Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model.

Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform.

The mild inflammatory response in febrile neutropenic lymphoma patients with low risk of complications is more pronounced in patients receiving tobramycin once daily compared with three times daily.

We evaluated inflammatory markers in febrile neutropenic lymphoma patients undergoing high-dose chemotherapy with autologous stem cell support. Based on MASCC scores, our patients had a low risk of serious complications and a perspective of a benign initial clinical course of the febrile neutropenia. We also studied the impact of tobramycin given once versus three times daily on these immune markers. Sixty-one patients participating in a Norwegian multicentre prospective randomized clinical trial, comparing tobramycin once daily versus three times daily, given with penicillin G to febrile neutropenic patients, constituted a clinically homogenous group. Four patients had bacteraemia, all isolates being Gram-positive. Thirty-two patients received tobramycin once daily, and 29 patients received tobramycin three times daily. Blood samples were taken at the onset of febrile neutropenia and 1-2 days later. All samples were frozen at -70 °C and analysed at the end of the clinical trial for C-reactive protein (CRP), procalcitonin (PCT), complement activation products, mannose-binding lectin (MBL) and 17 cytokines. We found a mild proinflammatory response in this series of patients. CRP was non-specifically elevated. Ten patients with decreased MBL levels showed the same mild clinical and proinflammatory response. Patients receiving tobramycin once daily showed a more pronounced proinflammatory response compared with patients receiving tobramycin three times daily. Overall, febrile neutropenic cancer patients with a benign clinical course show a mild proinflammatory immune response.

Phase I/II trial of melanoma therapy with dendritic cells transfected with autologous tumor-mRNA.

We have developed an individualized melanoma vaccine based on transfection of autologous dendritic cells (DCs) with autologous tumor-mRNA. Dendritic cells loaded with complete tumor-mRNA may generate an immune response against a broad repertoire of antigens, including unique patient-specific antigens. The purpose of the present phase I/II trial was to evaluate the feasibility and safety of the vaccine, and the ability of the DCs to elicit T-cell responses in melanoma patients. Further, we compared intradermal (i.d.) and intranodal (i.n.) vaccine administration. Twenty-two patients with advanced malignant melanoma were included, each receiving four weekly vaccines. Monocyte-derived DCs were transfected with tumor-mRNA by electroporation, matured and cryopreserved. We obtained successful vaccine production for all patients elected. No serious adverse effects were observed. A vaccine-specific immune response was demonstrated in 9/19 patients evaluable by T-cell assays (T-cell proliferation/interferon-gamma ELISPOT) and in 8/18 patients evaluable by delayed-type hypersensitivity (DTH) reaction. The response was demonstrated in 7/10 patients vaccinated intradermally and in 3/12 patients vaccinated intranodally. We conclude that immuno-gene-therapy with the described DC-vaccine is feasible and safe, and that the vaccine can elicit in vivo T-cell responses against antigens encoded by the transfected tumor-mRNA. The response rates do not suggest an advantage in applying i.n. vaccination.

Growth factor receptors in hematopoietic stem cells: EPH family expression in CD34+ and CD133+ cell populations from mobilized peripheral blood.

Cell-surface antigen expression of hematopoietic stem cells has a crucial role in characterizing cell subpopulation with distinct functional properties. The Eph receptors are the largest receptor tyrosine kinase family being involved in processes like vascular remodelling during development and physiological and pathological angiogenesis. Some Eph/Ephrin members are expressed in hematopoietic cells. The ability to isolate purified cell populations co-expressing CD34 and CD133 antigens as most commonly used markers for identification of hematopoietic progenitors has provided the opportunity to identify their surface-receptor profile. As positively expressed CD34 and CD133 cells take place not only in hematopoietic but also in endothelial differentiation, we aimed to define the Eph/Ephrin characteristic of these cells and relate these findings to new therapy strategies. Positive selections of CD34 and CD133 cells from PBPC in lymphoma patients were performed using magnetic beads and AutoMACS (Miltenyi Biotec) device. The purity of isolated cells was tested by flow cytometry. Immunocytochemistry was used to assess the Eph/Ephrin expression profile of positively selected samples. Our study revealed that all samples (10 from CD34+ and 8 from CD133+ cells) expressed one or more of Eph/Ephrin antigens in different proportions. All CD34+ cell samples, and 6 of 8 in the CD133+ cell fraction were strongly immunoreactive for EphA2. EphB2 was strongly expressed in all CD133+ cases, but 50% of the CD34 positive group lacked or weakly expressed this receptor. EphB4 was negative in 9 of 10 CD34+ cases and in all CD133+ cells. Thus, we have shown the surface marker profile of positively selected CD34 and CD133 cells in leukapheresis samples from lymphoma patients with regard to Eph/Ephrin receptors and discussed their biological clinical potential.

