The Experts below are selected from a list of 984 Experts worldwide ranked by ideXlab platform
Chien-hsing Chang - One of the best experts on this subject based on the ideXlab platform.
-
Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus
2016Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35 % of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and b7-integrin, and wit
-
Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus
PLOS ONE, 2014Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenstrom’s macroglobulinemia, Sjogren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced Trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.
-
anti cd22 cd20 bispecific antibody with enhanced Trogocytosis for treatment of lupus
PLOS ONE, 2014Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenstrom’s macroglobulinemia, Sjogren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced Trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.
-
Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus
Blood, 2013Co-Authors: Rosana B. Michel, Chien-hsing Chang, David M. GoldenbergAbstract: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, Waldenstrom's macroglobulinemia, Sjogren'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][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. View this table: Table 1 Percent reduction of B-cells antigens following overnight treatment of PBMCs 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. [1]: #T1
-
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
David M. Goldenberg - One of the best experts on this subject based on the ideXlab platform.
-
Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus
2016Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35 % of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and b7-integrin, and wit
-
Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus
PLOS ONE, 2014Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenstrom’s macroglobulinemia, Sjogren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced Trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.
-
anti cd22 cd20 bispecific antibody with enhanced Trogocytosis for treatment of lupus
PLOS ONE, 2014Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenstrom’s macroglobulinemia, Sjogren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced Trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.
-
Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus
Blood, 2013Co-Authors: Rosana B. Michel, Chien-hsing Chang, David M. GoldenbergAbstract: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, Waldenstrom's macroglobulinemia, Sjogren'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][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. View this table: Table 1 Percent reduction of B-cells antigens following overnight treatment of PBMCs 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. [1]: #T1
-
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
Edmund A. Rossi - One of the best experts on this subject based on the ideXlab platform.
-
Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus
2016Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35 % of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and b7-integrin, and wit
-
Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus
PLOS ONE, 2014Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenstrom’s macroglobulinemia, Sjogren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced Trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.
-
anti cd22 cd20 bispecific antibody with enhanced Trogocytosis for treatment of lupus
PLOS ONE, 2014Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. GoldenbergAbstract:The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenstrom’s macroglobulinemia, Sjogren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. 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 β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of Trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced Trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced Trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.
-
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
Denis Hudrisier - One of the best experts on this subject based on the ideXlab platform.
-
Preferential transfer of certain plasma membrane proteins onto T and B cells by Trogocytosis.
PloS one, 2010Co-Authors: Sandrine Daubeuf, Anne Aucher, Christine Bordier, Etienne Joly, Audrey Salles, Laurent Serre, Gérald Gaibelet, Jean-charles Faye, Gilles Favre, Denis HudrisierAbstract:T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed Trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during Trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by Trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRγ found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in Trogocytosis.
-
Suitability of various membrane lipophilic probes for the detection of Trogocytosis by flow cytometry.
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2009Co-Authors: Sandrine Daubeuf, Denis Hudrisier, Christine Bordier, Etienne JolyAbstract:Trogocytosis is a recently discovered phenomenon whereby lymphocytes capture fragments of the plasma membrane from antigen presenting cells (APCs). Using APCs labeled with widely used fluorescent lipophilic probes, we previously described a Trogocytosis analysis protocol (TRAP) useful to understand the mechanisms and biological consequences of this process and to identify lymphocytes reacting specifically with antigen-bearing APCs. We have compared the suitability of 22 different fluorescent lipophilic probes for use in TRAP assays with cytotoxic T lymphocytes (CTL). The criteria we used were: simple and efficient incorporation in APC membranes, minimal passive diffusion among cells but efficient transfer onto T cells during Trogocytosis. Sphingosin-based probes were found to incorporate inefficiently into cells. For others with unsaturated lipid chains, we found a tendency for extensive passive diffusion. In the end, about a third of the probes tested were found to be suitable in TRAP assays, which all carry either C16 or C18 saturated carbon chains, including some that can be excited with a red laser. Moreover, we found it possible to combine TRAP assays based on lipophilic probes with intracellular cytokine detection. We have identified a set of new lipophilic fluorescent probes suitable for TRAP assays in combination with intracellular staining.
