The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Chien-hsing Chang - One of the best experts on this subject based on the ideXlab platform.
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27 - B cells were more responsive than CD27 + cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Daniel J. Wallace, Edmund A. Rossi, R. Michel, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27- B cells were more responsive than CD27+ cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P <.02) lower for the epratuzumab group. Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
abstract 4744 cd22 targeting epratuzumab mediates trogocytosis of multiple cell surface markers on normal malignant and lupus b cells
Cancer Research, 2013Co-Authors: Edmund A. Rossi, Rosana B. Michel, Diane L. Rossi, Daniel J. Wallace, David M. Goldenberg, Chien-hsing ChangAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphoma and autoimmune diseases, demonstrating therapeutic activities in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy. Although epratuzumab is capable of depleting on average 35% of circulating B cells in patients, its in vivo mechanism of action (MOA) remains incompletely understood. We hypothesized that ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Methods. Peripheral blood mononuclear cells (PBMCs) from either healthy donors or lupus patients with flares, were incubated with epratuzumab, and the relative surface levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Results. Epratuzumab promptly induced a marked decrease of CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on the surface of B cells in PBMCs obtained from normal donors or treatment-naive lupus patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs. Although some Fc-independent loss of CD22 is expected from its internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcγR-expressing effector cells, including monocytes, NK cells, and granulocytes. Following incubation with epratuzumab, but not with an isotype control mAb, reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. In the absence of PBMCs, treatment of NHL cell lines (Daudi and Raji) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) reduction of CD19 and CD21. When these NHL cells (1 x 105 cells) were mixed with PBMCs (1 x 106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Under the conditions examined, rituximab at 10 μg/mL reduced the B-cell count by 50%, whereas epratuzumab did not cause significant B-cell depletion, either at 10 μg/mL or 1 mg/mL. Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. Whether the observed trogocytosis could be correlated with the depletion of malignant B cells in lymphoid tissues is currently under investigation. Citation Format: Edmund A. Rossi, David M. Goldenberg, Rosana Michel, Diane L. Rossi, Daniel J. Wallace, Chien-Hsing Chang. CD22-targeting epratuzumab mediates trogocytosis of multiple cell-surface markers on normal, malignant, and lupus B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2013-4744
-
horizontal transmission of malignancy in vivo fusion of human lymphomas with hamster stroma produces tumors retaining human genes and lymphoid pathology
PLOS ONE, 2013Co-Authors: David M. Goldenberg, Chien-hsing Chang, David V Gold, Meiyu Loo, Donglin Liu, Elaine S JaffeAbstract:We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5–6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.
-
cd22 targeting epratuzumab down regulates multiple bcr regulators on the surface of normal lupus and malignant b cells
Blood, 2012Co-Authors: Edmund A. Rossi, Rosana B. Michel, Chien-hsing Chang, Daniel J. Wallace, David M. GoldenbergAbstract:Abstract 1357 Background. The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenstrom9s macroglobulinemia, Sjogren9s syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason 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, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required. Methods and Results. PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab9)2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal ( Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosures: Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.
David M. Goldenberg - One of the best experts on this subject based on the ideXlab platform.
