Статті в журналах: "CD20 antibodies"

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Fuh, Franklin, Reina Fuji, Kirsten A. Poon, Dongwei Li, Clarissa David, Quyen Nguyen, Kathy Howell, et al. "Pharmacodynamic Effects of Administration of Maytansine Conjugated Anti-CD22 Monoclonal Antibodies to Cynomolgus Monkeys." Blood 112, no. 11 (November 16, 2008): 4996. http://dx.doi.org/10.1182/blood.v112.11.4996.4996.

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Abstract CD22 is a B cell-specific glycoprotein expressed on the cell surface of all mature B cells. A candidate therapeutic anti-CD22 antibody, 10F4v3, was conjugated to the anti-mitotic agent maytansine (10F4v3-DM1). DM1 disrupts cellular mitosis through inhibition of tubulin polymerization when internalized into cells. The anti-CD22 DM1 conjugate was shown to have significant potency in preclinical efficacy models of NHL. In order to further characterize this antibody-drug conjugate in preclinical studies, we first evaluated the binding characteristics of the 10F4v3 to peripheral blood B cells from various geographical sources of cynomolgus monkeys. 10F4v3 bound to peripheral blood B cells from all cynomolgus monkeys of Indonesian and Mauritian origins, but displayed only limited binding to cynomolgus monkeys of Chinese and Cambodian origins. Therefore, further pre-clinical evaluation of 10F4v3-DM1 was conducted in Indonesian cynomolgus monkeys to examine the safety, pharmacokinetic, and pharmacodynamic effects in monkeys dosed at 10, 20, and 30 mg/kg (2000, 4000, and 6000 mg/m2 DM1). Pharmacodynamic assessments of peripheral blood and lymphoid tissues included examination of B cells, B cell subsets, CD4+ T cells, CD8+ T cells, and CD3−CD20− (NK) cells. B cell subsets included CD20+, CD20+CD21+, CD20+CD21−, CD20+CD21+CD27+, CD20+CD21+CD27−, and CD20+CD21high lymphocytes which are phenotypically similar to human B cells, mature B cells, germinal center B cells, memory B cells, naïve B cells, and marginal zone B cells, respectively. B cells and B cell subsets were substantially depleted in peripheral blood at all doses, with no apparent dose-dependent effects. In lymphoid tissue, B cells were also depleted, with substantial depletion of CD20+CD21− and CD20+CD21high B cell subsets in spleen and bone marrow. Based on the nonclinical data, 10F4v3-DM1 exhibits an encouraging pharmacodynamic profile that supports clinical development for the potential treatment of non-Hodgkin’s lymphoma.

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Duperray, C., J. M. Boiron, C. Boucheix, J. F. Cantaloube, T. Lavabre-Bertrand, M. Attal, J. Brochier, D. Maraninchi, R. Bataille, and B. Klein. "The CD24 antigen discriminates between pre-B and B cells in human bone marrow." Journal of Immunology 145, no. 11 (December 1, 1990): 3678–83. http://dx.doi.org/10.4049/jimmunol.145.11.3678.

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Abstract When bone marrow (BM) lymphoid cells from 12 adult healthy donors were labeled by CD24 antibodies and analyzed by flow cytometry, two positive populations of cells were demonstrated in each sample (by a separated bimodal specific immunofluorescence). One population had intermediate CD24-Ag density (termed CD24+ cells) whereas the other had high CD24-Ag density (termed CD24(2+) cells). CD24+ cells represented 5.8 +/- 2.7% of the total lymphoid BM cells and CD24(2+) cells 5.6 +/- 2.5%. Using dual fluorescence analysis on eight samples, all CD24+ cells expressed the CD21 and CD37 mature B cell Ag and also surface IgM (sIgM), but this population lacked CD10 Ag. These cells also expressed CD19 Ag, and at a higher density than CD24(2+) cells. They were also positive for HLA-DR Ag. Conversely, CD24(2+) cells were shown to be early cells of the B cell lineage. While all the CD24(2+) cells were HLA-DR+ and CD19+, 64 +/- 16% of them expressed CD20 Ag (at a lower density than CD24+ cells), 65 +/- 21% CD10 Ag, and 22 +/- 8% were positive for cytoplasmic mu-chains (c mu). None of these cells expressed the CD21 and CD37 mature B cell Ag or sIgM. Additional experiments on four different healthy donors demonstrated that 30 +/- 9% of the CD24(2+) cells expressed the CD34 Ag and that the CD24+ cells did not express it. Thus, the CD24 Ag permits discrimination between two populations of the B cell lineage present in adult BM: 1) A CD24(2+) cell population including "pre" pre-B cells (HLA-DR+, CD19+, CD10+/-, CD20-, CD21-, CD34+, CD37-, c mu-), "intermediate" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu-), and "true" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu+). 2) A CD24+ cell population including B cells of the standard phenotype (HLA-DR+, CD19+, CD10-, CD20+, CD21+, CD34-, CD37+, c mu-, sIgM+).

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Rymkiewicz, Grzegorz, Renata Woroniecka, Katarzyna Blachnio, Barbara Pienkowska-Grela, and Jan Walewski. "Flow Cytometry and Fluorescence In Situ Hybridization Are Methods of Choice for Routine Diagnosis of Mantle Cell Lymphoma." Blood 106, no. 11 (November 16, 2005): 4659. http://dx.doi.org/10.1182/blood.v106.11.4659.4659.

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Abstract Mantle cell lymphoma (MCL) is a distinct clinicopathologic entity, characterized by expansion of lymphocytes with co-expression of CD5 and CD20 and frequent t(11;14) translocation. MCL and its morphological variants are frequently confused with other lymphoma subtypes. The aim of this study was to analyze a contribution of histopathology (HP), immunohistochemistry (IHC), flow cytometry (FCM) and cytogenetic analysis with fluorescence in situ hybridization (FISH) to ultimate diagnosis of MCL. We identified 66 pts diagnosed with MCL either by use of HP/IHC or/and FCM. Initial diagnosis was based on HP/IHC only, and was validated in 55 of 66 patients by combined use of HP/IH, FCM, and FISH. We examined paraffin sections IHC panel consisting of antibodies to CD3, CD5, CD20, CD23, CD43, and cyclin D1. FCM analysis was done by 3-colour direct immunofluorescence with extended panel of antibodies to CD5, CD10, CD11c, CD19, CD20, CD22, CD23, CD25, CD38, CD45, HLADR, FMC7, light and heavy chains. We used FISH to detect the t(11;14) in interphase cells of 24 cases of MCL. All 55 cases of MCL were CD20 positive and CD23 negative on IHC, 32 of 51 (63%) and 46 of 52 (88%) cases coexpressed the CD5 and CD43 antigens, respectively. Cyclin-D1 staining revealed nuclear positivity in 38 of 52 (73%) pts. Dual expression of CD5 and cyclin D1 - a finding highly reliable for MCL diagnosis on IHC, was seen in 49% (27/55) of pts. All MCL cases were CD20 (51/51) and CD19 (55/55) positive by FCM with higher intensity of CD20 expression compared to CD19 in 100% (51/51) of cases. CD5 and CD25 were coexpressed in 54 of 55 (98%) and in 43 of 49 (88%) pts respectively. CD22 (45/45) and HLADR (55/55) were positive in all cases. FMC7 and CD38 were found in 22 of 23 (96%) and in 19 of 23 (83%) patients, respectively. CD10, CD11c, CD23 were only seen on a subpopulation of cells with weak intensity in 15%(7/48), 13% (6/45), and 11% (5/46) pts., respectively. MCL cells expressed moderate intensity monoclonal surface light chains in 47 of 52 (90%) pts with L light chain predominance and IgM+/IgD+ in 16 of 17 (94%) pts. False or incomplete initial diagnosis based mainly on lymph node HP/IHC was found in 35 of 66 (53%) cases. False FCM diagnosis of MCL was found in 2 of 66 (3%) cases. Results of FCM and FISH were consistent with the MCL diagnosis in 92% (22/24) of patients, in our hands. Our data indicate that the phenotypic pattern of MCL by FCM is remarkably constant among patients. As MCL represents a frequent diagnostic challenge, a combined use of FCM and FISH may provide an optimum solution.

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4

Lones, Mark, and Ivan Kirov. "Cell Surface Targets for Monoclonal Antibody Therapy in Lymphoid Neoplasms of Children and Adolescents." Blood 104, no. 11 (November 16, 2004): 4544. http://dx.doi.org/10.1182/blood.v104.11.4544.4544.

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Abstract Recently, monoclonal antibodies have become available for treatment of lymphoid neoplasms in adults, but have not been studied in children and adolescents. These monoclonal antibodies are directed against cell surface antigens CD20 (Rituximab, Ibritumomab-Tiuxetan, Tositumomab), CD22 (Epratuzumab), CD52 (CAMPATH-1H), HLA-DR Beta-chain (Hu1D10), CD23 (IDEC-152), and CD33 (Gemtuzumab Ozogamicin). The objective of this study is to identify cell surface targets eligible for monoclonal antibody therapy in lymphoid neoplasms of children and adolescents. This is a retrospective analysis of lymphoid neoplasms evaluated by flow cytometry immunophenotyping at a single institution from January 2002 to July 2004. All patients were less than 21 years old at primary diagnosis. Flow cytometry immunophenotyping employed a 3-color method. Fluorochrome-conjugated monoclonal antibodies were utilized to detect cell surface antigens: CD20, CD22, CD23 (Becton-Dickinson), and CD52 (CALTAG) conjugated with PE; HLA-DR and CD33 (Becton-Dickinson) conjugated with FITC. For this study, a cell surface antigen was interpreted as positive when neoplastic cells exhibited moderate or bright intensity staining, or interpreted as negative when staining was dim or absent. A total of 95 patients are included in this study. Demographic data: Age <1 to 20 years (median 7); Male=52, Female=43. Diagnoses included: Precursor-B Acute Lymphoblastic Leukemia (Pre-B ALL) = 80, Precursor-T Acute Lymphoblastic Leukemia or Precursor-T Lymphoblastic Lymphoma (Pre-T ALL/LBL) = 11, Burkitt Lymphoma = 4. Total specimens = 105 (primary diagnosis = 82, relapse = 23). Immunophenotyping results for the number of specimens tested are in the Table. Table 1 Diagnosis CD20 CD22 CD52 HLA-DR CD23 CD33 Pre-B ALL 32/86 (37%) 90/90 (100%) 53/57 (93%) 87/87 (100%) 0/15 (0%) 4/90 (4%) Pre-T ALL/LBL 0/11 (0%) 0/11 (0%) 9/10 (90%) 2/11 (18%) 0/5 (0%) 0/11 (0%) Burkitt Lymphoma 4/4 (100%) 4/4 (100%) 3/3 (100%) 3/3 (100%) 0/2 (0%) 0/3 (0%) CD22 was positive (usually bright intensity) in all Pre-B ALL and Burkitt Lymphoma specimens. CD20 was positive in all Burkitt Lymphoma (bright intensity) and in a subset of Pre-B ALL (usually moderate intensity) specimens. CD22 and CD20 were negative in Pre-T ALL/LBL specimens. In a subset, CD52 was positive (moderate to bright intensity) in nearly all specimens. HLA-DR was positive (moderate to bright intensity) in all Pre-B ALL and Burkitt Lymphoma specimens. In a subset, CD23 was negative in all specimens. Also, CD33 was negative in nearly all specimens. In conclusion, lymphoid neoplasms in children and adolescents have cell surface antigens that are eligible targets for currently available monoclonal antibody therapy. Patients with Pre-B ALL are candidates for therapy directed to CD22, CD52, HLA-DR, and a subset to CD20, but not to CD23 or CD33. Patients with Burkitt Lymphoma are eligible for therapy to CD20, CD22, CD52, and HLA-DR, but not CD23 or CD33. Patients with Pre-T ALL/LBL are eligible for therapy to CD52, but not CD20, CD22, HLA-DR, CD23 or CD33. These results indicate that future clinical therapeutic trials can be designed for children and adolescents with lymphoid neoplasms to evaluate monoclonal antibody therapy directed to CD20, CD22, CD52, or HLA-DR, employing single or multiple antibodies as a new modality, in addition to chemotherapy.

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Paiva, Aldair Sousa, Alessandra Suelen Jardim Silva, Victor lima Soares, Gustavo Henrique de Medeiros Oliveira, Lenilton Silva DA Silva Júnior, Hugo Henrique de Freitas Ferreira, Rodrigo Villar Freitas, et al. "Importance of Flow Cytometry in the Differential Diagnosis of Hairy Cell Leukemia in the State of Rio Grande Do Norte, Brazil." Blood 136, Supplement 1 (November 5, 2020): 14–15. http://dx.doi.org/10.1182/blood-2020-143225.

