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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
    2016
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    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

  • Subcutaneous veltuzumab, a humanized anti-CD20 antibody, in the treatment of refractory pemphigus vulgaris.
    JAMA dermatology, 2014
    Co-Authors: Christoph T. Ellebrecht, David M. Goldenberg, Eun J. Choi, David Allman, Donald E. Tsai, William A. Wegener, Aimee S. Payne
    Abstract:

    Importance B-cell depletion with the anti-CD20 antibody rituximab is highly effective for pemphigus vulgaris (PV) treatment. However, most patients experience relapse, and intravenous rituximab infusions are expensive. Therefore, cost-effective anti-CD20 therapies are desirable. Observations A compassionate-use investigational new drug protocol was approved to administer veltuzumab, a second-generation humanized anti-CD20 antibody, to a patient with refractory PV. Veltuzumab was administered as two 320-mg (188 mg/m 2 ) subcutaneous doses 2 weeks apart, resulting in complete remission of disease off therapy. The disease relapsed 2 years after treatment. A second cycle of subcutaneous veltuzumab, using the same dosage regimen, again induced complete remission off therapy, which remained at 9 months. No serious adverse events occurred during 35 months of follow-up. Serum veltuzumab levels were 22 and 29 μg/mL 2 weeks after the first dose of each cycle, and the drug remained detectable in the serum for longer than 3 months. Relapse and response to veltuzumab generally correlated with desmoglein 3 enzyme-linked immunosorbent assay index values. Shortly after a relapse that occurred after a long-term remission, the patient demonstrated an elevated naive (CD19 + CD27 − ) to memory (CD19 + CD27 + ) B-cell ratio of 19.5 and transitional (CD19 + CD24 + CD38 + ) B-cell frequency of 12.5%. Conclusions and Relevance Subcutaneous veltuzumab may be a safe, effective, and more economical alternative to intravenous rituximab for PV therapy. Clinical trials of subcutaneous veltuzumab for PV are warranted.

  • anti cd22 cd20 bispecific antibody with enhanced trogocytosis for treatment of lupus
    PLOS ONE, 2014
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    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.

  • Analysis of trogocytosis by flow cytometry.
    2014
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    PBMCs were incubated overnight with 10 µg/mL of various mAbs or bsHexAbs prior to measurement of surface antigens by flow cytometry. (A) Gating of lymphocytes by forward vs. side scattering (Left) and B cells from the lymphocyte gate using CD19 and CD22 staining (Right) following treatment with control mAb (labetuzumab). (B) Example dot-plots comparing CD19 and CD22 staining on B cells following treatment of PBMCs with 22*-(20)-(20), epratuzumab and labetuzumab (Left) and histograms showing β7 integrin staining following treatment with the indicated mAbs or bsHexAbs (Right). (C) Trogocytosis mediated by Ck and CH3-based bsAbs. PBMCs were incubated overnight with 10 µg/mL 22*-(20)-(20), 22-(20)-(20), veltuzumab, epratuzumab or labetuzumab (control), prior to measurement of surface CD19, CD22 and CD21 by flow cytometry. Results are shown as the % MFI of the control treatment. Error bars, Std. Dev.

  • Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus
    Blood, 2013
    Co-Authors: Rosana B. Michel, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    Background The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases (AIDs), treating currently over 1500 cases of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemias, 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

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
    2016
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    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, 2014
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    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.

  • Analysis of trogocytosis by flow cytometry.
    2014
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    PBMCs were incubated overnight with 10 µg/mL of various mAbs or bsHexAbs prior to measurement of surface antigens by flow cytometry. (A) Gating of lymphocytes by forward vs. side scattering (Left) and B cells from the lymphocyte gate using CD19 and CD22 staining (Right) following treatment with control mAb (labetuzumab). (B) Example dot-plots comparing CD19 and CD22 staining on B cells following treatment of PBMCs with 22*-(20)-(20), epratuzumab and labetuzumab (Left) and histograms showing β7 integrin staining following treatment with the indicated mAbs or bsHexAbs (Right). (C) Trogocytosis mediated by Ck and CH3-based bsAbs. PBMCs were incubated overnight with 10 µg/mL 22*-(20)-(20), 22-(20)-(20), veltuzumab, epratuzumab or labetuzumab (control), prior to measurement of surface CD19, CD22 and CD21 by flow cytometry. Results are shown as the % MFI of the control treatment. Error bars, Std. Dev.

  • Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus
    Blood, 2013
    Co-Authors: Rosana B. Michel, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    Background The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases (AIDs), treating currently over 1500 cases of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemias, 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, 2013
    Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing Chang
    Abstract:

    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
    2016
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    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, 2014
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    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.

  • Analysis of trogocytosis by flow cytometry.
    2014
    Co-Authors: Edmund A. Rossi, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    PBMCs were incubated overnight with 10 µg/mL of various mAbs or bsHexAbs prior to measurement of surface antigens by flow cytometry. (A) Gating of lymphocytes by forward vs. side scattering (Left) and B cells from the lymphocyte gate using CD19 and CD22 staining (Right) following treatment with control mAb (labetuzumab). (B) Example dot-plots comparing CD19 and CD22 staining on B cells following treatment of PBMCs with 22*-(20)-(20), epratuzumab and labetuzumab (Left) and histograms showing β7 integrin staining following treatment with the indicated mAbs or bsHexAbs (Right). (C) Trogocytosis mediated by Ck and CH3-based bsAbs. PBMCs were incubated overnight with 10 µg/mL 22*-(20)-(20), 22-(20)-(20), veltuzumab, epratuzumab or labetuzumab (control), prior to measurement of surface CD19, CD22 and CD21 by flow cytometry. Results are shown as the % MFI of the control treatment. Error bars, Std. Dev.

  • op0118 epratuzumab mediates bcr antigen trogocytosis as potential mechanism of action in systemic lupus erythematosus sle
    Annals of the Rheumatic Diseases, 2013
    Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing Chang
    Abstract:

    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, 2013
    Co-Authors: David M. Goldenberg, Daniel J. Wallace, Edmund A. Rossi, R. Michel, Chien-hsing Chang
    Abstract:

    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

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, 2013
    Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing Chang
    Abstract:

    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, 2013
    Co-Authors: David M. Goldenberg, Daniel J. Wallace, Edmund A. Rossi, R. Michel, Chien-hsing Chang
    Abstract:

    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, 2013
    Co-Authors: Edmund A. Rossi, Rosana B. Michel, Diane L. Rossi, Daniel J. Wallace, David M. Goldenberg, Chien-hsing Chang
    Abstract:

    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, 2012
    Co-Authors: Edmund A. Rossi, Rosana B. Michel, Chien-hsing Chang, Daniel J. Wallace, David M. Goldenberg
    Abstract:

    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.

  • Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus
    Blood, 2013
    Co-Authors: Rosana B. Michel, Chien-hsing Chang, David M. Goldenberg
    Abstract:

    Background The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases (AIDs), treating currently over 1500 cases of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemias, 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, 2013
    Co-Authors: David M. Goldenberg, Rosana B. Michel, Edmund A. Rossi, Daniel J. Wallace, Chien-hsing Chang
    Abstract:

    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, 2013
    Co-Authors: Edmund A. Rossi, Rosana B. Michel, Diane L. Rossi, Daniel J. Wallace, David M. Goldenberg, Chien-hsing Chang
    Abstract:

    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, 2012
    Co-Authors: Edmund A. Rossi, Rosana B. Michel, Chien-hsing Chang, Daniel J. Wallace, David M. Goldenberg
    Abstract:

    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.