:NKG2A and the activating CD94:NKG2C complex were equally distributed. This preferential development of NKG2C-expressing NK cells in the CD3/19 group was paralleled by a loss of NKG2A-mediated inhibition of NK cell degranulation. These results show that the use of CD3(+)/CD19(+)-depleted grafts facilitates strong NK cell cytolytic responses directly after SCT, and the rapid emergence of an NK cell receptor phenotype that is more prone to activation.

Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products.

BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS: In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS: Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION: Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Long-term follow-up results after autologous haematopoietic stem cell transplantation for severe systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a generalised autoimmune disease, causing morbidity and a reduced life expectancy, especially in patients with rapidly progressive diffuse cutaneous SSc. As no proven treatment exists, autologous haematopoietic stem cell transplantation (HSCT) is employed as a new therapeutic strategy in patients with a poor prognosis. This study reports the effects on survival, skin and major organ function of HSCT in patients with severe diffuse cutaneous SSc. PATIENTS AND METHODS: A total of 26 patients were evaluated. Peripheral blood stem cells were collected using cyclophosphamide (4 g/m2) and rHu G-CSF (5 to 10 microg/kg/day) and were reinfused after positive CD34+ selection. For conditioning, cyclophosphamide 200 mg/kg was used. RESULTS: After a median follow-up of 5.3 (1-7.5) years, 81% (n = 21/26) of the patients demonstrated a clinically beneficial response. The Kaplan-Meier estimated survival at 5 years was 96.2% (95% CI 89-100%) and at 7 years 84.8% (95% CI 70.2-100%) and event-free survival, defined as survival without mortality, relapse or progression of SSc, resulting in major organ dysfunction was 64.3% (95% CI 47.9-86%) at 5 years and 57.1% (95% CI 39.3-83%) at 7 years. CONCLUSION: This study confirms that autologous HSCT in selected patients with severe diffuse cutaneous SSc results in sustained improvement of skin thickening and stabilisation of organ function up to 7 years after transplantation.

Three-color flowcytometric analysis of mature and immature hematological malignancies. A guideline of the Dutch Foundation for Immunophenotyping of Hematological Malignancies (SIHON).

Multiparameter flowcytometry offers an insight into differentiation pathways, maturation stages and abnormal features of cell (sub)populations thus helping to establish and classify hematological malignancies. The Dutch Foundation for Immunophenotyping of Hematological Malignancies (SIHON) has formulated a guideline for a rapid screening followed by confirmation and classification in a standardized way. For this aim seven carefully composed monoclonal antibody combinations are elucidated for screening the test sample in a first phase. In this phase a relative frequency distribution of the cells will be established and a decision will be made about abnormal cells present, as well as their mature or immature state and the cell lineage they belong to. In a second phase, panels with cell lineage dependent monoclonal antibody combinations may be used to confirm and classify the abnormal cell population indicated in phase 1, as well as to establish the presence or absence of an abberant immunophenotype.

Production of anti-CD3 and anti-CD7 ricin A-immunotoxins for a clinical pilot study.

This report describes the preparation of an immunotoxin-combination, consisting of an anti-CD3 and anti-CD7 monoclonal antibody (MoAb) both conjugated to the A-chain of plant toxin ricin, for the experimental treatment of graft-versus-host disease. MoAbs and toxin were conjugated by conventional biochemical and chromatographic techniques. Raw materials, intermediate and final products were evaluated in accordance with the relevant 'points to consider' of the FDA. Yields, purity and sterility of the two final products were all satisfactory. Preservation of MoAb-affinity and toxin-activity were confirmed in biological assays. The LD50, 25-45 mg immunotoxin-combination/kg mouse, equalled that of similar immunotoxins already in clinical use. Because in vitro cross-reactivity screening revealed an unexpected binding of the CD3-MoAb to the esophagus epithelium, human doses of immunotoxin-combination were administered to two cynomolgus monkeys. Clinically relevant serum concentrations were obtained without irreversible toxicities occurring. The T(1/2) varied between approximately 6 and 9 h and the C(max) ranged from 1.8 to 3.9 microg/ml. The main side effect was a transient rise of serum creatine kinase. Importantly, neither damage nor binding of the CD3-immunotoxin to the monkey esophagus epithelium could be demonstrated. It was concluded that sufficient material of proper quality and with an acceptable toxicity profile was produced, warranting the evaluation in a clinical pilot-study.

