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

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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 , 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 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 . 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 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; 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 initiation of CDC was more depen- vided with the UniProt reference sequence). These constructs were cloned into the pcDNA3.1/- (neo) expression vector (Invitrogen). dent on the expression level of EGFR than was NK-mediated ADCC. However, it has long been known that Ag specificity Cell transfections (and not simply Ab isotype or expression level) is an important A total of 10 3 106 CHO-S cells was transfected with 10 mg of plasmid DNA factor in determining the efficacy of CDC (26). Another obser- usinglipofectionwithFreeStyleMAXReagent (Invitrogen). A20 cells were vation is that the targets approved for are nucleofected with 2 mg of plasmid DNA using an Amaxa Cell Line Nucleo- Downloaded from generally small proteins (18, 27) or, if a larger protein (e.g., Her2), fector Kit T (Lonza). Following nucleofection, cells were cultured in the pres- the Ab binds relatively close to the cell membrane (28). Ac- ence of 1 mg/ml Geneticin (Life Technologies) to select for stable transfectants. cordingly, two of the most successful targets in oncology, and CDC assay highly represented in the list of approved reagents, are CD20 and 3 5 CD52, both of which are highly expressed surface receptors with Atotalof1 10 cells was labeled with diluting concentrations of Ab for 15 min at room temperature. Human serum (Sigma) was added at 15% final small extracellular domains. concentration (v/v) and incubated at 37˚C for 30 min. Cell death was assessed http://www.jimmunol.org/ Targeting small Ags, or membrane proximal epitopes, has been with propidium iodide inclusion by flow cytometry using a FACScan (BD). shown to be beneficial for chimeric AgR T cells; a decrease in the Nonopsonized cells were used to establish basal cell death, and the raw data T cell cytotoxicity was observed when binding domains were were adjusted to the transfection efficiency using the following equation: positioned further away from the target cell (22, 29). However, this CDC-specific lysis ¼½ð%PIþof sample 2 baseline=100 2 %PIþbaselineÞ property has not been investigated directly for mAb immuno- 3 100=%transfected cells 3 100 therapy. Therefore, we hypothesized that the distance between an epitope and the target cell membrane was important for mAb immunotherapy, particularly for the engagement of the Fc- ADCP assay mediated mechanisms CDC, ADCP, and ADCC. To investigate Target cells were labeled with 5 mM CFSE for 10 min at room temperature by guest on September 26, 2021 this directly, while avoiding the potential caveats arising from before washing in complete media. CFSE-labeled targets were then using mAbs with different affinities and/or targeting subtly dif- opsonized with diluting concentrations of Ab before coculturing at a 1:5 E: T ratio with BMDMs in 96-well plates for 1 h at 37˚C. BMDMs were then ferent mAb epitopes, we generated a panel of fusion proteins labeled with anti-F4/80–allophycocyanin for 15 min at room temperature based upon a CD137 scaffold displaying the exact target epitope and washed with PBS twice. Plates were kept on ice, wells were scraped to recognized by RTX or CAMPATH-1H at different distances from collect BMDMs, and phagocytosis was assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4/80+CFSE+ cells the target cell membrane. These proteins were then expressed in a + variety of target cells and used as model Ags in deletion assays within the F4/80 cell population. in vitro and in vivo. We found that the effector mechanisms en- Confocal imaging of ADCP gaged by anti-CD20 and anti-CD52 mAbs were dependent on the 3 5 fusion protein targeted and its distance from the cell surface. This BMDMs were plated overnight at 1 10 cells per milliliter onto poly-L- lysine (Sigma)-coated coverslips. Target cells were labeled with CFSE and work has important implications for the development of new 10 mg/ml of Ab, as described above (ADCP assay). Following the 1-h therapeutics that seek to exploit specific effector mechanisms and coculture, coverslips were washed in PBS before fixation in 2% parafor- elicit more effective deletion. maldehyde for 15 min at room temperature. A total of 100 mg/ml of rhodamine wheat germ agglutinin was added for 30 min at room tem- perature before washing in PBS. Lastly, nuclei were stained with DAPI Materials and Methods before mounting. Images were collected using a Leica TCS SP5 with Leica Abs software (LAS-AF v2; both from Leica Microsystems) and processed using Adobe Photoshop CC (version 7 SP1). F4/80-allophycocyanin was from Serotec (clone CI:A3-1), and B220-PerCP (clone RA3-6B2), anti-mouse Fc–Fab2–PE (clone Poly4053), CD55-PE ADCC assay (clone RIKO-3), and CD59-PE (clone mCD59.3) were purchased from BioLegend. RTX–human (h)IgG1 and RTX–mouse (m)IgG2a were pro- Target cells were labeled with calcein AM, followed by the addition of di- duced in-house from patented sequences (22). CAMPATH-1H was kindly luting concentrations of Ab. Target cells were cocultured with human PBMCs 3 provided by Dr. S. Cobbold and Dr. G. Hale; A20 anti-idiotype (clone 1G6) at a 50:1 E:T ratio for 4 h at 37˚C. The plate was centrifuged at 400 g for 5 (30) was provided by Prof. R. Levy; and anti-hCD137 mAbs, SAP1–3 and min to pellet the cells, and the supernatant was transferred to a white 96-well SAP3–6, were produced in-house as part of the Southampton Antibody plate. Calcein release was measured using a Varioskan (Thermo Scientific) at Discovery Program (Cancer Research UK). Polyclonal anti-hCD137 (clone 455 nm, and the percentage of maximal release was calculated as follows: ab197942) Ab, used for Western blotting, was from Abcam. %max release ¼ðsample=triton treatedÞ 3 100 Cell culture In vivo experiments Bone marrow–derived macrophages (BMDMs) were generated from the bone marrow of wild-type (WT) BALB/c female mice, collected, and All animal experiments were performed in accordance with the Animal cultured at 0.8 3 106 cells per milliliter in six-well plates for 8 d in RPMI (Scientific Procedures) Act 1986 within a Home Office–approved facility The Journal of Immunology 3 under project license 30/2964. WT female BALB/c mice younger than 4 in the protein expression of the different constructs, the degree of mo of age were used. The actual severity of all animal experiments was target cell lysis was corrected according to the level of expression, 3 6 recorded as mild in compliance with the project license. A total of 1 10 as described in the Materials and Methods. These experiments A20 cells was administered i.v. on day 0. On day 3, 100 mg of RTX- mIgG2a (22) or PBS was given by i.v. injection. The spleen, liver, and clearly showed that the ability of RTX to initiate CDC was di- inguinal lymph nodes were collected from all mice when the control group minished when targeted furthest away from the cell surface with developed terminal tumor burden (defined as visible signs of ill health, the R-8d construct (Fig. 2B). Less than half of the cells were lysed including expansion of the liver). Organs were homogenized into a single- under saturating (10 mg/ml) concentrations of Ab, with little-to-no cell suspension in 3 ml of sterile PBS and passed through a 100-mm cell strainer before analysis by flow cytometry. lysis observed when the Ab concentration was #0.4 mg/ml. Al- though the three other constructs were able to initiate CDC sim- Western blotting of cell lysates ilarly at the higher mAb concentrations, R-4d showed a trend A total of 6 3 106 cells was lysed in RIPA buffer (25 mM Tris-HCl, (although not statistically significant) toward being less effective 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS as a target at lower concentrations of Ab (,0.4 mg/ml) compared containing protease inhibitor mixture [Sigma], 50 mM NaF, and 0.2 mM with constructs targeting the mAb closer to the cell surface (R-1d Na3VO4). A total of 25 mg of lysate was loaded onto 10% Bis-Tris gels, and R-flush). and proteins were separated under constant voltage. The proteins were Next, we assessed the ability of the constructs to function as transferred onto a nitrocellulose membrane, blocked using a 5% milk so- lution (Marvel), and probed with a polyclonal rabbit anti-hCD137 Ab targets for phagocytosis using mouse BMDMs as effectors overnight at 4˚C. The membrane was washed, and bound Ab was detected (Fig. 2C). In these ADCP assays, targeting all of the constructs, using an HRP-conjugated anti-Rabbit secondary Ab (NA9340; GE with the exception of R-flush, resulted in effective phagocytosis of

