ALBERT

Description: Allogeneic stem cell transplantation can be successfully applied in the treatment of hematological malignancies and relies on the graft versus leukemia (GVL) effect mediated by donor T cells directed against minor histocompatibility antigens (mHag) selectively expressed on malignant hematopoietic cells of the patient. However, due to insufficient in-vivo priming of donor T cells the GVL response may not be adequately initiated or amplified. Vaccination strategies using immunogenic peptides derived from hematopoeisis specific mHag like HA-1 may form a strategy to initiate or boost the in-vivo GVL response. However, it has been reported that repetitive vaccination with HLA-class I binding 9-mer peptides can lead to the induction of T cell anergy. We hypothesized that repetitive strong priming of the mHag specific T cells may also lead to prolonged downregulation of the T cell receptor (TCR) resulting in inability of the T cells to subsequently attack tumor cells expressing the mHag, allowing tumor escape despite the presence of potentially effective T cells. We tested this hypothesis in an in-vitro model using CFSE-labeled HA-1+ CD34+ chronic myeloid leukemia (CML) cells as target/stimulator cells, and HA-1 specific T cells as effector cells. In previous studies we have demonstrated the resistance of a small population of quiescent CML stem cells to all high avidity T cells, allowing the subsequent outgrowth of malignant progeny from this population. To mimick a peptide vaccination strategy, we loaded CD34+ CML cells from an HA-1+ patient with various concentrations (E-12-E-6M) of the 9-mer HA-1 peptide, and investigated the direct and residual functional cytotoxic capacity of an HA-1 specific CD8+ T cell clone. In accordance with our previous results, we observed complete deletion of all proliferating CML precursor cells after 24–48 hours of exposure to the CTLs, whereas a small subpopulation of quiescent CD34+ cells was resistant to T cell attack. The exogenous peptide loading resulted in more rapid lysis and also attack of part of the quiescent stem cell population. However, in the next days malignant progeny was formed from the quiescent stem cell population in the conditions of high peptide stimulation despite the continuous presence of the T cells, suggesting impaired residual cytotoxic function of these T cells. Therefore, we analyzed the level of TCR downregulation after exposure to the HA-1 positive CML CD34+ cells in the absence or presence of E-12-E-6 M HA-1 peptide loaded to the target cells. We observed strong dose-dependent TCR downregulation as measured by specific tetramer staining (20%–78% decrease in fluorescence intensity after 24 hours of exposure to targets loaded with 0-E-6M HA-1 peptide). At high peptide concentrations it took 6–9 days before proper functional TCR expression could be again demonstrated. In conclusion, we here demonstrate that high affinity T cells show a prolonged TCR downregulation after vigorous stimulation by peptide loaded target cells. In this period the T cells showed a dramatic loss of function and allowed the outgrowth of a leukemic subpopulation expressing the HA-1 antigen. Milder vaccination strategies using longer peptides requiring uptake and processing by the target cells may lead to expression of more physiological levels of the mHag and less vigorous priming of the mHag specific T cells, thereby preserving their functional capacity and responsiveness.

