Inhibition of antibody-dependent cellular cytotoxicity and immunoglobulin synthesis by an antiserum prepared against a human B-cell Ia-like molecule.

Rabbit antisera to the human B-cell-specific antigen complex, p23,30, was used to define further the functional heterogeneity of isolated human lymphocyte subpopulations. Specific depletion of p23,30-bearing cells from Ig-negative cell populations and Ig-negative, E rosette-negative (Null) populations by either complement-mediated lysis or by physical separation on goat antirabbit Fab immunoabsorbent columns, eliminates the antibody-dependent cellular cytotoxic (ADCC) function. Furthermore, binding of anti-p23,30 serum to the effector cell surface inhibits ADCC but does not interfere with EA rosette formation. Apparently p23,30 represents a cell surface site which is distinct from the Fc receptor but which is important in the triggering of ADCC. In addition, depletion of p23,30-bearing cells from unfractionated cell populations, Ig-positive B-cell populations and Ig-negative, E rosette-negative (Null) populations eliminates the capacity of these populations to secrete immunoglobulin during subsequent culturing. Thus both the Ig-secreting cells and the ADCC effector cells within the Ig-negative, E rosette-negative (Null) population reside in the same population of cells which bears the p23,30 antigen.

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Trogocytosis of multiple B-cell surface markers by CD22 targeting with epratuzumab

Blood ◽

10.1182/blood-2012-12-473744 ◽

2013 ◽

Vol 122 (17) ◽

pp. 3020-3029 ◽

Cited By ~ 69

Author(s):

Edmund A. Rossi ◽

David M. Goldenberg ◽

Rosana Michel ◽

Diane L. Rossi ◽

Daniel J. Wallace ◽

...

Keyword(s):

B Cells ◽

Autoimmune Disease ◽

Cell Surface ◽

B Cell ◽

Regulatory Proteins ◽

Surface Markers ◽

B Cell Antigen Receptor ◽

Cell Surface Markers ◽

Effector Cells ◽

Key Points

Key Points Epratuzumab induces the reduction of multiple B-cell antigen receptor–modulating proteins on the surface of B cells via their trogocytosis to effector cells. Modulation of B cells by trogocytosis of key regulatory proteins may be an important mechanism of immunotherapy of autoimmune disease.

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Direct demonstration of an HLA-DR allotypic determinant on the low molecular weight (beta) subunit using a mouse monoclonal antibody specific for DR3

Journal of Experimental Medicine ◽

10.1084/jem.156.1.104 ◽

1982 ◽

Vol 156 (1) ◽

pp. 104-111 ◽

Cited By ~ 55

Author(s):

JP Johnson ◽

T Meo ◽

G Reithmuller ◽

DJ Schendel ◽

R Wank

Keyword(s):

Molecular Weight ◽

Monoclonal Antibody ◽

Cell Surface ◽

B Cell ◽

Beta Subunit ◽

Low Molecular Weight ◽

Β Subunit ◽

Direct Demonstration ◽

Hla Dr ◽

Human B Cell

A murine monoclonal antibody directed against a human B cell surface antigen with the characteristics of HLA-DR is described. The antigen detected is tightly linked to HLA and is correlated with the alloantigen HLA-Dw/DR3. Reactivity with a fraction of Dw/DRw6 cells is also observed. The determinant recognized by this antibody has been shown to be present on the smaller molecular weight β subunit of the HLA-DR antigen.

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Cell surface phenotype, cytokines, and antibody gene expression in immortalized human B cell lines

Human Antibodies ◽

10.3233/hab-1990-1305 ◽

1990 ◽

Vol 1 (3) ◽

pp. 145-153 ◽

Cited By ~ 7

Author(s):

K. James ◽

J. Gardner ◽

G. Skibinski ◽

M. McCann ◽

R. Thorpe ◽

...

Keyword(s):

Gene Expression ◽

Cell Surface ◽

B Cell ◽

Cell Lines ◽

Surface Phenotype ◽

Human B Cell ◽

Antibody Gene

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The monoclonality of human B-cell lymphomas.