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27 - B cells were more responsive than CD27 + cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Daniel J. Wallace, Edmund A. Rossi, R. Michel, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27- B cells were more responsive than CD27+ cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P <.02) lower for the epratuzumab group. Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
abstract 4744 cd22 targeting epratuzumab mediates trogocytosis of multiple cell surface markers on normal malignant and lupus b cells
Cancer Research, 2013Co-Authors: Edmund A. Rossi, Rosana B. Michel, Diane L. Rossi, Daniel J. Wallace, David M. Goldenberg, Chien-hsing ChangAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphoma and autoimmune diseases, demonstrating therapeutic activities in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy. Although epratuzumab is capable of depleting on average 35% of circulating B cells in patients, its in vivo mechanism of action (MOA) remains incompletely understood. We hypothesized that ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Methods. Peripheral blood mononuclear cells (PBMCs) from either healthy donors or lupus patients with flares, were incubated with epratuzumab, and the relative surface levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Results. Epratuzumab promptly induced a marked decrease of CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on the surface of B cells in PBMCs obtained from normal donors or treatment-naive lupus patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs. Although some Fc-independent loss of CD22 is expected from its internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcγR-expressing effector cells, including monocytes, NK cells, and granulocytes. Following incubation with epratuzumab, but not with an isotype control mAb, reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. In the absence of PBMCs, treatment of NHL cell lines (Daudi and Raji) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) reduction of CD19 and CD21. When these NHL cells (1 x 105 cells) were mixed with PBMCs (1 x 106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Under the conditions examined, rituximab at 10 μg/mL reduced the B-cell count by 50%, whereas epratuzumab did not cause significant B-cell depletion, either at 10 μg/mL or 1 mg/mL. Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. Whether the observed trogocytosis could be correlated with the depletion of malignant B cells in lymphoid tissues is currently under investigation. Citation Format: Edmund A. Rossi, David M. Goldenberg, Rosana Michel, Diane L. Rossi, Daniel J. Wallace, Chien-Hsing Chang. CD22-targeting epratuzumab mediates trogocytosis of multiple cell-surface markers on normal, malignant, and lupus B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2013-4744
-
horizontal transmission of malignancy in vivo fusion of human lymphomas with hamster stroma produces tumors retaining human genes and lymphoid pathology
PLOS ONE, 2013Co-Authors: David M. Goldenberg, Chien-hsing Chang, David V Gold, Meiyu Loo, Donglin Liu, Elaine S JaffeAbstract:We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5–6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.
-
cd22 targeting epratuzumab down regulates multiple bcr regulators on the surface of normal lupus and malignant b cells
Blood, 2012Co-Authors: Edmund A. Rossi, Rosana B. Michel, Chien-hsing Chang, Daniel J. Wallace, David M. GoldenbergAbstract:Abstract 1357 Background. The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenstrom9s macroglobulinemia, Sjogren9s syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason 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, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required. Methods and Results. PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab9)2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal ( Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosures: Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.
Edmund A. Rossi - One of the best experts on this subject based on the ideXlab platform.
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27 - B cells were more responsive than CD27 + cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Daniel J. Wallace, Edmund A. Rossi, R. Michel, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27- B cells were more responsive than CD27+ cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P <.02) lower for the epratuzumab group. Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
abstract 4744 cd22 targeting epratuzumab mediates trogocytosis of multiple cell surface markers on normal malignant and lupus b cells
Cancer Research, 2013Co-Authors: Edmund A. Rossi, Rosana B. Michel, Diane L. Rossi, Daniel J. Wallace, David M. Goldenberg, Chien-hsing ChangAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphoma and autoimmune diseases, demonstrating therapeutic activities in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy. Although epratuzumab is capable of depleting on average 35% of circulating B cells in patients, its in vivo mechanism of action (MOA) remains incompletely understood. We hypothesized that ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Methods. Peripheral blood mononuclear cells (PBMCs) from either healthy donors or lupus patients with flares, were incubated with epratuzumab, and the relative surface levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Results. Epratuzumab promptly induced a marked decrease of CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on the surface of B cells in PBMCs obtained from normal donors or treatment-naive lupus patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs. Although some Fc-independent loss of CD22 is expected from its internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcγR-expressing effector cells, including monocytes, NK cells, and granulocytes. Following incubation with epratuzumab, but not with an isotype control mAb, reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. In the absence of PBMCs, treatment of NHL cell lines (Daudi and Raji) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) reduction of CD19 and CD21. When these NHL cells (1 x 105 cells) were mixed with PBMCs (1 x 106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Under the conditions examined, rituximab at 10 μg/mL reduced the B-cell count by 50%, whereas epratuzumab did not cause significant B-cell depletion, either at 10 μg/mL or 1 mg/mL. Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. Whether the observed trogocytosis could be correlated with the depletion of malignant B cells in lymphoid tissues is currently under investigation. Citation Format: Edmund A. Rossi, David M. Goldenberg, Rosana Michel, Diane L. Rossi, Daniel J. Wallace, Chien-Hsing Chang. CD22-targeting epratuzumab mediates trogocytosis of multiple cell-surface markers on normal, malignant, and lupus B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2013-4744
-
cd22 targeting epratuzumab down regulates multiple bcr regulators on the surface of normal lupus and malignant b cells
Blood, 2012Co-Authors: Edmund A. Rossi, Rosana B. Michel, Chien-hsing Chang, Daniel J. Wallace, David M. GoldenbergAbstract:Abstract 1357 Background. The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenstrom9s macroglobulinemia, Sjogren9s syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason 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, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required. Methods and Results. PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab9)2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal ( Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosures: Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.