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Introduction:Hairy Cell Leukemia (HCL) is a B-cell non-Hodgkin's Lymphoma (B-NHL) representing about 2% of chronic leukemias, is manifested in adults with an average age of 55 years old or more and the ratio of male: female is 5:1, being more common among white people. It is characterized by the presence of neoplastic lymphocytes with cytoplasmic projections (villous cells), a characteristic commonly observed in other DLPCs such as variant HCL (HCL-v) and splenic villous cell lymphoma (SVCL), being the immunophenotyping by flow cytometry determinant in the differential diagnosis of these neoplasms. HCL is characterized by splenomegaly, hepatomegaly, pancytopenia in peripheral blood (PB) with leukopenia, anemia, neutropenia, monocytopenia, and thrombocytopenia. It has a low number of circulating tumor cells, spleen, liver, and bone marrow (BM) infiltration.Objective:To investigate, by flow cytometry, patients with lymphocytosis and presence of villous lymphocytes in the characterization of HCL and HCV-v and SMZL.Methodology:Were investigated samples of peripheral blood (SP) and bone marrow (MO) from 27 patients previously diagnosed with DLPC and presence of villous lymphocytes which were by flow cytometry with a panel of monoclonal antibodies (MoAb) conjugated to fluorochromes and targeted to T-lymphocytes: CD1a, CD2, CD3, CD5, CD7, subpopulation T-helper (CD3/CD4) and T-cytotoxic (CD3/CD8), in addition to TCR a/b and TCR g/d; Natural Killer cells: CD16-56; B-lymphocytes: CD19, CD20, CD21, CD22, CD23, CD79b, CD200, IgM, IgG, IgD, anti-kappa and anti-lambda, in addition to CD10, TdT (Terminal deoxynucleotidyl Transferase), CD103, CD123, CD11c, CD25, CD38, CD138, CD45 and CD14. At the same time, a complete blood count with differential white blood cell count and investigation of clinical and demographic data such as age, sex and ethnicity / race were also performed.Results:The distribution of patients according to ethnicity and gender, there was a predominance of white individuals and males. The age group most affected was in patients older than 60 years. All patients expressed pan-B antigens on leukemic cells with expression of CD19, CD22 / CD20 (Forte), sIgH, associated with clonal restriction for immunoglobulin light chain (kappa= 20 and lambda= 7), associated with FMC7 expression, HLADR, CD38 and CD45 strong and negativity to CD10, CD138, CD200, CD23, CD5, TdT and related T antigens. Sixteen cases were categorized as HCL, six HCLv and five SVCL. The immunophenotyping of HCL cases was positive for CD103, CD25, CD123 and CD11c. HCLv was negative for CD103 in three cases and CD25 and SVCL negative for CD103, CD123 and CD11c and CD25 in all cases.Conclusions:The precise diagnosis of HCL has fundamental importance because each NHL-B has a specific treatment, besides emphasizing the sensitivity and speed of the IFC regarding diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.

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Geisler, CH, JK Larsen, NE Hansen, MM Hansen, BE Christensen, B. Lund, H. Nielsen, T. Plesner, K. Thorling, and E. Andersen. "Prognostic importance of flow cytometric immunophenotyping of 540 consecutive patients with B-cell chronic lymphocytic leukemia." Blood 78, no. 7 (October 1, 1991): 1795–802. http://dx.doi.org/10.1182/blood.v78.7.1795.1795.

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Abstract Blood mononuclear cells from 540 newly diagnosed, unselected patients with B-cell chronic lymphocytic leukemia (CLL) were examined by immunofluorescence flow cytometry for a panel of surface membrane markers, including IgM and IgD, the monoclonal antibodies anti-CD3, -5, -20, -21, -22, -FMC7, and, for the final 125 patients, anti-CD23. There were 503 CD5+ and 37 CD5- cases. In the CD5+ cases, the cells typically expressed IgM, IgD, CD20, CD21, CD22, and CD23. In univariate analysis, age, clinical stage, IgM-fluorescence intensity, CD23, and FMC7 had significant prognostic importance, with high IgM-fluorescence intensity, high FMC7, and low CD23 expression being associated with a short survival. There was no significant difference in survival between 351 cases expressing IgMD and 55 cases expressing IgM without IgD, or between kappa and lambda light chain monoclonal cases. CD20, CD21, and CD22 had no prognostic importance. In Cox multiple regression analyses, age, CD23, IgM-fluorescence intensity, and clinical stage (International Workshop System) had independent prognostic importance. Thus, besides clinical variables, CD23 and IgM intensity might be useful prognostic markers in the management of CD5+, B-cell CLL. The survival of CD5- patients was on the borderline of being significantly shorter than that of CD5+ patients. The majority of the CD5- cases were FMC7+, CD23-, had strong IgM fluorescence, and had splenomegaly.

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Geisler, CH, JK Larsen, NE Hansen, MM Hansen, BE Christensen, B. Lund, H. Nielsen, T. Plesner, K. Thorling, and E. Andersen. "Prognostic importance of flow cytometric immunophenotyping of 540 consecutive patients with B-cell chronic lymphocytic leukemia." Blood 78, no. 7 (October 1, 1991): 1795–802. http://dx.doi.org/10.1182/blood.v78.7.1795.bloodjournal7871795.

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Blood mononuclear cells from 540 newly diagnosed, unselected patients with B-cell chronic lymphocytic leukemia (CLL) were examined by immunofluorescence flow cytometry for a panel of surface membrane markers, including IgM and IgD, the monoclonal antibodies anti-CD3, -5, -20, -21, -22, -FMC7, and, for the final 125 patients, anti-CD23. There were 503 CD5+ and 37 CD5- cases. In the CD5+ cases, the cells typically expressed IgM, IgD, CD20, CD21, CD22, and CD23. In univariate analysis, age, clinical stage, IgM-fluorescence intensity, CD23, and FMC7 had significant prognostic importance, with high IgM-fluorescence intensity, high FMC7, and low CD23 expression being associated with a short survival. There was no significant difference in survival between 351 cases expressing IgMD and 55 cases expressing IgM without IgD, or between kappa and lambda light chain monoclonal cases. CD20, CD21, and CD22 had no prognostic importance. In Cox multiple regression analyses, age, CD23, IgM-fluorescence intensity, and clinical stage (International Workshop System) had independent prognostic importance. Thus, besides clinical variables, CD23 and IgM intensity might be useful prognostic markers in the management of CD5+, B-cell CLL. The survival of CD5- patients was on the borderline of being significantly shorter than that of CD5+ patients. The majority of the CD5- cases were FMC7+, CD23-, had strong IgM fluorescence, and had splenomegaly.

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Escribano, Luis, Alberto Orfao, Jesús Villarrubia, Beatriz Díaz-Agustín, Carlos Cerveró, Agustín Rios, José L. Velasco, Juana Ciudad, José L. Navarro, and Jesús F. San Miguel. "Immunophenotypic Characterization of Human Bone Marrow Mast Cells. A Flow Cytometric Study of Normal and Pathological Bone Marrow Samples." Analytical Cellular Pathology 16, no. 3 (1998): 151–59. http://dx.doi.org/10.1155/1998/341340.

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The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC) from healthy controls and patients with hematologic malignancies (HM) based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogenous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p= 0.08). Three patterns of antigen expression were detected: (1) markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and FcεRI), (2) antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138), and (3) markers that were positive in a variable proportion of cases – CD11b (50%), CD11c (77%), CD13 (40%), CD18 (20%), CD22 (68%), CD35 (27%), CD40 (67%), CD54 (88%) and CD61 (40%). In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes.In summary, our results show that BMMC from both healthy controls and HM patients display a relatively heterogenous immunophenotype. Interestingly, we have observed clear differences between the immunophenotype of BMMC and MC from other tissues. This could be due either to the heterogeneity of human MC according to their tissue localization or to the sensitivity of the method used for antigen detection.

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Weitzman, James, Monica Betancur, Laurent Boissel, Arthur P. Rabinowitz, and Hans Klingemann. "Variable Contribution of Different Monocloncal Antibodies to NK Cell-Mediated ADCC Against Primary CLL Cells." Blood 110, no. 11 (November 16, 2007): 4715. http://dx.doi.org/10.1182/blood.v110.11.4715.4715.

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Abstract Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of the B-cell antigens CD19, 20 and 22, along with CD5 and CD23. These antigens make the malignant cells an ideal target for monoclonal antibody (mAb) therapy. Although the mechanism of action of mAbs is complex and not fully understood, one well-described action is antibody-dependent cellular cytotoxicity (ADCC). Binding of mAb to its target surface antigen initiates cytotoxicity through the interaction of the Fc portion of the mAb with the Fc receptor (FcR) on natural killer (NK) cells. This triggers release of perforin and granzymes from NK cells, and subsequent killing of the target cells. This study’s objectives were to measure ADCC of 4 different mAbs against primary CLL cells, and determine if cytotoxicity is dependent on antigen density. Methods: Mononuclear cells from 16 patients with untreated CLL were separated by density gradient separation and served as targets. The antigen density for CD20, 22 and 23 of each patient sample was quantified by flow cytometry. Effector cells for ADCC were NK-92 cells that do not express FcR, and a high affinity Fc receptor-expressing NK-92 variant (NK92.26.5) that also expresses inhibitory receptors (i.e. KIR). A fluourochrome-based flow cytometric assay determined ADCC by subtracting NK-92 induced cytotoxicity from NK-92.26.5 induced killing. Monoclonal antibodies tested were Rituximab (anti-CD20, Biogen/IDEC), Veltuzumab (anti-CD20, Immunomedics), Epratuzumab (anti-CD22, Immunomedics), and Lumiliximab (anti-CD23, Biogen/IDEC). Results: Mean ADCC of the four antibodies tested against 16 primary CLL cells were: 46.5% for Rituximab; 43.4% for Veltuzumab; 5.8% for Epratuzumab; 8.8% for Lumiliximab. Cytotoxicity of NK-92 and NK-92.26.5 against CLL cells without antibody ranged from 2–6%. Mean antigen density on the 16 CLL patient specimens were: CD20: 27,900 (range: 10,000–56,100); CD22: 820 (600–1300); CD23: 9870 (2340–14,800). Conclusions: We have developed a reliable in vitro assay to measure ADCC of mAbs against CLL cells. Our results indicate that ADCC contributes to cytotoxcity of CLL in vitro, and suggest that the magnitude of ADCC depends upon the surface antigen targeted. Anti-CD20 antibodies had significantly greater ADCC than the anti-CD22 and CD23 antibodies. This pattern was consistent in all patient cells tested. A similar pattern was observed with the surface antigen density on CLL cells tested, suggesting a correlation between cell surface antigen density and ADCC. Our results also suggest that resistance of primary CLL cells toward NK-mediated killing can be overcome by monoclonal antibodies even in the presence of inhibitory KIR expression on NK cells.

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Kusakabe, Manabu, Guillermo Simkin, Justin Meskas, Chaoran Zhang, Daisuke Ennishi, Merrill Boyle, David W. Scott, et al. "Single Cell Mass Cytometry for Phenotypic Analysis of Diffuse Large B-Cell Lymphoma." Blood 124, no. 21 (December 6, 2014): 2976. http://dx.doi.org/10.1182/blood.v124.21.2976.2976.

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Abstract Diffuse Large B-cell Lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin Lymphoma (NHL). Despite its improved outcome with R-CHOP chemotherapy, 40% of patients still suffer with relapsed or refractory disease. Further investigation is needed to understand the complexity of DLBCL. Time-of-flight mass cytometry (CyTOF) is a recently developed technology that combines traditional flow cytometry with mass spectrometry and supports analysis of up to 30-40 parameters simultaneously at the single cell level with minimal spectral overlap (Bendall et al, Science 2011). In this study, we sought to develop a CyTOF method for phenotypic analysis of DLBCL to obtain high resolution profiles of the malignant clone(s) represented in individual tumor samples and resolve any underlying population substructure that might be informative in understanding the clinical and biologic heterogeneity of this disease. We designed a two-tube assay for this study. Tube #1 contained 35 cell surface markers including CD45, CD19, CD20, CD22, CD79B, IgM, IgD, Ig Kappa, Ig Lambda, CD5, CD10, CD23, CD43, CD38, CD138, CD44, CD21, CD24, CD40, CD72, CD80, CD45RA, CD49D, CD49F, CD62L, CD25, CD27, CD30, CD127, CD184, CD194, CD200, CD34, HLA-DR, and CD3. Tube #2 contained 37 markers in total including 17 cell surface markers overlapping with Tube #1 plus an additional 20 intracellular markers (BCL2, BCL6, IRF4/MUM1, LMO2, MYC, MCL1, MEF2B, KAT3B/P300, CBP, FOXP1, RUNX1, Ikaros, EZH2, BMI1, NOTCH1, CARD11, IkBa, phospho-Rb, Ki67, and CyclinD2). Metal-conjugated antibodies were purchased from DVS Sciences or unconjugated antibodies labeled in-house using DVS MaxPar metal labeling kits. Data were acquired using a DVS CyTOF2 instrument. We examined both fresh and viably frozen single cell suspensions from diagnostic lymph node biopsy samples received for flow cytometric analysis at the BC Cancer Agency. We used SPADE (spanning-tree progression analysis of density-normalized events) for initial data analysis. We first performed preliminary validation studies including cross-comparison of mass cytometry (CyTOF2) vs. flow cytometry (BD Canto2) datasets and fresh vs. previously frozen cell suspension material. Although individual marker intensities using matched antibody clones were in general lower by CyTOF, eight-parameter surface staining results were qualitatively comparable between the two platforms. Also there were essentially no differences observed in CyTOF staining profiles between fresh and previously frozen samples. Preliminary clustering analysis of cell populations using SPADE revealed clear separation between normal and malignant B cell populations as well as apparent substructure to the malignant population in a subset of DLBCL samples. These findings suggest intratumoral heterogeneity can be resolved by high dimensional CyTOF analysis. Ongoing efforts will focus on determining if phenotypically defined subsets show enrichment for subclonal mutations. Disclosures No relevant conflicts of interest to declare.

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Qu, Zhengxing, David M. Goldenberg, Thomas M. Cardillo, Victoria Shi, Hans J. Hansen, and Chien-Hsing Chang. "Bispecific anti-CD20/22 antibodies inhibit B-cell lymphoma proliferation by a unique mechanism of action." Blood 111, no. 4 (February 15, 2008): 2211–19. http://dx.doi.org/10.1182/blood-2007-08-110072.