Description: We perform reduced intensity allogeneic stem cell transplantation (SCT) with HLA identical sibling and matched unrelated donors using a conditioning regimen consisting of fludarabine, busulphan and ATG combined with alemtuzumab added to the stem cell graft for extensive donor T cell depletion. With this regimen hardly any acute and chronic graft versus host disease (GVHD) are observed after SCT and mixed chimerism is induced in most patients (Barge et al, Exp Hematol 2003). In patients transplanted for malignant disease, donor lymphocytes (DLI) are administered in escalating doses from 6 to 9 months to convert mixed into complete donor chimerism and to induce graft versus tumor (GVT) responses. We report results of chimerism analysis, clinical antitumor response and GVHD after DLI. 41 patients (median age 54 years, range 37–65) transplanted with stem cells from sibling donors (29) or matched unrelated donors (12) for various malignancies received DLI with a median dose of 5 × 106 CD3+ cells/kg (range 1–5) at a median time point of 7 months after SCT (range 5.1–15). After DLI administration 35 of 38 evaluable patients (92%) developed an immune response as defined as a major decrease in patient chimerism. In 26 patients full donor chimerism was achieved, in 9 patients a low patient signal remained present (1–2%). Patient chimerism decreased from median 12% (range 1 to 86%) to 0% (range 0–2%). In most patients conversion occurred after the first DLI, in 4 patients after the second and in 1 patient after the third DLI. In 31 of 38 patients measurable disease was present before DLI. In 22 of these patients conversion from mixed to full donor chimerism coincided with clinical GVT responses. Seven patients with active myeloid disease all achieved complete remission (4 AML/MDS patients with 8–65% blasts, 2 patients with progressive CML and 1 with active CMMOL). All these patients are still in CR after a median time of 22 months. In 8 myeloma patients with measurable disease 5 complete and 1 partial response were observed. However, most responding myeloma patients subsequently developed bone or extramedullary relapses without evidence of bone marrow involvement. In 13 patients with lymphoid disease 9 responses were observed. One patient with an immunocytoma and one with T-PLL achieved CR. In 7 CLL patients 3 CR and 1 PR were observed, notably two CR occurred in patients with massive bone marrow involvement. Two of four patients with aggressive NHL showed a CR to DLI in combination with rituximab and one patient a PR to DLI. Although no regression of tumor was observed in three patients with renal cell carcinoma after DLI, disease progression was halted for several years. Grade 3–4 acute GVHD developed in 22% of patients, grade 1–2 in 39%. Limited or extensive chronic GVHD was observed in 30 and 23% of patients, respectively. GVHD responded to therapy in most patients, only in 9% of patients chronic GVHD did not resolve. Mortality due to acute and chronic GVHD was 10%. In conclusion, after T cell depleted reduced intensity SCT a state of mixed chimerism is induced in most patients, which can be converted into full donor chimerism with DLI in more than 90% of patients. Conversion to full donor chimerism is accompanied with clinical GVT responses in a high percentage of patients with acceptable GVHD.

Description: T-cell tolerance is mandatory for major histocompatibility complex (MHC)-mismatched stem-cell transplantation without cytoreduction. Here, we used a cytotoxicity assay based on the infusion of differentially carboxyfluorescein succinimidyl ester (CFSE)-labeled syngeneic and donor splenocytes to determine the survival of donor cells in vivo. In vivo cytotoxicity data showed that treatment with anti-CD40 ligand monoclonal antibody in combination with a low dose of MHC-mismatched bone marrow cells was sufficient to induce T-cell tolerance. However, CFSE-labeled donor cells were still eliminated. A similar elimination pattern was observed in T-cell and natural killer T-cell (NKT-cell)-deficient mice, suggesting the involvement of natural killer (NK) cells. Indeed, in vivo NK-cell depletion resulted in a prolonged survival of CFSE-labeled donor cells, confirming the role of NK cells in this process. Transplantation of a megadose of MHC-mismatched bone marrow cells was required for a complete survival of CFSE-labeled donor cells. This NK-cell tolerance was donor specific and was associated with mixed chimerism. Additional NK-cell depletion significantly enhanced engraftment and allowed long-term chimerism after transplantation of a relatively low dose of donor bone marrow cells. These data demonstrate the importance of NK cells in the rejection of MHC-mismatched hematopoietic cells and may explain the high numbers of bone marrow cells required for transplantation over MHC barriers. (Blood. 2005;106:2215-2220)

Description: Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8+ T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7–restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1) gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1 gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1–specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

Description: Minor histocompatibility antigens (mHags) play an important role in both graft-versus-tumor effects and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. We applied biochemical techniques and mass spectrometry to identify the peptide recognized by a dominant tumor-reactive donor T-cell reactivity isolated from a patient with relapsed multiple myeloma who underwent transplantation and entered complete remission after donor lymphocyte infusion. A frequently occurring single nucleotide polymorphism in the human ATP-dependent interferon-responsive (ADIR) gene was found to encode the epitope we designated LB-ADIR-1F. Although gene expression could be found in cells from hematopoietic as well as nonhematopoietic tissues, the patient suffered from only mild acute GVHD despite high percentages of circulating LB-ADIR-1F–specific T cells. Differential recognition of nonhematopoietic cell types and resting hematopoietic cells as compared with activated B cells, T cells, and tumor cells was demonstrated, illustrating variable LB-ADIR-1F expression depending on the cellular activation state. In conclusion, the novel mHag LB-ADIR-1F may be a suitable target for cellular immunotherapy when applied under controlled circumstances.

