The gene encoding the granulocyte colony-stimulating factor receptor is a target for deregulation in pre-B ALL by the t(1;19)-specific oncoprotein E2A-Pbx1.

Approximately 25-30% of childhood pre-B cell acute lymphoblastic leukemias (pre-B ALL) is characterized by the presence of a (1;19)(q23;p13.3) translocation. The presence of this translocation is generally accompanied by a poor prognosis. The chimeric gene resulting from this chromosomal rearrangement encodes a hybrid transcription factor, E2A-Pbx1. In an attempt to delineate the genetic cascade initiated by E2A-Pbx1, we sought to identify genes that are deregulated by this transcription factor in t(1;19) pre-B ALL. We show here that the gene encoding the granulocyte colony-stimulating factor receptor (G-CSFr) is specifically upregulated in pre-B cells expressing E2A-Pbx1. G-CSFr is also expressed in cell lines established from t(1;19) pre-B cell leukemia and on primary t(1;19) tumor cells, but not on control cells. These data indicate that G-CSFr gene is a target for deregulation by E2A-Pbx1.

Production of hybrid hybridomas based on HAT(s)-neomycin(r) double mutants.

A detailed procedure is described for the preparation of hybrid hybridomas, that produce bispecific antibodies. This is achieved by fusing two hybridoma cell lines that are phenotypically distinct (HAT(s)/neo(r) and HAT(r)/neo(s)) and thereby allow for the selection of the appropriate hybrid cells. HATs mutants were obtained from one of the two fusion partners by 8-azaguanine treatment; these mutant phenotypes were found in an unexpected high frequency. For the introduction of the dominant neo(r) marker gene in one of the HAT(s) fusion partners, a retroviral vector was used in order to obtain a high efficiency of gene transfer. Our method was very effective in the production of hybrid hybridomas, so-called quadromas. The detection of bispecific antibodies was based on simultaneous binding by one antibody of two different antigens, or on the presence of two different H chain isotypes in this molecule.

Putative myeloma precursor cells expressing 2,6 sialic acid-modified antigens actually belong to the erythroid lineage.

The Golgi enzyme alpha2,6-sialyltransferase modifies glycoconjugates by adding sialic acid. In lymphocytes, different epitopes that result from this modification have been identified by the B cell-related CDw75, CDw76, HB4 or HB6 Ab. We previously described positive staining with these Ab of a highly transferrin receptor-positive (CD71) cell type in the bone marrow of multiple myeloma patients. These cells were distinct from plasma cells, but did contain Ig of the same isotype and idiotype as seen in the plasma cells. We postulated a precursor role for this cell type in myeloma. Here, we report that this CD71+ (HB4/HB6/CDw75/CDw76)+ cell is an erythroid precursor cell instead. RT-PCR did not detect Ig mRNA, and from immuno electron microscopy Ig appeared to be endocytosed rather than synthesized by these cells. At their cell surface the erythroid/megakaryocytic markers CD36 and CD41, and the erythroid-specific glycophorin A can be detected, while haemoglobin can be detected antigenically in the cytoplasm. Finally, purified cells proliferate in vitro upon addition of erythropoietin. Uptake of Ig could be explained by the presence of Fc gammaRIII(CD16), which has also been found on other haematopoietic precursor cells.

Enrichment and selection of hybrid hybridomas by Percoll density gradient centrifugation and fluorescent-activated cell sorting.

Hybrid hybridomas, producing bi-specific monoclonal antibodies that react with horseradish peroxidase and human IgA1 were isolated by sorting the double-fluorescent cells on single-cell basis after fusion of two hybridomas, previously labelled green or red by octadecylamine-FITC or -TRITC, respectively. The double-fluorescent fused cells were significantly different in AXL (size) and RAS (internal structure) distribution compared with the (non-fused) mono-fluorescent cells. The percentage of double-fluorescent cells and the viability of these cells could be increased by Percoll density gradient centrifugation. As a result, there was an 8-fold increase of total isolated hybrid hybridomas (up to 30% of all tested clones) compared to isolations without Percoll density gradient centrifugation. All the isolated hybrid hybridoma clones had similar amounts of DNA, equal to the sum of the DNA of both parental hybridomas.