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Combination immunotherapy with anti-CD20 and anti-CD22 mAbs shows promising activity in non-Hodgkin lymphoma. Therefore, bispecific mAbs (bsAbs) were recombinantly constructed from veltuzumab (humanized anti-CD20) and epratuzumab (humanized anti-CD22) and evaluated in vitro and in vivo. While none of the parental mAbs alone or mixed had notable antiproliferative activity against Burkitt lymphoma cells when not cross-linked, the bsAbs [eg, anti-CD20 IgG-anti–CD22 (scFv)2] were inhibitory without cross-linking and synergistic with B-cell antigen (BCR)-mediated inhibition. The bsAbs demonstrated higher antibody-dependent cellulary cytoxicity (ADCC) activity than the parental mAbs, but not complement-dependent cytoxicity (CDC) of the parental CD20 mAb. Cross-linking both CD20 and CD22 with the bsAbs resulted in the prominent redistribution of not only CD20 but also CD22 and BCR into lipid rafts. Surprisingly, appreciable translocation of CD22 into lipid rafts was also observed after treatment with epratuzumab. Finally, the bsAbs inhibited Daudi lymphoma transplant growth, but showed a significant advantage over the parental anti-CD20 mAb only at the highest dose tested. These results suggest that recombinantly fused, complementary, bispecific, anti-CD20/22 antibodies exhibit functional features distinct from their parental antibodies, perhaps representing new candidate therapeutic molecules.

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Chuksina, J. J., E. V. Kataeva, and T. A. Mitina. "Features of immunophenotypic finding B-cell lymphoproliferative diseases by flow cytometry." Kazan medical journal 101, no. 1 (February 11, 2020): 145–52. http://dx.doi.org/10.17816/kmj2020-145.

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Aim. To assess the information content of conventional and additional immunophenotypic markers (CD200, CD305) in the differential diagnosis B-cell lymphoproliferative diseases by flow cytometry. Methods. An immunophenotypic study using 4-color flow cytometry was performed in 204 patients with different variants of B-cell non-Hodgkin's lymphomas. The study material included peripheral blood and bone marrow. The expression of CD45, CD19, CD20, CD22, CD79b, CD79a, CD5, CD10, CD23, FMC7, CD43, CD38, CD11c, CD103, CD25, CD 200, CD 305, light chains of immunoglobulins (kappa/lambda) using monoclonal antibodies (Becton Dickinson, USA) was evaluated. The intensity of antigen expression was assessed using mean fluorescence intensity (y. e.). Results. Conventional FMC7-positive expression revealed only half patients with different variants of leukemization of non-Hodgkin's lymphomas, whereas atypical positive expression of CD23 was observed in patients with marginal spleen lymphoma and follicular lymphoma in 27.3 and 28.6% of cases, respectively. In mantle cell lymphoma, expression of CD200 in B-cell was detected in a significantly smaller number of observations, accompanied by a significant decrease in the average intensity of CD200 fluorescence compared to B-cell chronic lymphocytic leukemia (B-CLL) cells. The mean fluorescence intensity (MFI) of CD305 in hairy cell leukemia is significantly higher than in splenic marginal zone lymphoma (SMZL) with villous lymphocytes. Conclusion. Different levels of the information content of some conventional markers were revealed in differential immunophenotypic diagnosis of B-cell lymphoproliferative diseases by flow cytometry; the use of additional markers CD200 and CD305 was highly informative in differential diagnostics between different variants of B-cell lymphoproliferative diseases with similar immunophenotypic and morphological characteristics of lymphoid elements.

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Jabbour, Elias, Susan O’Brien, Farhad Ravandi, and Hagop Kantarjian. "Monoclonal antibodies in acute lymphoblastic leukemia." Blood 125, no. 26 (June 25, 2015): 4010–16. http://dx.doi.org/10.1182/blood-2014-08-596403.

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Abstract With modern intensive combination polychemotherapy, the complete response (CR) rate in adults with acute lymphoblastic leukemia (ALL) is 80% to 90%, and the cure rate is 40% to 50%. Hence, there is a need to develop effective salvage therapies and combine novel agents with standard effective chemotherapy. ALL leukemic cells express several surface antigens amenable to target therapies, including CD20, CD22, and CD19. Monoclonal antibodies target these leukemic surface antigens selectively and minimize off-target toxicity. When added to frontline chemotherapy, rituximab, an antibody directed against CD20, increases cure rates of adults with Burkitt leukemia from 40% to 80% and those with pre-B ALL from 35% to 50%. Inotuzumab ozogamicin, a CD22 monoclonal antibody bound to calicheamicin, has resulted in marrow CR rates of 55% and a median survival of 6 to 7 months when given to patients with refractory-relapsed ALL. Blinatumomab, a biallelic T cell engaging the CD3-CD19 monoclonal antibody, also resulted in overall response rates of 40% to 50% and a median survival of 6.5 months in a similar refractory-relapsed population. Other promising monoclonal antibodies targeting CD20 (ofatumumab and obinutuzumab) or CD19 or CD20 and bound to different cytotoxins or immunotoxins are under development. Combined modalities of chemotherapy and the novel monoclonal antibodies are under investigation.

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Paietta, Elisabeth, Xiaochun Li, Sue Richards, Janis Racevskis, Gordon W. Dewald, Jacob M. Rowe, Martin S. Tallman, et al. "Implications for the Use of Monoclonal Antibodies in Future Adult ALL Trials: Analysis of Antigen Expression in 505 B-Lineage (B-Lin) ALL Patients (pts) on the MRC UKALLXII/ECOG2993 Intergroup Trial." Blood 112, no. 11 (November 16, 2008): 1907. http://dx.doi.org/10.1182/blood.v112.11.1907.1907.

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Abstract MRC UKALLXII/ECOG2993 accrued 739 ECOG pts over 13 years. ECOG’s reference laboratories centrally characterized 613 (91%) and 505 were B-Lin ALLs. Expression of 32 antigens (Ags), including 9 myeloid-associated Ags, was determined by multiparameter flow cytometry on gated blasts, both as percentage of Ag expressing blasts and intensity of antibody staining (equivalent of Ag density). To test for the potential therapeutic efficacy of monoclonal antibodies, such as rituximab, epratuzumab, alemtuzumab, or gemtuzumab in future trials, expression of Ags for these antibodies (CD20, CD22, CD52, or CD33, respectively) was determined. In E2993, 14% of pts typed as Pro-B (CD10 negative), 71% as Early Pre-B (CD10 positive), 13% as Pre-B (intracytoplasmic mu positive), and 2% as Mature B-ALL (surface mu chains positive). CD20 expression correlated with the stage of B-lymphoid maturation (p=5.4E-13) (see table). Median CD20 intensity of fluorescence was lowest in Pro-B and highest in Mature B-ALL. Membrane expression of CD22 was lower in Pro-B compared with the other three groups, Early Pre-B, Pre-B and Mature B (p=1.0E-5) but indistinguishable among the latter three (p=0.21). While levels of CD20 and CD22 had no effect on complete remission (CR) and overall survival (OS) univariatly, higher CD22 expression was significantly correlated with better OS (p=0.02) in a bivariate Cox model of CD20 and CD22. With respect to myeloid Ags, CD33/CD13 expression was associated with Early Pre-B (p=0.02), and CD65(s)/CD15(s) with Pro-B ALL (p<0.0001). While CD65(s) and CD15(s) did not affect outcome, the combined expression of CD33/ CD13 conferred an inferior prognosis; this effect was due to the association of CD33/ CD13 with BCR/ABL positivity (p<0.0001). BCR/ABL negative B-Lin ALL (N=350) expressed CD33 on a median of only 4% of blasts, compared with a median of 54% in BCR/ABL positive ALL (N=152) (p=3.5E-09). However, intensity of CD33 staining was significantly lower in both BCR/ABL negative and positive blasts (median 3.1) compared with leukemic myeloblasts (median 56) (p=<0.001). Median CD52 expression was higher in BCR/ABL positive versus negative B-Lin ALL (p=3.5E-06); in neither group was CD52 associated with outcome. We conclude that antibodies to CD52 are better suited for BCR/ ABL positive than negative B-Lin ALL. Since expression of CD33 in ALL is weak, CD33- independent effects of gemtuzumab must be investigated. Low CD20 combined with high CD22 expression confers superior OS. The expression of these Ags in most B-Lin ALL pts justifies the incorporation of antibodies into frontline chemotherapy regimens. B-Lin ALL Stage CD20 Percentage Median Intensity CD22 Percentage Median Intensity ≥20% ≥50% ≥75% ≥20% ≥50% ≥75% Pro-B 21 10 3 2 88 72 55 12 Early PreB 66 48 37 21 96 90 84 28 Pre-B 61 46 37 21 100 90 81 37 Mature B 87 80 64 106 92 84 80 9

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Chang, Chien-Hsing, Edmund A. Rossi, and David M. Goldenberg. "Monoclonal antibodies targeting CD20." mAbs 5, no. 3 (May 2013): 335–36. http://dx.doi.org/10.4161/mabs.24106.

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Amu, Sylvie, and Mikael Brisslert. "Phenotype and Function of CD25-Expressing B Lymphocytes Isolated from Human Umbilical Cord Blood." Clinical and Developmental Immunology 2011 (2011): 1–9. http://dx.doi.org/10.1155/2011/481948.

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Background. We have shown that approximately 30% of human peripheral blood B-cells express CD25. B cells expressing CD25 display a mature phenotype belonging to the memory B-cell population and have a better proliferative and antigen-presenting capacity. The aim of the present study was to characterize the CD25-expressing subset of B cells in human cord blood.Material and Methods. Mononuclear cell fraction from human cord blood (n=34) and peripheral adult blood (n=22) was sorted into CD20+CD25+and CD20+CD25-B-cell populations. Phenotype and function of these B-cell populations were compared using flow cytometry, proliferation, cytokine production, and immunoglobulin secretion.Results. CD25-expressing B cells are a limited population of cord blood mononuclear cells representing 5% of the CD20+B cells. They are characterised by high expression of CD5 in cord blood and CD27 in adult blood. CD25-expressing B cells express a functional IL-2 receptor and high levels of CC-chemokine receptors and spontaneously produce antibodies of IgG and IgM subclass.Conclusions. CD25 expression is a common denominator of a specific immunomodulatory B-cell subset ready to proliferate upon IL-2 stimulation, possibly ready to migrate and home into the peripheral tissue for further differentiation/action.

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Loken, MR, VO Shah, KL Dattilio, and CI Civin. "Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development." Blood 70, no. 5 (November 1, 1987): 1316–24. http://dx.doi.org/10.1182/blood.v70.5.1316.1316.

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Abstract A panel of B lymphoid-reactive monoclonal antibodies was used to analyze the phenotypic changes that accompany B lymphocyte development in normal human bone marrow. The B lymphoid cells were identified using light scattering and the expression of CD19 on a flow cytometer. Quantitative three-color immunofluorescence was then used to correlate other cell surface antigens on these cells identified as B lymphoid in normal marrow. CD10 and CD20 identified almost exclusive populations and provided a convenient means of discriminating between the less and more mature B lineage cells. The CD10+ cells could be further subdivided using CD34. The population of CD19+, CD10+, CD34+ cells comprised only 0.6% of marrow cells, but these contained the majority of terminal deoxynucleotidyl transferase (TdT+) cells. In the assessment of class II antigens, HLA-DR was expressed on all B lineage cells whereas HLA-DP preceded HLA-DQ in appearance during the developmental process. Among the later antigens expressed on B lineage cells, cell surface IgM, CD20, and HLA-DQ were expressed at essentially the same time. Cell surface CD10 was lost at the time when CD21 and CD22 were acquired on the cell surface. These data illustrate that multiparameter flow cytometry can be used to define a continuous progression of stages of B lymphocyte development based on cell surface antigen expression even though these cells represent a minor fraction of normal marrow cells.

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Loken, MR, VO Shah, KL Dattilio, and CI Civin. "Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development." Blood 70, no. 5 (November 1, 1987): 1316–24. http://dx.doi.org/10.1182/blood.v70.5.1316.bloodjournal7051316.

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A panel of B lymphoid-reactive monoclonal antibodies was used to analyze the phenotypic changes that accompany B lymphocyte development in normal human bone marrow. The B lymphoid cells were identified using light scattering and the expression of CD19 on a flow cytometer. Quantitative three-color immunofluorescence was then used to correlate other cell surface antigens on these cells identified as B lymphoid in normal marrow. CD10 and CD20 identified almost exclusive populations and provided a convenient means of discriminating between the less and more mature B lineage cells. The CD10+ cells could be further subdivided using CD34. The population of CD19+, CD10+, CD34+ cells comprised only 0.6% of marrow cells, but these contained the majority of terminal deoxynucleotidyl transferase (TdT+) cells. In the assessment of class II antigens, HLA-DR was expressed on all B lineage cells whereas HLA-DP preceded HLA-DQ in appearance during the developmental process. Among the later antigens expressed on B lineage cells, cell surface IgM, CD20, and HLA-DQ were expressed at essentially the same time. Cell surface CD10 was lost at the time when CD21 and CD22 were acquired on the cell surface. These data illustrate that multiparameter flow cytometry can be used to define a continuous progression of stages of B lymphocyte development based on cell surface antigen expression even though these cells represent a minor fraction of normal marrow cells.

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Santos, Marcelo Antônio Oliveira, and Marinus de Moraes Lima. "CD20 role in pathophysiology of Hodgkin’s disease." Revista da Associação Médica Brasileira 63, no. 9 (2017): 810–13. http://dx.doi.org/10.1590/1806-9282.63.09.810.

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Summary Hodgkin’s lymphoma (HL) is a tumor comprising non-malignant and malignant B-cells. Classical HL expresses CD15+ and CD30+ antigens, and 20 to 40% of patients are CD20+. This antigen is a ligand free protein present in B lymphocyte cells and its function is not well known. Some studies suggest that expression of CD20 may play a major role in Hodgkin’s disease pathophysiology and may affect the patients’ treatment prognosis, as well as relapse and refractory response. In the past few years, development of monoclonal anti-CD20 antibodies changed drastically the treatment for non-Hodgkin lymphomas in which CD20 is expressed. HL treatment is essentially composed of radiotherapy and chemotherapy; however, monoclonal anti-CD20 antibodies applicability is not well delimitated due to lack of information about clinical outcomes with anti-CD20 monotherapy or combined drug therapy using a classic regimen, as well as about CD20 pathophysiology mechanisms in B-cells tumors. The objective of our review is to discuss CD20 function in Hodgkin’s lymphoma development, its influence on disease evolution and outcomes, as well as its effects on therapeutics and patients’ prognostic.