Journal of Experimental Medicine ◽

10.1084/jem.145.4.1014 ◽

1977 ◽

Vol 145 (4) ◽

pp. 1014-1028 ◽

Cited By ~ 166

Author(s):

R Levy ◽

R Warnke ◽

R F Dorfman ◽

J Haimovich

Keyword(s):

B Cell ◽

Lymphoid Hyperplasia ◽

Cell Populations ◽

Reactive Lymphoid Hyperplasia ◽

Frozen Sections ◽

B Cell Lymphomas ◽

Lymphoid Follicles ◽

Human Lymphoma ◽

Double Label ◽

Human B Cell

Human tissues involved with lymphoma have been examined in frozen sections for immunoglobulin-bearing cells by a technique involving double-label immunofluorescence with mixed anti-kappa and anti-lambda antibodies. F (ab')2 fragments of purified antibodies were employed to avoid any binding via Fc receptors. B cell lymphomas were shown to be composed of monoclonal populations of Ig bearing cells, whereas normal or reactive lymphoid follicles contained a mosaic of Ig-bearing cells derived from multiple clones. Nodules of lymphoma were often surrounded by normal polyclonal B cell populations. We anticipates that the approach described here will be useful in the diagnosis of lymphoma, differentiating it from reactive lymphoid hyperplasia by the demostration of monoclonality. In addition, it should provide a sensitive and reliable tool for investigating the immunobiology of human lymphoma.

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BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

Journal of Experimental Medicine ◽

10.1084/jem.140.4.877 ◽

1974 ◽

Vol 140 (4) ◽

pp. 877-894 ◽

Cited By ~ 86

Author(s):

Argyrios N. Theofilopoulos ◽

Frank J. Dixon ◽

Viktor A. Bokisch

Keyword(s):

Cell Surface ◽

B Cell ◽

Cell Lines ◽

Immune Complexes ◽

Surface Membrane ◽

Raji Cells ◽

Human Lymphoblastoid Cell ◽

C Cell ◽

A Cell ◽

Soluble Immune Complexes

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells.

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A Non-Internalizing Anti-CD40 Antibody, CHIR-12.12, Blocks CD40L-Induced Cytokine Production and Mediates Greater ADCC Than Rituximab in Primary CLL Cells.

Blood ◽

10.1182/blood.v106.11.2964.2964 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2964-2964

Author(s):

Xia Tong ◽

Sharon Lea Aukerman ◽

Karen Lin ◽

Natasha Aziz ◽

Cheryl Goldbeck ◽

...

Keyword(s):

Cell Surface ◽

B Cell ◽

Cytokine Production ◽

Ex Vivo ◽

Lymphocytic Leukemia ◽

Target Cells ◽

Adcc Activity ◽

Effector Cells ◽

Cells Cultured

Abstract CD40 is expressed on chronic lymphocytic leukemia (CLL) cells, and CD40 activation leads to signaling critical for cell survival and proliferation. We have previously described a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, generated in XenoMouse® mice (Abgenix, Inc.), and have demonstrated that it inhibits normal human B cell proliferation and survival and mediates potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. In this study, we examined the ability of CHIR-12.12 to modulate cytokine production by primary CLL cells and compared the ADCC activity of CHIR-12.12 with rituximab against primary CLL cells. Primary CLL cells stimulated with CD40L produced a variety of cytokines, including IL-10, TNF-α , IL-8, GM-CSF, IL-6, MCP-1, and MIP-1β. Addition of CHIR-12.12 to primary CLL cells inhibited CD40L-mediated production of these cytokines. Cytokine production by primary CLL cells cultured with CHIR-12.12 alone in the absence of CD40L did not exceed levels produced by CLL cells cultured in medium. These data suggest that CHIR-12.12 is a potent antagonist for CD40L-mediated cytokine production by primary CLL cells and shows no agonistic activity by itself. We next compared the relative ADCC activity of CHIR-12.12 and rituximab against ex vivo primary CLL cells from 8 patients. CHIR-12.12 exhibited greater ADCC than rituximab against CLL cells from all patients. The average percent of maximum lysis by CHIR-12.12 and rituximab were 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was greater than 10-fold more potent than rituximab, as measured by ED50 values (14.1 pM versus 155.5 pM, respectively). Quantitative CD20 and CD40 density on CLL cells and the degree of antibody internalization were investigated as potential reasons for the difference in ADCC activity. The greater ADCC potency and efficacy of CHIR-12.12 was not dependent on a higher density of cell surface CD40 molecules, as there were 1.3 to 14-fold higher numbers of CD20 than CD40 molecules on the cell surface. Antibody internalization studies using primary CLL cells conducted by flow cytometry and confocal microscopy show that upon binding to CD40 at 37°C, CHIR-12.12 remains uniformly distributed on the cell surface, even after 3 hours. In contrast, after binding at 37°C, rituximab is redistributed into caps and internalized. These data suggest that the potent ADCC activity of CHIR-12.12 may be partly related to its ability to remain on the surface of target cells uniformly, allowing optimal interaction with effector cells. Taken together, these results suggest that CHIR-12.12 may be effective at mediating potent ADCC against CLL cells in vivo. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.

