Breton et al. Journal of Hematology & Oncology 2014, 7:33 http://www.jhoonline.org/content/7/1/33
RESEARCH
JOURNAL OF HEMATOLOGY & ONCOLOGY
Open Access
A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies Caroline S Breton1, Aimable Nahimana1, Dominique Aubry1, Julie Macoin2, Pierre Moretti2, Martin Bertschinger2, Samuel Hou2, Michel A Duchosal1*† and Jonathan Back2*†
Abstract Background: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. Methods: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. Results: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. Conclusion: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies. Keywords: B cell malignancies, GBR 401, Anti-CD19 monoclonal antibody, ADCC, Therapeutic antibody
Background The last decade has seen an increased interest in the use of antibody-based therapeutic approaches for cancer treatment with clear efficacy and safety profiles [1-3]. In this context, anti-CD20 monoclonal antibodies (mAbs), such as the first in line Rituximab (RTX), have been used to target human B cell malignancies, improving the treatment * Correspondence: [email protected]; [email protected] † Equal contributors 1 Service and Central Laboratory of Hematology, University Hospital of Lausanne, Rue du Bugnon 46, 1011- CHUV, Lausanne, Switzerland 2 Glenmark Pharmaceuticals S.A, Chemin de la Combeta 5, 2300 La Chaux de-Fonds, Switzerland
of this group of diseases [4,5]. Anti-CD20 mAbs exert their antitumor activities through various modes of cell death induction including antibody dependent cellular cytotoxicity (ADCC) [6], complement-dependent cytotoxicity (CDC) [7] and direct induction of cell death [4]. However, despite this success, some patients do not respond to this targeted mAb therapy. Some of the main molecular mechanisms underlying the resistance to anti-CD20 mAbs are (a) low expression levels of CD20 (such as in chronic lymphocytic leukemia-CLL) [8]; (b) down regulation of CD20 expression during the treatment [9,10]; (c) expression of CD20 transcript variants lacking the RTX epitope sequence [11] and
© 2014 Breton et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Breton et al. Journal of Hematology & Oncology 2014, 7:33 http://www.jhoonline.org/content/7/1/33
(d) complement depletion associated with the loss of component C2 [12]. Developing novel and potent mAbs to circumvent such drug resistances is of great importance. An interesting development is to target CD19, a specific B cell marker, expressed early during pre-B cell ontogeny and until terminal differentiation into early plasma cells, with a potential efficacy on a large panel of B cell malignancies [13]. Indeed, the majority of B cell lineage malignancies (more than 90%) express CD19, including notably non-Hodgkin’s lymphoma, CLL and acute lymphoblastic leukemia (ALL) [14,15]. Finally, tumor B cells that have lost the expression of CD20 after anti-CD20 mAb therapy, maintain the expression of CD19. Given its biology and broad expression pattern, CD19 represents an attractive chemotherapeutic target to treat B cell malignancies. Consequently, efforts are now being deployed to develop novel anti-CD19 mAbs to treat various hematological malignancies and emerging results from preclinical studies are encouraging [16-19]. In the past few years, the knowledge of how the Fc fragment of antibodies can trigger cytotoxic mechanisms has improved substantially. In particular, glycosylation analyses have identified fucose residues as negatively influencing the binding of the Fc to FcγRIIIa. This receptor is expressed by natural killer (NK) cells, neutrophils and monocytes and is known to trigger ADCC. Antibodies bearing low levels of fucose residues have been shown to display enhanced ADCC potential [20,21]. Here we describe GBR 401, a partially defucosylated humanized mAb targeting the domain II of CD19 and derived from the parental mouse anti human CD19 antibody clone FMC63 [19]. Both in vitro and in vivo data showed that GBR 401 was highly effective at depleting human malignant B cells mainly via ADCC. It also exhibited a direct killing effect on human B cell malignancies. Finally, benchmarking done against RTX, demonstrated a remarkably superior killing capacity of GBR 401. Our preclinical results suggest GBR 401 to be an efficacious therapeutic agent for human B lymphoma and leukemia and warrant further clinical studies of GBR 401 in these diseases.