HB4 antibody recognizes a carbohydrate structure on lymphocyte surface proteins related to HB6, CDw75, and CD76 antigens.

Cells regulate the specificity of the carbohydrate chains on their membrane-bound glycoconjugates by differential expression of glycosyltransferases. In lymphocytes, beta-galactoside alpha 2,6-sialyltransferase is reportedly involved in the generation of epitopes recognized by HB6, CDw75, and CD76 mAb. The HB4 mAb binds to an Ag present on subpopulations of B and NK cells. We now show that this Ag represents another member of a set of neuraminidase-sensitive, alpha 2,6-sialyltransferase-generated sugar Ag. Transient expression of a cDNA encoding this enzyme in COS cells generated a minor population of HB4+ cells that was completely contained within the HB6+ COS cell population. Using various proteinases and an inhibitor of N-linked carbohydrate processing, we show both epitopes to represent components of N-glycosylated membrane proteins. Remarkably, porcine thyroglobulin, an alpha 2,6-NeuAc+ glycoprotein, is specifically recognized by both mAb. These data underline a close relationship between HB4 and HB6 epitopes and imply further that both mAb react with oligosaccharide chains irrespective of the carrier molecule nature. Thus, the terminal sugar residue sialic acid plays a pivotal role in at least four distinct epitopes that are expressed differentially in immune cells. This may point at an important role for these epitopes in biologic recognition.

Absence of preferential homologous H/L chain association in hybrid hybridomas.

Bispecific mAb contain two Ag-combining sites each composed of a different combination of H and L chains. The resulting ability to react with and cross-link two different Ag makes these molecules a novel tool for application in biology and medicine. Intact bispecific mAb can be made only by biologic means, e.g., by fusion of two established hybridomas. Appropriate assembly of bispecific mAb by these hybrid cells depends on H = L chain behavior: strong preferential homologous H-L pairing would benefit the yield of bispecific antibodies. We have analyzed the Ig species produced by eight hybrid hybridomas (quadromas). Quadroma-produced IgG was fractionated and characterized for H and L chain content. The Ag reactivities were verified by using ELISA and immunofluorescence. Preferential homologous pairing was seen only with a minority of H-L chain pairs; L chains associated on average in a random fashion with H chains. This indicates that in the B cells from which the parental hybridomas were obtained, no strong selection had occurred on H-L recombination. Our results extend recent biochemical competitive H-L reassociation experiments, where on average an at random association of L chains with H chains was found; evidently this random association occurs in our biologic system as well. For the biologic production of bispecific antibodies this means that only in a small number of cases the "ideal" producer will be met. From the viewpoint of generation of antibody diversity, our results favor a large freedom for combinatorial binding of H and L chains during B cell ontogeny.

The efficacy of CD3 x CD19 bispecific monoclonal antibody (BsAb) in a clonogenic assay: the effect of repeated addition of BsAb and interleukin-2.

To evaluate the potency by which human T cells are targeted and activated by bispecific monoclonal antibodies (BsAbs) to lyse tumor cells, a clonogenic assay was developed. The efficacy of a CD3 x CD19 BsAb binding to both the CD3 T-cell antigen and the CD19 B-cell antigen was already proven in 51Cr-release assays and in 3-day activation cultures. To achieve more quantitative results, a 14-day clonogenic assay, based on limiting-dilution, was performed for the determination of the initial and residual number of clonogenic units obtained with a CD19+ pre-pre-B acute lymphoblastic leukemia (ALL-B) cell line. Elimination of up to 5 logs of ALL-B cells by freshly isolated peripheral blood mononuclear cells (PBMCs) cultured with BsAb plus interleukin-2 (IL-2) could be detected. The presence of human IgG did not abolish the effect. Repeated addition of each of the two agents was necessary, because a single treatment produced only a 1- to 2-log kill. CD3 monoclonal antibody and IL-2 stimulation ("lymphokine-activated killer cell" conditions) resulted in only a 2-log kill. The number of T cells proved critical in lysis of ALL-B cells, with a 5-log kill using a T-cell:B-cell ratio of 3:1 but with only a 1-log kill using a ratio of 1:1. PBMCs isolated from patients with non-Hodgkin's lymphoma, both in relapse or remission, proved to be as competent as those from healthy donors in removing ALL-B cells. This clonogenic assay shows the importance of repeated administration of CD3 x CD19 BsAb and IL-2 and offers the possibility to compare it with other therapies in B-cell malignancy.