A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease.

This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10(-8 )mol/L (about 1.8 microg/mL). Because most natural killer (NK) cells are CD7(+), NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m(2) IT combination, administered intravenously at 48-hour intervals. The T(1/2) was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells. (Blood. 2000;95:3693-3701)

Relationship of the CD22 immunotoxin dose and the tumour establishment in a SCID mice model.

Immunotoxins (ITs) may be very potent to erradicate tumour growth in vivo. We investigated the influence of the IT-dose, in relation to the establishment of the tumour, on the anti-tumour activity of CD22-recombinant (rec) ricin A for a disseminated tumour (Ramos) in SCID mice. Furthermore, the enhancement of the IT cytotoxicity in vivo by chloroquine was assessed. CD22-rec ricin A appeared to be highly effective. Paralysis of the hind legs was significantly delayed by a very low IT-dose of 2 microg administered intravenously (i.v.) 7 days after i.v. inoculation of the tumour cells. Even a dose of 30 microg administered 21 days after inoculation of the target cells significantly delayed the onset of paralysis up to 8 days compared with the median paralysis time (MPT) of the control group. The efficacy of treatment was obviously influenced by the establishment of the tumour, the tumour load and localisation. The anti-tumour activity of 10 and 30 microg IT diminished when the IT was administered after increasing the time lag following inoculation of tumour cells. Delaying IT administration resulted in growth of solid tumours. This implies that cells migrate to sanctuaries protected from the IT indicating that the anti-tumour activity was influenced by the accessibility of the IT to the target cells. The in vivo anti-tumour activity of CD22-rec ricin A could not be enhanced by simultaneously administered chloroquine, despite the continuous infusion with an intraperitoneally (i.p.) implanted mini-osmotic pump. Ex vivo experiments revealed that the maximally tolerated serum concentration (3.9 microM) was too low to be effective. In conclusion, CD22-rec ricin A is highly effective for in vivo treatment of B-cell malignancies, in particular if treatment is started when the tumour load is low and before migration takes place to poorly accessible sanctuaries.

Persistent symptoms in former UNTAC soldiers are not associated with shifted cytokine balance.

BACKGROUND: The pathogenesis of post-combat syndromes, such as Gulf War syndrome, is poorly understood. Recently, it has been postulated that the symptoms of veterans with such syndromes are due to a disturbed cytokine balance shifted towards a T-helper (Th) 2 profile. We investigated this hypothesis in 21 symptomatic former UNTAC soldiers and compared their results with those obtained in 21 healthy former UNTAC soldiers matched for age, sex and military force. DESIGN: The numbers of intracellular interleukin 4 (IL-4) and interferon gamma- (IFN-gamma) producing CD4+ and CD8+ T lymphocytes (CD3+) were determined after in vitro stimulation with phorbol myristate acetate and calcium ionophore in the presence of brefeldin to block secretion of induced cytokines. Circulating concentrations and lipopolysaccharide- (LPS) or phytohaemagglutinin- (PHA) stimulated whole-blood production of the proinflammatory cytokines IL-1beta, IL-1ra, tumour necrosis factor alpha (TNF-alpha) and IL-10 and IFN-gamma were measured. RESULTS: The numbers of CD4+ and CD8+ T lymphocytes positive for IL-4 or IFN-gamma production were not significantly different in patients and control subjects. After stimulation with LPS or PHA, the in vivo circulating concentration and concentration of IL-10 and IFN-gamma were also similar. CONCLUSIONS: The present study demonstrates that there is no shift in cytokine balance towards a Th2 profile in former UNTAC soldiers with symptoms similar to those of the Gulf War syndrome.