Results GBR 401 is a partially defucosylated mAb
GBR 401 is a mAb with enhanced affinity for FcγRIIIa due to its low fucose content. The humanization, binding characteristics and engineering performed to produce GBR 401 are described in Skegro et al. (manuscript in preparation). GBR 401 is produced in a recombinant CHO cell line allowing the expression of mAbs with a reduced level of α1-6 fucose linked to the N-acetylglucosamines in the Nglycan core. The glycosylation of GBR 401 can be seen by HPLC run (Figure 1) and is compared to its fully fucosylated parent GBR 401(F) antibody. Whereas GBR 401(F)
Page 2 of 13
shows a normal CHO glycosylation profile with biantennary complex N-oligosaccharides G0F, G1F, G1F’ and G2F, GBR 401 shows a high level of defucosylated glycans G0, G1, G1’ and G2 (Figure 1A). The overall defucosylation level of GBR 401 reaches approximately 50% versus 50 cells) were scored under an inverted microscope after 14 days at 37°C with 5% CO2. Statistics
Data were expressed as mean plus or minus standard deviation (SD). Student’s t-tests or Wilcoxon ranked tests were performed to determine differences between samples. GraphPad Prism version 6.02 (GraphPad Software, San Diego, CA) was used for statistical analysis. P values
Received: 10 December 2013 Accepted: 7 April 2014 Published: 14 April 2014 References 1. Gokbuget N, Hoelzer D: Treatment with monoclonal antibodies in acute lymphoblastic leukemia: current knowledge and future prospects. Ann Hematol 2004, 83:201–205. 2. Bassan R, Hoelzer D: Modern therapy of acute lymphoblastic leukemia. J Clin Oncol 2011, 29:532–543. 3. Fanale MA, Younes A: Monoclonal antibodies in the treatment of non-Hodgkin’s lymphoma. Drugs 2007, 67:333–350. 4. Maloney DG: Anti-CD20 antibody therapy for B-cell lymphomas. N Engl J Med 2012, 366:2008–2016. 5. Hoelzer D: Novel antibody-based therapies for acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program 2011, 2011:243–249. 6. Tedder TF, Baras A, Xiu Y: Fcgamma receptor-dependent effector mechanisms regulate CD19 and CD20 antibody immunotherapies for B lymphocyte malignancies and autoimmunity. Springer Semin Immunopathol 2006, 28:351–364.
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doi:10.1186/1756-8722-7-33 Cite this article as: Breton et al.: A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies. Journal of Hematology & Oncology 2014 7:33.
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RESEARCH
JOURNAL OF HEMATOLOGY & ONCOLOGY
Open Access
A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies Caroline S Breton1, Aimable Nahimana1, Dominique Aubry1, Julie Macoin2, Pierre Moretti2, Martin Bertschinger2, Samuel Hou2, Michel A Duchosal1*† and Jonathan Back2*†
Abstract Background: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. Methods: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. Results: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. Conclusion: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies. Keywords: B cell malignancies, GBR 401, Anti-CD19 monoclonal antibody, ADCC, Therapeutic antibody
Background The last decade has seen an increased interest in the use of antibody-based therapeutic approaches for cancer treatment with clear efficacy and safety profiles [1-3]. In this context, anti-CD20 monoclonal antibodies (mAbs), such as the first in line Rituximab (RTX), have been used to target human B cell malignancies, improving the treatment * Correspondence: [email protected]; [email protected] † Equal contributors 1 Service and Central Laboratory of Hematology, University Hospital of Lausanne, Rue du Bugnon 46, 1011- CHUV, Lausanne, Switzerland 2 Glenmark Pharmaceuticals S.A, Chemin de la Combeta 5, 2300 La Chaux de-Fonds, Switzerland
of this group of diseases [4,5]. Anti-CD20 mAbs exert their antitumor activities through various modes of cell death induction including antibody dependent cellular cytotoxicity (ADCC) [6], complement-dependent cytotoxicity (CDC) [7] and direct induction of cell death [4]. However, despite this success, some patients do not respond to this targeted mAb therapy. Some of the main molecular mechanisms underlying the resistance to anti-CD20 mAbs are (a) low expression levels of CD20 (such as in chronic lymphocytic leukemia-CLL) [8]; (b) down regulation of CD20 expression during the treatment [9,10]; (c) expression of CD20 transcript variants lacking the RTX epitope sequence [11] and
© 2014 Breton et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Breton et al. Journal of Hematology & Oncology 2014, 7:33 http://www.jhoonline.org/content/7/1/33
