CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering

Simone C Oostindie, Hilma J van der Horst, Margaret A Lindorfer, Erika M Cook, Jillian C Tupitza, Clive S Zent, Richard Burack, Karl R VanDerMeid, Kristin Strumane, Martine E D Chamuleau, Tuna Mutis, Rob N de Jong, Janine Schuurman, Esther C W Breij, Frank J Beurskens, Paul W H I Parren, Ronald P Taylor

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo. Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.
Original languageEnglish
Pages (from-to)1841-1852
JournalHaematologica
Volume104
Issue number9
Early online date21 Feb 2019
DOIs
Publication statusPublished - 2019

Cite this

Oostindie, Simone C ; van der Horst, Hilma J ; Lindorfer, Margaret A ; Cook, Erika M ; Tupitza, Jillian C ; Zent, Clive S ; Burack, Richard ; VanDerMeid, Karl R ; Strumane, Kristin ; Chamuleau, Martine E D ; Mutis, Tuna ; de Jong, Rob N ; Schuurman, Janine ; Breij, Esther C W ; Beurskens, Frank J ; Parren, Paul W H I ; Taylor, Ronald P. / CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering. In: Haematologica. 2019 ; Vol. 104, No. 9. pp. 1841-1852.
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title = "CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering",
abstract = "CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo. Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.",
author = "Oostindie, {Simone C} and {van der Horst}, {Hilma J} and Lindorfer, {Margaret A} and Cook, {Erika M} and Tupitza, {Jillian C} and Zent, {Clive S} and Richard Burack and VanDerMeid, {Karl R} and Kristin Strumane and Chamuleau, {Martine E D} and Tuna Mutis and {de Jong}, {Rob N} and Janine Schuurman and Breij, {Esther C W} and Beurskens, {Frank J} and Parren, {Paul W H I} and Taylor, {Ronald P}",
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year = "2019",
doi = "10.3324/haematol.2018.207266",
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Oostindie, SC, van der Horst, HJ, Lindorfer, MA, Cook, EM, Tupitza, JC, Zent, CS, Burack, R, VanDerMeid, KR, Strumane, K, Chamuleau, MED, Mutis, T, de Jong, RN, Schuurman, J, Breij, ECW, Beurskens, FJ, Parren, PWHI & Taylor, RP 2019, 'CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering' Haematologica, vol. 104, no. 9, pp. 1841-1852. https://doi.org/10.3324/haematol.2018.207266, https://doi.org/10.3324/haematol.2018.207266

CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering. / Oostindie, Simone C; van der Horst, Hilma J; Lindorfer, Margaret A; Cook, Erika M; Tupitza, Jillian C; Zent, Clive S; Burack, Richard; VanDerMeid, Karl R; Strumane, Kristin; Chamuleau, Martine E D; Mutis, Tuna; de Jong, Rob N; Schuurman, Janine; Breij, Esther C W; Beurskens, Frank J; Parren, Paul W H I; Taylor, Ronald P.

In: Haematologica, Vol. 104, No. 9, 2019, p. 1841-1852.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering

AU - Oostindie, Simone C

AU - van der Horst, Hilma J

AU - Lindorfer, Margaret A

AU - Cook, Erika M

AU - Tupitza, Jillian C

AU - Zent, Clive S

AU - Burack, Richard

AU - VanDerMeid, Karl R

AU - Strumane, Kristin

AU - Chamuleau, Martine E D

AU - Mutis, Tuna

AU - de Jong, Rob N

AU - Schuurman, Janine

AU - Breij, Esther C W

AU - Beurskens, Frank J

AU - Parren, Paul W H I

AU - Taylor, Ronald P

N1 - Copyright © 2019, Ferrata Storti Foundation.

PY - 2019

Y1 - 2019

N2 - CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo. Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.

AB - CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo. Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.

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UR - https://www.ncbi.nlm.nih.gov/pubmed/30792198

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DO - 10.3324/haematol.2018.207266

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JO - Haematologica

JF - Haematologica

SN - 0390-6078

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