Description: Abstract 4475 Allogeneic stem cell transplantation (alloSCT) is frequently complicated by life-threatening graft versus host disease (GVHD). Previous studies demonstrated that T cell depletion (TCD) of the graft significantly decreases the incidence and severity of GVHD, and is associated with a higher percentage of patients with mixed chimerism (MC). In most studies chimerism analysis is performed on the total bone marrow (BM) leukocyte fraction, and changes in chimerism are related to engraftment. In this study we investigated whether MC in the total BM leukocyte fraction truly reflects engraftment or if it is influenced by survival and expansion of donor and recipient residual mature T cells, and whether hematopoietic lineage specific chimerism analysis is therefore a better method to determine engraftment. It is likely that chimerism analysis of the stem cell compartment is best reflected in peripheral blood (PB) in those cells that are continuously produced and short lived, such as monocytes and granulocytes, and therefore PB myeloid chimerism primarily reflects engraftment. In contrast, previous studies have shown by T cell receptor excision circle analysis that T cell neogenesis is virtually absent in the first 6 months after alloSCT, and that predominantly memory T cells are present in PB and BM. Therefore, we hypothesize that MC of these long lived T cells merely reflects survival and expansion of recipient and donor residual T cells. Since the life span of B and NK cells is longer than myeloid cells, but shorter than T cells, we anticipate that in the first 6 months after alloSCT, B and NK cell chimerism reflects a combination of survival and neogenesis. To analyze these hypotheses we performed hematopoietic lineage specific chimerism analysis on PB cells of 22 patients (median age 52 years, range 23-73, 11 males) receiving a TCD alloSCT between June and November 2008 after a myeloablative (n=11) or non myeloablative conditioning regimen (n=11) for AML, ALL, high risk MDS, multiple myeloma, CML, CLL or NHL. At intervals of 6 weeks PB was collected, and monocytes, granulocytes, B and NK cells, CD4+ and CD8+ T cells were sorted. The total leukocyte fraction was obtained by erythrocyte lysis of BM. DNA was isolated to perform chimerism analysis using short tandem repeats - PCR. Our results show that in the BM leukocyte fraction 47% of the patients were MC at 3 months after alloSCT, with a median frequency of patient cells of 4%. However, of the patients with MC in the total leukocyte fraction, 67% was complete chimeric in the myeloid subsets and MC in the T cell compartment. In the PB myeloid subsets (monocytes and granulocytes) less than 28% of the patients were MC during the first 6 months after alloSCT with a median frequency of patient cells less than 5%. In the B and NK cell subsets, at most time points more patients were MC (7-43%) with higher frequencies of patient cells (2-14%) compared to the myeloid subsets. The CD4 and CD8 T cell subsets showed the highest frequencies of MC in numbers of patients (31-61%) as well as the highest MC frequencies of patient cells (13-80%). Phenotypic analysis of the T cell compartment showed that 98% of the CD4 and CD8 T cells were memory cells during the first 6 months after alloSCT. Preliminary data indicate that the median percentage of donor derived T cells increased during the first 6 months after alloSCT, correlating with development of mild GVHD, suggesting that T cell chimerism is influenced by immunogenic triggers. In conclusion, these results illustrate that for engraftment and neogenesis of donor hematopoiesis, myeloid chimerism analysis provides more accurate information than total BM leukocyte chimerism analysis, since the results are greatly influenced by T cell chimerism. Since almost all T cells were memory cells within the first 6 months after alloSCT, T cell chimerism analysis reflects survival and expansion of mature donor as well as recipient T cells, and can therefore not be used to measure engraftment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4084 Poster Board III-1019 Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T-cells recognizing minor histocompatibility antigens (mHags) encoded by single nucleotide polymorphisms (SNPs) on malignant cells of the recipient. Donor T-cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues. The aim of this study was to investigate the specificity and diversity of mHags recognized by T-cells in Graft-versus-Leukemia (GvL) reactivity. Activated (HLA-DR+) CD8+ and CD4+ T-cell clones were isolated from a patient successfully treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. GvL reactivity in this patient was accompanied with mild GvHD of the skin. Isolated