Cellular distribution of a B-cell specific surface antigen (gp54) detected by a monoclonal antibody (anti-BL4)

A monoclonal antibody (anti-BL4) recognizing a previously characterized Mr 54,000 glycoprotein (gp54) was developed by immunizing BALB/c mice with cells from a precursor B-cell line (Josh-7). In normal individuals, this antigenic molecule was present on tonsillar B-cells (60-80%) and on a fraction of peripheral blood B-cells (5-25%). BL4 (gp54) expression was investigated in 186 patients with a variety of hematological malignancies using indirect immunofluorescence and flow cytometric analysis. Twenty-six of 37 cases of B-cell chronic lymphocytic leukemia (CLL) and 18 of 33 cases of B-cell non-Hodgkin's lymphoma were BL4 positive. Surface expression of BL4 on reactive cases of CLL and non-Hodgkin's lymphoma was brighter than those of B1, B2, and B4, BL4 positive CLL cases expressed a higher proportion of mouse rosette forming cells and Leu-1 positive cells than the BL4 negative subgroup and were not associated with elevated serum immunoglobulin levels. Four of 7 BL4 negative CLL cases were associated with increased serum levels of immunoglobulin M. Lymphoblasts from 14 of 14 cases of non-T acute lymphoblastic leukemia and 3 of 3 pre-B lymphoid blast crisis of chronic myeloid leukemia were BL4 negative. Neoplastic cells from 2 of 3 cases of Waldenstrom's macroglobulinemia and 4 of 7 cases of hairy cell leukemia were BL4 reactive. None of 7 cases of multiple myeloma and plasma cell leukemia were BL4 positive. All 11 T acute lymphoblastic leukemia cases, 6 other T-cell malignancies, 5 cases of Hodgkin's disease, 51 cases of acute nonlymphocytic leukemia, and 9 cases of chronic myeloid leukemia in chronic phase thus far studied were BL4 negative. An in vitro induction experiment using phorbol ester on a case of B-CLL demonstrated disappearance of BL4 accompanied with further B-cell differentiation. Our study further substantiates the previous finding that gp54 is a differentiation antigen restricted to the B-cell lineage and expressed during the intermediate stage of B-cell ontogeny.     

Phenotypic characterization of 'non-T, non-B' acute lymphoblastic leukemia by a new panel (BL) of monoclonal antibodies

Peripheral blood and/or bone marrow leukemic cell suspensions from 49 patients with 'non-T, non-B' acute lymphoblastic leukemia (ALL) were analysed by flow cytometry using a new panel of four monoclonal antibodies. Anti-BL1 and anti-BL2 originating from NALM-6 and B35M lymphoblastoid cell lines, respectively. These antibodies recognize B-cell differentiation antigens: a heat stable non-immunoprecipitable antigenic determinant, and a 68 000 daltons glycoprotein molecule, respectively. BL5 and BL6 were derived by immunization with the promyelocytic cell line HL-60, recognizing antigens present on early hematopoietic cells: an 85 000 daltons MW glycoprotein (Pro-Im 1) and a heat stable antigen (Pro-Im2), respectively. All ALL patients studied had L1 or L2 morphology by the FAB classification and a blast count exceeding 50 per cent. There were 25 males and 24 females. Median age was 8 years (range 1-67 years). Thirty-nine cases were studied at initial presentation and 10 at relapse. Cells from 46/49 cases expressed BL2 and/or BL1, but were not reactive with BL5 or BL6. Three of 49 cases did not express BL1 or BL2. However, a small percentage of blasts from one case was positive for BL5 (13 per cent) and the other 2 cases were reactive with BL6 (20 per cent and 36 per cent, respectively). These were one adult and 2 pediatric patients that had other ALL markers and achieved a complete remission with appropriate ALL therapy. One of the BL6+ cases relapsed after 19 months with a change in phenotype to BL1+ BL2+ BL5- BL6-. This analysis shows that the majority of 'non-T, non-B' ALL's do express B-cell associated antigens (BL1/BL2) argumentative of their B-cell origin. A small subgroup does not express such antigens and may arise from a more immature cell, since they expressed antigens on early hematopoietic stem cells.     

