Antibody Distance from the Cell Membrane Regulates Antibody Effector Mechanisms Kirstie L. S. Cleary, H. T. Claude Chan, Sonja James, Martin J. Glennie and Mark S. Cragg This information is current as of September 26, 2021. J Immunol published online 12 April 2017 http://www.jimmunol.org/content/early/2017/04/12/jimmun ol.1601473 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/04/12/jimmunol.160147 Material 3.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 26, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 12, 2017, doi:10.4049/jimmunol.1601473 The Journal of Immunology Antibody Distance from the Cell Membrane Regulates Antibody Effector Mechanisms Kirstie L. S. Cleary, H. T. Claude Chan, Sonja James, Martin J. Glennie, and Mark S. Cragg Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits dif- ferent levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the afore- mentioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains Downloaded from to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab- dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells http://www.jimmunol.org/ through specific effector mechanisms and provide more effective therapeutic agents. The Journal of Immunology, 2017, 198: 000–000. n the last decade, mAb immunotherapy has become an cascade continues, resulting in inflammation via C3 or, ultimately, established treatment for cancer. In hematology, based upon insertion of the membrane attack complex into the target cell I the success of the anti-CD20 mAb, rituximab (RTX) (1), membrane and potential cell lysis. In addition, soluble compo- mAbs are now becoming central to the treatment of many solid nents, such as C3a and C5a, are produced that facilitate the re- cruitment of immune effector cells, such as neutrophils and and difficult-to-treat cancers (2). mAbs, such as RTX, are referred to by guest on September 26, 2021 as direct-targeting mAbs and, as such, bind directly to the tumor cell macrophages, which interact with complement receptors to initiate target, facilitating tumor clearance via an Fc-independent mechanism activation and phagocytosis (8, 9). ADCC and ADCP are mediated (direct cell death) (3, 4) or Fc-dependent mechanisms (complement- via activatory FcgR expressed on immune cells, such as NK cells dependent cytotoxicity [CDC], Ab-dependent cellular phagocytosis and myeloid cells (10). For ADCC, binding of FcgR on NK cells [ADCP], and Ab-dependent cellular cytotoxicity [ADCC]) (5). Un- culminates in the release of cytotoxic granules (containing per- derstanding what facilitates or impairs these mechanisms is pivotal for forin and granzymes) that enter the targeted cell, resulting in cell death (11). In contrast, for ADCP, FcgR activation results in cy- the development of more effective therapeutics. toskeletal rearrangements, allowing the formation of a phagosome CDC involves initiation of the classical complement pathway following the interaction of C1q with at least two Ab Fc regions in (12). The mAb-coated cell is then endocytosed and degraded by lysosomal enzymes present within (13). close proximity (6), with hexameric conformation of Ab com- Which effector mechanism is most important for the efficacy of plexes demonstrated to be highly efficient (7). The proteolytic direct-targeting mAbs in humans is widely debated. Pivotal work in 2000 demonstrated that the therapeutic activity of anti-CD20 mAbs Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University was dependent on the presence of activatory FcgR in mice and was of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom limited by the presence of the inhibitory FcgRIIB, CD32B (14). ORCIDs: 0000-0003-0530-9480 (H.T.C.C.); 0000-0003-2077-089X (M.S.C.). This dependence on FcgR is not restricted to anti-CD20 mAb; it has Received for publication August 24, 2016. Accepted for publication March 16, 2017. also been reported for anti-EGFR mAb directed at solid tumors This work was supported by studentships from the Biotechnology and Biological (15). More recently, there is a growing body of work that proposes Sciences Research Council to K.L.S.C. and by Programme Grants from Bloodwise myeloid cells as the FcgR-expressing effectors and ADCP as the (12050) and Cancer Research UK (A20537). predominant effector mechanism for this class of mAbs (16, 17). Address correspondence and reprint requests to Prof. Mark S. Cragg, Faculty of Since the initial generation of mAbs, it has been known that Medicine, University of Southampton, Southampton General Hospital, Tremona Road, Southampton, Hampshire SO16 6YD, U.K. E-mail address: [email protected] different mAbs to the same target can elicit widely different ac- The online version of this article contains supplemental material. tivities (18), including differences in the effector mechanisms Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; ADCP, engaged. For example, type II anti-CD20 mAbs do not elicit CDC Ab-dependent cellular phagocytosis; BMDM, bone marrow–derived macrophage; efficiently, whereas type I mAbs do (19, 20), and ofatumumab has CDC, complement-dependent cytotoxicity; h, human; m, mouse; RTX, rituximab; greater CDC than RTX (21). These mechanistic differences are T , regulatory T cell; WT, wild-type. reg not unique to anti-CD20 mAbs; trastuzumab is reported to initiate Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 ADCC better than pertuzumab (18). Importantly, these differences www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601473 2 EPITOPE DEFINES ANTIBODY EFFECTOR MECHANISMS are not a simple consequence of the Ab isotype (22); therefore, it 1640 media supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium is logical to conclude that the nature of the epitope bound influ- pyruvate, and 20% L929-conditioned media (containing GM-CSF) at 37˚C ences the effector mechanism engaged. in 5% CO2. CHO-S cells (Invitrogen) were cultured in serum-free FreeStyle CHO media in a shaking incubator at 37˚C in 5% CO2. A20 cells (31) were As of July 2015, 22 mAbs were approved or pending approval for cultured in RPMI 1640 media supplemented with 10% FCS, 2 mM glu- the treatment of cancer in the United States and the European Union tamine, 1 mM pyruvate, and 100 IU/ml penicillin and streptomycin (all (23). Nearly two thirds are direct-targeting mAbs against only from Life Technologies) at 37˚C in 5% CO2. eight Ags. Many more targets have been assessed in oncology, so Cloning of fusion constructs it is unclear what makes these targets so effective as Ags for mAb- mediated immunotherapy. Various explanations for the relation- Fusion constructs were cloned through overlap-extension PCR (32) using ship between the Ag and mAb effector mechanisms have been WT hCD137 (UniProt: Q07011; NCBI ref: NM_001561), hCD52 (Uni- Prot: P31358; NCBI ref: NM_001803.2), or the Rp3/Cp11 epitope (kind proposed that relate to Ag density, size, position on the target, and gift of Prof. M. Pule and Dr. B. Philip, University College London) (33, mobility. Ag-expression levels have been correlated with anti- 34). Each fusion protein consisted of the CD52 leader sequence, Rp3/ CD20 mAb efficacy (21, 24), and work performed by Derer Cp11, and CD137 (with domains removed based on the annotations pro- et al. (25) demonstrated that