New indirect approach to the therapeutic use of immunotoxins.

To improve the applicability of immunotoxins (ITs), we have developed a new two-step indirect procedure. The target cells to be killed are first incubated with cell-specific mouse monoclonal antibodies (MAbs). After removal of excess unbound antibody, the cells are incubated in the presence of lactose with an indirect IT, a conjugate of whole abrin and sheep anti-mouse immunoglobulin (SAM) that binds only to cells having primary mouse MAbs on their surfaces. The SAM-abrin IT is affinity purified before use to remove molecules with exposed B-chain-binding sites; it was nontoxic in the absence of the specific mouse MAbs, demonstrating the specificity of the two-step method. We compared the indirect approach, using four different primary MAbs, with the conventional method, in which abrin is coupled directly to the mouse MAbs. In three human cell lines--the melanoma line FEMX, the Burkitt cell line Rael, and the leukemia cell line KM3--the cell kill, measured by a clonogenic assay, was consistently greater with the indirect than with the direct method. In the melanoma and Rael cells, the indirect method gave a higher cell kill than even native abrin. With a mixture of two different antibodies an additive effect was observed with the indirect but not with the direct method. The new approach greatly simplifies the therapeutic application in vitro of ITs, because it permits the use of different primary antibodies, singly or in mixtures, in conjunction with only one or a few general indirect ITs. In efforts to further improve the usefulness of the indirect method, other indirect ITs containing different toxin moieties are being examined. The possibility of employing the indirect principle in vivo is being explored.

Successful clinical use of an anti-HLA-DR monoclonal antibody for autologous bone marrow transplantation.

It has been widely assumed that anti-HLA-DR antibodies react with pluripotent stem cells and cannot be used in bone marrow purging. We report a case of non-Hodgkin's lymphoma in which an anti-HLA-DR antibody (AB4) was used for immunomagnetic purging and the subsequent autologous bone marrow transplantation resulted in rapid marrow engraftment with no serious complications. The results indicate that the AB4 antibody, which binds to an antigen encoded by the B3 gene of the DR region, can be safely used in the clinic in the purging of bone marrow from patients with AB4-positive tumors (non-T-cell acute lymphocytic leukemia, non-Hodgkin's lymphoma, and some cases of acute myelogenous leukemia.

Phase I study of the plant protein ricin.

A Phase I study was carried out with ricin, a plant toxin acting by inhibiting protein synthesis, on 54 cancer patients with advanced disease. Ricin was given as i.v. bolus injections every two weeks at dose levels ranging from 4.5 to 23 micrograms/sq m of estimated body surface area. Ricin was well tolerated at doses up to 18 to 20 micrograms/sq m. At these levels and at higher levels, flu-like symptoms with fatigue and muscular pain appeared and, in some patients, nausea and vomiting occurred also. No myelo-suppression was seen. Antibodies to ricin were detected in serum after two to three ricin injections. Ricin was eliminated from blood according to first order kinetics. At each dose level, the plasma concentrations, as well as the side effects, showed only minor differences between patients. The highest dose given, 23 micrograms/sq m, gave plasma concentrations twice those found previously to be therapeutically effective in tumor-bearing mice. Of 38 evaluable patients, one patient with lymphoma had a partial response. Stable disease was observed in four patients with renal cancers, in two with soft tissue sarcomas, and in one patient each with mesothelioma, thyroid, and rectal cancer. A dose of 23 micrograms/sq m is recommended for Phase II trials of ricin.