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Paiva, Aldair Sousa, Alessandra Suelen Jardim Silva, Lenilton Silva DA Silva Júnior, Gustavo Henrique de Medeiros Oliveira, Hugo Henrique de Freitas Ferreira, Maria das Graças Pereira Araujo, Victor lima Soares, et al. "Importance of Flow Cytometry in the Immunophenotyping Characterization of Mantle Cell Lymphoma in State of Rio Grande Do Norte, Brazil." Blood 136, Supplement 1 (November 5, 2020): 15. http://dx.doi.org/10.1182/blood-2020-143328.

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Introduction:Mantle Cell Lymphoma (MCL) is a disease of a neoplastic nature characterized by proliferation in lymphoid tissues, classified as non-Hodgkin's lymphoma of B lymphocytes (NHBL). The nomenclature derives from the specific pathophysiology of this disease, that proliferates in the marginal area of the mantle of the germinal center of the lymph nodes.Objective:Carry out a detailed assessment of the immunophenotypic profile using the flow cytometry (CF) technique in 26 patients, NHBL to confirm mantle lymphoma.Materials and Methods:Biological samples from 26 patients, these being from peripheral blood (SP) or bone marrow (MO), of patients with suspected MCL were marked with monoclonal antibodies conjugated to fluorochromes and subsequently analyzed by FC.Results:all patients with a confirmed diagnosis of LCM were positive for pan-B antigens CD19, CD20 (strong) and CD22 (strong), CD19+/CD5+ and ciclin-D1 in 100% of cases. In addition to these, CD23, CD56, CD38, CD79b, Ki-67, Bcl-2, FMC-7 and HLA-Dr also obtained positive staining, as well as the immunoglobulin heavy chain (IgH) in all cases, and isotypes of immunoglobulins, IgD, IgG and IgM in most cases, with a predominance of the heavy chain of the lambda light chain in 79% of cases. Antigens related to T lymphocytes such as CD1a, CD2, CD3, CD7, TCR alpha/beta, TCR gamma/delta, CD4 and CD8, as well as antigens related to NK cells (CD16-56) and lymphoid progenitor cells (CD10 and Terminal- deoxynucleotidyl-transferase) were negative, as were CD200, antigens related to hairy cell leukemia (CD103, CD11c and CD123) and multiple myeloma (CD138).Discussion:According to the expression of the antigens, the antibodies were positive for: Cyclin D, CD19, CD20, CD22, CD38, Ki-67, Bcl-2, FMC-7 and CD79b, which are relevant to the diagnosis of this disease. Conclusion: Flow cytometry immunophenotyping is the technique currently used to confirm the diagnosis of neoplasms, such as LCM, which, due to its specific phenotype, helps in the correct selection of treatment. Disclosures No relevant conflicts of interest to declare.

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Jiao, Cheng, and P. A. Zeynalova. "Prognostically significant bone marrow cellular content changes in diffuse large B-cell lymphoma." Russian Journal of Biotherapy 17, no. 4 (January 11, 2019): 58–63. http://dx.doi.org/10.17650/1726-9784-2018-17-4-58-63.

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Introduction. Increasing of blast cell percentage in bone marrow of diffuse large B-cell lymphoma patients is a sign of unfavourable prognosis. We estimated the frequency of this phenomenon and made attempts to idenify minimal bone marrow involvement in DLBCL by flow cytometry.Materials and methods. Study has been done in 60 DLBCL patients. Diagnosis in all cases was done according to WHO (2008) criteria. Bone marrow study was done on smears (myelogram). Immunophenotypic study was done with a large panel of B-lineage monoclonal antibodies as well as kappa and lambda light chains to membrane immunoglobulins.Results. Increase in blast cell content in bone marrow was noted in 73.3 % of DLBCL patients. That group of patients had lower surviv al. We used the following methods for identification of monoclonal B-lymphocytes: monoclonality according to membrane light immu noglobuline chains within mature B-lymphocytes and aberrancy of immunophenotype. Levels of different B-cell antigen expression – CD20, CD21, CD22, CD24. It was identified B-lymphocytes with aberrant expression of СD21.Conclusion. The most informative methods of identification of minimal bone marrow involvement in DLBCL are estimation of clonality according to light chain expression of mature B-lymphocytes (CD45++CD20+CD5–), as well as assessement of aberrancy of CD21 expression of mature B-lymphocytes.

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Turaj, Anna H., Vikki L. Field, Claude H. T. Chan, Christine A. Penfold, Jinny H. Kim, Sonya James, Kerry L. Cox, et al. "Anti-CD27 Enhances Lymphoma Immunotherapy through Profound Myeloid Cell Recruitment." Blood 128, no. 22 (December 2, 2016): 3024. http://dx.doi.org/10.1182/blood.v128.22.3024.3024.

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Abstract Direct-targeting monoclonal antibodies (mAb) such as anti-CD20 mAb are thought to elicit their anti-tumor function through antibody-dependent cellular phagocytosis (ADCP) mediated by myeloid cells (monocytes and macrophages), with little involvement of T cells. In contrast, immunomodulatory mAbs to TNFR superfamily members, CD27, OX40 and CD137, function by augmenting T-cell responses. We examined the therapeutic potential of combining anti-CD20 mAb with a panel of immunomodulatory mAbs (OX40, CD137, CD27, TIGIT, GITR, CTLA4, PD-1). In the syngeneic BCL1 B-cell lymphoma mouse model only an agonistic mAb to CD27, provided a synergistic effect when combined with anti-CD20. Anti-CD20 and anti-CD27 mAb individually provided modest therapeutic benefit (median survival 33 days and 62 days, respectively), but mice treated with the combination survived beyond 100 days. Similar synergistic survival benefit was observed in another B-cell lymphoma model, A31, and in BCL1-bearing human CD27 transgenic mice, when anti-CD20 was combined with varlilumab, an anti-human CD27 mAb currently under clinical investigation. We observed that in mice treated with anti-CD27, there was an early and substantial increase in intra-tumoral monocyte, neutrophil and macrophage infiltration. CD27 is expressed constitutively on T and NK cells but not myeloid cells or the tumor itself. To investigate whether CD27 agonism promotes intra-tumoral myeloid cell infiltration through T cells, we depleted T cells in the BCL1model. Surprisingly, CD4 or CD8 T-cell depletion had no effect on the survival of anti-CD20 and anti-CD27-treated mice, suggesting that the remaining CD27+ immune effector cells, NK cells, are required. To further probe the relative importance of these two sub-sets, we performed experiments in γ chain knockout mice, where activatory FcγR are not expressed. Here, anti-CD27 mediated T-cell activation can still occur via crosslinking from the inhibitory FcγRII, but effector function through NK cells, mediated through activatory FcγR, is abrogated. In this model, the therapeutic benefit of anti-CD27 was completely abolished, thereby supporting the requirement for NK cells. We hypothesize that anti-CD27 stimulates CD27+ NK cells to release chemokines that draw myeloid cells into the tumor, where they subsequently perform augmented anti-CD20 mediated ADCP. These data demonstrate the clear therapeutic potential of combining direct targeting and immunomodulatory mAb but that the therapeutic mechanism of action may differ to that expected; here involving a previously unheralded effect of anti-CD27 on myeloid infiltration. Based upon these data, we have implemented a phase II clinical trial examining rituximab and varlilumab in B-cell lymphoma, which will commence recruitment shortly. Disclosures Keler: Celldex Therapeutics: Employment, Equity Ownership. Johnson:Celldex Therapeutics: Research Funding. Al-Shamkhani:Celldex Therapeutics: Patents & Royalties: On therapeutic use of antibodies targeting anti-CD27 and another applied for anti-CD20/anti-CD27 use, Research Funding. Glennie:Celldex Therapeutics: Patents & Royalties: Patent on therapeutics use of antibodies targeting human CD27 and patent for anti-CD20+anti-CD27 applied. Cragg:Baxalta: Consultancy; Gilead Sciences: Research Funding; GSK: Research Funding; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding. Lim:Celldex Therapeutics: Patents & Royalties: Patent for anti-CD20+anti-CD27 applied, Research Funding.

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Rossi, Edmund A., Rosana Michel, Chien-Hsing Chang, and David M. Goldenberg. "Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus." Blood 122, no. 21 (November 15, 2013): 2282. http://dx.doi.org/10.1182/blood.v122.21.2282.2282.

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Abstract Background The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases (AIDs), treating currently over 1500 cases of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemias, Waldenström's macroglobulinemia, Sjögren's syndrome, and systemic lupus erythematosus (SLE). Because epratuzumab, which is currently in worldwide Phase III registration trials for SLE, reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity (ADCC) and negligible complement-dependent cytotoxicity (CDC) when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. Instead, ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, activation, homing, and re-circulation, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and b7 integrin, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or SLE patients, and of NHL cells spiked into normal PBMCs (Rossi et al., Blood 2013 PMID: 23821660). Rituxmab has clinical efficacy in SLE, but failed to achieve primary endpoints in a Phase III trial. Here we show for the first time that a bispecific hexavalent antibody (bsHexAb), comprising epratuzumab and veltuzumab (humanized anti-CD20), exhibits enhanced trogocytosis compared to epratuzumab, with considerably less B-cell depletion than observed with anti CD20 mAbs. Methods and Results A pair of bsHexAbs were generated using DOCK-AND-LOCKTM (DNLTM) to comprise epratuzumab fused with four additional Fab fragments of either veltuzumab [designated 22*-(20)-(20)] or of a humanized anti-CD19 mAb [22*-(19)-(19)]. PBMCs were incubated with the bsHexAbs or the parental mAbs (10 µg/mL) overnight, and the relative surface levels of the key antigens were analyzed by flow cytometry. The 22*-(20)-(20) exhibited the broadest and most extensive trogocytosis, reducing each of CD22, CD20, CD19, CD21, CD79b, CD44, CD62L, and Beta-7 integrin more than epratuzumab, and to a similar extent as veltuzumab, except for CD22, which was much lower with the 22*-(20)-(20) (Table 1). In general, 22*-(19)-(19) showed intermediate trogocytosis, with less antigen reduction than 22*-(20)-(20), but more than epratuzumab. Veltuzumab and rituximab caused considerable (40-50%) B-cell depletion in the ex-vivo assay. Alternatively, epratuzumab, hA19, and both bsAbs did not significantly deplete B cells. ADCC, which is presumably, the primary mechanism of B-cell depletion in the ex-vivo assay, is less potent for 22*-(20)-(20), compared to veltuzumab. CDC, which along with ADCC is an important mechanism for B-cell depletion in vivo, is ∼25-fold less potent for 22*-(20)-(20) compared to veltuzumab. Epratuzumab has minimal CDC and ADCC. Conclusion The bsHexAb 22*-(20)-(20) is an excellent candidate for treatment of SLE and other AIDs due to its ability to mediate potent trogocytosis without wholesale depletion of B cells, which leads to increased risk of serious infections associated with anti-CD20 therapy. Disclosures: Rossi: Immunomedics, Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties.

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Mishima, Yuji, Yasuhito Terui, Kengo Takeuchi, Yuko Mishima, and Kiyohiko Hatake. "Characterization of Mechanisms for Acquiring Resistant Phenotype Using a Novel Screening Strategy for Detecting Rituximab-Resistant Mutations." Blood 120, no. 21 (November 16, 2012): 1550. http://dx.doi.org/10.1182/blood.v120.21.1550.1550.

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Abstract Abstract 1550 Background: We previously reported that mutations of CD20 gene were found in patients with B-cell non-Hodgkin's lymphoma, and we proposed that C-terminal deletion mutations of CD20 might be involved in relapse/resistance after rituximab containing therapy. Most of the patients that had mutation in the C-terminal leagion were diagnosed as CD20 negative by immunohistochemistry using L26 monoclonal antibody. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. So at first we determined the binding site of L26 antibody on CD20 protein. Then we developed novel diagnostic antibodies that recognize wide variety of CD20 molecular subtypes including those having mutations. Methods: To determine the epitope of L26 antibody, we established six sub-lines expressing various length of C-terminal truncated CD20 using an originally CD20 negative myeloma cell line. Then we carried out epitope-mapping using these cell lines. To detect comprehensive CD20 molecules including that having mutation in C-terminal region, we developed antibodies that recognize near the amino terminus of CD20 molecules (CD20N antibody). CD20N antibody is the only monoclonal antibody that recognizes N-terminal region of CD20 so far. Using these antibodies, we screened the specimens of the cases diagnosed as CD20 negative determined by L26-based immunohistochemistry. Results: The epitope-mapping revealed that L26 recognizes near the C-terminus of CD20. This suggested that most of CD20 molecules with the C-terminal deletion mutation or frame-shift mutation could not be recognized by L26. Then we screened previously diagnosed specimens and found several cases that having the cells stained by our novel antibody but not by L26. Genetic analysis revealed that all these cells had a mutation in the C-terminal cytoplasmic region of CD20. One of these cases, we successfully analyzed the phenotype of lymphoma cells with mutated CD20 in detail using cryopreserves living specimens. In this case, a frame shift mutation occurred due to one base nucleotide deletion, resulting in the translation of peptide of another reading frame of 41 amino acids with premature stop at the amino acid position 250. Interestingly, mutant CD20 molecule expressed adjacent to the plasma membrane, but rituximab could not bind to these cells. DNA sequencing study about genome and mRNA of CD20 gene suggested that the lymphoma cells of this patient had one normal and one mutated CD20 allele. Discussions: The C-terminal region of CD20 may undertake a pivotal role in presentation of the large loop where the rituximab binding site locates. Thus, deletion or frame-shift mutation of CD20 in C-terminal cytoplasmic region impairs the antigenicity against rituximab and it may cause resistance to rituximab therapy. The resistance caused by gene mutation thought to be irreversible. And it should be discriminated from transient downregulation of antigen expression. We propose here that immunohistochemical screening using CD20N antibody is very rapid and effective screening stategy that find out irreversible rituximab resistant cases. Disclosures: No relevant conflicts of interest to declare.