Counterflow centrifugation allows addition of appropriate numbers of T cells to allogeneic marrow and blood stem cell grafts to prevent severe GVHD without substantial loss of mature and immature progenitor cells.

Using counterflow centrifugation elutriation (CCE) lymphocytes can be separated from CD34+ populations based on size. Immature progenitors tend to be smaller than mature cells suggesting that CCE introduces loss of stem cells. We compared the separation of 12 PBSC with 16 BM transplants. Cells were separated in 12 fractions (3000-2200 r.p.m.) and the rotor off (RO) fraction. Separation patterns of BM and PBSC were comparable. B cells were collected in the high speed fractions followed by T and NK cells. In contrast, progenitor cells were collected in lower speed fractions. By adding successively T cell-depleted fractions to the RO fraction a BM transplant could be composed containing 0.7 x 10(6) T cells/kg and 90%, 89% and 68% recovery of CD34+, CFU-GM and BFU-E. PBSC were separated in four CCE runs inducing higher numbers of T cells in the graft (4.4 x 10(6)/kg) and 54% CD34+, 46% CFU-GM and 37% BFU-E recovery. Time of engraftment was not delayed and no graft failure was observed. The higher number of T cells was not associated with higher incidence of GVHD. Acute GVHD > or = grade III occurred in 0 of 16 BM and two of 12 PBSC recipients; extensive chronic GVHD was observed in four of 15 and three of nine recipients, respectively. To study immature cells in the graft, CD34 subpopulations and cells with long-term repopulating ability, determined using cobble-stone area formation (CAFC assay), were evaluated in each fraction. The separation patterns in BM and PBSC were comparable. Cells with mature and immature phenotype were enriched in lower speed fractions (mean recovery of 74% CD34+/CD13-/DR-). The CAFC week 2, 4 and 6 were also enriched in these fractions. These data show that the used CCE procedure is a reliable method to deplete T cells from stem cell transplants without substantial loss of immature and mature progenitors.

Influence of cytotoxicity enhancers in combination with human serum on the activity of CD22-recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells.

Despite the strong in vitro activity of some immunotoxins (ITs), clinical application did not result in complete cure. The outcome of therapy may be improved by combining ITs with IT-cytotoxicity enhancing agents. We studied the effect of various agents that influence the intracellular routing of ITs on the activity of the anti-B cell IT CD22-recombinant (rec) ricin A. In protein synthesis inhibition assays the carboxylic ionophores monensin and nigericin enhanced the activity of the IT 117- and 382-fold, respectively, against the cell line Daudi, and 81- and 318-fold, respectively, against the cell line Ramos. IT activity to Daudi and Ramos was enhanced to a lesser extent by the lysosomotropic amines chloroquine (14- and 11-fold, respectively) and NH4Cl (nine- and 10-fold, respectively). However, the combination of NH4Cl and chloroquine induced more than an additive effect (145- and 107-fold, respectively). Cytotoxicity was not influenced by brefeldin A, all-trans retinoic acid (ATRA), verapamil and perhexiline maleate. Bacitracin enhanced the IT cytotoxicity in contrast to the other protease inhibitors aprotinin, leupeptin and soybean trypsin inhibitor, albeit enhancement was weak (two-fold). The enhancers exerted only a negligible effect on bone marrow progenitor cells. We recently developed a flow cytometric cytotoxicity assay in which cell elimination can be assessed. In order to detect enhancement in this assay, we used 5 x 10(-11) M IT (approximately the 50% protein synthesis inhibiting dose (ID50)). This concentration killed 41% of the Daudi cells and 42% of the Ramos cells. In the presence of 10 nM monensin the IT killed 74% and 99% and in the presence of 10 nM nigericin 96% and 99% of the Daudi and Ramos cells, respectively. At 10(-8) M, CD22-rec ricin A eliminated malignant cells originating from three patients with B-CLL (0.42 log) and two with B-ALL (0.19 log) patients. Cytotoxicity to malignant cells was enhanced by NH4Cl, chloroquine, monensin and nigericin. The combination of NH4Cl and chloroquine enhanced the activity most effectively (up to 2.06 log). To determine the applicability of the IT in combination with enhancers in vivo we investigated the effect of human serum. Human serum inhibited IT activity which could not be restored by monensin and nigericin because of complete inhibition of these enhancers by serum. In contrast, chloroquine partially restored the activity of CD22-rec ricin A in the presence of human serum. We conclude that monensin, nigericin and the combination of NH4Cl and chloroquine can be used instead of NH4Cl to potentiate CD22-rec ricin A activity in purging autologous bone marrow transplants contaminated with malignant B cells. Chloroquine might be a promising enhancer of CD22-rec ricin A for treating patients in vivo.

