ACCEPTED ARTICLE PREVIEW
Accepted Article Preview: Published ahead of advance online publication A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells
Kristina Nalivaiko, Martin Hofmann, Karina Kober,Nadine Teichweyde, Peter H. Krammer, Hans-Georg Rammensee, Ludger GrosseHovest,Gundram Jung
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Cite this article as: Kristina Nalivaiko, Martin Hofmann, Karina Kober,Nadine Teichweyde, Peter H. Krammer, Hans-Georg Rammensee, Ludger Grosse-Hovest,Gundram Jung, A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells, Molecular Therapy accepted article preview online 19 November 2015; doi:10.1038/mt.2015.209
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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
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Received 06 July 2015; accepted 30 October 2015; Accepted article preview online 19 November 2015
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells Kristina Nalivaiko1, Martin Hofmann1,3, Karina Kober1, Nadine Teichweyde1, Peter H. Krammer2,4, Hans-Georg Rammensee1,4, Ludger Grosse-Hovest1,3, Gundram Jung1,4 Affiliations: 1 Department of Immunology, Eberhard Karls Universität Tübingen, Germany 2 Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Present address: Synimmune GmbH, Auf der Morgenstelle 15, Tübingen, Germany 4 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Contributions: K.N. performed most of the experiments, and contributed to data presentation and editing of the paper. M.H. developed the FACS method used for the cell depletion experiments and contributed to the analysis of the respective experiments. K.K. was involved in the initial construction of recombinant variants of BS9520. N.T. generated the recombinant CD20 antibodies (together with L.G.H.). P.H.K contributed the method used for measuring antibody production by PWM activated cells. H.G.R suggested some of the experiments. L.G.H. invented the Fabsc-format and was involved in the construction of BS9520 and BS95Mel. G.J. designed the concept of the study, most of the experiments (together with K.N.) and wrote the manuscript.
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Competing financial interests: LGH and GJ have filed patent applications covering the stimulation of CD95 with recombinant, bispecific antibodies. Patent rights have been licensed to Baliopharm AG, Jülich, Germany. GJ is a member of the scientific advisory board of Baliopharm. The remaining authors declare no competing financial interests.
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Acknowledgements: This work was supported by a fellowship from the Catholic Academic Student Exchange Service (Katholischer Akademischer Ausländer-Dienst, KAAD), Germany, to K.N. and, in part, by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, SFB773, project C4). We thank Dr. O. Planz for help with the animal experiments and B. Pömmerl for skillful technical assistance. Corresponding author: Gundram Jung Department of Immunology Eberhard Karls Universität Tübingen E-mail: [email protected] Phone: x49-7071-29-87621 FAX: x49-7071-29-5653
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Monoclonal antibodies directed to the B-cell specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here we compare the anti-B-cell activity of three different antibodies directed to CD20, (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof and (iii) a bispecific CD20XCD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant- as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from PBMC cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its non-optimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20XCD95-antibody might become a new, prototypical reagent for the treatment of Bcell mediated autoimmune disease.
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Chimeric or humanized second generation antibodies recognizing CD20, a protein expressed on normal and malignant B-cells, have already been prototypical in several respects: Rituximab, a chimeric version of the parental mouse antibody 2B8, was the first antibody to demonstrate convincing anti-tumor effects in humans and has become a cornerstone of current lymphoma treatment1. In addition, given the activity of CD20 antibodies not only against malignant- but also against normal B-cells, those reagents have been used to treat B-cell associated autoimmune diseases2, such as rheumatoid arthritis, Wegeners granulomatosis, Sjögrens syndrome3 and, more recently, multiple sclerosis4,5. For immunosuppression, Rituximab as well as newly developed CD20 antibodies, such as Ocrelizumab and Ofatumumab, were used. The latter reagents are derived from the parental antibodies 2H76 and B-Ly1, respectively, and are directed against different but overlapping epitopes of the CD20 molecule. CD20 antibodies were also the first to benefit from Fc-optimization7, a promising strategy to further improve the therapeutic activity of