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies.

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.

Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer.

PURPOSE: This study was performed to disclose the clinical impact of isolated tumor cell (ITC) detection in bone marrow (BM) in breast cancer. PATIENTS AND METHODS: BM aspirates were collected from 817 patients at primary surgery. Tumor cells in BM were detected by immunocytochemistry using anticytokeratin antibodies (AE1/AE3). Analyses of the primary tumor included histologic grading, vascular invasion, and immunohistochemical detection of c-erbB-2, cathepsin D, p53, and estrogen receptor (ER)/progesterone receptor (PgR) expression. These analyses were compared with clinical outcome. The median follow-up was 49 months. RESULTS: ITC were detected in 13.2% of the patients. The detection rate rose with increasing tumor size (P =.011) and lymph node involvement (P <.001). Systemic relapse and death from breast cancer occurred in 31.7% and 26.9% of the BM-positive patients versus 13.7% and 10.9% of BM-negative patients, respectively (P <.001). Analyzing node-positive and node-negative patients separately, ITC positivity was associated with poor prognosis in the node-positive group and in node-negative patients not receiving adjuvant therapy (T1N0). In multivariate analysis, ITC in BM was an independent prognostic factor together with node, tumor, and ER/PgR status, histologic grade, and vascular invasion. In separate analysis of the T1N0 patients, histologic grade was independently associated with both distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS), ITC detection was associated with BCSS, and vascular invasion was associated with DDFS. CONCLUSION: ITC in BM is an independent predictor of DDFS and BCSS. An unfavorable prognosis was observed for node-positive patients and for node-negative patients not receiving systemic therapy. A combination of several independent prognostic factors can classify subgroups of patients into excellent and high-risk prognosis groups.

Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells.

Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.

Detection of isolated tumor cells in bone marrow in early-stage breast carcinoma patients: comparison with preoperative clinical parameters and primary tumor characteristics.

UNLABELLED: PURPOSE/EXPERIMENTAL DESIGN: The importance of detection of disseminated isolated tumor cells (ITCs) in bone marrow (BM) is still not settled. BM aspirates from 920 patients with primary breast cancer were analyzed for tumor cells by standardized direct immunocytochemical analysis (ICC) of 2 x 10(6) mononuclear cells (MNCs) using anticytokeratin monoclonal antibody (AE1/AE3). Samples (637) were analyzed by negative immunomagnetic enrichment (IMS) followed by ICC (10 x 10(6) MNCs). Analyses of the primary tumor specimens have been performed, including histomorphology, tumor-node-metastasis (TNM) staging, grading, and immunohistochemical analyses. RESULTS: Of the patients with infiltrating carcinoma, 63% were node negative (N0) and 33%, node positive (N+). The results show the presence of tumor cells in 13.4% of the evaluable patients after direct ICC analysis. The presence of tumor cells correlated to the nodal- and tumor stage, showing BM positivity in 9.9% of the N0 cases and 20.6% in the N+ group (P < 0.0005), 11.2% of the stage T(1) were positive, and 15.0% and 22.6% were positive in the T(2) and T(3/4) groups, respectively (P = 0.013). No correlation between detection of ITC and detection of p53 and cathepsin D expression was found. Vascular invasion and c-erbB2 expression were associated with ITCs in BM (P = 0.045 and P = 0.024, respectively). Node-negative patients with estrogen receptor (ER)+ and/or progesterone receptor (PgR)+ tumors had lower frequency of ITCs than ER-/PgR- (P = 0.004). The use of negative IMS increased the frequency of positive BM by 63% (P < 0.0005). CONCLUSIONS: The direct ICC detection of ITCs in BM correlated with primary tumor stage, nodal stage, vascular invasion, c-erbB2 expression, and ER/PgR status. Analysis of larger BM samples by negative IMS resulted in increased number of ITC-positive patients.