Daniel J. Wallace - One of the best experts on this subject based on the ideXlab platform.
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27 - B cells were more responsive than CD27 + cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Daniel J. Wallace, Edmund A. Rossi, R. Michel, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27- B cells were more responsive than CD27+ cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P <.02) lower for the epratuzumab group. Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
abstract 4744 cd22 targeting epratuzumab mediates trogocytosis of multiple cell surface markers on normal malignant and lupus b cells
Cancer Research, 2013Co-Authors: Edmund A. Rossi, Rosana B. Michel, Diane L. Rossi, Daniel J. Wallace, David M. Goldenberg, Chien-hsing ChangAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphoma and autoimmune diseases, demonstrating therapeutic activities in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy. Although epratuzumab is capable of depleting on average 35% of circulating B cells in patients, its in vivo mechanism of action (MOA) remains incompletely understood. We hypothesized that ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Methods. Peripheral blood mononuclear cells (PBMCs) from either healthy donors or lupus patients with flares, were incubated with epratuzumab, and the relative surface levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Results. Epratuzumab promptly induced a marked decrease of CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on the surface of B cells in PBMCs obtained from normal donors or treatment-naive lupus patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs. Although some Fc-independent loss of CD22 is expected from its internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcγR-expressing effector cells, including monocytes, NK cells, and granulocytes. Following incubation with epratuzumab, but not with an isotype control mAb, reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. In the absence of PBMCs, treatment of NHL cell lines (Daudi and Raji) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) reduction of CD19 and CD21. When these NHL cells (1 x 105 cells) were mixed with PBMCs (1 x 106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Under the conditions examined, rituximab at 10 μg/mL reduced the B-cell count by 50%, whereas epratuzumab did not cause significant B-cell depletion, either at 10 μg/mL or 1 mg/mL. Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. Whether the observed trogocytosis could be correlated with the depletion of malignant B cells in lymphoid tissues is currently under investigation. Citation Format: Edmund A. Rossi, David M. Goldenberg, Rosana Michel, Diane L. Rossi, Daniel J. Wallace, Chien-Hsing Chang. CD22-targeting epratuzumab mediates trogocytosis of multiple cell-surface markers on normal, malignant, and lupus B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2013-4744
-
cd22 targeting epratuzumab down regulates multiple bcr regulators on the surface of normal lupus and malignant b cells
Blood, 2012Co-Authors: Edmund A. Rossi, Rosana B. Michel, Chien-hsing Chang, Daniel J. Wallace, David M. GoldenbergAbstract:Abstract 1357 Background. The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenstrom9s macroglobulinemia, Sjogren9s syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason 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, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required. Methods and Results. PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab9)2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal ( Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosures: Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.
Rosana B. Michel - One of the best experts on this subject based on the ideXlab platform.