Reactivity of acute lymphoblastic leukemia and normal bone marrow cells with the monoclonal anti-B-lymphocyte antibody, anti-Y 29/55

Malignant lymphocyte populations in peripheral blood of patients with B-cell chronic lymphocytic leukemia, leukemic variant of B-cell non-Hodgkin's lymphoma, and hairy cell leukemia can be characterized by the use of a monoclonal murine antibody (anti-Y 29/55) which is directed against a cell membrane component normally confined to the sessile nonrecirculating cells of the B-lymphocyte population in lymphoid tissues. The present report describes the reactivity of the anti-Y 29/55 antibody with bone marrow cells obtained from children with acute lymphoblastic leukemia using an indirect immunofluorescence method in combination with morphological and cytokinetic studies. In 25 patients (acute lymphoblastic leukemia subtype: 14 common; 4 pre-B-cell; 4 null; and 3 T-cell), a maximum of 2% of cells (small lymphocytes) were stained. One patient presented with blasts exhibiting cytoplasmic and surface immunoglobulin M (IgM) (pre-B-B-cell acute lymphoblastic leukemia). About 11% of this patient's blast cells showed a positive reaction with anti-Y 29/55. They could not be differentiated by morphological criteria from the anti-Y 29/55-negative blast cell population. In another patient with pre-B-B-cell acute lymphoblastic leukemia, only 1% of anti-Y 29/55-positive cells was found. In bone marrow of children with relative lymphocytosis, 1.4 to 8.7% of mononuclear cells reacted with anti-Y 29/55. Morphologically, these cells were small lymphocytes and predominantly expressed surface IgM. In two of these children, a further subdivision of bone marrow cells could be achieved by combining anti-Y 29/55 and cytoplasmic IgM reactivity with [3H]thymidine pulse labeling. These studies revealed that the actively proliferating, normal pre-B-cell population was anti-Y 29/55-nonreactive, whereas a nonproliferating population of anti-Y 29/55-reactive, cytoplasmic IgM-positive cells probably represented B-cells with surface immunoglobulin M reacting when cytoplasmic IgM was assessed. We conclude that the reactivity of the monoclonal anti-B-cell antibody (anti-Y 29/55) is restricted to surface immunoglobulin-positive bone marrow cells and that neither leukemic or normal pre-B-cells nor common, null-cell, or T-cell acute lymphoblastic leukemia blasts react.     

Novel Therapeutic Strategies in Adult Acute Lymphoblastic Leukemia – A Focus on Emerging Monoclonal Antibodies

The outcomes in adult B-cell acute lymphoblastic leukemia (ALL) remain inferior to those achieved in pediatric populations. Targeted therapy with monoclonal antibodies may improve outcomes in adult B-cell ALL without significant additive toxicity. Rituximab is the best known monoclonal antibody and is routinely used in combination chemo-immunotherapy for treatment of adult B-cell ALL and Burkitts leukemia. A number of other monoclonal antibodies are currently under investigation for treatment of adult B-cell ALL including unconjugated antibodies (eg., ofatumumab, alemtuzumab and epratuzumab), antibodies conjugated to cytotoxic agents (eg., inotuzumab ozogamycin and SAR3419), antibodies conjugated to toxins such Pseudomonas or Diptheria toxins (eg., BL22 and moxetumomab pasudotox), and T-cell engaging bi-specific antibodies that redirect cytotoxic T lymphocytes to lyse target ALL cells (eg., blinatumomab). In this article we review the therapeutic implications, current status and results of monoclonal antibody-based therapy in adult B-cell ALL.     

Antibody fusion proteins: anti-CD22 recombinant immunotoxin moxetumomab pasudotox

Recombinant immunotoxins are fusion proteins that contain the cytotoxic portion of a protein toxin fused to the Fv portion of an antibody. The Fv binds to an antigen on a target cell and brings the toxin into the cell interior, where it arrests protein synthesis and initiates the apoptotic cascade. Moxetumomab pasudotox, previously called HA22 or CAT-8015, is a recombinant immunotoxin composed of the Fv fragment of an anti-CD22 monoclonal antibody fused to a 38-kDa fragment of Pseudomonas exotoxin A, called PE38. Moxetumomab pasudotox is an improved, more active form of a predecessor recombinant immunotoxin, BL22 (also called CAT-3888), which produced complete remission in relapsed/refractory hairy cell leukemia (HCL), but it had a <20% response rate in chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL), diseases in which the leukemic cells contain much lower numbers of CD22 target sites. Compared with BL22, moxetumomab pasudotox is up to 50-fold more active on lymphoma cell lines and leukemic cells from patients with CLL and HCL. A phase I trial was recently completed in HCL patients, who achieved response rates similar to those obtained with BL22 but without dose-limiting toxicity. In addition to further testing in HCL, moxetumomab pasudotox is being evaluated in phase I trials in patients with CLL, B-cell lymphomas, and childhood ALL. Moreover, protein engineering is being used to increase its activity, decrease nonspecific side effects, and remove B-cell epitopes.     