Healthcare) and ECL substrate (Thermo Scientific) before chemilumi- RTX-opsonized targets (Fig. 2D). Although R-8d was less sensi- Downloaded from nescence film was applied and developed using a Xograph Imaging Sys- tems Compact X4. tive to ADCP compared with the R-1d and R-4d at lower con- centrations of Ab (,1 mg/ml), this was not statistically significant. Statistics Importantly, expression of the R-flush domain was equivalent to Data were analyzed using one- or two-way ANOVA, where appropriate. All the other constructs (Fig. 1B) and was sufficient for effective CDC statistical analyses were performed using GraphPad Prism (version 7.01). (Fig. 2B), indicating that displaying the Ag too close to the cell

surface was specifically detrimental to phagocytosis, as opposed to http://www.jimmunol.org/ Results simply being inaccessible to mAb binding. The ability of the various constructs to mediate ADCC activity Generation of a model Ag system was also evaluated. Human PBMCs were used as the source of A panel of engineered molecules was designed to assess the re- effector cells in these assays, with the resident NK cells previously lationship between the epitope targeted along a target protein (from shown to be the main mediators of cytotoxic activity (36). Tar- its membrane proximal to distal domains) and the effector geting R-flush resulted in the greatest level of ADCC, whereas mechanisms engaged. These first constructs contained a small targeting R-8d elicited the lowest level of killing (Fig. 2E), similar [previously identified to be bound by the anti-CD20 mAb to the trend seen with CDC.