-
Capture of plasma membrane fragments from target cells by Trogocytosis requires signaling in T cells but not in B cells
Blood, 2008Co-Authors: Anne Aucher, Etienne Joly, Eddy Magdeleine, Denis HudrisierAbstract:Upon recognition of their respective cellular partners, T and B cells acquire their antigens by a process of membrane capture called Trogocytosis. Here, we report that various inhibitors of actin polymerization or of kinases involved in intracellular signaling partially or fully inhibited Trogocytosis by CD8+ and CD4+ T cells, whereas they had no effect on Trogocytosis by B cells. Similarly, Trogocytosis by T cells was inhibited at 4°C, whereas in B cells it was independent of temperature, indicating that Trogocytosis by B cells does not rely on active processes. By contrast, most inhibitors we tested impaired both T-cell and B-cell activation. The differential effect of inhibitors on T-cell and B-cell Trogocytosis was not due to the higher affinity of the B-cell receptor for its cognate antigen compared with the affinity of the T-cell receptor for its own antigen, but it correlated tightly with the abilities of T cells and B cells to form conjugates with their target cells in the presence of inhibitors. Trogocytosis thus has different requirements in different cell types. Moreover, the capture of membrane antigen by B cells is identified as a novel signaling-independent event of B-cell biology.
-
capture of target cell membrane components via Trogocytosis is triggered by a selected set of surface molecules on t or b cells
Journal of Immunology, 2007Co-Authors: Denis Hudrisier, Anne Aucher, Annelaure Puaux, Christine Bordier, Etienne JolyAbstract:Key events of T and B cell biology are regulated through direct interaction with APC or target cells. Trogocytosis is a process whereby CD4 + T, CD8 + T, and B cells capture their specific membrane-bound Ag through the acquisition of plasma membrane fragments from their cellular targets. With the aim of investigating whether the ability to trigger Trogocytosis was a selective property of Ag receptors, we set up an assay that allowed us to test the ability of many different cell surface molecules to trigger Trogocytosis. On the basis of the analysis of a series of surface molecules on CD4 + T, CD8 + T, and B cells, we conclude that a set of cell type-specific surface determinants, including but not limited to Ag receptors, do trigger Trogocytosis. On T cells, these determinants include components of the TCR/CD3 as well as that of coreceptors and of several costimulatory molecules. On B cells, we identified only the BCR and MHC molecules as potentials triggers of Trogocytosis. Remarkably, latrunculin, which prevents actin polymerization, impaired Trogocytosis by T cells, but not by B cells. This was true even when the same Abs were used to trigger Trogocytosis in T or B cells. Altogether, our results indicate that although Trogocytosis is performed by all hemopoietic cells tested thus far, both the receptors and the mechanisms involved can differ depending on the lineage of the cell acquiring membrane materials from other cells. This could therefore account for the different biological consequences of Ag capture via Trogocytosis proposed for different types of cells.
-
Capture of membrane components via Trogocytosis occurs in vivo during both dendritic cells and target cells encounter by CD8(+) T cells.
Scandinavian journal of immunology, 2007Co-Authors: Joëlle Riond, Denis Hudrisier, J. Elhmouzi, Jean Edouard GairinAbstract:Cytotoxic T lymphocytes recently stimulated by antigen-presenting cells (APC) display major histocompatibility class (MHC) I and II molecules inherited from APC. We have previously reported that, in vitro, transfer of MHC molecules and several other proteins occurs through Trogocytosis, i.e. the active acquisition of target cell membrane fragments by T lymphocytes. Here, using the model of viral antigen LCMVgp33-41 recognition in transgenic P14 mice, we show that CD8(+) T cells perform Trogocytosis in vivo, as detected by the capture of biotin- or fluorescence-labeled components of the APC surface. Trogocytosis occurs during interactions of CD8(+) T cells with at least two kinds of cells: target cells and dendritic cells (DC). In lymph nodes, CD8(+) T cells having performed Trogocytosis with DC express the CD69 activation marker indicating that Trogocytosis detects recently activated cells. Taken together, our findings suggest that Trogocytosis may be a new in vivo marker of the recent interaction between a CD8(+) T cell and its cellular partners or targets.