T-cell clones were shown to recognize 13 different mHags. CD8+ T-cell clones were specific for HA-1 and HA-2 in HLA-A*0201, one unknown mHag in B*0801 and 4 unknown mHags in B*4001. CD4+ T-cell clones were specific for one unknown mHag in HLA-DQ and 5 unknown mHags in DR. By screening plasmid (class I) and bacteria (class II) cDNA libraries, we identified a mHag in HLA-DQ encoded by the PI4K2B gene (Griffioen et al., PNAS 2008), 4 mHags in HLA-DR encoded by the PTK2B, MR-1, LY75 and MTHFD1 genes (Stumpf et al., Blood 2009) and a mHag in B*4001 encoded by the TRIP10 gene. For the 3 T cell clones recognizing unknown mHags in B*4001, we performed Whole Genome Assocation scanning (WGAs). A panel of 60 EBV-LCL was retrovirally-transduced with B*4001 and tested for T-cell recognition. In parallel, genomic DNA was isolated and more than one million single nucleotide polymorphisms (SNPs) were determined by the Illumina beadchip array. Statistical analysis revealed significant association between T-cell recognition of EBV-LCL and the presence of coding SNPs in the SON DNA-binding protein and SWAP-70 genes. To get more insight into the role and potential use of the mHags in GvL reactivity and GvHD, all T-cell clones were analyzed in detail for reactivity against hematopoietic and non-hematopoietic cells. Hematopoietic cells included peripheral blood cells (monocytes, B-cells and T-cells), professional antigen presenting cells (APC) and leukemic cells (CML, ALL and AML). All CD8+ T-cell clones recognized (subsets of) peripheral blood cells as well as CML cells, except for the T-cell clone for TRIP10. Recognition of (subsets of) peripheral blood cells was also observed for all CD4+ T-cell clones, but CML cells were differentially recognized. CML cells were strongly recognized by the T-cell clones for MTHFD1 and the unknown mHag in HLA-DR, whereas no or low reactivity was observed for all other CD4+ T-cell clones. All CD8+ and CD4+ T-cell clones strongly recognized professional APC, including monocyte-derived dendritic cells and in vitro differentiated CML cells with APC phenotype. All T-cell clones were also capable of recognizing AML and ALL, except for the T-cell clone for TRIP10, which showed restricted recognition of AML-M4 and -M5 of monocytic origin. As non-hematopoietic cells, patient-derived fibroblasts were cultured with and without IFN-γ and tested for T-cell recognition. In the absence of IFN-γ, all T-cell clones failed to recognize fibroblasts, except for the T-cell clone for the unknown mHag in B*0801. After treatment with IFN-γ, additional reactivity was observed for the T-cell clones for SON DNA-binding protein and the unknown mHag in B*4001. Our data showed the specificity and diversity of mHags recognized by T-cells induced in a patient successfully treated with DLI for relapsed CML. The T-cell response was directed against 13 different mHags, of which 10 mHags in HLA class I and class II have now been identified by different techniques. Detailed analysis of T-cell recognition of hematopoietic and non-hematopoietic cells provides evidence that the mHags played different roles in the onset and execution of GvL and GvHD. Moreover, only one of the 10 identified mHags was expressed on fibroblasts after treatment with IFN-γ, indicating the characterization of mHags with potential relevance for T-cell based immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: To study the biology of ALL, primary leukemic cells were cultured in a longterm serum-free culture system. In 10 out of 25 primary precursor B-ALL cells we succeeded to establish ALL cell lines, phenotypically identical to their original clone. The cultured leukemic cells proliferated for more than two years with a constant doubling time of 2.0 ± 0.3 days, but only in cultures of moderate to high densities, suggesting the production of an autocrine proliferation inducing factor. To study the possible autocrine production of a proliferation inducing factor low cell concentrations (1,000 cells/ml/well) were cultured in the absence or presence of autologous conditioned medium(CM, 20%v/v). In the absence of CM, no proliferation was observed as determined by 3H-thymidine uptake as well as in a limiting dilution assay in 9 out of 10 ALL samples. In the presence of CM, proliferation was induced in all ALL cell lines. CM generated from a variety of other unrelated cell sources, as well as CM derived from lysed cells also induced identical proliferation in all ALL samples demonstrating that the samples proliferated and responded to a factor present in all cell populations.To characterize this autocrine factor CM was prepared from ALL cell lysates. Chemical analysis of this CM revealed that the factor was a heat stable molecule with