RTX (34)] attached to the N-terminal domain of hCD137. hCD137 by guest on September 26, 2021 contains four fairly equivalently sized extracellular cysteine-rich Assessment of Ab effector engagement with CAMPATH-1H domains (Fig. 1A) and so is ideal for modular manipulation (35). To confirm that the results generated above were related to Ab Through the use of overlap-extension PCR, extracellular domains distance and were not a unique property of the RTX epitope, a were removed or replicated, resulting in a panel of fusion proteins second panel of fusion proteins was generated. These contained the designed to display the RTX epitope at varying distances from the same CD137 backbone as before (Fig. 1A), but the RTX epitope cell surface, estimated to range from 0.3 to 16 nm away (Fig. 1A). was replaced by one recognized by the anti-CD52 Ab, CAMPATH-1H These constructs were transiently transfected into CHO-S cells, () (33). These proteins were expressed in CHO-S with protein expression confirmed by flow cytometry using Abs cells and were recognized specifically by CAMPATH-1H directed against the RTX epitope and individual domains of (Fig. 3A) and not RTX (data not shown). When used as targets CD137 (Fig. 1B). Importantly, the position of the epitope did not in the three in vitro assays (Fig. 3B, 3C), the same trends in terms appear to affect binding, because RTX bound equally well at all of distance and the effector mechanism engaged were identified, positions along CD137. Western blotting was also performed, in such that CDC and ADCC were ineffective when targeted fur- particular to confirm the size difference between the R-4d and thest away from the target cell membrane (C-8d), and ADCP R-8d constructs, which was not possible by flow cytometry because was suboptimal when targeted too close to the cell membrane of the duplication of the same extracellular domains in CD137 (C-flush). (Fig. 1C). Stable expression compared with transient expression systems Assessment of Ab effector engagement with RTX The engagement of Ab effector mechanisms, such as CDC, can be CHO-S cells expressing each of the fusion proteins were used as influenced by the expression levels of the targeted Ag (21, 24, 25). targets to assess whether the ability of RTX to initiate CDC, ADCP, The transient transfection approach presented previously resulted or ADCC was altered depending on the distance that the epitope in a heterogeneous population containing nontransfected cells and was held from the cell membrane. The first mechanism investigated cells with a range of expression levels for each fusion protein, was CDC; cells were cultured with RTX in the presence of human including a proportion of cells expressing extremely high levels serum, and cell death was recorded by flow cytometry (Fig. 2A). In of target (Figs. 1B, 3A). Although this approach is useful for preliminary experiments, using CHO-S cells expressing the R-4d avoiding potential selection artifacts, it may obscure more subtle construct as targets, a serum titration was performed and found observations at lower expression levels that occur as a conse- that maximal killing could be achieved at 15% serum, with no quence of distance. To determine whether engagement of the further improvement, even when the serum concentration was FcgR-dependent effector mechanisms was affected by the Ag increased to 45% (Supplemental Fig. 1). As such, 15% serum was expression level, the murine cell line A20 was transfected with used in all subsequent CDC assays. To adjust for any differences R-4d, and stable clones of different expression levels were isolated 4 EPITOPE DEFINES ANTIBODY EFFECTOR MECHANISMS

FIGURE 1. Generation and ex- pression of fusion proteins display- ing epitopes at different distances from the plasma membrane. (A) Il- lustration representing the fusion proteins generated for use in this study. The resolved structure of TNFR1 was used as a model for the structure of CD137 and highlights its four clear cysteine-rich domains. The image was generated in PyMol.1.3 using the uploaded Protein Data Bank file 1TNR. A peptide recog- nized by the anti-CD20 mAb RTX

was attached to the N-terminal do- Downloaded from main of hCD137 that contains four cysteine-rich domains (R-4d). Ex- tracellular domains of CD137 were then replicated to generate an eight- domain construct (R-8d) or removed to produce a single-domain protein

(R-1d), as indicated. R-flush consists http://www.jimmunol.org/ of the RTX epitope attached to only the transmembrane (Tm) and intra- cellular domain of CD137. The do- mains bound by the anti-CD137 Abs (SAP1–3 and SAP3–6) that were used to confirm expression are pro- vided for reference. (B) CHO-S cells were transfected with each of the constructs detailed, and cell surface expression was assessed by flow by guest on September 26, 2021 cytometry using Abs toward the RTX epitope and CD137 backbone 24 h later. (C) Confirmation of the size difference between R-4d and R-8d was achieved by Western blot using polyclonal anti-CD137 Ab on whole- cell lysates of the transfected CHO-S cells. a-Tubulin was used as a load- ing control. NT, nontransfected CHO-S cell lysate.