Katherine S Ralston - One of the best experts on this subject based on the ideXlab platform.
-
direct and high throughput assays for human cell killing through Trogocytosis by entamoeba histolytica
Molecular and Biochemical Parasitology, 2020Co-Authors: Akhila Bettadapur, Katherine S RalstonAbstract:Entamoeba histolytica is the causative agent of amoebiasis. Pathogenesis is associated with profound damage to human tissues. We previously showed that amoebae kill human cells through Trogocytosis. Trogocytosis is likely to underlie tissue damage during infection, although the mechanism is still unknown. Trogocytosis is difficult to assay quantitatively, which makes it difficult to study. Here, we developed two new, complementary assays to measure Trogocytosis by quantifying human cell death. One assay uses CellTiterGlo, a luminescent readout for ATP, as a proxy for cell death. We found that the CellTiterGlo could be used to detect death of human cells after co-incubation with amoebae, and that it was sensitive to inhibition of actin or the amoeba surface Gal/GalNAc lectin, two conditions that are known to inhibit amoebic Trogocytosis. The other assay uses two fluorescent nuclear stains to directly differentiate live and dead human cells by microscopy, and is also sensitive to inhibition of amoebic Trogocytosis through interference with actin. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of Trogocytosis and amoebiasis pathogenesis.
-
Biting Off What Can Be Chewed: Trogocytosis in Health, Infection, and Disease
Infection and immunity, 2020Co-Authors: Akhila Bettadapur, Hannah W. Miller, Katherine S RalstonAbstract:Trogocytosis is part of an emerging, exciting theme of cell-cell interactions both within and between species, and it is relevant to host-pathogen interactions in many different contexts. Trogocytosis is a process in which one cell physically extracts and ingests "bites" of cellular material from another cell. It was first described in eukaryotic microbes, where it was uncovered as a mechanism by which amoebae kill cells. Trogocytosis is potentially a fundamental form of eukaryotic cell-cell interaction, since it also occurs in multicellular organisms, where it has functions in the immune system, in the central nervous system, and during development. There are numerous scenarios in which Trogocytosis occurs and an ever-evolving list of functions associated with this process. Many aspects of Trogocytosis are relevant to microbial pathogenesis. It was recently discovered that immune cells perform Trogocytosis to kill Trichomonas vaginalis parasites. Additionally, through Trogocytosis, Entamoeba histolytica acquires and displays human cell membrane proteins, enabling immune evasion. Intracellular bacteria seem to exploit host cell Trogocytosis, since they can use it to spread from cell to cell. Thus, a picture is emerging in which Trogocytosis plays critical roles in normal physiology, infection, and disease.
-
Inhibition of Amebic Cysteine Proteases Blocks Amebic Trogocytosis but Not Phagocytosis.
The Journal of infectious diseases, 2020Co-Authors: Allissia A. Gilmartin, Katherine S Ralston, William A PetriAbstract:Background Entamoeba histolytica kills human cells by ingesting fragments of live cells until the cell eventually dies, a process termed amebic Trogocytosis. In a previous study, we showed that acidified amebic lysosomes are required for both amebic Trogocytosis and phagocytosis, as well as cell killing. Methods Amebic cysteine proteases (CPs) were inhibited using an irreversible inhibitor, E-64d. Results Interfering with amebic CPs decreased amebic Trogocytosis and amebic cytotoxicity but did not impair phagocytosis. Conclusions We show that amebic CPs are required for amebic Trogocytosis and cell killing but not phagocytosis. These data suggest that amebic CPs play a distinct role in amebic Trogocytosis and cell killing.