Monoclonal antibody against a membrane antigen characterizing leukemic human B-lymphocytes

In the diagnosis of non-Hodgkin's lymphomas, the ready characterization of the neoplastic cell lineage by analysis of cell surface markers is of great importance. We present evidence for the existence of a human B-leukemia-associated antigen recognized by a complement-fixing monoclonal antibody (anti-Y 29/55). A hybridoma was produced by fusing mouse myeloma cells and splenocytes of a mouse immunized against lymphoid cells of a patient with B-cell chronic lymphocytic leukemia. Characterization of anti-Y 29/55-reactive normal and malignant leukocytes was demonstrated by cytolysis and indirect immunofluorescence. This revealed reactivity with an antigen on B-lymphoma cells (11 patients), on leukemic lymphocytes in B-cell chronic lymphocytic leukemia (13 patients), and on malignant cells in hairy-cell leukemia (two patients) but not on leukemic cells of T-cell acute lymphoblastic leukemia (one patient), on T-lymphoma cells (one patient), on cells of acute myeloblastic leukemia (four patients), or of chronic myeloid leukemia (four patients). No specific cytolysis occurred with B- and T-peripheral blood lymphocytes from (a) healthy donors (16 individuals), (b) patient with reactive lymphocytosis (one patient), (c) nonleukemic multiple myeloma (six patients), or (d) Hodgkin's disease (three patients). Surface immunoglobulin-positive, sheep RBC-negative lymphocytes isolated from human spleen (three individuals), tonsils (seven individuals), and lymph nodes (one individual), however, were recognized. It is concluded that leukemic B-cells carry a marker characteristic of nonrecirculating sessile B-lymphocytes.     

68例成人白血病免疫分型特点分析

[目的]分析68例成人白血病免疫分型特点.[方法]采用单克隆双色或三色直接荧光标记,流式细胞仪检测以CD45-SSC辅助设门,分型根据抗体积分系统.并与FAB分型进行比较.[结果](1)62例成人白血病中未分化型占6.45%(4例),变异型占12.9%(8例).其中急性髓细胞白血病(AML)占69.35%(43例),B细胞型急性淋巴细胞白血病(B-ALL)占16.13%(10例),T细胞型急性淋巴细胞白血病(T-ALL)占6.45%(4例),AML/ALL为3.07:1.CD34的阳性率在62例成人急性白血病和6例慢性粒细胞性白血病急变患者中分别为58.06%和83.33%;免疫分型与FAB分型的符合率为83.9%.[结论]在流式细胞仪上采用CD45-SSC设门法多参数分析白血病免疫分型,并利用抗体积分系统诊断标准可为临床诊断提供重要依据.     

Immunologic, morphologic and chromosomal characterization of a cell line (TC78) established from a child with acute lymphoblastic leukemia

We characterized a cell line established from bone marrow cells from a child with acute lymphoblastic leukemia. This cell line, TC78, had lymphoblastic morphology and was cytoplasmic peroxidase and esterase negative. The cells did not have T- or B-cell properties such as E- or EAC-rosette forming ability, reactivity with monoclonal T-cell or B2 antibodies, or immunoglobulin synthesis. We concluded that TC78 was a pre-pre B-cell line based on the following monoclonal antibody staining pattern: BA-I⁺, BA-2⁺, cALLa⁺, Ia⁺, 2H7⁺ and OKB2⁺. Growth in ‘Dickie’ culture and reactivity with 1G10 myeloid antibody suggested coexpression of lymphoid and myeloid characteristics. However, 1G10 expression proved dependent on culture conditions, illustrating one caveat in application of monoclonal antibodies in lineage determination.     