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Brauner, Jonathan, Ingrid Beukinga, Zoulikha Amraoui, Zaina Kassengera, Michel Toungouz, and Olivier Pradier. "Flow Cytometry Primary 10 Colours Panel for Chronic Lympho Proliferative Diseases Diagnosis." Blood 118, no. 21 (November 18, 2011): 5190. http://dx.doi.org/10.1182/blood.v118.21.5190.5190.

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Abstract Abstract 5190 Objectives: Definition of the primary antibodies panel for 10 colours flow cytometry able to describe normal and clonal T, B lymphocytes and plamocytes in blood and bone marrow. Once clonalities are detected, the complete characterisation of Chronic Lymphoproliferative Diseases (CLPD) is supported by secondary panels chosen based on the results of CD5/CD10 expression for clonal B lymphocytes, CD27/CD38 for plasmatocytes and CD3/CD27 for clonal T cells. Materials and Methods: Blood and bone marrow of patients (N=50) with CLPD (mainly B-CLL). Samples are enumerated by haematology analyzer DxH 800 then 106 cells are washed three times, stained with the antibodies combination and red blood cells lysed with Versalyse (TM. Beckman Coulter). The samples were analysed on a 10 colours Navios flow cytometer (Beckman Coulter Fullerton, CA). The staining panel consists of 14 antibodies (CD45, CD8, CD4, CD5, CD3, CD19, CD38, λ, κ, CD23, CD5, CD10, CD14, CD27) conjugated with 10 different fluorochromes. The fixed gating strategy allows linking Navios analysis software to the middleware Remisol which drives the choice of the secondary panel. In some cases a third tube is performed for Ki67 or Zap-70 intra-cytoplasmic staining. Results: Monocytes are removed on the basis of their CD14/CD4 expression. B lymphocytes are CD19 positive. Normal naïve/memory B cells, hematogones and plasma cells are defined by their CD27, CD10 and CD38 expression. Eventual monoclonality is sought by analysis of the distribution of Kappa and Lambda light chains. A first classification of B cell lymphoma is achieved with the CD5 and CD10 expression of the clone (CD5+/CD10−: B-CLL MCL and few MZL, CD5−/CD10−: MZL and related, CD5−/CD10+ DLBCL and FL). Analysis of CD27, CD20 and CD23 expression allows discriminating between CD5+/CD10- lymphomas. All the 50 samples were correctly detected as CLPD and the automated Remisol choice of the second panel fit to the final diagnosis of all the cases of this small series. T lymphocytes are defined by their CD3 and CD5 expression. The analysis of CD4/CD8 balance and CD27/CD5 distribution are first line test when T cell clonality is suspected. There is a special gating to detect CD3-CD4+ T cell lymphoma and double negativity of CD4 and CD8 is a surrogate marker for gamma/delta T cells. NK cells are mentioned as not-T not-B lymphocytes, without specific staining. Conclusion/Discussion:This 10 colours 14 antibodies panel allows describing in one tube normal T and B cells, hematogones, memory and naives B cells plasma cells and detects T and B clonalities. This panel follows a similar logic than the Euroflow LST tube but with 10 colours and with Beckman Coulter's technology and antibodies. Moreover, this combination helps discriminating rapidly the CD5+/CD10- lymphomas while the complete characterisation of CD5 negative lymphomas only require less than 6 antibodies second tube. This is a paperless (all the process is driven and controlled by Remisol), fast and inexpensive diagnostic approach (always less than 20 antibodies required). Disclosures: Pradier: Beckman Coulter: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Sapra, Puja, Gary Kikuchi, Ashwini Venkatasamy, Marianne K. Hayes, Christina M. Satterwhite, Darren Warren, Mark D. Walker, David M. Goldenberg, and Ivan D. Horak. "Preclinical Pharmacology and Toxicology of Humanized Anti-B-Cell Antibodies (Anti-CD22 and Anti-CD20) in Cynomolgus Monkeys (CM)." Blood 106, no. 11 (November 16, 2005): 1471. http://dx.doi.org/10.1182/blood.v106.11.1471.1471.

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Abstract hLL2 (epratuzumab) and hA20 are humanized IgG1 monoclonal antibodies (MAbs) recognizing B-cell antigens, CD22 and CD20, respectively. CD22 and CD20 are expressed at all stages of B-cell development, except progenitor stem cells and plasma cells. Both hLL2 and hA20 have shown promising clinical activity in patients with non-Hodgkin’s lymphomas (NHL). Further, hLL2 has demonstrated clinical efficacy in patients with autoimmune diseases, such as systemic lupus erythematosus and Sjögren’s syndrome. Here, we discuss preclinical toxicology studies and compare the pharmacodynamics (PD) and pharmacokinetics (PK) of hA20 and hLL2 in CD20/CD22-crossrecative CM. For hLL2, we performed two toxicology studies. In the first 14-Day tolerability study, CM received hLL2 as two intravenous (IV) infusions at 0 (vehicle), 10, 60 and 160 mg/kg given one week apart, followed by necropsy on Day 14. In a second 6-month PK/PD study, hLL2 was administered in 2 cycles of 4-weekly infusions, with a 1-month treatment-free period between cycles followed by necropsy at the end of the second cycle. For hA20, CM received 4-weekly infusions of hA20 at 0 (vehicle), 8, 24, 80, and 240 mg/kg-doses and were either sacrificed 1 week after the last infusion or followed for recovery. Both hA20 and hLL2 were well tolerated. There were no drug-related clinical signs or changes in body weight, food intake, ophthalmic condition, EKG, blood pressure, and standard hematology, serum chemistry, coagulation or urinalysis parameters. Also, there were no changes in organ weights, gross necropsy, or histopathology findings. For hLL2, PD indicated a mean reduction from baseline for all B-lymphocytes (CD20+B-cells, or activated CD20+HLA-DR+ and CD20+CD27+ B-cells) of approximately 30–50%, starting on Day 3 in both studies, with no apparent dose-response. In the cyclical regimen study, this effect continued through Day 113 (day after the end of 2nd cycle). In contrast, for hA20, almost 85–95% B cell depletion was observed in all treatment groups on Day 6 and there was a trend towards dose-related depletion. Both hLL2 and hA20 had little discernable effect on T cell subsets, monocytes, or plasma cells. hLL2 and hA20 had a t1/2 of 3–5 days after the first infusion, and hLL2 had a t1/2 of 6–7 days after the fourth infusion. There was no significant difference between PK of hLL2 after the first and second cycles. With regard to immunogenicity, primate antibodies to either hA20 or hLL2 were not detected. In conclusion, hA20 demonstrates significantly higher B-cell depletion, similar to Rituximab, compared to hLL2 in CM. Clinical results obtained with hLL2 as well as studies of in vitro mechanism of action suggest that partial B-cell modulation by hLL2 in autoimmune diseases may provide benefit, and may function by different mechanisms than B-cell depletion related to hA20. In NHL, combined therapy of CD20 and CD22 may be more effective than single-MAb treatment, as has been suggested clinically.

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Niederfellner, Gerhard J., Alfred Lammens, Manfred Schwaiger, Guy Georges, Kornelius Wiechmann, Andreas Franke, Wolfgang Schaefer, et al. "Crystal Structure Analysis Reveals That the Novel Type II Anti-CD20 Antibody GA101 Interacts with a Similar Epitope as Rituximab and Ocrelizumab but in a Fundamentally Different Way." Blood 114, no. 22 (November 20, 2009): 3726. http://dx.doi.org/10.1182/blood.v114.22.3726.3726.

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Abstract Abstract 3726 Poster Board III-662 CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all therapeutic CD20 antibodies are directed against the two relatively small extracellular loops of CD20, such antibodies can be classified into Type I CD20 antibodies like Rituximab, Ofatumumab or Ocrelizumab or Type II CD20 antibodies like the novel glycoengineered humanized CD20 antibody GA101 or the murine antibody Tositumumab. Type I and Type II antibodies differ significantly in their mode of action and mechanisms of killing malignant B-cells. The molecular basis of this is not understood. We use data from epitope mapping, X-ray crystallography, isothermal titration calorimetry, and point mutagenesis i) to accurately map the epitopes of different anti-CD20 antibodies, in particular GA101, and ii) to compare the molecular interactions involved in their binding. Although the epitope regions of these antibodies largely overlap, the crystal structure shows that GA101 binds CD20 in a completely different orientation from Rituximab, Ocrelizumab and Ofatumumab and that its binding also involves a larger surface area. In agreement with predictions based on the crystallographic data, point mutagenesis of single amino acid residues confirmed that exchanges at certain positions in CD20 affect binding of Rituximab and GA101 differently. Our data suggest that engagement of CD20 by these antibodies favors different conformations of CD20, which could form the molecular basis for the observed differences in cellular signals triggered by the respective antibodies. Disclosures: Niederfellner: Roche: Employment. Schwaiger:Roche: Employment. Georges:Roche: Employment. Wiechmann:Roche: Research Funding. Franke:Roche: Research Funding. Schaefer:Roche: Employment. Jenewein:Roche: Employment. Slootstra:Pepscan: Employment, Patents & Royalties. Moessner:Glycart: Employment, Equity Ownership, Patents & Royalties. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Hopfner:Roche: Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

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Rossi, Edmund A., Chien-Hsing Chang, Thomas M. Cardillo, Rhona Stein, Diana Pilas, Diane L. Nordstrom, John Kopinski, and David M. Goldenberg. "Bispecific Anti-CD20/Anti-CD22 Antibodies with Potent Lymphoma Cytotoxicity." Blood 112, no. 11 (November 16, 2008): 1591. http://dx.doi.org/10.1182/blood.v112.11.1591.1591.

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Abstract BACKGROUND: Multivalent bispecific antibodies (bsMAbs) have shown improved efficacy in a number of preclinical studies. We made a pair of hexavalent bsMAbs based on epratuzumab (hLL2, anti-CD22) and veltuzumab (hA20, anti-CD20) by the Dock-and-Lock (DNL) method, and compared their properties to each other and their parental MAbs. METHODS AND RESULTS: DNL is used for the site-specific and covalent assembly of modular components, and was utilized to generate stably tethered hexavalent bispecific complexes, each composed of 4 Fab fragments conjugated to an IgG at the latter’s carboxyl termini of the heavy chain. DNL-1 has 4 veltuzumab Fabs tethered to epratuzumab IgG; DNL-2 has 4 epratuzumab Fabs bound to veltuzumab IgG. DNL made each construct as a single, defined, homogeneous structure that is stable in serum. All of the constituent Fab fragments are functional, with binding affinities similar to the parental MAbs. In vitro analyses using human lymphoma cell lines demonstrated that, unlike the parental MAbs, the bsMAbs each induced translocation of both CD22 and CD20 into lipid rafts and also strong cell-cell adhesion. DNL1 or DNL2 treatment resulted in increased apoptosis vs. the parental MAbs alone or combined. DNL1 and DNL2 inhibited the growth of Ramos, Raji and Daudi Burkitt lymphoma cell lines without the requirement of crosslinking, and more potently than the combination of the parental MAbs. For Daudi cells, DNL1 and DNL2 showed similar activity, which was approximately 50-fold more potent than the combination of the parental MAbs. For Raji and Ramos, DNL1 was 8–10-fold more potent than DNL2, which in turn was 8–10-fold more potent than the combined parental MAbs. The results suggest that crosslinking of CD20 and CD22 at the cell surface is required for enhanced cytotoxicity. Veltuzumab, but neither epratuzumab nor either bsMAb, displayed CDC activity. Veltuzumab and DNL2 induced a similarly high degree of ADCC. DNL1 induced ADCC to an intermediate level between veltuzumab/DNL2 and epratuzumab. In an ex-vivo assay using fresh whole blood mixed with either Daudi or Raji, DNL1 and DNL2 each demonstrated selective killing of lymphoma cells over normal B-cells compared to veltuzumab or rituximab; the latter depleted normal B-cells with greater efficiency than the bsMAbs. PK studies in mice demonstrated that despite their large size, the bsMAbs have a significantly shorter serum half-life than the IgGs. Even without CDC activity and with a considerably shorter serum half-life, DNL2 had anti-lymphoma efficacy in the Daudi Burkitt lymphoma model in mice that was equivalent to veltuzumab. DNL1 was less potent than DNL2 in vivo, but more effective than epratuzumab and control bsMAbs comprising either epratuzumab-IgG-AD2 (22-14) or 4 veltuzumab-Fab-DDD2 groups (734-22) combined with 4 non-binding Fab-DDD2 groups or a non-binding IgG-AD2, respectively. The anti-tumor efficacy of both DNL1 and DNL2 was abolished in tumor-bearing mice in which their ADCC potential was diminished by depletion of neutrophils and NK cells. These findings suggest that ADCC is the most critical mechanism of action for lymphoma killing in these murine models. DNL1 is more potent than DNL2 in vitro yet DNL2, having the stronger ADCC activity, is more potent than DNL1 in vivo. CONCLUSIONS: These findings suggest that the DNL method can be used to make a variety of multivalent bsMAbs with potent anti-tumor activity, and having distinct properties dependent on their arrangement and composition. CD20/CD22 bsMAbs appear to have different functions than their parental MAbs, even when these were combined, and appear to be potent anti-B-cell lymphoma therapeutics.

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Bazinet, Alexandre, Ryan N. Rys, Claudia M. Wever, Amadou Barry, Celia Greenwood, Christian Young, Alma Mendoza, et al. "A 10-Color Flow Cytometry Panel for Both Diagnosis and Minimal Residual Disease Measurement in Chronic Lymphocytic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 5451. http://dx.doi.org/10.1182/blood-2019-123904.