antibodies, that largely depends on the interaction of antibody Fc parts and Fc-receptors (FcRs) expressed on immune cells. In principle, Fcoptimization of an IgG antibody may be achieved by genetic engineering of the glycosylation machinery8 or the amino acid sequence of the CH2 domain9, the region responsible for Fc-FcR interaction. Both of these techniques have been used by the industry to develop third generation antibodies up to the stage of clinical trials: Roche’s glyco-engineered CD20antibody GA101 is the successor of the prototypical Rituximab antibody10. Xencor introduced the amino acid modifications S239D and I332E (SDIE-modification) into the CH2 domain of XmAb 5574, an antibody directed to CD19, an alternative B-cell associated antigen11. GA10112, 13 as well as XmAb 557411, exerted remarkably enhanced antibody dependent cytotoxicity (ADCC) against malignant lymphoma cells if compared to the respective nonoptimized antibody variants. Recently, a clinical trial with more than 700 CLL patients demonstrated superior therapeutic activity of GA101 compared to that of Rituximab14, and a phase I trial with XmAB 5574 demonstrated safety and efficicacy in relapsed CLL15. As CD19 and CD20, the death receptor CD95 (Apo-1, Fas) is expressed on malignant- as well as on normal B-cells. Normal cells, however, express only marginal amounts of the protein, unless they undergo activation that is accompanied by a greatly enhanced CD95 expression16. Previously, we have demonstrated that bispecific CD20xCD95-Fab2-antibodies, generated by chemical hybridization, are capable of inducing CD95-mediated apoptosis selectively in malignant cells expressing CD2017. Later, we found that induction of apoptosis depends on
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© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
mutual bi-cellular crosspresentation of bispecific antibodies by cells expressing the relevant target antigen18. For the work described here we have used the CD20 antibody 2H76, contained in Ocrelizumab, to construct a bispecific CD20xCD95 antibody (BS9520) in a newly developed, recombinant format, termed Fabsc19. For comparison, we have also generated a chimeric CD20 antibody (CH20) as well as an Fc-optimized version of this reagent carrying the SDIE modification described above (SDIE20, Fig.1). We have evaluated the cytolytic activity of these reagents against malignant lymphoma cells as well as normal resting and activated B-cells. We have also explored the consequences of this activity, that is, suppression of tumor growth in a SCID mouse model and inhibition of antibody production by normal B-cells, in order to assess and compare the anti-B cell activity of the different reagents.
RESULTS Construction and characterization of the antibodies Fig. 1 depicts the antibodies compared in this paper. All reagents contain the CD20 antibody 2H7, either as a chimeric version (CH20) or as an Fc-optimized, SDIE-modified variant thereof (SDIE20). For construction of the bispecific CD20XCD95 molecule (BS9520), we used the Fabsc-format consisting of an N-terminal Fab-part linked to a C-terminal single chain antibody by an Fc-attenuated CH2 domain (Fig. 1c)19. Fc-attenuation is required to prevent multimerization of the CD95 arm –and, thus, CD95 stimulation- on the surface of FcRexpressing cells 17, 18. We also produced a Fabsc-control antibody, designated BS95Mel, directed to the melanoma associated chondroitin sulfate proteoglycan CSPG4. Previously, we have compared bispecific antibodies with FLT3xCD3-specificity expressed in the Fabsc- as well as the widely used bispecific single chain (bssc)-format, also termed BiTE, and have found that the Fabsc-format is superior with respect to (i) increased production rates, (ii) preserved affinity of the N-terminal Fab-arm and (iii) diminished aggregation19. In fact, the latter advantage became strikingly evident during the development of BS9520: we did not succeed in constructing a functional bispecific antibody containing the CD95 binding part as a C-terminal single chain molecule due to extensive aggregation of this moiety. Only when we expressed the CD95 antibody as the N-terminal Fab-part within the Fabsc-format a monomeric and functional bispecific molecule was obtained (Supplementary Fig. 1). Not unexpectedly, expression of the CD20 antibody in the single chain format resulted in a significant loss of avidity if compared to the intact, monospecific and bivalent CD20 antibodies. In contrast, the avidity of BS9520 towards CD95 is higher and only moderately reduced if compared to the parental CD95 antibody Apo-1 (Supplementary Fig. 2) . In accordance with these findings we observed only weak binding of BS9520 to resting normal B cells due to the low avidity of its CD20 binding part. In contrast, binding to activated cells was considerably more pronounced because the CD95 expression on these cells allows binding of the high affinity CD95 binding part of the molecule (Supplementary Fig.3).