Depletion of T lymphocytes from human bone marrow. Use of magnetic monosized polymer microspheres coated with T-lymphocyte-specific monoclonal antibodies.

A new technique for depletion of T cells from bone marrow is presented. Bone marrow cells (BMC) were rosetted with magnetic monosized polystyrene microspheres coated with monoclonal antibodies (MAbs) specific for T cell CD2 and CD3 antigens. Rosetted T cells were subsequently removed from non-T cells with the aid of a magnet. This immunomagnetic separation procedure was carried out in less than 40 min and reproducibly removed T cells, leaving a maximum of 0.025% sheep-red-blood-cell (SRBC) rosette-forming cells and less than 0.02% T cells as detected by a T cell limiting dilution assay. The efficacy of the depletion procedure was further shown by flow cytometry data, by effective removal of cells from a T cell line added to the BMC prior to immunomagnetic separation, and by abrogation of interleukin 2 (IL-2)-producing capacity in T-cell-depleted BMC (BMC-T). The T cell depletion procedure provided a 43-74% recovery of non-T cells present in the Isopaque-Ficoll-isolated bone marrow mononuclear cell fraction and did not disturb the growth potential of stem cells, as assayed by hematopoietic stem cell assays.

Intra-arterial infusion of mitomycin C in treatment of breast cancer: occurrence of skin necrosis in irradiated patients.

Eight previously irradiated breast cancer patients with local recurrences were treated with intra-arterial infusions of 8 mg/m2 mitomycin C given at 3-week intervals. The mean time interval between radiotherapy and intra-arterial chemotherapy was 38 months (range 2-60). In five cases a temporary reduction in tumour size was observed. However, in 3 of the 8 patients severe local pain, starting immediately after the third course of treatment, was followed 4 weeks later by the development of deep necrotic ulcers of the chest wall. These cases are reported here and discussed in relation to the results of previous studies.

Removal of cells from a malignant B-cell line from bone marrow with immunomagnetic beads and with complement and immunoglobulin switch variant mediated cytolysis.

In this report we describe the generation of complement (C') fixing IgG2b CD19 and CD22 monoclonal antibodies (mAbs) by the isolation of immunoglobulin (Ig) class switch variants using a simple and efficient method for the selection of spontaneously mutating hybridomas. The aim of this study was to compare the efficacy of C'-mediated cytolysis vs immunomagnetic (IB) depletion of tumor cells from mixtures of malignant B cells and normal bone marrow. In a clonogenic assay employing the B-cell lines Namalwa and OCI.LY1, we found that the use of immunomagnetic beads warranted a highly efficient tumor cell removal independent of the Ig isotype of the mAbs used. Elimination of up to 4 log was achieved using a cocktail consisting of CD19, CD20, CD22 and CD37 B-cell mAbs. A less efficient killing of 2 log was obtained by C' lysis using IgM mAbs, while only about 1 log tumor cell elimination was obtained using IgG2b mAbs. Immunomagnetic purging, besides being more effective than C'-mediated cytolysis, is easier to handle and more rapid.

Granulocyte colony-stimulating factor improves myelopoiesis and host defense in fulminant intra-abdominal sepsis in rats.

The therapeutic efficacy of granulocyte colony-stimulating factor (G-CSF) was studied in a model of fulminant sepsis in rats. Polymicrobial peritonitis was induced by a 4 mm cecal perforation and 10 micrograms/kg recombinant human G-CSF was given intravenously every 12 h, with the first dose at sepsis induction or 4 h post-induction. Rats were sacrificed at various intervals throughout sepsis to measure levels of neutrophil progenitors in the bone marrow and neutrophils and bacteria in blood and peritoneal fluid. Sepsis gave a sustained neutropenia and bacteremia, but did not affect numbers of blast- or GM-colonies, and only a delayed and moderate proliferation of G-clones was seen. Treatment with G-CSF at sepsis induction improved myelopoiesis by doubling the numbers of GM- and G-progenitors at 12 and 24 h post-induction. Concentrations of neutrophils increased twofold in blood and 5-fold in peritoneal fluid, while bacteria counts in the same compartments declined logarithmically. Mortality was 92% in untreated sepsis and declined to 46% when G-CSF therapy was started at sepsis induction, and to 42% following 4 h delayed therapy.