-
direct and high throughput assays for human cell killing through Trogocytosis by entamoeba histolytica
bioRxiv, 2019Co-Authors: Akhila Bettadapur, Katherine S RalstonAbstract:Abstract Entamoeba histolytica is a microbial eukaryote and causative agent of the diarrheal disease amoebiasis. Pathogenesis is associated with profound damage to human tissues, and treatment options are limited. We discovered that amoebae attack and kill human cells through a cell-nibbling process that we named Trogocytosis (trogo-: nibble). Trogocytosis is likely to underlie tissue damage during infection and it represents a potential target for therapeutic intervention, although the mechanism is still unknown. Assays in current use to analyze Trogocytosis by amoebae have not been amenable to studying different types of human cells, or to high-throughput analysis. Here, we developed two complementary assays to measure Trogocytosis by quantifying human cell viability, both of which can be used for suspension and adherent cells. The first assay uses CellTiterGlo, a luminescent readout for cellular ATP levels, as a proxy for cell viability. We found that the CellTiterGlo signal is proportional to the quantity of viable cells, and can be used to detect death of human cells after co-incubation with amoebae. We established a second assay that is microscopy-based and uses two fluorescent stains to directly differentiate live and dead human cells. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of amoebiasis pathogenesis.
-
Trogocytosis by entamoeba histolytica mediates acquisition and display of human cell membrane proteins and evasion of lysis by human serum
Mbio, 2019Co-Authors: Hannah W. Miller, Rene L Suleiman, Katherine S RalstonAbstract:We previously showed that Entamoeba histolytica kills human cells through a mechanism that we termed Trogocytosis ("trogo-" means "nibble"), due to its resemblance to Trogocytosis in other organisms. In microbial eukaryotes like E. histolytica, Trogocytosis is used to kill host cells. In multicellular eukaryotes, Trogocytosis is used for cell killing and cell-cell communication in a variety of contexts. Thus, nibbling is an emerging theme in cell-cell interactions both within and between species. When Trogocytosis occurs between mammalian immune cells, cell membrane proteins from the nibbled cell are acquired and displayed by the recipient cell. In this study, we tested the hypothesis that through Trogocytosis, amoebae acquire and display human cell membrane proteins. We demonstrate that E. histolytica acquires and displays human cell membrane proteins through Trogocytosis and that this leads to protection from lysis by human serum. Protection from human serum occurs only after amoebae have undergone Trogocytosis of live cells but not phagocytosis of dead cells. Likewise, mutant amoebae defective in phagocytosis, but unaltered in their capacity to perform Trogocytosis, are protected from human serum. Our studies are the first to reveal that amoebae can display human cell membrane proteins and suggest that the acquisition and display of membrane proteins is a general feature of Trogocytosis. These studies have major implications for interactions between E. histolytica and the immune system and also reveal a novel strategy for immune evasion by a pathogen. Since other microbial eukaryotes use Trogocytosis for cell killing, our findings may apply to the pathogenesis of other infections.IMPORTANCE Entamoeba histolytica causes amoebiasis, a potentially fatal diarrheal disease. Abscesses in organs such as the liver can occur when amoebae are able to breach the intestinal wall and travel through the bloodstream to other areas of the body. Therefore, understanding how E. histolytica evades immune detection is of great interest. Here, we demonstrate for the first time that E. histolytica acquires and displays human cell membrane proteins by taking "bites" of human cell material in a process named Trogocytosis ("trogo-" means "nibble"), and that this allows amoebae to survive in human serum. Display of acquired proteins through Trogocytosis has been previously characterized only in mammalian immune cells. Our study suggests that this is a more general feature of Trogocytosis not restricted to immune cells and broadens our knowledge of eukaryotic biology. These findings also reveal a novel strategy for immune evasion by a pathogen and may apply to the pathogenesis of other infections.