a molecular size less than 500 Dalton. The factor was further purified on a carbograph column and finally on a Superdex Peptide size-exclusion column. The active fractions were analysed by NMR spectroscopy and the pattern of the spectrum strongly suggested the presence of a heterocycle purine. CE-MS analysis confirmed this observation by detecting the presence of a molecule with a mass of 136.05, identical to the theoretical mass of Hypoxanthine. By MS/MS fragmentation analysis of both the isolated compound and synthetic Hypoxanthine the compounds were found to be identical. Addition of the chemically synthetised Hypoxanthine (0.45 μM) to low cell concentrations of ALL cell cultures as well as to cultures of primary ALL cells induced proliferation comparable to cell cultures in the presence of CM. Proliferation of ALL cells could also be induced by Deoxyinosine or Inosine comparable to Hypoxanthine but only moderate to low proliferation was observed after addition of Adenine or Adenosine and no proliferation in the presence of Guanine, Guanosine or Inosinemonophosphate. In conclusion, we developed a culture system allowing identification of the requirements of ALL for proliferation in vitro to study the biology of these leukemic cells. Using this culture system, we illustrated that ALL cells appeared to be highly dependent on an exogenous source of Hypoxanthine or its metabolic component. This dependency may open new perspectives in the development of treatment for ALL, and may result in a more specific control of the different metabolic pathways of purines in malignant cells.

Description: HLA disparity between patient and donor increases the risk and the severity of graft versus host disease (GVHD). In order to safely administer HLA mismatched stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), it is important to understand the mechanisms underlying GVHD in HLA mismatched settings. The degree of patient chimerism, especially of the antigen presenting cells, at the time of DLI has been shown to influence the development of GVHD. This is suggested by the correlation between the time after SCT of DLI administration and the occurrence of GVHD. In mouse models the underlying role of the presence of host APCs in the occurrence of GVHD has been demonstrated in both MHC matched and mismatched models. In this study, the alloimmune response in a patient suffering from GVHD after an HLA class I mismatched DLI was characterized. The patient received a T-cell depleted SCT followed by two DLIs from the same donor. The SCT and first DLI, administered for mixed chimerism, did not result in any signs of GVHD. In contrast, the second DLI led to severe GVHD. Patient chimerism in different cell subsets present prior to the two DLIs showed no significant difference in patient T-cell chimerism and there were no patient B-cell, monocytes or dendritic cells present prior to each DLI. However, a significant difference in the presence of patient leukemic blasts was found at the time of the two DLIs. Prior to the first DLI no leukemic cells could be demonstrated, whereas 9% leukemic blasts were present in bone marrow prior to the second DLI. We showed that the leukemic blasts expressed HLA class II. Correlating with the absence of GVHD, the first DLI did not lead to activation of donor T-cells while the second DLI led to severe GVHD accompanied by a vigorous CD8 and CD4 T-cell response. Activated donor derived CD8 and CD4 T-cells from this response were isolated by single cell sort based on HLA-DR expression. Expanded clones were characterized for alloreactivity, HLA restriction, specificity and clonality. We showed that 50 of the 53 expanded CD8 T-cells were alloreactive and that all alloreactive CD8 T-cells were restricted to the mismatched HLA-A2. Additionally, by sequencing the T-cell receptor (TCR) Vβ CDR3 region, we showed that all alloreactive CD8 clones expressed a different CDR3 region, illustrating polyclonality. Of the 88 expanded CD4 clones, 21 were alloreactive and the majority of the alloreactive CD4 T-cell clones were directed against an HLA-A2 derived peptide presented in HLA-DR1. By Vβ analysis we showed that the CD4 T-cell response was also polyclonal. Finally, to investigate the role of the leukemic blasts in the initiation of the GVHD, the capacity of the different cell subsets present prior to DLI 1 or 2 to stimulate donor CD8 and CD4 T-cells was analyzed. This showed that only the leukemic blasts were able to highly stimulate both the CD8 and the CD4 T-cells. Our data indicate that the leukemic blasts by acting as host APCs triggered a combined CD4 and CD8 allo-immune response directed against the mismatched HLA, and thus initiated not only anti-leukemic reactivity but also lethal GVHD.