Immunomagnetic removal of B-lymphoma cells using a novel mono-sized magnetizable polymer bead, M-280, in conjunction with primary IgM and IgG antibodies.

The efficacy of a novel monosized and magnetizable polymer bead, denoted M-280, in immunomagnetic removal of Rael B-lymphoma cells was compared with that of M-450 beads, previously used in bone marrow purging. The M-280 beads which are smaller (diameter 2.8 microns) and contain less iron than the M-450 beads were coated with polyclonal IgG sheep antimouse (SAM) antibody. The two types of immunobeads were equally efficacious when used together with the mouse monoclonal IgG antibodies HH1 or FN1, giving tumor cell depletions of about 3 logs in one cycle of operation. However, when used together with the primary IgM monoclonal antibodies (MoAbs) AB1 or HH2, the new immunobeads were significantly more efficacious than the M-450 immunobeads. To elucidate the underlying mechanism flow cytometric studies and measurements of the binding of the labeled primary MoAbs to the cellular antigens as well as to the immunobeads were carried out. Competition experiments showed that in the case of IgG MoAbs, the SAM beads bind predominantly to the Fc portion, whereas in the case of the IgM MoAbs, the Fab part plays a relatively greater role in the binding. The results indicate that if M-280 immunobeads are used, IgM MoAbs may profitably be included in antibody cocktails together with IgG antibodies in immunomagnetic purging of B-lymphoma cells. They suggest that in the case of cell bound MoAbs, the epitopes on IgG are more accessible to SAM beads than those of surface bound IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

High-dose therapy in patients with Hodgkin's disease: the use of selected CD34(+) cells is as safe as unmanipulated peripheral blood progenitor cells.

Register data suggest that patients with Hodgkin's disease (HD) given high-dose therapy (HDT) with peripheral blood progenitor cells (PBPC) have a less favourable prognosis as compared to those given bone marrow as stem cell support. Since this can be due to infusion of tumour cells contaminating the PBPC grafts, we initiated a feasibility study in which PBPC grafts from HD patients were purged by CD34(+) cell enrichment. Controversy exists about whether the use of CD34(+) enriched stem cells leads to a delayed haematological and immune reconstitution. We compared these parameters, including risk of infections and clinical outcome after HDT, in patients with HD given either selected CD34(+) cells or unmanipulated PBPC as stem cell support. From October 1994 to May 2000, 40 HD patients with primary refractory disease or relapse were treated with HDT and supported with either selected CD34(+) cells (n = 21) or unmanipulated PBPC (n = 19) as stem cell support. All patients had chemosensitive disease at the time of transplantation. A median of 5.8 (range 2.7-20.0) vs 4.5 (range 2.3-17.6) x 10(6) CD34(+) cells per kilo were reinfused in the CD34(+) group and PBPC group, respectively. No difference was observed between the two groups with regard to time to haematological engraftment, reconstitution of B cells, CD56(+) cells and T cells at 3 and 12 months and infectious episodes after HDT. Two (5%) treatment-related deaths, one in each group, were observed. The overall survival at 4 years was 86% for the CD34(+)group and 74% for the PBPC group with a median follow-up of 37 months (range 1-61) and 46 months (range 4-82), respectively (P = 0.9). The results of this study demonstrate that the use of CD34(+) cells is safe and has no adverse effects either with respect to haematological, immune reconstitution or to infections after HDT.

Combination chemotherapy with mitoguazon, ifosfamide, MTX, etoposide (MIME) and G-CSF can efficiently mobilize PBPC in patients with Hodgkin's and non-Hodgkin's lymphoma.