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Background: Chronic lymphocytic leukemia (CLL) has a classic immunophenotype, consisting of light chain restriction, CD5+, CD19+, dim CD20, CD23+, CD43+, CD200+, CD10- and CD79b-. This distinguishes it from normal B cells and other lymphoproliferative disorders (LPDs). Antibodies targeting these antigens are included in two 8-color flow cytometry panels developed by the Euroflow consortium for the work up of B cell LPDs. Combining these antibodies into one 10-color panel would be more cost-effective. Furthermore, new CLL therapies can induce deep remissions, creating an increasing demand to measure minimal residual disease (MRD), defined as having over 1 residual leukemic cell per 10,000 leukocytes (10-4). The current international standardized approach for measuring MRD established by the European Research Initiative on CLL (ERIC) uses a panel of antibodies targeting CD3, CD5, CD19, CD20, CD22, CD43, CD79b and CD81. However, these antibody-fluorochrome combinations are different than those used by the Euroflow diagnostic panels. Thus laboratories considering implementing MRD testing would need to purchase antibodies for 3 different panels (2 diagnostic and 1 MRD). We expanded the Euroflow 8-color lymphocyte screening tube (LST) to include CD200 and CD23, such that CLL can be detected in one 10-color tube, at levels as low as 0.01%. The goal of this study was to determine the potential cost savings in implementing this new panel and to determine if it is sensitive enough to detect MRD. Methods: We calculated the number of samples analyzed with our modified 10-color LST tube (mLST1, obtained lyophilized) from April 2018 to March 2019 to rule out an LPD and the number of antibody aliquots saved using this approach compared to the standard 2-tube Euroflow method. We also created a version of the above-mentioned panel (mLST2) using liquid antibodies to increase the generalizability of our results, substituting CD38 with CD43 to see if this improved MRD detection (see panels below). For MRD testing, we used CLL samples from 24 different patients to produce 60 MRD samples at various concentrations of leukemic cells. Samples were prepared by spiking CLL cells into suspensions of normal leukocytes at approximate concentrations of 0.1%, 0.01% and 0.001%. Each sample was aliquoted and stained with the three panels: ERIC, mLST1 and mLST2. Data was acquired using a BD FACSCanto II or a BD FACSAria Fusion and analysed using BD FACSDiva software. CLL cells were identified based on differential expression of key markers and MRD was calculated as the number of CLL cells/total leukocytes. MRD positivity was defined as ≥ 0.01%. Agreement between the panels was assessed using the Bland-Altman plot method. We also calculated the percentage agreement between the panels in identifying MRD positivity. Results: In 1 year, mLST1 was performed on 474 samples, of these 220 had an LPD and 123 (56%) had a classic CLL phenotype, obviating the need for further testing. This resulted in the net savings of 476 antibody aliquots. For MRD assessment, differential expression of CD5 and CD20 were the most significant contributors in distinguishing CLL from normal B cells using the mLST1 and mLST2. We identified one CLL case with an atypical immunophenotype (dim CD5, bright CD20) which proved difficult to gate using a mLST panel. There was agreement in MRD results obtained with the mLST panels and the ERIC panel. For values above the limit of quantification, the 95% limit of agreement was ±0.3369 log for the ERIC vs mLST1 comparison and ±0.3485 log for the ERIC vs mLST2 comparison. Thus, variability in MRD levels between the panels was less than 2-fold the majority of the time, which we considered clinically acceptable as MRD is measured on an exponential scale. The ERIC panel and the mLST1 had 88.3% agreement in distinguishing MRD-positive versus MRD-negative samples. Agreement was 93.3% between the ERIC panel and the mLST2. Conclusions: Using a modified 10-color LST panel for both diagnosis and MRD measurement of CLL is feasible. The advantages are increased familiarity with the antibodies and potential cost savings, making MRD accessible to more cytometry laboratories. Atypical CLL cases without the usual CD5 positivity and dim CD20 are very difficult to gate using an LST panel. In these cases, the ERIC panel is clearly superior as CD22, CD79b, CD81 and CD43 can still provide separation between the malignant and normal lymphocytes. Disclosures Bazinet: BD Biosciences: Other: Provided a significant amount of the antibodies used in this project free of cost.. Wever:Teva Canada Innovation: Employment. Gimmig:BD Biosciences: Employment. Johnson:Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Merck: Consultancy, Honoraria; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; BMS: Consultancy, Honoraria; Seattle Genetics: Honoraria.

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30

Cassano, Carlos, Swetlana Mactier, Stephen P. Mulligan, Larissa Belov, Pauline Huang, and Richard I. Christopherson. "Cladribine and Fludarabine Nucleoside Change the Levels of CD Antigens on B-Lymphoproliferative Disorders." International Journal of Proteomics 2010 (May 5, 2010): 1–9. http://dx.doi.org/10.1155/2010/964251.

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The purine analogs, fludarabine nucleoside (FdA), and cladribine (CdA) (1 μM, 24 hours), significantly changed the levels of some surface antigens on the human B-cell lines MEC2 and Raji. Changes in the surface proteins were identified using a Cluster of Differentiation (CD) antibody microarray that captures live cells and confirmed by flow cytometry. For Raji cells, CdA up-regulated CD10, CD54, CD80, and CD86, with repression of CD22, while FdA up-regulated CD20, CD54, CD80, CD86 and CD95. For MEC2 cells, CdA up-regulated CD11a, CD20, CD43, CD45, CD52, CD54, CD62L, CD80, CD86, and CD95, but FdA had no effect. Up-regulation of particular CD antigens induced on a B-cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing.

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31

Niederfellner, Gerhard, Alfred Lammens, Olaf Mundigl, Guy J. Georges, Wolfgang Schaefer, Manfred Schwaiger, Andreas Franke, et al. "Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies." Blood 118, no. 2 (July 14, 2011): 358–67. http://dx.doi.org/10.1182/blood-2010-09-305847.

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Abstract CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30° wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies.

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Nalivaiko, Kristina, Martin Hofmann, Ludger Grosse-Hovest, Peter H. Krammer, Hans-Georg Rammensee, Helmut R. Salih, and Gundram Jung. "Inhibition of Antibody Production in Vitro with Bispecific CD20 X CD95 Antibodies." Blood 118, no. 21 (November 18, 2011): 1114. http://dx.doi.org/10.1182/blood.v118.21.1114.1114.

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Abstract Abstract 1114 Antibodies directed against the B-cell associated CD20 surface antigen can target normal as well as malignant B cells. They are sucessfully used for the treatment of B-cell derived leukemia and lymphoma and antibody mediated autoimmune disease, respectively. We have previously described that bispecific antibodies with specificity for CD20 and the death receptor CD95 are capable of inducing CD95 mediated apoptosis selectively in CD20-positive lymphoma cells. We now show that CD20 X CD95 hybrid antibodies induce apoptosis in pokeweed mitogen (PWM) activated B cells expressing CD95, but not in resting cells lacking it. Antibody production induced by PWM in vitro is profoundly inhibited. These results indicate that bispecific CD20 X CD95 antibodies may be used for the treatment of antibody mediated autoimmune disease. Compared to monospecific CD20 antibodies these reagents offer a new effector principle and specificity for activated rather than resting B cells. Disclosures: No relevant conflicts of interest to declare.

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Mason, DY, H. Stein, J. Gerdes, KA Pulford, E. Ralfkiaer, B. Falini, WN Erber, K. Micklem, and KC Gatter. "Value of monoclonal anti-CD22 (p135) antibodies for the detection of normal and neoplastic B lymphoid cells." Blood 69, no. 3 (March 1, 1987): 836–40. http://dx.doi.org/10.1182/blood.v69.3.836.836.

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Abstract Two monoclonal antibodies (To15 and 4KB128) specific for the B cell- associated CD22 antigen (135,000 mol wt) are described. On immunoenzymatic analysis of cryostat tissue sections, these antibodies strongly label both mantle zone and germinal center B lymphoid cells in secondary lymphoid follicles (and also scattered extrafollicular lymphoid cells) but are unreactive with other cell types (with the exception of weak reactivity with some epithelioid histiocytes). These reactions differ from those of monoclonal antibodies B1 and B2 (anti- CD20 and CD21) but are similar to those of the pan-B antibody B4 (anti- CD19). One of the anti-CD22 antibodies (To15) has been tested extensively by immunoenzymatic labeling on greater than 350 neoplastic lymphoid and hematological samples. The CD22 antigen was found in tissue sections in most B cell-derived neoplasms, the major exceptions being myeloma (all cases negative) and a small proportion of high-grade lymphoma (6% of cases negative). In cell smears, the antigen could be found on neoplastic cells in most B cell lymphoproliferative disorders, including common acute lymphoblastic leukemia (ALL) (90% positive) and B cell chronic lymphocytic leukemia (CLL) (89% positive). We conclude that anti-CD22 antibodies are of value for identification of human B cell lymphoproliferative disorders (especially when used in conjunction with anti-CD19 antibodies). Previous reports that the CD22 antigen is absent from many B cell neoplasms are probably due to its being expressed within the cytoplasm of immature B cells rather than on their surface.

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Mason, DY, H. Stein, J. Gerdes, KA Pulford, E. Ralfkiaer, B. Falini, WN Erber, K. Micklem, and KC Gatter. "Value of monoclonal anti-CD22 (p135) antibodies for the detection of normal and neoplastic B lymphoid cells." Blood 69, no. 3 (March 1, 1987): 836–40. http://dx.doi.org/10.1182/blood.v69.3.836.bloodjournal693836.

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Two monoclonal antibodies (To15 and 4KB128) specific for the B cell- associated CD22 antigen (135,000 mol wt) are described. On immunoenzymatic analysis of cryostat tissue sections, these antibodies strongly label both mantle zone and germinal center B lymphoid cells in secondary lymphoid follicles (and also scattered extrafollicular lymphoid cells) but are unreactive with other cell types (with the exception of weak reactivity with some epithelioid histiocytes). These reactions differ from those of monoclonal antibodies B1 and B2 (anti- CD20 and CD21) but are similar to those of the pan-B antibody B4 (anti- CD19). One of the anti-CD22 antibodies (To15) has been tested extensively by immunoenzymatic labeling on greater than 350 neoplastic lymphoid and hematological samples. The CD22 antigen was found in tissue sections in most B cell-derived neoplasms, the major exceptions being myeloma (all cases negative) and a small proportion of high-grade lymphoma (6% of cases negative). In cell smears, the antigen could be found on neoplastic cells in most B cell lymphoproliferative disorders, including common acute lymphoblastic leukemia (ALL) (90% positive) and B cell chronic lymphocytic leukemia (CLL) (89% positive). We conclude that anti-CD22 antibodies are of value for identification of human B cell lymphoproliferative disorders (especially when used in conjunction with anti-CD19 antibodies). Previous reports that the CD22 antigen is absent from many B cell neoplasms are probably due to its being expressed within the cytoplasm of immature B cells rather than on their surface.

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Rossi, Edmund A., David M. Goldenberg, Thomas M. Cardillo, Rhona Stein, and Chien-Hsing Chang. "Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma." Blood 113, no. 24 (June 11, 2009): 6161–71. http://dx.doi.org/10.1182/blood-2008-10-187138.

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Abstract The dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a pair of humanized hexavalent anti-CD20/22 bispecific antibodies (bsAbs) derived from veltuzumab (v-mab) and epratuzumab (e-mab). The 22-20 was made by site-specific conjugation of e-mab to 4 Fabs of v-mab; 20-22 is of the opposite configuration, composing v-mab and 4 Fabs of e-mab. Each bsAb translocates both CD22 and CD20 into lipid rafts, induces apoptosis and growth inhibition without second-antibody crosslinking, and is significantly more potent in killing lymphoma cells in vitro than their parental antibodies. Although both bsAbs triggered antibody-dependent cellular toxicity, neither displayed complement-dependent cytotoxicity. Intriguingly, 22-20 and 20-22 killed human lymphoma cells in preference to normal B cells ex vivo, whereas the parental v-mab depleted malignant and normal B cells equally. In vivo studies in Daudi tumors revealed 20-22, despite having a shorter serum half-life, had antitumor efficacy comparable with equimolar v-mab; 22-20 was less potent than 20-22 but more effective than e-mab and control bsAbs. These results indicate multiple advantages of hexavalent anti-CD20/22 bsAbs over the individual parental antibodies and suggest that these may represent a new class of cancer therapeutics.

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Gerasimova, E., D. Tabakov, A. Aleksankin, R. Shayakhmetova, T. Popkova, and A. Avdeeva. "AB0032 CHARACTERIZATION OF PERIPHERAL BLOOD B-CELL SUBSET IN UNTREATED PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND SYSTEMIC SCLEROSIS." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 1150.2–1151. http://dx.doi.org/10.1136/annrheumdis-2022-eular.3739.