(d) complement depletion associated with the loss of component C2 [12]. Developing novel and potent mAbs to circumvent such drug resistances is of great importance. An interesting development is to target CD19, a specific B cell marker, expressed early during pre-B cell ontogeny and until terminal differentiation into early plasma cells, with a potential efficacy on a large panel of B cell malignancies [13]. Indeed, the majority of B cell lineage malignancies (more than 90%) express CD19, including notably non-Hodgkin’s lymphoma, CLL and acute lymphoblastic leukemia (ALL) [14,15]. Finally, tumor B cells that have lost the expression of CD20 after anti-CD20 mAb therapy, maintain the expression of CD19. Given its biology and broad expression pattern, CD19 represents an attractive chemotherapeutic target to treat B cell malignancies. Consequently, efforts are now being deployed to develop novel anti-CD19 mAbs to treat various hematological malignancies and emerging results from preclinical studies are encouraging [16-19]. In the past few years, the knowledge of how the Fc fragment of antibodies can trigger cytotoxic mechanisms has improved substantially. In particular, glycosylation analyses have identified fucose residues as negatively influencing the binding of the Fc to FcγRIIIa. This receptor is expressed by natural killer (NK) cells, neutrophils and monocytes and is known to trigger ADCC. Antibodies bearing low levels of fucose residues have been shown to display enhanced ADCC potential [20,21]. Here we describe GBR 401, a partially defucosylated humanized mAb targeting the domain II of CD19 and derived from the parental mouse anti human CD19 antibody clone FMC63 [19]. Both in vitro and in vivo data showed that GBR 401 was highly effective at depleting human malignant B cells mainly via ADCC. It also exhibited a direct killing effect on human B cell malignancies. Finally, benchmarking done against RTX, demonstrated a remarkably superior killing capacity of GBR 401. Our preclinical results suggest GBR 401 to be an efficacious therapeutic agent for human B lymphoma and leukemia and warrant further clinical studies of GBR 401 in these diseases.
Results GBR 401 is a partially defucosylated mAb
GBR 401 is a mAb with enhanced affinity for FcγRIIIa due to its low fucose content. The humanization, binding characteristics and engineering performed to produce GBR 401 are described in Skegro et al. (manuscript in preparation). GBR 401 is produced in a recombinant CHO cell line allowing the expression of mAbs with a reduced level of α1-6 fucose linked to the N-acetylglucosamines in the Nglycan core. The glycosylation of GBR 401 can be seen by HPLC run (Figure 1) and is compared to its fully fucosylated parent GBR 401(F) antibody. Whereas GBR 401(F)
Page 2 of 13
shows a normal CHO glycosylation profile with biantennary complex N-oligosaccharides G0F, G1F, G1F’ and G2F, GBR 401 shows a high level of defucosylated glycans G0, G1, G1’ and G2 (Figure 1A). The overall defucosylation level of GBR 401 reaches approximately 50% versus 50 cells) were scored under an inverted microscope after 14 days at 37°C with 5% CO2. Statistics
Data were expressed as mean plus or minus standard deviation (SD). Student’s t-tests or Wilcoxon ranked tests were performed to determine differences between samples. GraphPad Prism version 6.02 (GraphPad Software, San Diego, CA) was used for statistical analysis. P values
Received: 10 December 2013 Accepted: 7 April 2014 Published: 14 April 2014 References 1. Gokbuget N, Hoelzer D: Treatment with monoclonal antibodies in acute lymphoblastic leukemia: current knowledge and future prospects. Ann Hematol 2004, 83:201–205. 2. Bassan R, Hoelzer D: Modern therapy of acute lymphoblastic leukemia. J Clin Oncol 2011, 29:532–543. 3. Fanale MA, Younes A: Monoclonal antibodies in the treatment of non-Hodgkin’s lymphoma. Drugs 2007, 67:333–350. 4. Maloney DG: Anti-CD20 antibody therapy for B-cell lymphomas. N Engl J Med 2012, 366:2008–2016. 5. Hoelzer D: Novel antibody-based therapies for acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program 2011, 2011:243–249. 6. Tedder TF, Baras A, Xiu Y: Fcgamma receptor-dependent effector mechanisms regulate CD19 and CD20 antibody immunotherapies for B lymphocyte malignancies and autoimmunity. Springer Semin Immunopathol 2006, 28:351–364.
Breton et al. Journal of Hematology & Oncology 2014, 7:33 http://www.jhoonline.org/content/7/1/33
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doi:10.1186/1756-8722-7-33 Cite this article as: Breton et al.: A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies. Journal of Hematology & Oncology 2014 7:33.
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