Anti-GD3 monoclonal antibody analysis of childhood T-cell acute lymphoblastic leukemia: detection of a target antigen for antibody-mediated cytolysis

We have demonstrated in previous studies significant quantitative differences in the ganglioside content of leukemia cell membranes within immunological subclasses of acute lymphoblastic leukemia (ALL): The disialoganglioside GD3 (disialolactosylceramide) is increased in lymphoblasts with a T-cell immunophenotype compared to non-T-ALL blasts. Utilizing an indirect immunofluorescence assays with a monoclonal antibody to GD3(R24), pretreatment leukemic lymphoblasts from 80 children with ALL were assayed for GD3 expression. GD3 was observed in 75% of leukemic samples in which lymphoblasts exhibited a T-cell phenotype, whereas none of the 33 non-T-ALL samples tested exhibited GD3. Correlation between the expression of GD3 and various antigenic determinants of T-cell differentiation was restricted to CD2; 75% of CD2-negative T-cell ALL blasts failed to express GD3. Anti-GD3 immunoreactivity to T-ALL samples was not restricted to R24 in that two other monoclonal anti-GD3 antibodies were similarly reactive to T-ALL blasts. In vitro incubation of T-cell lymphoblasts with the anti-GD3 antibodies, R24 and C281 and human serum resulted in significant cytotoxicity, and R24 also mediated antibody-dependent cellular cytotoxicity by normal effector cells. Cytotoxicity was specific for those T-ALL blast cell populations which reacted with anti-GD3 as assessed by immunofluorescence microscopy. Since immunoreactivity with monoclonal antibody to GD3 was exclusively observed in a large population of immunophenotypically defined T-cell leukemic lymphoblasts, these studies suggest a possible immunodiagnostic and immunotherapeutic potential for anti-GD3 monoclonal antibodies in T-cell lymphoblastic malignancies.     

Two mouse monoclonal antibodies detecting two different epitopes of an activated lymphocyte antigen on adult T-cell leukemia cells

Mouse monoclonal antibodies were produced against MT-2 cell line derived from adult T-cell leukemia or human T-cell leukemia virus-rich fraction therefrom. Two IgG1 antibodies, Ta60a and Ta60b, were found to be reactive not only with cell lines derived from adult T-cell leukemia or cutaneous T-cell lymphomas, but also with activated peripheral blood lymphocytes, suggesting the similarity of Ta60 antigen group to Tac antigen which is present on interleukin 2 receptor. Thus, the relationship among these antigens was studied. Two Ta60 antibodies and Tac antibody immunoprecipitated the molecule with almost identical electrophoretic mobility, approximately a Mr 60,000 antigen from [3H]glucosamine-labeled activated peripheral blood lymphocytes or MT-2, MT-1, or ATN-1 cells from adult T-cell leukemia and a Mr 53,000 antigen from HUT-102 cells derived from cutaneous T-cell lymphomas. Further, Tac antibody was found to immunoprecipitate Ta60b molecule on 125I-labeled MT-2 cells by sequential immunoprecipitation, indicating that these two epitopes are on the same molecule. Antibody binding inhibition assays with either 3H-labeled Ta60a or Ta60b antibody demonstrated that Ta60a and Tac are the same epitope, but different from Ta60b. Thus, at least two epitopes were demonstrated to be present on interleukin 2 receptor molecule. However, Ta60b antibody showed almost no blocking effects on proliferation of an interleukin-2-dependent cell line, whereas Ta60a antibody did. Various hematopoietic tumor cells were typed with these two antibodies, but the results with Ta60b antibody were described, because they showed a similar specificity. Ta60b antibody reacted with all adult T-cell leukemia cases, but did not react with T-cell acute lymphoblastic leukemia, lymphoblastic lymphoma, or mature T-cell lymphoma. Interestingly, 3 of 12 acute myeloblastic leukemia and 2 of 5 chronic myelocytic leukemia in blastic crisis showed positive reactions. One-third of B-cell chronic lymphocytic leukemia and B-cell lymphoma as well as a few B-cell lines were also weakly reactive with this antibody. A part of the results with direct tests was confirmed by the absorption tests. The results obtained demonstrated the presence of Ta60b on a certain fraction of malignant hematopoietic cells of other than T-cell origin.     