Heterodimeric complex formation with CD8 and TCR by bispecific antibody sustains paracrine IL-2-dependent growth of CD3+ CD8+ T cells.

During physiologic activation of mature CD8+ T cells, TCR and CD8 bind to the same Ag-complexed MHC class I molecule. Thereby, close proximity is induced between CD8 and the TCR/CD3 complex. During this engagement, CD8 may deliver TCR-independent signals via its associated protein tyrosine kinase, p56lck. We studied the potential biologic effects of close association between CD8 and TCR/CD3 complexes by using a bispecific antibody (bsAb) directed against both TCR and CD8 molecules. This hybrid hybridoma (quadroma)-produced bsAb binds as a monomeric molecule to CD3+ CD8+ but not CD3+ CD4+ T cells. The bsAb proved capable of inducing the cytotoxic effector function of cloned CD3+ CD8+ T cells but not of CD3+ CD4+ T cells. When the bsAb was presented to resting T cells by monocytes, proliferation of the CD3+ CD4+ but not the CD3+ CD8+ subset of T lymphocytes was induced. Parental anti-TCR antibody induced vigorous growth of cells of both subsets. Essentially identical results were obtained when bsAb was presented in an immobilized fashion. The unresponsiveness of the CD3+ CD8+ T cells with respect to mitogenesis could be restored by exogenous rIL-2. The data suggest that bsAb-induced activation differs from activation by monospecific anti-TCR antibody. The former appears to more closely mimic physiologic Ag-induced signaling, because it leads to a similar paracrine IL-2-dependent growth pattern. The bsAb may, therefore, be instrumental in studying T cell signaling pathways, in particular the role of CD8-associated p56lck therein.

Evaluation of Fc gamma receptor mediated T-cell activation by two purified CD3 x CD19 bispecific monoclonal antibodies with hybrid Fc domains.

Two bispecific monoclonal antibodies (BsAb), differing in H chain isotype combination, were made for treatment of B-cell leukaemia/lymphoma; QAI-2, CD3-mouse-IgG1 x CD19-mouse-IgG2a and QAI-3, CD3-mouse-IgG1 x CD19-mouse-IgG2b. Both purified BsAb proved equally effective for their ability to target pre-activated T cells towards CD19 positive tumour cells. In T-cell proliferation assays, the capacity of Fc gamma RIa (CD64), Fc gamma RIIa-R131 and Fc gamma RIIa-H131 (CD32) transfected fibroblasts was tested to present the BsAb. The BsAb combining mouse (m) IgG1 and mIgG2a promoted T-cell activation in combination with the Fc gamma RIa transfectant; the mIgG1-mIgG2b BsAb was only marginally active. Both BsAb could not induce T-cell activation when presented by either of the Fc gamma RIIa transfectants. Similar results were obtained using PBMC cultures, containing Fc gamma RIa+/Fc gamma RIIa+ monocytes as accessory cells. The importance of Fc gamma R-dependent BsAb-mediated T-cell activation emerged from experiments with T cells and CD19 positive B-cell lines, showing that cross-linking via CD19+ target cells alone did not induce T-cell proliferation. Therefore, BsAb with functionally different Fc domains represent alternative strategies in BsAb therapy, the efficacy of which deserves to be compared in vivo.

Unprimed CD4+ and CD8+ T cells can be rapidly activated by a CD3 x CD19 bispecific antibody to proliferate and become cytotoxic.