Rationale for the use of immunotoxins in the treatment of HIV-infected humans.

The first step in the replication of human immunodeficiency virus (HIV) is selective binding of the envelope glycoprotein (gp120) to CD4 receptors on T cells or macrophages. After penetration in these cells, the genome of the virus is integrated in the human genome. HIV-infection causes depletion of CD4-positive cells resulting in a severe immunosuppression. It is believed that eliminating HIV-infected cells is crucial in limiting further reduction of CD4-positive cells and thus, preventing disease progression. The most commonly used drugs, such as zidovudine (AZT), appeared to be not completely effective. Therefore many investigators are searching for alternative treatment modalities. The use of immunotoxins (ITs) to eliminate HIV-infected cells is discussed. ITs are chimeric molecules in which cell-binding ligands are coupled to toxins and can specifically eliminate undesired cells. The cell-binding carriers of anti-HIV ITs have been directed against different regions of the HIV envelope glycoprotein (gp120 and gp41) and surface antigens (e.g CD4, CD25). The ITs have been composed of different ribosome-inactivating proteins (RIPs) like pokeweed antiviral protein (PAP), Pseudomonas exotoxin (PE), Diphtheria toxin (DT), or ricin. In in vitro studies, several of these ITs have been shown to be effective and specific in killing acute and persistently HIV-infected cells. The ITs were effective at concentrations (ID50 range from 10(-9) M to 10(-12) M) that were not toxic to uninfected cells or cells without the antigen. The IT CD4(178)PE40, a fusion protein directed against the CD4 binding site of gp120, has been investigated in two in vivo trials. The results were disappointing considering the antiviral activity in vitro. This was thought to be due to the rapid clearance of the IT and the differential resistance of clinical HIV isolates. Use of a panel of ITs is likely to be more effective because multiple approaches cover the intrinsic variability of HIV and the presence of IT-resistant or latently infected cells, as well as the blocking presence of neutralizing anti-HIV antibodies and the immunogenicity of most ITs. It may be possible to control the virus completely with a panel of ITs in combination with other antiviral or immunosuppressive agents such as RT inhibitors (e.g AZT), interferon alpha, or cyclosporine. More research will be necessary to develop such a combined therapy.

The role of the different CD34 epitopes in detection and positive selection of CD34+ bone marrow and peripheral blood stem cells.

Positive selection of CD34+ cells is an attractive approach to reduce tumour cell contamination in bone marrow (BM) and peripheral blood progenitor cell (PBPC) autografts in malignancies not expressing CD34. All current selection methods use monoclonal antibodies (MoAbs) specific for the class I or class II CD34 epitopes, while for detection most investigators use class III MoAbs. Since the distribution of the different CD34 epitopes on haematopoietic progenitors differs, we studied their significance in CD34+ selection procedures. Testing MoAbs against class I, II and III CD34 epitopes on normal BM we observed that +/- 23% of class III positive cells was class I negative. A higher expression of the class III epitope compared with classes I or II was observed on the KG1 cell line, whereas no differences in binding capability were found. The class III epitope anti-CD34, 561, was compared with the class I epitope anti-CD34, BI-3C5, both coupled to M450 Dynabeads. The yield of CD34+ cells obtained with the 561 beads was 1.7% of the mononuclear cells versus 0.95% using the class I epitope, a 1.95-fold increase (1.3-2.7), whereas the purity was similar (96% in both cases). The absolute number of CD34+ cells was therefore twofold higher after 561 selection, including cells with a more mature phenotype. In single cell assay comparable numbers of highly proliferative progenitors but higher numbers of differentiated colonies per phenotypic subfraction were measured. In conclusion, M450 beads coated with the 561 anti-class III CD34 epitope are more efficient in isolating CD34+ cells from bone marrow, probably due to a broader distribution of the class III epitope.