(Fig. 4A). These clones (referred to as R-4d–Low, R-4d–Med, and CHO-S system (i.e., CDC and ADCC were diminished when R-4d–High) were assessed in the FcgR-mediated assays: ADCP targeting the cells displaying the R-8d construct, and ADCP was (Fig. 4B) and ADCC (Fig. 4C). In both cases, there was a clear poor when targeting the R-flush–transfected cells). These data decrease in the ability to engage these mechanisms when targeting provide further evidence that the effector mechanisms engaged by Ag was expressed at lower levels, although the difference be- an Ab are directly influenced by the epitope distance from the tween the high and medium expressing clones is not statistically target cell membrane. significant. Having established these principles in a controlled system, we Having established this clear relationship, to exclude this var- then sought to validate them using a more typical scenario in iable when comparing between the panel of proteins, the A20 cell which Abs are available to different epitopes on the same target line was transfected with the RTX-binding fusion constructs molecule. This approach has the added advantage that the ex- (R-flush, R-4d, and R-8d) to produce a panel of stable cell lines pression profile of the target molecule, and the target cell itself, is with equivalent expression levels (Fig. 5A). These cells were used exactly the same for both of the mAbs used. Using a recently as targets in the three effector assays. As seen in Fig. 5B and 5D, generated pair of anti-CD137 mAbs with similar affinities the results confirmed the observations reported for the transient that are known to bind the most proximal (SAP3–6) or distal The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Comparison of cytotoxic activity engaged by RTX when targeting CHO-S cells displaying the RTX epitope at different distances from the plasma membrane. CHO-S cells transfected with each of the fusion proteins (Fig. 1) were used as targets to investigate Ab effector mechanisms in vitro.(A and B) Transfected CHO-S cells were labeled with RTX or an isotype control (ISO) and incubated with 15% human serum for 30 min. CDC was measured by flow cytometry using propidium iodide inclusion as a measure of cell lysis. Representative data (A) and results from n = 3 independent experiments (B). (C and D) CFSE-labeled targets were opsonized with RTX or ISO before use as targets in an ADCP assay with murine BMDMs. The percentage of BMDMs that phagocytosed the target cells (defined as the F4/80+CFSE+ population) was recorded and plotted. (C) Representative flow cytometry data and confocal imaging confirming phagocytosis of CFSE-labeled targets. CFSE-labeled targets are green, wheat germ agglutinin (WGA)-stained surface membranes are red, and DAPI-stained nuclei are blue (original magnification 363). (D) Results from n = 3 independent experiments. (E) Calcein-AM–labeled targets were opsonized with RTX or ISO before acting as targets in ADCC assays with human PBMCs. For all experiments, the mean and SD of three independent experiments are presented. *p , 0.05, **p , 0.005, ***p , 0.001, ****p , 0.0001, two-way ANOVA.

(SAP1–3) domains of CD137 (Fig. 1A), we explored their rel- Resolving the conflict between Cp11-flush and WT CD52 in ative efficacy in CDC, ADCP, and ADCC. In the CDC and ADCP assay ADCC assays, the most membrane-proximal mAb, SAP3–6, The work presented thus far demonstrated that engagement of the was more effective than the distal mAb (Fig. 6A, 6C), partic- mAb effector mechanisms was influenced by the distance that an ularly in ADCC. In contrast, in our ADCP assay (Fig. 6B), there Ab binds from the target cell membrane, initially with RTX and was no clear difference between the two Abs; both were able to subsequently with CAMPATH-1H. hCD52, the Ag for CAMPATH- elicit significant phagocytosis compared with the isotype con- 1H, consists of a 12-aa extracellular peptide tethered to the cell trol. The similarity observed between SAP1–3 and SAP3–6 in the ADCP assay was comparable to that reported earlier be- membrane via a GPI anchor (37, 38). This is similar to the C-flush tween the four-domain and one-domain constructs (Figs. 2D, construct generated within this study, which contained an 11-aa 3C). This indicates that, although SAP3–6 binds the bottom extracellular peptide tethered via a protein transmembrane domain domain of CD137, it is at a similar distance from the cell (Fig. 1A). In vitro, CAMPATH-1H is highly effective at initiating membrane as the R-1d construct (rather than R-flush) and so is CDC to delete target cells (39), whereas in vivo, human and ideal for ADCP. These data further corroborate the distance murine studies indicate that the effectiveness and prolonged hypothesis that binding close to the target cell membrane is clearance observed are primarily mediated by FcgR (40, 41). preferable for ADCC and CDC. Given the key role for macrophages in Ab immunotherapy (17, 42), 6 EPITOPE DEFINES ANTIBODY EFFECTOR MECHANISMS

it is logical to assume that CAMPATH-1H is also effective in ADCP. However, we demonstrated that CAMPATH-1H was unable to effectively elicit phagocytosis when targeting the C-flush construct (Fig. 3C). Knowing that the engagement of CDC and ADCC by CAMPATH-1H when binding the C-flush construct fitted the expected mechanism of action for this mAb (Fig. 3B, 3D), the poor phagocytosis when binding an Ag similar to its natural target warranted further investigation. This was not the consequence of the CHO-S transient expression system used, because transfecting these cells with WT CD52, followed by CAMPATH-1H, resulted in highly effective ADCP (Supplemental Fig. 2A), indicating that the C-flush construct must differ in some other key property. Both proteins consist of small extracellular domains; however, CD52 is glycosylated just outside of the CAMPATH-1H epitope, whereas the C-flush construct is not (37, 43). Therefore, it was postulated that the presence of the glycan on CD52 may stabilize the interaction of CAMPATH-1H, leading to preferential ADCP