Many centers use CY and G-CSF to mobilize PBPC. In this study we explored whether a standard chemotherapy regimen consisting of mitoguazon, ifosfamide, MTX and etoposide (MIME) combined with G-CSF was capable of mobilizing PBPC in lymphoma patients. Twelve patients with Hodgkin's disease (HD) and 38 patients with non-Hodgkin's lymphoma (NHL) were mobilized with MIME/G-CSF. Most patients were heavily treated with different chemotherapy regimens receiving a median of 11 cycles (range 3 to 20) of chemotherapy prior to mobilization. It was found that the optimal time of PBPC harvest was at days 12 and 13 after initiating the mobilization regimen. The median number of collected CD34+ cells per kg body weight was 7.1 x 10(6) (range 0.5-26.2). More than 2.0 x 10(6) CD34+ cells/kg were achieved in 69% of the patients after one apheresis. When additional cycles of apheresis were done, only 6% failed to harvest this number of CD34+ cells. There was a statistically significant inverse correlation between the number of prior chemotherapy cycles and CD34+ cell yield (P = 0.003). No such association was found between CD34+ cell yield and prior radiotherapy. When MIME/G-CSF was compared with Dexa-BEAM/G-CSF, it was found that MIME/G-CSF tended to be more efficient in mobilizing PBPC in spite of being less myelotoxic. All patients transplanted with MIME/G-CSF mobilized PBPC had fast and sustained engraftment. These results demonstrate that an ordinary salvage chemotherapy regimen, such as MIME combined with G-CSF can be successfully used to mobilize PBPC.

Persistent changes in the immune system 4-10 years after ABMT.

The aim of the present study was to investigate whether the early changes in the immune system observed after ABMT would persist over years. Eighty-five patients with malignant lymphoma were treated with ABMT in Norway from 1987 until 1993. Of the 46 patients in CR by 1997, 36 were enrolled in our study. Median time from ABMT was 5 years (4-10 years). Immunophenotyping showed an increase in the median number of B cells (0.35 x 109/l in patients vs 0.28 x 109/l in controls), and a decrease in T cells (1.08 vs 1.35 x 109/l). Furthermore, a lower median count of CD4+ T cells (0.54 x 109/l in patients vs0.87 x 109/l in controls) resulted in reduced CD4/CD8 ratios (0.8 in patients vs 1.6 in controls). The subgroup of CD4+ T cells expressing the 'naive' phenotype CD45RA was 19.5% in patients vs 38% in controls. In contrast, the fraction expressing the 'memory' phenotype CD45RO was higher in the ABMT group (76% vs 54%). When stimulated, larger fractions of CD3+CD4+ cells in patients produced IFN-gamma (32% vs 16%) or IL-4 (7% vs 1%) compared to controls; thus a differentiation into the functionally separate subgroups Th1 and Th2, with a dominant Th2 response. Our data further suggest that the decrease in CD4+ T cell counts and the imbalance between CD45RA+ and CD45RO+ subsets persists 4-10 years after ABMT.

Immunomagnetic removal of B-lymphoma cells from human bone marrow: a procedure for clinical use.

B-lymphoma cells were purged from human bone marrow by incubating the cell suspension with a cocktail of three different pan-B cell mouse IgG1 monoclonal antibodies, and then with immunobeads charged with sheep anti-mouse antibody, followed by magnetic separation. The primary antibodies used, HD37 (CD19), HD6 (CD22), and HH1 (CD37), bind to a very high percentage of the cells in non-Hodgkin's lymphomas of poor prognosis. The secondary antibody is directed against the Fc portion of the IgG antibodies. In model experiments Burkitt's lymphoma cells (Rael) were admixed to mononuclear bone marrow cells in the ratio 1/9. With a ratio of immunobeads/total antibody-binding B cells of 50/1 in a first treatment cycle and repeating the procedure with the same number of beads in a second cycle, a tumor cell depletion of more than 5 logs was achieved, as judged by a clonogenic assay. The concomitant reduction of CFU-GM and CFU-GEMM was about 20%. The purging procedure has been scaled up to clinical use. Equipment suitable for purging patients' marrow specimens, employing standard transfusion facilities, is described. With this equipment the efficacy of tumor cell removal was the same as in the model experiments, and the whole magnetic separation could be completed in 2 hours.