Highly potent CD22-recombinant ricin A results in complete cure of disseminated malignant B-cell xenografts in SCID mice but fails to cure solid xenografts in nude mice.

The highly specific cytotoxic action of ribosome-inactivating protein (RIP) containing immunotoxins (ITs) makes IT therapy a promising approach to eliminating residual malignant cells. We investigated the cytotoxicity of the IT CD22-recombinant ricin A to the B-cell line Ramos in vitro and in vivo. Cytotoxicity of CD22-recombinant ricin A in vitro was very high as expressed by the very low 50% inhibition dose (ID50) of 3.5 x 10(-11) M. Cytotoxicity was increased 7 times in the presence of the cytotoxicity enhancer NH4Cl. The ultimate kill of Ramos cells by CD22-recombinant ricin A was high (2.7-log kill) and was increased strongly in the presence of NH4Cl (4.2-log kill). Anti-tumor activity in vivo was investigated by i.v. treatment of solid s.c. Ramos xenografts in nude BALB/c mice. A single dose did not inhibit tumor growth. Treatment on 5 consecutive days resulted in evident tumor reduction. In one mouse, tumor could no longer be detected on the 6th day after starting treatment. However, after 8 days tumor volumes increased again. Antitumor activity was more pronounced in a disseminated tumor model in SCID mice. IT treatment (i.v.) 7 days after i.v. inoculation with Ramos cells resulted in cure of all mice. Non-specific toxicity was low. Alanine aminotransferase (ALAT) levels in serum were elevated temporarily. Serum values of gamma-glutamyl transferase (gamma-GT), bilirubin and creatinin did not change. Body weight was also transiently reduced. The LD50 in SCID mice after i.v. administration was high (0.626 mg IT per mouse). The clearance rate in SCID mice, as determined by ELISA, was biphasic.

Cytotoxic potency of CD22-ricin A depends on intracellular routing rather than on the number of internalized molecules.

Cytotoxicity of immunotoxins (ITs) varies considerably depending on factors like the capability of the target antigen to internalize IT molecules, intracellular processing and routing of the IT. We studied factors that may influence cytotoxicity of CD22-ricin A IT to several B cell lines. The antigen density varied from 5.9 x 10(3) to 6.0 x 10(4) molecules/cell. The ID50, determined by protein synthesis inhibition, varied from 2.1 x 10(-12) to 3.8 x 10(-11) M IT in absence and from 2.8 x 10(-14) M to 5.2 x 10(-12) M IT in presence of the cytotoxicity enhancer NH4Cl (6 mM). In absence as well as in presence of NH4Cl no correlation could be found between antigen density and ID50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low antigen density, such as KM3, protein synthesis was quickly inhibited. In order to investigate whether the cytotoxicity was dependent on the number of internalized molecules the kinetics of internalization and exocytosis of degraded 125I-labelled CD22 molecules were studied. After 24 h the number of internalized CD22 molecules was highest in Ramos (154,500), followed by Daudi (110,300) and KM3 (69,900). However, despite the higher internalization rate of Daudi the rate of cytotoxicity of 10(-8) M IT was comparable with KM3. NH4Cl did not influence the number of internalized molecules but postponed degradation of CD22. In conclusion, CD22-ricin A is a very potent and fast acting IT even for elimination of target cells that express low numbers of antigen. These results may have implication for treatment of different B cell malignancies with CD22-ricin A.