compared with the C-flush fusion protein. A mutant form of CD52 Downloaded from (referred to as CD52-NQ), in which the Asn was mutated to a Gln, was generated, expressed on CHO-S cells (Supplemental Fig. 2B), and assessed in the ADCP assay (Fig. 7A). The nonglycosylated mutant was still able to elicit ADCP following CAMPATH-1H treatment, demonstrating that glycosylation of CD52 was not

important for the effective induction of ADCP by CAMPATH-1H. http://www.jimmunol.org/ We next considered whether the method of membrane attach- ment was influencing the efficiency of ADCP. To test this, a GPI- anchored version of the CAMPATH-1H epitope was generated. The C-GPI construct contained the same leader and C-terminal se- quences as hCD52, as well as the Pro-Ser of the CD52 GPI- attachment site (Fig. 7B). This was based on previous work that proposed that 2 aa prior to the GPI attachment site and the carboxy sequence were important for mediating the posttranslational at- tachment of the protein to the GPI anchor (44, 45). The C-GPI by guest on September 26, 2021 construct was expressed in CHO-S cells (Supplemental Fig. 2C), bound by CAMPATH-1H, and compared in ADCP assays along- side C-flush and WT CD52 (Fig. 7C). These experiments clearly demonstrated a rescue in ADCP function when the CAMPATH- 1H epitope was tethered through the GPI format (C-GPI), leading to a construct that was at least as efficient as WT CD52 and far more effective than C-flush. These data indicate that the GPI an- chor likely overcomes the restriction on ADCP for targets that are extremely close to the cell surface. In vivo function of RTX targeting different distances Investigating the effector mechanisms in individual effector assays in vitro clearly identified differences between the most proximal and distal epitope constructs, R-flush and R-8d, respectively. To explore the relevance of these findings for immunotherapy in vivo where all mechanisms might be in play, we used A20 cells stably transfected with R-flush or R-8d (Fig. 5A). These were injected i.v. into WT female BALB/c mice, and the resulting tumors were detected and distinguished from endogenous B cells using flow cytometry with an A20-specific anti-idiotype mAb (Fig. 8A). At terminal tumor burden, the A20 cells were located predominantly FIGURE 3. Comparison of cytotoxic activity initiated by CAMPATH-1H in the spleen and liver. when targeting CHO-S cells displaying the CAMPATH epitope at different To compare the capacity of these two Ags to facilitate tumor A distances from the plasma membrane. ( ) CHO-S cells were transfected with deletion, a single dose of PBS or RTX-mIgG2a was administered each of the same CD137 backbone constructs as illustrated in Fig. 1A but with 3 d following tumor inoculation (Fig. 8B), and the presence of A20 the RTX epitope replaced with an epitope recognized by CAMPATH-1H. Cell surfaceexpressionwasconfirmed24hlaterbyflowcytometryusingFITC- cells expressing each construct was determined in the spleen and conjugated CAMPATH-1H. CHO-S cells expressing each of the fusion pro- liver. Although all groups had high tumor burden in the liver, teins were then used as targets to assess sensitivity to CDC (B), as well as for resulting in them becoming terminal, when comparing the per- ADCP (C)andADCC(D) assays. The mean and SD of three independent centage of tumor cells present in the spleens, mice that received experiments are presented. *p , 0.05, **p , 0.005, two-way ANOVA. tumor cells expressing R-flush followed by RTX-mIgG2a were The Journal of Immunology 7

FIGURE 4. Importance of expression level for engagement of FcgR-mediated mechanisms. A20 cells were transfected with the R-4d construct, and stable clones expressing different levels at the cell surface were isolated. (A) The clones were labeled with RTX-FITC or ISO-FITC, and expression levels

were measured by flow cytometry. The three cell lines (R-4d–Low, R-4d–Med, and R-4d–High) were used as targets for ADCP (B) and ADCC (C). The Downloaded from mean and SD of three independent experiments are presented. *p , 0.05, one-way ANOVA.

effectively cleared compared with those receiving PBS (Fig. 8C). ability to cluster and redistribute into lipid raft areas of the plasma