-
op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
Annals of the Rheumatic Diseases, 2013Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing ChangAbstract:Background Epratuzumab, a humanized monoclonal antibody targeting CD22, has demonstrated therapeutic activity in clinical trials of patients with SLE, yet an understanding of its mechanism of action (MOA) is still emerging. Because epratuzumab reduces on average 35% of circulating B cells in patients, its therapeutic efficacy involves MOA beyond B-cell depletion. Modulation of CD22 and other surface molecules that regulate B-cell antigen receptor (BCR) signaling may alter B-cell functions and ultimately mitigate symptoms of the underlying disease. Objectives Using an experimental ex-vivo analysis, we identified trogocytosis as a novel MOA for epratuzumab, which may be important for therapy of SLE and other autoimmune disorders. Clinical specimens of SLE patients were evaluated for evidence of trogocytosis induced by epratuzumab in vivo . Methods PBMCs from either healthy donors or SLE patients were incubated with epratuzumab, and the relative surface levels of CD22, CD19, CD21, and CD79b on B cells were analyzed by flow cytometry (FCM). Trogocytosis was studied with FCM and fluorescence microscopy using B-cell NHL cell lines (Daudi and Raji) mixed with PBMCs, T cells, monocytes, or granulocytes. We further measured the relative levels of CD22, CD19, CD21, and CD79b on B cells from five SLE patients who were receiving epratuzumab, four treatment-naive SLE patients, and two receiving belimumab. Results Epratuzumab promptly induced a marked decrease of surface CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on B cells in PBMCs obtained from normal donors or treatment-naive SLE patients. CD27 - B cells were more responsive than CD27 + cells. Within a few hrs, B-cell surface proteins were reduced to a similar level over a broad concentration range (0.01 – 100 µg/mL) of epratuzumab. Although some Fc-independent loss of CD22 is expected from internalization, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcgR-expressing effector cells, including monocytes, NK cells, and granulocytes. Reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. Epratuzumab-induced transfer of membrane components from Daudi cells to monocytes was also evident by fluorescence microscopy. Unlike rituximab, which reduced the B-cell count by 50% in the ex-vivo assay, epratuzumab did not cause significant B-cell depletion. Analysis of SLE patient samples suggests that similar epratuzumab-mediated trogocytosis, as observed ex-vivo , also occurs clinically. As expected, CD22 was significantly ( P 80%) on the B cells of epratuzumab-treated patients. Notably, CD19, CD21 and CD79b were each also significantly ( P Conclusions This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in SLE, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosure of Interest D. Goldenberg Shareholder of: Immunomedics, Inc., Employee of: Immunomedics, Inc., E. Rossi Employee of: Immunomedics, Inc., R. Michel Employee of: Immunomedics, Inc., D. Wallace: None Declared, C.-H. Chang: None Declared
-
abstract 4744 cd22 targeting epratuzumab mediates trogocytosis of multiple cell surface markers on normal malignant and lupus b cells
Cancer Research, 2013Co-Authors: Edmund A. Rossi, Rosana B. Michel, Diane L. Rossi, Daniel J. Wallace, David M. Goldenberg, Chien-hsing ChangAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphoma and autoimmune diseases, demonstrating therapeutic activities in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy. Although epratuzumab is capable of depleting on average 35% of circulating B cells in patients, its in vivo mechanism of action (MOA) remains incompletely understood. We hypothesized that ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Methods. Peripheral blood mononuclear cells (PBMCs) from either healthy donors or lupus patients with flares, were incubated with epratuzumab, and the relative surface levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Results. Epratuzumab promptly induced a marked decrease of CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on the surface of B cells in PBMCs obtained from normal donors or treatment-naive lupus patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs. Although some Fc-independent loss of CD22 is expected from its internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcγR-expressing effector cells, including monocytes, NK cells, and granulocytes. Following incubation with epratuzumab, but not with an isotype control mAb, reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. In the absence of PBMCs, treatment of NHL cell lines (Daudi and Raji) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) reduction of CD19 and CD21. When these NHL cells (1 x 105 cells) were mixed with PBMCs (1 x 106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Under the conditions examined, rituximab at 10 μg/mL reduced the B-cell count by 50%, whereas epratuzumab did not cause significant B-cell depletion, either at 10 μg/mL or 1 mg/mL. Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. Whether the observed trogocytosis could be correlated with the depletion of malignant B cells in lymphoid tissues is currently under investigation. Citation Format: Edmund A. Rossi, David M. Goldenberg, Rosana Michel, Diane L. Rossi, Daniel J. Wallace, Chien-Hsing Chang. CD22-targeting epratuzumab mediates trogocytosis of multiple cell-surface markers on normal, malignant, and lupus B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2013-4744
-
cd22 targeting epratuzumab down regulates multiple bcr regulators on the surface of normal lupus and malignant b cells
Blood, 2012Co-Authors: Edmund A. Rossi, Rosana B. Michel, Chien-hsing Chang, Daniel J. Wallace, David M. GoldenbergAbstract:Abstract 1357 Background. The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenstrom9s macroglobulinemia, Sjogren9s syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason 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, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required. Methods and Results. PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab9)2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal ( Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosures: Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.