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Anti-CD20-Interferon-α Fusion Protein Demonstrates Potent Direct Growth Inhibition, Antibody-Dependent Cellular Cytotoxicity, and Enhanced Complement-Dependent Cytotoxicity Against Human B Cell Lymphomas In Vitro, and Activity Against Multiple Human NHL Xenografts In Vivo

Blood ◽

10.1182/blood.v118.21.2724.2724 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2724-2724

Author(s):

Reiko E Yamada ◽

Kristopher K Steward ◽

Gataree Ngarmchamnanrith ◽

Sanjay Khare ◽

Raj Sachdev ◽

...

Keyword(s):

Fusion Protein ◽

Growth Inhibition ◽

B Cell ◽

Antibody Dependent Cellular Cytotoxicity ◽

Complement Dependent Cytotoxicity ◽

Direct Growth ◽

Cd20 Antibody ◽

Anti Cd20 ◽

Human B Cell

Abstract Abstract 2724 The type 1 interferons (IFNα and IFNβ) are potent regulators of malignant cell growth. IFNα has anti-proliferative and pro-apoptotic effects against many cancers, including non-Hodgkin lymphomas (NHL), and immunostimulatory effects including activation of natural killer cells, dendritic cells, and T cell anti-tumor immunity. Until now however, the clinical use of these agents has been limited by the inability to achieve effective concentrations of IFN at sites of tumor without causing systemic toxicity. We recently reported the ability of an anti-CD20 antibody-IFNα fusion protein to induce apoptosis and promote in vivo eradication of CD20-expressing mouse and human B cell lymphomas (C. Xuan et al, Blood 115:2864, 2010). We now report on the preclinical anti-lymphoma activity of a recombinant anti-CD20-human IFNα (IgG1 anti-CD20-hIFNα) fusion protein derived from rituximab variable region sequences. Anti-CD20-hIFNα was evaluated against a large panel of human B cell NHL lines representing aggressive histologies including Burkitt (Daudi, Raji, Ramos), diffuse large B cell (SUDHL-4, OCI-Ly2, OCI-Ly3, OCI-Ly19, HBL-1, RC-K8), and mantle cell (Granta-519) lymphomas. Proliferation was measured by [3H]-thymidine incorporation in quadruplicate 48 hour cultures, apoptosis measured by Annexin-V and propidium iodide (PI) staining, complement-dependent cytotoxicity (CDC) measured by PI flow cytometry, and antibody-dependent cellular cytotoxicity (ADCC) measured by LDH release using peripheral blood mononuclear cell effectors. NHL xenografts Daudi, Raji, and Namalwa were grown in SCID mice. Equimolar doses of rituximab and fusion protein were compared in each assay. Against IFN-sensitive CD20-negative U266 tumor cells, anti-CD20-hIFNα fusion protein had 10–15% IFNα bioactivity when compared to conventional recombinant IFNα (rIFNα). However, when targeting CD20-positive Daudi cells the inhibitory growth activity is significantly enhanced over rIFNα. Anti-CD20-hIFNα fusion protein induced stronger direct growth inhibition than rituximab (23.3–93.1% vs. 0.0–39.8%); particularly against Burkitt (44.7–93.1% vs. 0.0–10.4%) and germinal center-type diffuse large B cell (59.0–88.8% vs. 10.5–39.8%) NHLs. Tumor growth inhibition by anti-CD20-hIFNα was associated with substantial apoptosis in some cell lines. The ADCC activity of fusion protein against Daudi, Ramos, and Raji cells was identical to that of rituximab. Against established human NHL xenografts (Daudi, Raji, and Namalwa), fusion protein achieved improved survival compared to rituximab. Surprisingly, anti-CD20-hIFNα exhibited superior CDC compared to rituximab against Daudi (62.0% vs. 28.3%), Ramos (81.0% vs. 57.3%), and Raji (78.0% vs. 54.6%) cells. We hypothesized that the increased CDC activity of the fusion protein might result from enhanced target cell avidity due to the IFNα moeity binding to IFNα receptors (IFNAR) on the tumor cell surface. However, this was not the case, as flow cytometric studies demonstrated no improvement of fusion protein binding over rituximab, and comparable Kd values. Also, antibody blockade of IFNAR-IFNα interactions, or pre-incubation with excess free IFNα did not prevent the increased CDC activity of anti-CD20-hIFNα fusion protein against Ramos cells. Moreover, the enhanced CDC depended upon linkage of IFNα to the anti-CD20 antibody, as CDC using rituximab plus equimolar free IFNα was equivalent to rituximab alone, suggesting superior complement fixation by anti-CD20-hIFNα. In conclusion, we have demonstrated that anti-CD20-hIFNα has substantially stronger direct anti-proliferative and CDC activities than rituximab against human lymphomas, while retaining potent ADCC activity. The greater effects of IFNα targeted to NHL via fusion protein suggest a broader therapeutic index than rIFNα. Anti-CD20-hIFNα fusion protein is also superior to rituximab in vivo against multiple human NHL xenografts. These results support the further development of anti-CD20-hIFNα fusion protein for the treatment of B cell malignancies. Disclosures: Khare: ImmunGene, Inc.: Employment. Sachdev:ImmunGene, Inc.: Employment. Grewal:ImmunGene, Inc.: Employment.