In contrast, the tumors expressing the R-8d construct were not membrane (19). CD137 is an integral membrane protein expressed http://www.jimmunol.org/ cleared in either location, indicating inefficient targeting of the on T cells, and it is a member of the TNFR superfamily (50), relevant effector mechanisms. forming trimers with its ligand for activation (51). Cross-linking of CD137 was subsequently shown to cause its redistribution to Discussion lipid raft regions at the contact site with ligand-expressing cells Although mAbs are being used increasingly in the clinic to treat an (52); therefore, like CD20 and CD52, CD137 may reflect a par- ever-expanding array of diseases, there is still much that we do not ticularly potent target molecule that can become redistributed and know about this class of therapeutics. The diversity of molecules concentrated within the plasma membrane to augment mAb ef- targeted by mAbs has increased exponentially over the last decade fector mechanisms. (23), but, surprisingly, we are still ignorant of some of the most Using a combination of approaches involving transient and by guest on September 26, 2021 basic rules for their behavior. Perhaps the simplest class of ther- stable expression systems, both in vitro and in vivo, we were able to apeutic mAbs to understand are the direct-targeting mAbs. These reveal differences in the ability to engage the three Fc-mediated reagents bind directly to the tumor cell target and elicit anticancer effector mechanisms depending upon the position of the mAb effects by inducing changes to signaling (direct cell death) or by epitope from the cell surface. For two different specificities (anti- engaging Fc-mediated effector functions, such as CDC, ADCP, CD20 and anti-CD52), CDC was ineffective when targeting the and ADCC. Although it is accepted that different mAb binding to epitope furthest away (R-8d, C-8d) from the membrane and was the same target can elicit striking differences in therapeutic effi- less sensitive at lower mAb concentrations when binding cells cacy, evidence for why this occurs is often scarce. expressing the four-domain constructs (R-4d and C-4d, Figs. 2B, mAb properties, such as FcgR affinity (46), isotype (47), and 3B). A similar trend was observed for ADCC (Figs. 2E, 3D). binding level (24, 25), have well-established effects on mAb ef- ADCP displayed different requirements; a minimum distance of a fector systems and can certainly help to explain why some mAbs single extracellular domain was required before phagocytosis of are more active than others. However, for other mAbs, more subtle the target cell was achieved, and membrane-distal epitopes (R-8d differences can also have a major impact. For example, in the case and C-8d) were still relatively effective as targets (Figs. 2D, 3C). of the anti-CD20 mAb, type I and II engage different effector Importantly, the ineffective phagocytosis of the membrane-flush functions (4, 22) while binding to an overlapping, cross-blocking epitopes (R-flush and C-flush) was not due to an inability of the epitope (48). Given that several successful and approved anti- Ab to bind these targets, as demonstrated by flow cytometry tumor mAbs bind to surface-proximal Ags, such as CD20 and (Figs. 1B, 3A) and their efficacy in the other effector assays CD52, in this study we examined the effect of position along a (ADCC and CDC). target molecule, from membrane proximal to distal, on the ef- The effect of membrane distance on CDC is intuitively logical. fector mechanisms engaged by direct-targeting Abs. The classical pathway of complement is known to be self-regulated The target molecule chosen, CD137, has four extracellular by the short half-life of the active components at all stages of the cysteine-rich domains (35). Its atomic structure has not been re- cascade (53). Being activated far away from the target cell solved, but, modeled upon the TNFR1 crystal structure (49), the membrane means that the activated complement components C2a four domains would appear to gravitate away from the plasma and C4b have a reduced chance of stabilizing on the cell surface as membrane in a relatively linear fashion (Fig. 1A); therefore, it is a result of the extended diffusion through the extracellular matrix. likely that the positions along CD137 correspond to increasing Therefore, the greater the distance from the cell surface, the lower distances from the target cell surface. However, it is important to the efficiency in the establishment of the C3 convertase complex, a note that some of our findings may have been influenced by our key determinant in the cascade that leads to cell lysis via the choice of receptor. For example, our previous data highlighted the formation of the membrane attack complex. This same reasoning particular potency of CD20 as a target molecule as a result of its was used to explain ofatumumab’s superior initiation of CDC 8 EPITOPE DEFINES ANTIBODY EFFECTOR MECHANISMS Downloaded from

FIGURE 5. Comparison of cytotoxic activity engaged by RTX when targeting A20 cells stably displaying the RTX epitope at different distances from the plasma membrane.

(A) A20 cells were stably transfected to express the fusion http://www.jimmunol.org/ proteins R-flush, R-4d, and R-8d and then clones with equivalent levels of expression were selected. The flow cytometry data show that the three cell lines express similar levels of the fusion protein when detected with FITC- conjugated RTX. The filled line graphs reflect staining with the ISO control. These cells were then used to assess CDC (B), ADCP (C), and ADCC (D) in vitro. The mean and SD of three independent experiments are presented. *p , 0.05, ***p , 0.001, two-way ANOVA. by guest on September 26, 2021

compared with RTX: it binds a more membrane-proximal loop of movement of the Ag:Ab complexes (7). All of the constructs hCD20 (5, 21). generated in this study contained identical transmembrane and More recent work has demonstrated that activation of the intracellular domains; therefore, it was assumed that they would classical complement pathway is better facilitated through a have equivalent ability to redistribute across the cell surface and hexameric association of Ab Fc regions. Although the formation of form hexamers. However, the extracellular domains of each con- hexamers can be engineered via Fc modification (54), they can struct vary in size. Therefore, differing levels of steric hindrance form naturally upon the surface of the target cell via lateral and interactions with other endogenous cell surface proteins could The Journal of Immunology 9