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BackgroundB-cells play a critical role in the regulation of systemic autoimmune diseases pathogenesis, that extends beyond antibodies production.ObjectivesTo examine В-cell subsets in peripheral blood of patients (pts) with untreated systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), to analyze of their associations with disease-specific autoantibodies.MethodsThirty six untreated SLE pts (31F/5M) and 32 untreated SSc pts (25F/7M) were enrolled. SLE pts had median age of 37(range)(32-41)years, disease duration of 3.5(1-11)years, SLEDAI 2K of 7(4-8), BILAG of 14,5(10,2-23). SSc pts had median age of 42(38-50)years, disease duration of 3.5(1-10)years, Disease Activity EScSG of 2.5 (1-3.2). The control group consisted of 29 volunteers (23F/6M, median age 38(35-48)years. CD19+B cells, memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), non-switched memory B cells (CD19+CD27+IgD+), naive (CD19+CD27-IgD+), double-negative (CD19+CD27-IgD-), transitional (CD19+CD38++CD10+IgD+CD27-) B cells, and plasmablasts (CD19+СD38+++CD27+IgD-CD20-) were analyzed using multicolor flow cytometry.ResultsDifferences in the median percentage and of absolute B-cells subset levels in untreated pts with RA and SLE were not found. The absolute counts of memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), transitional B cells (CD19+CD38++CD10+IgD+CD27-), and plasmablasts (CD19+СD38+++CD27+IgD-CD20-) were higher in SLE and SSc pts compared to healthy donors, p<0,01 for all cases. The absolute counts of double-negative B cells (CD19+CD27-IgD-) were lower in SLE pts than in donors, р=0,03 (Table 1). At significant correlation was found in SLE pts between anti-dsDNA levels and absolute counts of the following B-cell subtypes: CD19+B cells (r=0,72), memory B cells (CD19+CD27+) (r=0,76), switched memory B cells (CD19+CD27+IgD-) (r=0,75) and naive B cells (CD19+CD27-IgD+) (r=0,73); the a-Sm levels and absolute counts of the switched memory B cells (CD19+CD27+IgD-) (r=0,51); the antibodies to cardiolipin (aCL) IgG levels and absolute counts of CD19+B cells (r=0,64), and with naive B cells (CD19+CD27-IgD+) (r=0,59), p<0,01 for all cases. There was a correlation between the anti-topoisomerase-1 antibodies (a-Topo-1) and the absolute counts of double-negative B cells (CD19+CD27-IgD-) (R=0,62, p=0,01) in SSc pts.Table 1.Levels of the blood B-cell subsets in SLE pts, SSc pts and in control.Parameters, n (х109/l)SLESScControl groupCD19+B cells0,15 (0,12-0,25)0,14 (0,12-0,20)0,1 (0,08-0,2)memory B cells (CD19+CD27+)0,036(0,031-0,006) *0,042(0,027-0,053) *0,003(0,001-0,007) *switched memory B cells (CD19+ CD27+IgD-)0,02 (0,016-0,046) *0,026 (0,017-0,034) *0,01 (0,005-0,02) *non-switched memory B cells (CD19+CD27+IgD+)0,012(0,007-0,027)0,016(0,01-0,027)0,02(0,01-0,04)naive B cells (CD19+CD27-IgD+)0,09 (0,06-0,2)0,09 (0,06-0,16)0,1 (0,06-0,1)double-negative B cells (CD19+CD27-IgD-)0,011 (0,006-0,014) *0,02 (0,01-0,03)0,02 (0,01-0,02) *transitional B cells (CD19+CD38++CD10+IgD+CD27-)0,011 (0,004-0,026) *0,011 (0,005-0,012) *0,0001 (0-0,0003) *plasmablasts (CD19+СD38+++CD27+IgD-CD20-)0,001 (0,001-0,002) *0,001 (0-0,002) *0,0001 (0,00006-0,0003) *Note: * - Differences in B-cell subset of the control group with SLE and SSc groups.ConclusionImmunophenotyping showed similar levels of B-cell subset in untreated SLE and SSc pts, an increase in the absolute counts of memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), transitional B cells (CD19+CD38++CD10+IgD+CD27-), and plasmablasts (CD19+СD38+++CD27+IgD-CD20-) in untreated SLE and SSc pts compared with healthy subjects. Positive correlation between the counts of В-cell subsets and values of disease-specific autoantibodies (anti-dsDNA, a-Sm, aCLIgG, a-Topo-1) suggests that B-lymphocytes may be involved in SLE and SSc pathogenesis.This work was supported by the Russian Science Foundation (Grant № 22-25-00358).Disclosure of InterestsNone declared

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van Oers, Marinus H. J. "CD20 antibodies: type II to tango?" Blood 119, no. 22 (May 31, 2012): 5061–63. http://dx.doi.org/10.1182/blood-2012-04-420711.

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Although the chimeric anti-CD20 monoclonal antibody (mAb) rituximab has revolutionized the treatment of B-cell non-Hodgkin lymphoma (NHL), still many patients relapse and an increasing number become refractory to rituximab-containing therapy. This has initiated intense research to develop more potent anti-CD20 antibodies.

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38

Cragg, Mark S. "CD20 antibodies: doing the time warp." Blood 118, no. 2 (July 14, 2011): 219–20. http://dx.doi.org/10.1182/blood-2011-04-346700.

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39

Gopal, Ajay K., and Oliver W. Press. "Clinical applications of anti-CD20 antibodies." Journal of Laboratory and Clinical Medicine 134, no. 5 (November 1999): 445–50. http://dx.doi.org/10.1016/s0022-2143(99)90164-6.

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Klein, Christian, Alfred Lammens, Wolfgang Schäfer, Guy Georges, Manfred Schwaiger, Ekkehard Mössner, Karl-Peter Hopfner, Pablo Umaña, and Gerhard Niederfellner. "Response to: Monoclonal antibodies targeting CD20." mAbs 5, no. 3 (May 2013): 337–38. http://dx.doi.org/10.4161/mabs.24108.

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41

Mishima, Yuji, Yasuhito Terui, Yuko Mishima, and Kiyohiko Hatake. "Anti-N-Terminal CD20 Monoclonal Antibody and L26 Dual Staining Identifies An Irreversible Resistant Mutant Genes." Blood 116, no. 21 (November 19, 2010): 4145. http://dx.doi.org/10.1182/blood.v116.21.4145.4145.

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Abstract Abstract 4145 [Introduction] Recently, we reported that gene mutations of CD20 were involved in resistance to rituximab therapy, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy. Many of these cases were diagnosed as CD20 negative by the immunohistochemical analysis using the L26 monoclonal antibody used routinely in most clinical laboratories. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. So, we could not distinguish whether protein expression of CD20 extremely decreased or whether the epitope of the antibody was lost by these mutations. To make this clear, we determined the binding site of L26 antibody on CD20 protein in the present study. In addition, we developed new antibodies that recognize amino acid sequence close to the amino terminal of CD20 molecule. Then we investigated clinical specimens with these antibodies together with L26 to elucidate characteristics of CD20 molecules having C-terminal mutations. [Methods] To determine the binding site of L26 antibody on CD20, we made a series of constructs of the CD20 molecules with deletion mutations in the C-terminal cytoplasmic domain and introduced them into retrovirus vectors. A CD20 negative multiple myeloma cell line, KMS12PE cells were then transformed, and we established six kinds of sub-lines with the various C-terminal deletion mutations of CD20 and used them for epitope-mapping. On the other hand, we screened the CD20 gene sequence of the clinical specimen of rituximab-resistant patients and identified several cases with the mutation in the C-terminal cytoplasm region. The immunochemistry using L26 and newly developed antibodies, as well as membrane expression of CD20 molecules using the rituximab were analyzed. [Results] The epitope analysis of L26 antibody using a series of CD20 deletion mutations revealed that L26 recognizes near the C-terminus of CD20 cytoplasmic region. These results showed that most of CD20 molecules with the C-terminal deletion mutation and frame-shift mutation could not be recognized by L26. The immunohistochemical analysis performed for clinical specimens revealed that the cells that were stained by antibodies recognizing N-terminal region of CD20 but not by L26 were indeed included in some rituximab-resistant cases. DNA sequencing analysis revealed that all these cases had mutated CD20 genes in its C-terminal cytoplasmic region. In addition, a cell-surface expression analysis using flowcytometry demonstrated that the cells having these mutations has reduced cell surface expression of CD20 compared with those of normal CD20. [Discussion] In this study, we determined the recognition site of L26 and demonstrated that L26 couldn't recognize CD20 with the resistant mutations. In contrast, newly developed antibodies against N-terminal region of CD20 could stain even these CD20 molecules. These results suggest that combination use of these antibodies and L26 enables to detect the onset of irreversible rituximab-resistant clones with the CD20 mutations. Disclosures: Hatake: Chugai Pharmaceutical Co., Ltd: Honoraria, Research Funding.

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Kumar, Anand, Cyril Planchais, Rémi Fronzes, Hugo Mouquet, and Nicolas Reyes. "Binding mechanisms of therapeutic antibodies to human CD20." Science 369, no. 6505 (August 13, 2020): 793–99. http://dx.doi.org/10.1126/science.abb8008.

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Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo–electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting CD20.

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Gordeychuk, I. V., A. I. Tukhvatulin, S. P. Petkov, M. A. Abakumov, S. A. Gulyaev, N. M. Tukhvatulina, T. V. Gulyaeva, M. I. Mikhaylov, D. Y. Logunov, and M. G. Isaguliants. "Assessment of the Parameters of Adaptive Cell-Mediated Immunity in Naïve Common Marmosets (Callithrix jacchus)." Acta Naturae 10, no. 4 (December 15, 2018): 63–69. http://dx.doi.org/10.32607/20758251-2018-10-4-63-69.

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Common marmosets are small New World primates that have been increasingly used in biomedical research. This report presents efficient protocols for assessment of the parameters of adaptive cell-mediated immunity in common marmosets, including the major subpopulations of lymphocytes and main markers of T- and B-cell maturation and activation using flow cytometry with a multicolor panel of fluorescently labelled antibodies. Blood samples from eight common marmosets were stained with fluorescently labeled monoclonal antibodies against their population markers (CD45, CD3, CD20, CD4, CD8) and lymphocyte maturation and activation markers (CD69, CD62L, CD45RO, CD107a and CD27) and analyzed by flow cytometry. Within the CD45+ population, 22.75.5% cells were CD3- CD20+ and 67.66.3% were CD3+CD20-. The CD3+ subpopulation included 55.75.5% CD3+CD4+CD8- and 34.33.7% CD3+CD4-CD8+ cells. Activation and maturation markers were expressed in the following lymphocyte proportions: CD62L on 54.010.7% of CD3+CD4+ cells and 74.412.1% of CD3+CD8+ cells; CD69 on 2.71.2% of CD3+CD4+ cells and 1.20.5% of CD3+CD8+ cells; CD45RO on 1.60.6% of CD3+CD4+ cells and 1.80.7% of CD3+CD8+ cells; CD107a on 0.70.5% of CD3+CD4+ cells and 0.50.3% of CD3+CD8+ cells; CD27 on 94.62.1% of CD3+ cells and 8.93.9% CD20+ cells. Female and male subjects differed in the percentage of CD3+CD4+CD45RO+ cells (1.90.5 in females vs 1.10.2 in males; p 0.05). The percentage of CD20+CD27+ cells was found to highly correlate with animals age (r = 0.923, p 0.005). The basal parameters of adaptive cell-mediated immunity in nave healthy marmosets without markers of systemic immune activation were obtained. These parameters and the described procedures are crucial in documenting the changes induced in common marmosets by prophylactic and therapeutic immune interventions.

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Habermann, Thomas M. "Antibody Therapy in Aggressive Lymphomas." Hematology 2007, no. 1 (January 1, 2007): 257–64. http://dx.doi.org/10.1182/asheducation-2007.1.257.

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AbstractThe aggressive lymphomas are potentially curable. The natural history of certain aggressive lymphomas has been altered by monoclonal antibody therapy. Targeted monoclonal antibody therapy to the CD20 antigen has altered the outcome of patients with diffuse large B-cell lymphoma in patients of all ages. Anti-CD20–based radioimmunoconjugates are being evaluated as radioimmunotherapy approaches in patients who have relapsed and in stem cell transplant settings. Antibody-directed therapy to the B-cell–specific antigen CD22 are ongoing. New approaches include different CD20 antibodies and an antibody to the CD40 antigen, which is a member of the tumor necrosis factor (TNF) receptor family, which is expressed on B-cells. Antibody therapy has been incorporated into CHOP (cyclophosphamide, adriamycin, vincristine, prednisone) therapy and other regimens such as EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) and HyperCVAD (cyclophosphamide, vincristine, adriamycin, dexamethasone). Single-agent anti-CD20 therapy is active in the post-transplantation lymphoproliferative disorders. T-cell antibodies are under evaluation in a number of T-cell lymphoproliferative disorders. Targeted therapy has changed the natural history of a number of aggressive non-Hodgkin lymphomas. This review will describe the contributions of antibody therapies to the treatment of these diseases.

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Vilventhraraja, Emma, Tetyana Klymenko, Jennifer Edelmann, John Gribben, and Andrejs Ivanov. "Reduction of Mitochondrial Membrane Potential Leads to Enhancement of Type-II CD20-Antibody Cytotoxicity in Diffuse Large B-Cell Lymphoma." Blood 128, no. 22 (December 2, 2016): 1763. http://dx.doi.org/10.1182/blood.v128.22.1763.1763.

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Abstract Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies. The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function. In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced. We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines. Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. Disclosures No relevant conflicts of interest to declare.

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46

Kretschmer, Anna, Stefan Lohse, Thies Rösner, Marco J. H. Jansen, Christian Kellner, Matthias Peipp, Mark S. Cragg, et al. "CD20 Antibodies of Human IgA Isotype Mediate CDC, and ADCC By Myeloid Effector Cells." Blood 128, no. 22 (December 2, 2016): 1835. http://dx.doi.org/10.1182/blood.v128.22.1835.1835.