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Activity against malignant B-cells Fig. 2 depicts the anti-proliferative activity of the three antibodies and -for comparison- of the prototypic CD20 antibody Rituximab towards various lymphoma cells in the presence of different amounts of PBMC as a source of effector cells. The antibody dependent cellular cytotoxicity (ADCC) of Rituximab and CH20 was comparable and clearly less pronounced than the activity of the Fc-optimized SDIE20 antibody. At concentrations
Accepted Article Preview: Published ahead of advance online publication A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells
Kristina Nalivaiko, Martin Hofmann, Karina Kober,Nadine Teichweyde, Peter H. Krammer, Hans-Georg Rammensee, Ludger GrosseHovest,Gundram Jung
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Cite this article as: Kristina Nalivaiko, Martin Hofmann, Karina Kober,Nadine Teichweyde, Peter H. Krammer, Hans-Georg Rammensee, Ludger Grosse-Hovest,Gundram Jung, A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells, Molecular Therapy accepted article preview online 19 November 2015; doi:10.1038/mt.2015.209
s u n
This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
d e t
a m
p e c c A
Received 06 July 2015; accepted 30 October 2015; Accepted article preview online 19 November 2015
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells Kristina Nalivaiko1, Martin Hofmann1,3, Karina Kober1, Nadine Teichweyde1, Peter H. Krammer2,4, Hans-Georg Rammensee1,4, Ludger Grosse-Hovest1,3, Gundram Jung1,4 Affiliations: 1 Department of Immunology, Eberhard Karls Universität Tübingen, Germany 2 Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Present address: Synimmune GmbH, Auf der Morgenstelle 15, Tübingen, Germany 4 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Contributions: K.N. performed most of the experiments, and contributed to data presentation and editing of the paper. M.H. developed the FACS method used for the cell depletion experiments and contributed to the analysis of the respective experiments. K.K. was involved in the initial construction of recombinant variants of BS9520. N.T. generated the recombinant CD20 antibodies (together with L.G.H.). P.H.K contributed the method used for measuring antibody production by PWM activated cells. H.G.R suggested some of the experiments. L.G.H. invented the Fabsc-format and was involved in the construction of BS9520 and BS95Mel. G.J. designed the concept of the study, most of the experiments (together with K.N.) and wrote the manuscript.
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Competing financial interests: LGH and GJ have filed patent applications covering the stimulation of CD95 with recombinant, bispecific antibodies. Patent rights have been licensed to Baliopharm AG, Jülich, Germany. GJ is a member of the scientific advisory board of Baliopharm. The remaining authors declare no competing financial interests.
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Acknowledgements: This work was supported by a fellowship from the Catholic Academic Student Exchange Service (Katholischer Akademischer Ausländer-Dienst, KAAD), Germany, to K.N. and, in part, by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, SFB773, project C4). We thank Dr. O. Planz for help with the animal experiments and B. Pömmerl for skillful technical assistance. Corresponding author: Gundram Jung Department of Immunology Eberhard Karls Universität Tübingen E-mail: [email protected] Phone: x49-7071-29-87621 FAX: x49-7071-29-5653
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Monoclonal antibodies directed to the B-cell specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here we compare the anti-B-cell activity of three different antibodies directed to CD20, (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof and (iii) a bispecific CD20XCD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant- as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from PBMC cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its non-optimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20XCD95-antibody might become a new, prototypical reagent for the treatment of Bcell mediated autoimmune disease.
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Chimeric or humanized second generation antibodies recognizing CD20, a protein expressed on normal and malignant B-cells, have already been prototypical in several respects: Rituximab, a chimeric version of the parental mouse antibody 2B8, was the first antibody to demonstrate convincing anti-tumor effects in humans and has become a cornerstone of current lymphoma treatment1. In addition, given the activity of CD20 antibodies not only against malignant- but also against normal B-cells, those reagents have been used to treat B-cell associated autoimmune diseases2, such as rheumatoid arthritis, Wegeners granulomatosis, Sjögrens syndrome3 and, more recently, multiple sclerosis4,5. For immunosuppression, Rituximab as well as newly developed CD20 antibodies, such as Ocrelizumab and Ofatumumab, were used. The latter reagents are derived from the parental antibodies 2H76 and B-Ly1, respectively, and are directed against different but overlapping epitopes of the CD20 molecule. CD20 antibodies were also the first to benefit from Fc-optimization7, a promising strategy to further improve the therapeutic activity of antibodies, that largely depends on the