demonstrate that hIgG1 and hIgG3 were best at engaging CDC and ADCC. One interesting point is that, although hIgG3 is better at binding C1q, this binding does not translate directly to im- proved CDC (26, 47). Other work reported that a chimeric Ab, containing the CH1 and hinge region of hIgG1 attached to a hIgG3 Fc domain, was better able to initiate CDC compared with WT hIgG3 (56). One main difference between the hIgG1 and hIgG3 classes is the length of the hinge region, with hIgG3 having a hinge region approximately four times that of hIgG1 (55). In of our work presented in this article, the improved lytic ability of the hIgG1/3 hybrid may be due to the far shorter hinge region, in turn bringing the complement activation closer to the cell surface for the initiation of CDC. It would be interesting to determine whether combining this isotype chimera with epitopes directed to the membrane-proximal region of Ags would result in even more potent reagents. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Comparison of cytotoxic activity initiated by mAb binding different domains of CD137. CHO-S cells expressing R-4d were labeled with different concentrations of SAP1–3 (binds membrane-distal domain of CD137) or SAP3–6 (binds membrane-proximal domain of CD137) and used as targets in CDC (A), ADCP (B), and ADCC (C) assays. In (A), the mean and SD of three samples in a single experiment are plotted. In (B), the mean and SD of three independent experiments are plotted. Data in (C) are representative of three independent experiments. *p , 0.05, ****p , 0.0001, two-way ANOVA. n.s., not significant. influence membrane redistribution and restrict the hexameric complexes from forming. FIGURE 7. Importance of the GPI anchor in WT CD52 for ADCP All of the work presented in this article used the hIgG1 isotype ability. (A) A nonglycosylated mutant of hCD52 (CD52-NQ) was gener- (or mIgG2a in vivo as the closest mouse parallel) (10), because this ated and expressed in CHO-S cells. Its ability to facilitate ADCP of target reflects the isotype typically adopted for direct-targeting mAbs in cells when opsonized with CAMPATH-1H was compared with CHO-S B the clinic (23). Whether other isotypes will follow the same rules cells expressing C-flush or WT CD52. ( ) Gene schematics for the gen- eration of C-GPI consisting of the peptide CAMPATH-1H epitope attached remains to be determined, because it is well established that they to the same GPI anchor as CD52. (C) The C-GPI construct was generated differ in their ability to engage the various effector mechanisms. and expressed in CHO-S cells. Its ability to facilitate ADCP of target cells Recently, hIgG3 has been investigated as an alternative to hIgG1 when opsonized with CAMPATH-1H was compared with CHO-S cells because of its superior engagement of FcgR (55) and binding of expressing C-flush or WT CD52. The mean and SD of three independent C1q (47). Bruggemann€ et al. (47) used a panel of chimeric Abs experiments are shown. *p , 0.05, **p , 0.005, ***p , 0.001, two-way containing the same specificity but class-switched H chains to ANOVA. n.s., not significant. 10 EPITOPE DEFINES ANTIBODY EFFECTOR MECHANISMS

FIGURE 8. In vivo clearance of A20 cells expressing different RTX constructs following RTX therapy. (A) WT female BALB/c mice were inoculated with 1 3 106 A20 cells i.v. and screened for tumor development within the liver, spleen, and lymph nodes in terminal ani- mals by flow cytometry, as idiotype+B220+ cells. (B) WT BALB/c mice were inoculated with A20 cells expressing R-flush or R-8d on day 0, with 100 mg Ritm2a or PBS given on day 3. (C) Once mice had become terminal, the liver Downloaded from and spleen were harvested, and the percentage of tumor cells was identified by flow cytometry (idiotype+B220+). The percentage of tumor present for each individual mouse was nor- malized against the mean tumor burden within the control group in each experiment. The data

show the results from six mice from two inde- http://www.jimmunol.org/ pendent experiments. ****p , 0.0001, one- way ANOVA. n.s., not significant. by guest on September 26, 2021