Description: Acute lymphoblastic leukemia (ALL) in adults has a poor prognosis, despite intensive chemotherapy or allogeneic stem cell transplantation. Further treatment intensification is limited by treatment-related toxicity. Monoclonal antibodies display a relatively favorable toxicity profile. Rituximab recognizing CD20, and alemtuzumab recognizing CD52, have shown clinical activity in hematological malignancies. Precursor-B ALL (preB-ALL) may express CD20 and/or CD52. However, little is known on the in vivo activity of these antibodies in preB-ALL. We evaluated the activity of rituximab and alemtuzumab on preB-ALL in a preclinical model. We first evaluated the expression of CD20 and CD52 on primary preB-ALL cells. For this, 18 primary samples were randomly selected, stained with rituximab or alemtuzumab, and secondary anti-human-Ig antibodies. Mean fluorescence intensity (MFI) was analyzed by flow cytometry. Ten samples expressed CD20 (56%, median MFI 102, range 97 to 951), eight samples did not (44%, median MFI: 6.9, range 6.3 to 8.3). Twelve samples expressed CD52 (67%, median MFI: 324, range 111–633), six samples did not (33%, median MFI: 8.1, range 6.5–8.8). All CD20 positive samples expressed CD52. Subsequently, we evaluated the in vivo activity of rituximab and alemtuzumab, alone and in combination.We selected 2 primary ALL (coded COA and VBK) that expressed CD20 and CD52. NOD/scid mice were engrafted with COA or VBK cells. Three weeks after inoculation, treatment was started by daily injection of either 250 ug rituximab or 250 ug alemtuzumab, or 250 ug of both antibodies. Throughout the treatment period, therapeutic concentrations of antibody were measured in serum of treated animals (rituximab: median 399 ug/mL, range 200–739 ug/mL, alemtuzumab: median 152ug/mL, range 51–199 ug/mL). After four weeks of treatment, animals were sacrificed. Peripheral blood (PB), spleen (SPL) and bone marrow (BM) were isolated and analyzed by flow cytometry. In animals treated with single antibody, leukemic cells persisted though at lower levels when compared to control animals, suggesting limited activity. After engraftment of COA leukemia, in rituximab treated animals 1, 6 and 59%, in alemtuzumab treated animals 1, 8 and 19%, and in control treated animals 37, 80 and 70% leukemic cells were present in PB, SPL and BM, respectively. After engraftment of VKB leukemia in rituximab treated animals 42, 45 and 75%, in alemtuzumab treated animals 2, 2 and 24%, and control treated animals 50, 59 and 86% leukemic cells were found in PB, SPL and BM, respectively. In contrast, combination treatment with rituximab and alemtuzumab induced complete remissions in all of 7 COA-engrafted, and in 2 of 3 VBK-engrafted animals. The VBK-engrafted animal that did not reach a remission showed less than 1% leukemic cells in BM only. Cells that persisted during single antibody treatment were analyzed by flow cytometry. In all animals treated with rituximab or alemtuzumab alone, the persisting leukemic cells did not have therapeutic antibody present on their surface, nor could the targeted antigen be detected. These observations suggested that persistence of leukemic cells was due to loss of the targeted antigen. In the control animals no change of the expression of CD20 or CD52 during leukemic outgrowth was observed. These results suggest that combined treatment with rituximab and alemtuzumab may be far more effective than treatment with either agent alone in a significant number of preB-ALL patients.