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Abstract Introduction: Three unconjugated CD20 antibodies have been approved for the treatment of lymphoma patients. All three are of human IgG1 isotype, but nevertheless they differ in their modes of action: type I antibodies (e.g. rituximab and ofatumumab) trigger effective complement-dependent cytotoxicity (CDC), whereas type II antibodies (e.g. obinutuzumab) are potent inducers of direct cell death, while both type I and II antibodies can induce antibody-dependent cellular cytotoxicity (ADCC). Studies in syngeneic mouse models suggest that myeloid cells are the predominant effector cell type for CD20 antibodies (Uchida et al. J Exp Med 199:1659, 2004). However, human myeloid cells, particularly PMN, are activated more effectively by human IgA than by IgG1 antibodies (Dechant et al. Blood 100:4574, 2002) - especially when the latter are engineered for enhanced FcγRIII affinity (Peipp et al. Blood 112:2390, 2008). Antibodies of IgA isotype constitute an integral part of the mucosal immune system, and differ from IgG antibodies in their pharmacokinetic properties and immune effector mechanisms (Boross et al. EMBO Mol Med 5:1213, 2013, Lohse et al. Cancer Res 76:403, 2016). Here, we compared the efficiency of IgG1 and IgA2 isotype variants of the type I CD20 antibody 1F5 in killing lymphoma cells in vitro and in vivo. Methods: Recombinant antibodies against human CD20 were produced by co-transfecting BHK cells with vectors encoding the 1F5 variable, Igα2 or Igγ1 heavy, and κ light chain constant regions, respectively. The resulting isotype variants were compared for their biochemical characteristics as well as Fab- and Fc- mediated effector functions using human lymphoma cell lines as targets. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice xenotransplanted with human RAJI lymphoma cells were employed to investigate the therapeutic efficacy of human CD20 antibodies. Additionally, in vivo depletion of human CD20 transgenic B cells was evaluated in a syngeneic B cell depletion model using wildtype, human FcαRI transgenic and C3 knock-out mice. Results and Discussion: In vivo studies with xenotransplanted RAJI cells in NSG mice, which lack functional T and NK cells, demonstrated significantly prolonged survival of treated as compared to non-treated mice, indicating that myeloid effector cells may contribute to the therapeutic efficacy of CD20 antibodies against human lymphoma cells. In vitro, neither IgG1 nor IgA2 variants of 1F5 showed efficient Fab-mediated effects such as direct cell death induction and homotypic aggregation compared to type II antibodies. However, human IgA2 but not IgG1 antibody variants against CD20 effectively triggered ADCC by human PMN, the most numerous myeloid effector cell population. Although IgA does not bind C1q, CD20 IgA antibodies also triggered CDC against several lymphoma cell lines. CDC was predominantly mediated by the alternative pathway, as evidenced by the kinetics of lysis, the requirement for higher serum concentrations and inhibition by the C3 inhibitor compstatin. Further in vivo experiments demonstrated that 1F5-IgA2 effectively depleted B cells in a syngeneic human CD20 transgenic B cell depletion model. However, studies in human FcαRI transgenic or C3 knock-out mice indicated that B cell depletion was not mediated by FcαRI or complement - suggesting that other currently undefined mechanisms contribute to the in vivo efficacy of IgA antibodies against CD20. Conclusions: Together, the presented results suggest that CD20 antibodies of human IgA isotype constitute promising immunotherapeutic reagents with unique effector functions. Additional studies are required to further elucidate their effector mechanisms in vitro and in vivo. Disclosures Cragg: Roche: Consultancy, Research Funding; Gilead Sciences: Research Funding; Baxalta: Consultancy; Bioinvent International: Consultancy, Research Funding; GSK: Research Funding.

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Qu, Zhengxing, Thomas M. Cardillo, Victoria Shi, Diana L. Delaney, Hans J. Hansen, Chien-Hsing Chang, and David M. Goldenberg. "Recombinant Bispecific Monoclonal Antibody (bsMAb) Against CD20 and CD22 Active In Vitro and In Vivo Against B-Cell Lymphomas." Blood 108, no. 11 (November 16, 2006): 2521. http://dx.doi.org/10.1182/blood.v108.11.2521.2521.

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Abstract Background: Preclinical and clinical results suggest improved anti-lymphoma activity of combining anti-CD20 and anti-CD22 MAbs. Our aim was to develop a recombinant bispecific MAb against CD20 and CD22 antigens, and to evaluate its anti-tumor potency compared to the parental MAbs. Methods: Tetravalent anti-CD20/CD22 bsMAb in the form of anti-CD20 IgG linked to two anti-CD22 scFv’s was prepared recombinantly. The ability of the bsMAb to inhibit cell growth and mediate CDC and ADCC was evaluated by cell-based assays. Phosphorylation and distribution of CD22 in B-lymphoma cells treated with the bsMAb were studied by immunoblotting. The extension of survival of disseminated Daudi lymphoma cells in SCID mice also was evalauted. Results: In contrast to the parental anti-CD22 MAb, epratuzumab, the bsMAb did not internalize in Ramos cells. In CDC tests, the bsMAb showed no cytotoxic effects, similar to epratuzumab, although it did bind C1q complement protein, unlike epratuzumab. In ADCC, the bsMAb was as potent as the parental humanized anti-CD20 Mab, hA20, in inducing target-cell lysis. The anti-CD20/CD22 bsMAb has distinct effects on NHL B-cell lines compared to each parental monospecific MAb or in combination: <10 nM bsMAb causes cells to aggregate; in the soluble, non-crosslinked form, while none of the parental MAbs alone or mixed had significant anti-proliferative activity, the recombinant bsMAb was strongly anti-proliferative; the anti-proliferation activities induced by an anti-IgM Ab and the bsMAb were synergistic; and the bsMAb resulted in rapid redistribution of CD22 into detergent-resistant membrane microdomains on the cell surface. Initial animal studies show that the bsMab significantly extended survival over control SCID mice. Conclusion: The recombinantly fused anti-CD20/CD22 bsMAb has new properties compared to the parental monospecific MAbs, and may represent a new class of potential therapeutic agents that could replace binary combinations of antibodies.

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Luong, Quang Trong, Kouki Morizono, Irvin SY Chen, and Sven De Vos. "Construction of Lentiviruses Pseudotyped with Sindbis E2-Single Chain Antibody (SCA) Fusions or Membrane-Anchored SCAs for Targeted Therapies in Lymphoma." Blood 114, no. 22 (November 20, 2009): 3571. http://dx.doi.org/10.1182/blood.v114.22.3571.3571.

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Abstract Abstract 3571 Poster Board III-508 Previously, we demonstrated specific targeting of CD20+ and CD30+ cells can be achieved in vitro using vectors of Morizono et al. [Cell Cycle 2005; Nature Medicine 2005]. These lentiviruses expressed modified Sindbis E2 proteins that contain the Fc receptor binding domain (ZZ) of protein A. The lentiviruses could be specifically and effectively directed to target cells by conjugation of an antibody to the modified ZZ Sindbis viral envelope (m168). CD30+ HL cell lines and CD20+ non-Hodgkin lymphoma (NHL) cell lines were used. Anti-CD30-labeled viruses (m168-αCD30) specifically transduced CD30+ HL cells while avoiding CD30− NHL cells; in addition, transduction of CD20+ NHL cells was achieved using m168-αCD20 viruses (unpublished data). However, these viruses were unsuitable for in vivo studies because of the unstable conjugation of antibody and envelope protein, and the ambiguity of the ZZ domains to bind Fc receptors. We developed two targeting approaches. For the first approach, we constructed vectors containing single chain antibodies (SCA) directly fused to the Sindbis E2 spike glycoprotein. Approximately 100 different construct-variants have been produced so far, that contain SCA-E2 fusions with a variety of flexible linkers to allow for adequate folding and expression of the fusion proteins. These constructs contain either anti-CD30-E2 (T405 or T215) or anti-CD20-E2 sequences. The second approach was to construct membrane-anchored SCA vectors that can be expressed on the envelope of the lentiviruses. We produced in excess of 30 variants containing anti-CD30 or anti-CD20 sequences with transmembrane domains of CD4 or VSV-G, or a Glycosylphosphoinositol (GPI)-anchor. Potentially, these constructs are more stable and might allow an in vivo targeting approach. We have tested these constructs for expression of SCA-E2 or membrane-anchored SCA by Western analyses. The expression of SCA-E2 fusion proteins was dependent on the length of the linker used with greater expression observed with longer linkers. The linker length should contain a minimum of 20 amino acids either side of the cloning/fusion site to permit adequate expression of SCA-E2 fusion proteins. SCA-GPI-anchor proteins, on the other hand, are not fused to the Sindbis E2 protein but contain similar length flexible linkers to permit proper folding of the SCA and GPI proteins. We tested whether the viruses produced with these modified envelope proteins or membrane-anchored proteins were able to infect CD30 or CD20 expressing target cells. We achieved up to 40% infection of 293-T CD20+ cells with anti-CD20-E2 constructs but only about 10% infection of 293-T CD30+ cells using T405-E2 (anti-CD30) constructs. In contrast, we found that T405-GPI (anti-CD30) constructs were able to infect 60% of target cells while anti-CD20-GPI infected less than 10% of target cells. Additionally, we found that T215-E2 (anti-CD30) pseudotyped viruses were not able to infect CD30+ target cells. We conclude from our data (1) that while single chain antibody sequences were the functional components that determine specificity of pseudotyped lentiviruses, not all single chain antibodies will be functional in these systems; and (2) the success of the different anchoring techniques seems to depend on the single chain antibody and the target antigen. These vectors will now be subjected to in vivo targeting. Disclosures: No relevant conflicts of interest to declare.

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49

Sharman, Jeff P. "Targeting CD20: teaching an old dog new tricks." Hematology 2019, no. 1 (December 6, 2019): 273–78. http://dx.doi.org/10.1182/hematology.2019000031.

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Abstract Rituximab was the first monoclonal antibody used for the treatment of a malignancy. In the 22 years since initial approval, it has become a vital component of therapy for a multitude of B-cell malignancies. Within the last several years, however, there has been a robust development of novel agents targeting CD20, including second generation anti-CD20 antibodies, biosimilar antibodies, and subcutaneous formulations that have been approved. The era of passive immunotherapy is now yielding to therapeutic approaches that actively engage the immune system. Emerging approaches leverage immunomodulatory drugs or novel checkpoint inhibitors to enhance CD20 therapy. Recent data sets on bispecific CD3/CD20 antibodies demonstrate exciting early findings, and CD20-directed chimeric antigen receptor T-cell therapies are now entering clinical trials. Anti-CD20 therapies are a vital component of the treatment of B-cell malignancies, and there is a dynamic therapeutic environment with multiple new data sets reviewed here.

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Newell, Laura, Joseph Tuscano, Yunpeng Ma, Shiloh Martin, and Robert o'Donnell. "The Bispecific Anti-CD20/CD22 Antibody (Bs20x22) Has More Lymphomacidal Activity Than the Parent Antibodies Both Alone or In Combination." Blood 116, no. 21 (November 19, 2010): 425. http://dx.doi.org/10.1182/blood.v116.21.425.425.

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Abstract Abstract 425 Background: Over 400,000 people in the United States are living with non-Hodgkin's lymphoma (NHL). While survival rates have improved, over 20,000 persons die annually from NHL. Cytotoxic chemotherapies are initially effective, but resistance can develop and dose limiting toxicities are problematic. Monoclonal antibody (mAb) therapy with rituximab, a chimeric anti-CD20 mAb, has shown benefit alone and in combination with chemotherapy, and can improve overall survival. New mAbs are being tested in combination with rituximab, including bispecific antibodies (BsAb) that simultaneously target CD20 and CD22. CD22 is a B-lymphocyte specific adhesion molecule expressed on nearly all mature B-cells, including the majority of B cell-NHLs. HB22.7 is an anti-CD22 mAb that specifically blocks the interaction of CD22 with its ligand, initiates CD22-mediated signal transduction, and has direct cytotoxic effects. Anti-CD22 mAbs that do not block ligand binding possess only modest functional effects; prior BsAbs contained non-ligand binding anti-CD22 mAbs. Therefore, we constructed a BsAb (Bs20x22) using rituximab and HB22.7, and evaluated its cell binding, signaling patterns, and lymphomacidal activity using a human NHL xenograft model. Methods: Bs20x22 was constructed from F(ab')2 fragments of rituximab and HB22.7 using the ImmunoPure F(ab')2 Preparation Kit (Pierce). Cell binding studies used the CD20/CD22 double positive human Burkitt's B-cell lymphoma lines Raji and Ramos, and the CD20/CD22 double negative human embryonic kidney cell line, 293T. In vitro cytotoxicity was assessed by trypan blue exclusion. In vitro apoptosis assays were performed on plated Raji or Ramos cells which were then treated with HB22.7, rituximab, HB22.7 plus rituximab, or Bs20x22. The total number of cells and the number of apoptotic cells were counted, with results expressed as the % of control (% apoptotic cells treated / % apoptotic cells untreated). Results: Bs20x22 specifically bound CD20 and CD22, similar to the parent mAbs rituximab and HB22.7. In vitro cytotoxicity assays showed that Bs20x22 was three times more effective than either parent mAb alone and twice as effective as a combination of both parent mAb used at equimolar concentrations. Additionally, Bs20x22 was nearly four times more effective at inducing apoptosis than either mAb alone, with the percentage of apoptotic cells greatest for Bs20x22 treatment (20.1%), compared to combination rituximab plus HB22.7 (7.5%), rituximab (6.7%), and HB22.7 (6.5%). Examination of the MAPK and SAPK signaling cascades revealed that treatment with Bs20x22 resulted in significant activation of p38, while treatment with either parent mAb did not. In an in vivo human NHL xenograft model, treatment with Bs20x22 resulted in significantly greater tumor shrinkage resulting in the smallest tumor volume of any group. Bs20x22 treated mice had the best survival rate (88%) compared to the combination of rituximab plus HB22.7 (75%), rituximab (50%), HB22.7 (25%), and PBS control (0%). Significantly greater efficacy was also found when Bs20x22 was administered prior to the development of visible tumors versus treatment of established tumors, with tumors 89% smaller in mice treated pre-emptively. Conclusions: This study demonstrates the feasibility of constructing a bispecific antibody targeting both CD20 and CD22, using an anti-CD22 mAb with ligand-blocking ability. Efficacy was demonstrated by in vitro cytotoxicity and apoptosis assays, p38 activation, and human NHL xenograft models. Our results suggest that Bs20x22 is more efficacious than the combination of rituximab and HB22.7, and use of Bs20x22 eliminates the need for sequential administration of two separate mAbs. Disclosures: No relevant conflicts of interest to declare.

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