We previously reported that a CD3 x CD19 bispecific antibody (bsAb) can induce efficient killing of tumour cells by preactivated T cells isolated from patients with B cell malignancy. For future intravenous application we investigated whether resting T cells from peripheral blood can be stimulated to proliferate and become cytotoxic with the CD3 x CD19 bsAb alone. Indeed peripheral blood mononuclear cells, isolated from healthy donors or patients with B cell malignancy, started to proliferate within 1 day in response to CD3 x CD19 bsAb. Within the same time span cytotoxic activity against CD19-positive tumour cells was already detectable. Maintenance of cytotoxic activity was seen during 3 days of culture but optimal lysis of the target cells then required fresh CD3 x CD19 bsAb in the cytotoxicity assay. Essentially the same results for proliferation and cytotoxicity were found when separated CD4-positive and CD8-positive T cells were activated by the bsAb in the presence of autologous monocytes. These results may be relevant for the in vivo application of the bsAb when used as immunotherapy in patients with B cell malignancy.

Killing of autologous B-lineage malignancy using CD3 x CD19 bispecific monoclonal antibody in end stage leukemia and lymphoma.

To develop an effective tumor immunotherapy for B-lineage non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), a bispecific monoclonal antibody (BsAb) has been generated with the first specificity for the CD3 epsilon-chain and the second for the CD19 antigen. Peripheral blood mononuclear cells (PBMCs) isolated from patients with NHL or ALL during remission or relapse rapidly proliferated (up to 179-fold increase) on in vitro activation combining phytohemagglutinin or CD3 monoclonal antibody with interleukin-2. After 3 weeks of stimulation, more than 90% of the PBMCs was CD3+ and CD8+, even when cultures were started with only 5% CD3+ cells. Cytotoxic activity against autologous malignant B cells was markedly enhanced (from 5% baseline to 70% lysis) by the addition of the CD3 x CD19 BsAb in all samples tested. Immunophenotypic examination of a series of tumor target cells showed that all samples examined showed CD54 (intercellular adhesion molecule-1) and HLA class I, but showed no B7 expression. CD11a (lymphocyte function-associated antigen-1) expression was heterogeneous. Various types of experiments showed that efficient CD3 x CD19 BsAb-mediated cytolytic capacity was not dependent on expression of either of these surface proteins. This contrasts with normal major histocompatibility complex-restricted antigen-specific cytotoxicity and may be essential for effective in vivo application of this BsAb.

Analysis of CD2 and TCR-beta gene expression in Jurkat cell mutants suggests a cis regulation of gene transcription.

Thirty CD2- J32 stable clones, derived by mutagenesis and subsequent immunoselection with anti-CD2 Ab, were used to study the regulation of CD2 and TCR gene expression. Analysis of RNA expression revealed that the loss of surface expression of CD2 was due to a lack of expression of CD2 mRNA and was associated with a lack of expression of VDJ TCR-beta transcripts in 12 of these mutants, sparing the expression of DJ TCR-beta, TCR-alpha, CD3 gamma, delta, epsilon, and zeta RNA. The expression of other differentiation molecules was unaffected, except for CD1, CD4, and CD5, which were either decreased or absent in most of these mutants. A gain in the expression of TCR-gamma transcripts was observed in each of these mutants, while, as expected, no TCR-gamma transcripts were detected in wild-type J32 cells. Several mutants were able to use the human CD2 enhancer and the murine TCR-beta enhancer and promoter to activate transcription from reporter genes in the context of heterologous promoters, indicating that the mutation(s) does not affect transcription pathways. Consistent with this finding is the adequate expression in these mutants of several lineage-specific transcription factors. The expression of CD2 in several of these mutants was rescued by gene transfer using a genomic 28.5-kb CD2 fragment, suggesting that the enhancer function of this gene may be dependent on the enhancer site. These observations suggest that the coordinate expressions of CD2 and TCR-beta genes share common regulatory mechanisms involving factors regulating chromatin structure and accessibility.