interaction of antibody Fc parts and Fc-receptors (FcRs) expressed on immune cells. In principle, Fcoptimization of an IgG antibody may be achieved by genetic engineering of the glycosylation machinery8 or the amino acid sequence of the CH2 domain9, the region responsible for Fc-FcR interaction. Both of these techniques have been used by the industry to develop third generation antibodies up to the stage of clinical trials: Roche’s glyco-engineered CD20antibody GA101 is the successor of the prototypical Rituximab antibody10. Xencor introduced the amino acid modifications S239D and I332E (SDIE-modification) into the CH2 domain of XmAb 5574, an antibody directed to CD19, an alternative B-cell associated antigen11. GA10112, 13 as well as XmAb 557411, exerted remarkably enhanced antibody dependent cytotoxicity (ADCC) against malignant lymphoma cells if compared to the respective nonoptimized antibody variants. Recently, a clinical trial with more than 700 CLL patients demonstrated superior therapeutic activity of GA101 compared to that of Rituximab14, and a phase I trial with XmAB 5574 demonstrated safety and efficicacy in relapsed CLL15. As CD19 and CD20, the death receptor CD95 (Apo-1, Fas) is expressed on malignant- as well as on normal B-cells. Normal cells, however, express only marginal amounts of the protein, unless they undergo activation that is accompanied by a greatly enhanced CD95 expression16. Previously, we have demonstrated that bispecific CD20xCD95-Fab2-antibodies, generated by chemical hybridization, are capable of inducing CD95-mediated apoptosis selectively in malignant cells expressing CD2017. Later, we found that induction of apoptosis depends on
d e t
a m
s u n
i r c
p e c c A
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
mutual bi-cellular crosspresentation of bispecific antibodies by cells expressing the relevant target antigen18. For the work described here we have used the CD20 antibody 2H76, contained in Ocrelizumab, to construct a bispecific CD20xCD95 antibody (BS9520) in a newly developed, recombinant format, termed Fabsc19. For comparison, we have also generated a chimeric CD20 antibody (CH20) as well as an Fc-optimized version of this reagent carrying the SDIE modification described above (SDIE20, Fig.1). We have evaluated the cytolytic activity of these reagents against malignant lymphoma cells as well as normal resting and activated B-cells. We have also explored the consequences of this activity, that is, suppression of tumor growth in a SCID mouse model and inhibition of antibody production by normal B-cells, in order to assess and compare the anti-B cell activity of the different reagents.
RESULTS Construction and characterization of the antibodies Fig. 1 depicts the antibodies compared in this paper. All reagents contain the CD20 antibody 2H7, either as a chimeric version (CH20) or as an Fc-optimized, SDIE-modified variant thereof (SDIE20). For construction of the bispecific CD20XCD95 molecule (BS9520), we used the Fabsc-format consisting of an N-terminal Fab-part linked to a C-terminal single chain antibody by an Fc-attenuated CH2 domain (Fig. 1c)19. Fc-attenuation is required to prevent multimerization of the CD95 arm –and, thus, CD95 stimulation- on the surface of FcRexpressing cells 17, 18. We also produced a Fabsc-control antibody, designated BS95Mel, directed to the melanoma associated chondroitin sulfate proteoglycan CSPG4. Previously, we have compared bispecific antibodies with FLT3xCD3-specificity expressed in the Fabsc- as well as the widely used bispecific single chain (bssc)-format, also termed BiTE, and have found that the Fabsc-format is superior with respect to (i) increased production rates, (ii) preserved affinity of the N-terminal Fab-arm and (iii) diminished aggregation19. In fact, the latter advantage became strikingly evident during the development of BS9520: we did not succeed in constructing a functional bispecific antibody containing the CD95 binding part as a C-terminal single chain molecule due to extensive aggregation of this moiety. Only when we expressed the CD95 antibody as the N-terminal Fab-part within the Fabsc-format a monomeric and functional bispecific molecule was obtained (Supplementary Fig. 1). Not unexpectedly, expression of the CD20 antibody in the single chain format resulted in a significant loss of avidity if compared to the intact, monospecific and bivalent CD20 antibodies. In contrast, the avidity of BS9520 towards CD95 is higher and only moderately reduced if compared to the parental CD95 antibody Apo-1 (Supplementary Fig. 2) . In accordance with these findings we observed only weak binding of BS9520 to resting normal B cells due to the low avidity of its CD20 binding part. In contrast, binding to activated cells was considerably more pronounced because the CD95 expression on these cells allows binding of the high affinity CD95 binding part of the molecule (Supplementary Fig.3).
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Activity against malignant B-cells Fig. 2 depicts the anti-proliferative activity of the three antibodies and -for comparison- of the prototypic CD20 antibody Rituximab towards various lymphoma cells in the presence of different amounts of PBMC as a source of effector cells. The antibody dependent cellular cytotoxicity (ADCC) of Rituximab and CH20 was comparable and clearly less pronounced than the activity of the Fc-optimized SDIE20 antibody. At concentrations