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Characterization of Early Human B Cell Development by Gene Expression Profiling.

Blood ◽

10.1182/blood.v108.11.1352.1352 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1352-1352

Author(s):

Marit E. Hystad ◽

Trond H. Bo ◽

Edith Rian ◽

June H. Myklebust ◽

Einar Sivertsen ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Cell Development ◽

B Cell Development ◽

Cell Populations ◽

Early Human ◽

Human B Cell

Abstract B cells develop from hematopoietic stem cells (HSC) in the bone marrow (BM) through a number of distinct stages before they migrate to the periphery as naïve mature B lymphocytes. These developmental stages can be identified by expression of cell surface antigens and Ig gene rearrangement status. The aim of this study was to characterize the earliest steps of normal human B cell development by gene expression profiling. Immunomagnetic selection and subsequent fluorescence-activated cell sorting (FACS) were used to isolate five populations from adult human BM: CD34+CD38− (HSC), CD34+CD10+CD19− early lymphoid progenitor cells (ELP), CD34+CD10+CD19+IgM− progenitor B cells (pro-B), CD34−CD10+CD19+IgM− precursor B cells (pre-B) and CD34−CD10+CD19+IgM+ immature B cells (IM). Total RNA was extracted from the purified cell populations, amplified and hybridized to Lymphochip cDNA microarrays. Six independent experiments from different donors were performed for each cell population. Expression of the genes encoding the selection markers confirmed the validity of the approach. Interestingly, genes necessary for the V(D)J-recombination such as RAG-1, RAG-2, TdT and ADA showed higher gene expression in the ELP population than in the HSC. In contrast, the transcription factors E2A, EBF, and Pax-5, which are all essential for early B-cell development, were first turned on in pro-B cells, in accordance with the B-cell lineage commitment. The ELP did not express B, T or NK lineage markers, except for a higher expression level of CD2 in the ELP population than in the four other cell populations. Taken together, the expression pattern of CD2 and the V(D)J-recombination genes in the ELP population, indicate that these cells have developed a lymphocyte potential, but are not fully committed to B-lineage cells. Hierarchical cluster analysis of the 758 differentially expressed genes (differences in relative expression by a factor of two or more and with maximum10% FDR) revealed a pattern that clearly separated the five consecutive cell populations. Furthermore, we created expression signatures based on information from Gene Ontology (GO) http://source.stanford.edu/cgi-bin/source/sourceSearch. One of the clearest distinctions between the gene expressions of the five developmental populations involved genes associated with proliferation, and showed that the HSC and IM populations are relatively indolent while the pro-B and pre-B populations comprised high expression levels of nearly all the proliferation associated genes. Finally, we examined in further detail the transitions between HSC, ELP and pro B cells. We found 25 genes to be differently expressed in the ELP population in comparison to the HSC and pro-B populations, including IGJ, BCL2 and BLNK. To identify combinations of markers that could better discriminate the ELP population, we also performed a gene pair class separation test. This resulted in 68 gene pairs with score above 10 that were denoted very good discriminators. For several of the markers the differences in gene expression were verified at the protein level by five colour FACS analysis. Taken together, these results provide new insight into the molecular processes that take place in the early human B cell differentiation, and in particular provide new information regarding expression of genes in the ELP population.