Production and characterization of a human monoclonal antibody recognizing a new antigen expressed on some lymphoid cells

A cell line secreting a human monoclonal antibody was established by Epstein-Barr virus transforming B cells derived from an enlarged cervical lymph node excised from a patient bearing a carotid body tumor. The reactivity of the monoclonal antibody, designated as mNISP, was tested on various cells and cell lines. An antigen defined by the mNISP was expressed on some Burkitt's lymphoma cell lines and on a non-T non-B acute lymphoblastic leukemia cell line. Furthermore, this antigen was expressed on leukemic cells from 2 of 8 patients with chronic myelocytic leukemia, 2 of 10 patients with acute myeloblastic leukemia, one of 13 patients with acute lymphoblastic leukemia, and two patients with adult T cell leukemia, but it was not expressed on normal T, B and adherent (macrophage) cells. In addition, mNISP reacted with T cells obtained from human T-cell leukemia virus type I carriers. We found that the antigen defined by mNISP was distinct from any previously reported antigen in terms of its pattern of cellular expression and molecular weight, suggesting that mNISP recognizes a new antigen expressed on some lymphoid cells.     

Immunodiagnosis of childhood ALL with monoclonal antibodies to myeloid and lymphoid associated antigens

Sixty-five cryopreserved leukemic samples from children diagnosed and treated as having acute lymphocytic leukemia (ALL) were retrospectively examined for the presence of lymphoid and myeloid associated antigens by indirect immunofluorescence using monoclonal antibodies. Expectedly, the majority of these specimens expressed antigens known to be expressed on lymphoid, and not myeloid malignancies. These included the common acute lymphoblastic leukemia antigen (CALLA), the p32 B-cell associated antigen, and T-cell associated antigens. Leukemic cells from the 8 remaining patients expressed antigens known to be present on both myeloid and lymphoid leukemias. These included HLA/DR, and the antigens identified by BA-1 and BA-2. Cells from 2 of these 8 patients reacted with antibodies that define antigens present on normal and malignant myeloid cells. Both specimens reacted with 1G10, an anti-granulocyte antibody, and one reacted with 5F1 which reacts with monocytes, nucleated red blood cells, megakaryocytes and platelets. One of these patients relapsed while receiving ALL therapy, and the morphology of her leukemic cells became characteristic of acute monocytic leukemia (AMoL). The second patient failed ALL therapy but responded to standard acute nonlymphocytic leukemia (ANLL) therapy, clearing her peripheral blasts. Thus these studies confirm that cell surface phenotyping with monoclonal antibodies can recognize ALL cells that express myeloid rather than lymphoid associated antigens and demonstrate that the malignant cells display a clinical behavior consistent with the diagnosis of ANLL.     

Production and Characterization of a Human Monoclonal Antibody Recognizing a New Antigen Expressed on Some Lymphoid Cells

A cell line secreting a human monoclonal antibody was established by Epstein-Barr virus transforming B cells derived from an enlarged cervical lymph node excised from a patient bearing a carotid body tumor. The reactivity of the monoclonal antibody, designated as mNISP, was tested on various cells and cell lines. An antigen defined by the mNISP was expressed on some Burkitt's lymphoma cell lines and on a non-T non-B acute lymphoblastic leukemia cell line. Furthermore, this antigen was expressed on leukemic cells from 2 of 8 patients with chronic myelocytic leukemia, 2 of 10 patients with acute myeloblastic leukemia, one of 13 patients with acute lymphoblastic leukemia, and two patients with adult T cell leukemia, but it was not expressed on normal T, B and adherent (macrophage) cells. In addition, mNISP reacted with T cells obtained from human T-cell leukemia virus type I carriers. We found that the antigen defined by mNISP was distinct from any previously reported antigen in terms of its pattern of cellular expression and molecular weight, suggesting that mNISP recognizes a new antigen expressed on some lymphoid cells.     

CD9 antigen on acute non-lymphoid leukemia cells: preferential expression by promyelocytic (M3) subtype

A monoclonal antibody (S17-12), previously described to recognize subsets of myelomonocytic cells, is demonstrated to bind CD9 antigen, a surface marker of B-cell precursors, platelets and several non-hematopoietic tissues. The proportion of S17-12, CD9-positive leukemic cells was determined in 102 patients with acute non-lymphocytic leukemia. It was found to be above 75% in 14/22 M3 cases (including 3/3 of microgranular variant) and only in 1/80 cases of different FAB subtype. Complete reactivity (greater than 95%) of leukemic clonogenic cells with CD9 MoAB was restricted to 4/18 M3 cases. Therefore, a high proportion of CD9-positive cells seems to be peculiar to M3 cases. This may help in the diagnosis of cases that lack typical morphological features.     

B-cell maturation stages of Burkitt's lymphoma cell lines according to Epstein-Barr virus status and type of chromosome translocation

This study addressed the possible relationship between B-cell maturation stage of Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV) status, ethnic group, or type of chromosome translocation. Fifty-seven cell lines obtained at the International Agency for Research on Cancer from 51 patients were studied. Cytogenetic analyses of 54 cells lines were available. Cell size, surface immunoglobulins (sIgs), cytoplasmic immunoglobulins (cIgs), mouse red blood cell receptors, and reactivity with various monoclonal antibodies were assessed. Immunoglobulin (Ig) class secretions were measured in the supernatant of 2- and 5-day cultures from 33 cell lines, with the use of a sensitive enzyme-linked immunosorbent assay technique. From this study, BL appears to cover a broad range of the B-cell differentiation sequence, since the following Ig phenotypes were observed: null cells (sIg-, cIg-), large pre-B-cells (intracytoplasmic mu-chains), small B-cells (sIg+, cIg-), and various types of secreting B-cells (sIg+, cIg+). Among the latter, various patterns of cIg could be defined (perinuclear, paranuclear, and vesicular). B-cell maturation stages were correlated with the amount of secreted Ig. In sIg+ cell lines, different classes of Ig were found: 35 IgM, 10 IgM plus IgD, 4 IgG, and 1 IgA. None of the different monoclonal antibodies used was specific to a precise stage of maturation. The stages of maturation were correlated with neither the type of chromosome translocations of BL nor the presence of EBV genome, but the most immature cell lines were all EBV positive and most of them originated from African patients. In contrast with acute lymphoblastic leukemia, the common acute lymphocytic leukemia antigen (CD10) was expressed on nearly all BL cell lines of intermediate maturation stages but only on half of the pre-B ones. In addition, none of the cell lines tested was found to react with CD5 antibodies, which recognize most of the chronic lymphocytic leukemia of the same stage of maturation as that in the B-lymphocyte lineage.     

Release of platelet-activating factor in human leukemia

Cellular release of platelet-activating factor (PAF) was assessed in a series of human acute and chronic lymphoid and myeloid leukemias at presentation or in an active phase of the disease. PAF-like material, showing physicochemical properties similar to those of synthetic PAF and of PAF released from IgE-sensitized rabbit basophils, was found in cultures of cells from 5 of 6 acute lymphoblastic leukemias (ALL) (2 of 2 T-ALL and 3 of 4 common ALL) and from 13 of 24 B-cell chronic lymphocytic leukemias after stimulation with ionophore A23187 with or without phytohemagglutinin in the presence of acetyl coenzyme A. On the other hand, PAF was released only from 2 of 10 acute myeloblastic leukemias; both of them were of the more mature monoblastic subtype or M5 according to the French-American-British classification. Cells from all three cases of chronic myeloid leukemia studied were also capable of producing PAF. In eight cases of acute lymphoid and myeloid leukemia, the in vivo release of PAF was assessed by testing the plasma levels of this mediator. Only in two cases (one ALL and one acute myeloblastic leukemia) could detectable levels of circulating PAF be demonstrated; it is of interest that both of these cases showed clinical and hematological features of disseminated intravascular coagulation. No PAF was documented in the plasma of the five chronic leukemias tested (four B-cell chronic lymphocytic leukemias and one chronic myeloid leukemia). These findings indicate that lymphoid and myeloid leukemic cells have a different capacity of releasing PAF, possibly related to the level of cell differentiation rather than to an intrinsic property of the neoplastic cells. Furthermore, in some cases, an intravascular release of PAF may occur.     

Expression of Tac antigen on human immature B-cell lineage leukemic cells

Expression of interleukin 2 (IL-2) receptor (Tac antigen) on leukemic cells from 4 patients with acute lymphocytic leukemia (ALL) and 3 patients with blastic crisis of chronic myelogenous leukemia (CML-BC) was examined. Cells from 6 out of 7 patients did not carry immunoglobulins on their surfaces, but reacted with monoclonal antibodies (mAb) detecting B-cell related antigens such as B1, OKB2 and Leu12. Cells from two cases expressed Tac antigen immediately after cell separation. Phorbol 12-myristate 13-acetate (PMA) could induce Tac antigen in all cases. SDS-PAGE analysis of immunoprecipitates by anti-Tac mAb demonstrated that the molecular weight of Tac antigen on most of the leukemic cells was similar to that of Tac antigen present on Con A-stimulated normal lymphocytes. These leukemic cells poorly responded to exogenous recombinant IL-2. These findings suggest that IL-2 may interact with the immature and mature B cells, and play an important role in the differentiation of B lymphocytes.     