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Efficacy of an Antagonistic Anti-CD40 Monoclonal Antibody, HCD122 (CHIR-12.12), in Preclinical Models of Human Non-Hodgkin’s Lymphoma and Hodgkin’s Disease.

Blood ◽

10.1182/blood.v108.11.230.230 ◽

2006 ◽

Vol 108 (11) ◽

pp. 230-230 ◽

Cited By ~ 6

Author(s):

Li Long ◽

Montesa Patawaran ◽

Xia Tong ◽

Seema Kantak ◽

Sharon L. Aukerman ◽

...

Keyword(s):

Antitumor Activity ◽

B Cell ◽

Cell Lines ◽

Xenograft Model ◽

Preclinical Models ◽

B Cell Malignancies ◽

Effector Cells ◽

Mantle Cell ◽

Xenograft Models ◽

Human B Cell

Abstract Non-Hodgkin’s lymphoma (NHL) and Hodgkin’s disease (HD) account for about 9% of new cancer cases annually or 64,000 cases per year in the United States. Although the survival rate has significantly improved recently due to new combination therapy regimens, an unmet medical need remains for refractory or resistant patients. HCD122 is a fully human antagonistic anti-CD40 therapeutic monoclonal antibody (mAb) with a dual mechanism of action: blocking CD40 and CD40 ligand (CD40L) interactions and mediating antibody-dependent cellular cytotoxicity (ADCC). CD40 is expressed in all human B-cell malignancies, and the CD40/CD40L interaction is important for tumor cell proliferation and survival. Previously HCD122 was shown to potently inhibit CD40L-induced human B-cell and follicular NHL cell proliferation, mediate ADCC against CD40-positive human malignant B-cell lines and inhibit tumor growth in Burkitt’s lymphoma and multiple myeloma xenograft models. In this study the antitumor activity of HCD122 was assessed in preclinical models of HD and other subtypes of human NHL, such as Mantle cell and Follicular lymphoma. CD40 was expressed in 5 of 7 established human HD and 11 of 12 NHL tumor cell lines tested, including Hs445, HDLM-2, KM-H2, L428, L1236, Jeko-1 and WSU-NHL. Using purified human NK cells as effector cells, HCD122 mediated potent ADCC against these cell lines in vitro with a picomolar EC50. When human macrophages were used as effector cells, HCD122 also induced antibody-dependent cellular phagocytosis (ADCP) against the NHL Daudi cell line and the HD cell line Hs445. The antitumor activity of HCD122 was further evaluated in vivo in EBV-negative NHL and HD xenograft models. When tested in a staged human Mantle cell lymphoma Jeko-1 s.c. xenograft model in which treatment was initiated when the mean tumor volume reached 100 mm3, HCD122 was highly efficacious and induced complete tumor regression in 70% (7/10) of treated animals when administered intraperitoneally at 1 mg/kg weekly for 4 weeks. In a staged human HD L428 s.c. xenograft model, which expresses CD20 as well as CD40, the antitumor activity of HCD122 was compared to rituximab. HCD122 was highly efficacious and induced a mean 74 % tumor growth inhibition (TGI) when administered at 0.1 mg/kg weekly for 3 weeks (p<0.001). At the same dose and schedule, rituximab achieved only 40% TGI (HCD122 vs. rituximab: p<0.001). These data combined with our previous studies in multiple myeloma and EBV-positive Burkitt’s lymphoma models show that HCD122 is a potent anti-CD40 antibody with pronounced antitumor activity in both EBV-positive and EBV-negative malignant B cell preclinical models. HCD122 is currently in Phase I clinical trials in B-cell malignancies.

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