Exposure by desialylation of myeloid antigens on acute lymphoblastic leukemia cells

The 3-fucosyl-N-acetyllactosamine structure, a sugar sequence contained in the human milk oligosaccharide lacto-N-fucopentaose III, is recognized by most of the granulocyte-specific monoclonal antibodies (MoAb) reported in the literature, including the six MoAb from our laboratory. Blast cells from patients with acute myeloblastic leukemia (AML) displayed a heterogeneous reaction pattern when they were exposed to MoAb against this moiety, and the proportion of reactive cells in individual cell samples was highly variable. The intensity of the reaction was strongly enhanced by neuraminidase treatment of AML blasts, and reactive structures were exposed on previously negative AML blast cells. Surprisingly, this granulocyte-associated antigen was exposed by desialylation not only on malignant myeloid precursor cells but also on common acute lymphoblastic leukemia cells. No such effect was seen when normal peripheral blood lymphocytes, lymphocytes from patients with chronic lymphatic leukemia, or blast cells from patients with B-cell acute lymphoblastic leukemia, acute erythroid leukemia, and acute megakaryoblastic leukemia were treated with neuraminidase.     

The product of the proto-oncogene c-kit (P145c-kit) is a human bone marrow surface antigen of hemopoietic precursor cells which is expressed on a subset of acute non-lymphoblastic leukemic cells.

A monoclonal antibody (17F11) was raised by immunization of a Balb/c mouse with leukemic blasts from a patient with acute non-lymphocytic leukemia (ANLL). This antibody recognizes most leukemic blasts of myeloid but not of lymphoid lineage and no peripheral blood cells. By screening NIH-3T3 fibroblasts transfected with the human proto-oncogene c-kit (NIH-3T3/hckit) it could be shown that 17F11 specifically recognizes the gene product P145c-kit. Immunofluorescence analysis on normal hemopoietic cells revealed that 17F11 weakly stains 1-3% of bone marrow mononuclear cells (BMMNC). By FACS sorting and colony assays it could be shown that granulocyte--macrophage progenitor cells could be enriched 10-20-fold, granulocyte progenitors 50-80-fold, and erythroid and multipotential progenitor cells 15-20-fold, in the 17F11 positive fraction. Double fluorescence analysis revealed that P145c-kit is co-expressed on 40-60% of the CD34 positive BMMNC. Finally, these data show that P145c-kit is expressed on blast cells from most patients with ANLL (26/30) and chronic myeloid leukemia in blast crisis (7/9), but is absent on blasts from patients with acute lymphoblastic leukemia expressing the T-, B-lineage, or common ALL phenotypes.     

Production and analysis of HH 10 monoclonal antibodies reactive to immature hematopoietic cells and their use for monitoring acute leukemia cells

A clone of murine monoclonal antibody HH 10 (IgM) was raised by fusing NS-1 myeloma cells and spleen cells of a mouse hyperimmunized with acute promyelocytic leukemia cells. Serological analysis by means of immune adherence assays showed that HH 10 reacts with immature hematopoietic cells including thymocytes and myeloid precursor cells (defined as colony-forming units in culture assays). The antibodies were not reactive to either peripheral blood cells or bone marrow cells obtained from normal individuals. Lymphoid blasts induced with phytohemagglutinin, pokeweed mitogen, or concanavalin A were also non-reactive, and all non-hematopoietic cultured cells examined were also negative. The antibodies were, however, reactive to leukemia cells in 67 cases out of 91 cases (74%) of acute leukemia. In patients with acute lymphoblastic leukemia, 43 out of 52 cases (83%) were positive and, in particular, all of 12 T-cell-type acute leukemias were reactive to HH 10. Comparison of percent HH 10 positive cells in the bone marrow of patients with acute leukemia with the results of cytological studies showed a good correlation. Analysis of sequentially collected bone marrow cells of acute leukemia patients using HH 10 revealed its usefulness for monitoring leukemia cells.