Perhaps more surprising was the contrasting dependence on furthest away would fit this model, because the eight-domain fu- distance seen with the FcgR-mediated mechanisms ADCP and sion protein exceeds the maximum synapse size by ∼2 nm (58). ADCC. ADCP was significantly reduced when binding the For phagocytosis, whether FcgR-mediated or via other means, a membrane-flush constructs compared with those that were held at high level of intracellular reorganization is required to form the least one extracellular domain away (Figs. 2D, 3C). ADCC was phagosome and provide the means to engulf the targeted cell (12). effective for these constructs, but activity was lost when targeting This process is likely to last longer/require more continuous sig- an epitope positioned distal to the target cell membrane (Figs. 2E, naling compared with that for cytotoxic release; therefore, the 3D). The approach undertaken within this study used the same multimeric complex among the Ag, mAb, and FcgR would need epitope and mAb pairing; as such, the binding affinities remained to be more stable and to mediate the sustained signaling required. consistent among all of the constructs investigated. In addition, the Therefore, it is likely that, when targeting the membrane-flush same Ab was used, with no changes to the isotype or Fc region; constructs, although the binding kinetics remain similar to those therefore, the kinetics behind Fc:FcgR interactions would also be of the more distant epitopes, the endogenous proteins present equivalent among systems. These observations reveal fundamental within the cell membrane (not including the fusion protein itself) differences in the requirements of activation for these mecha- may provide steric resistance and pressures on the binding, nisms. Although ADCC and ADCP require the engagement of resulting in an overall less stable multimeric complex and leading activatory FcgR on the cell surface, the downstream effects and to less efficient ADCP. Phagocytosis is proposed to proceed outcomes are clearly very different, although the reasons for this through a zippering mechanism whereby integrins promote the remain unanswered. efficient binding of FcgR:Fc complexes and serve to integrate Release of cytotoxic granules by NK cells following FcgR signals from multiple, potentially disparate receptors (12). Be- activation has been demonstrated to use relatively small immune cause zippering requires the formation of sequential FcgR:Fc synapses, analogous to those induced during T cell–mediated complexes for continuing phagocytic cup generation and closure, cytotoxicity (11). There is evidence that a T cell synapse needs to it could be possible that, within this process, engagement of be ,14 nm in size to exclude any inhibitory signaling elements surface-proximal “flush” constructs do not facilitate appropriate (such as the phosphatase CD45) from preventing activation, integrin distribution or efficient zippering for the reasons listed according to the kinetic-segregation model (57, 58). Assuming above. that the requirement for NK cell killing is similar, the fact that Ag density has long been correlated with the effectiveness of there is poor activation of ADCC when targeting the epitope held CDC engagement in vitro and in clinical studies (21, 24). High The Journal of Immunology 11 expression levels allow for a better chance of juxtaposed Ab Fc tumor targets, this may benefit reagents seeking to deplete immune- regions being present in the correct orientation for favorable en- suppressive cells themselves (e.g., regulatory T cells [Tregs]) (62). gagement of C1 (6, 26). However, the relationship between Ag Indeed, the two anti-CD137 mAbs used in this study (SAP1–3 and expression level and engagement of the FcgR-mediated mecha- SAP3–6) demonstrated an application of our findings (Fig. 6). nisms are less defined. Derer et al. (25) indicated that NK and CD137 is expressed on resting Tregs and is upregulated following polymorphonuclear leukocyte–mediated killing was less depen- activation (63, 64); therefore, it may serve as a useful target for Treg dent on Ag expression compared with CDC; however, there deletion. SAP1–3 and SAP3–6 have the same isotype and bind at seemed to be a minimum level required before cell death was equivalent levels and with similar affinities toward CD137 (data not observed. It is worth noting that, in that study, different clones of shown). Although they were broadly equivalent with regard to their Ab were used that were potentially directed to different epitopes induction of ADCP, differences in the engagement of the effector and with different affinities, rather than a single epitope and Ab mechanisms ADCC and CDC were apparent, particularly in relation targeted in this study, making it difficult to directly compare the to the minimum concentration required to elicit cell death. Because efficacy of each epitope location. Nonetheless, by using a range of both mAbs are equivalent in all other measurable ways, the differ- cell lines stably expressing different levels of the R-4d fusion ence likely lies in the bound epitope and its relation to the target cell protein, it was clear that the percentage of ADCP and ADCC that membrane. occurred in response to RTX treatment positively correlated with As detailed above, the tumor can manipulate its local micro- the expression level (Fig. 4). This finding agrees with the con- environment and has the capacity to impair the various effector clusions made by Derer et al. (25) with regard to CDC and mechanisms to circumvent mAb-mediated killing. For example, a

ADCC and supports other work from our group in which the highly suppressive macrophage environment might exist with a low Downloaded from Ag expression level correlates positively with the amount of activatory/inhibitory FcgR ratio but where complement compo- ADCP observed (L. Dou, S.L. Buchan, S.N. Dunn, S.G. Booth, nents are plentiful. Similarly, if the environment is driving high C. Lai, M. Semmrich, I. Teige, H.J. Kim, J.E. Willoughby, levels of FcgRIIB, NK cell–mediated ADCC may remain func- L.N. Dahal, K.L. Cleary, P. Kannisto, M. Jernetz, E.L. Williams, E. tional, because they, unlike macrophages, , and den- Healy, J.S. Verbeek, B. Frende´us, M.S. Cragg, M.J. Glennie, J.C. dritic cells, do not express this inhibitory receptor. Using the

Gray, A. Al-Shamkhani, and S.A. Beers, manuscript in preparation). knowledge gained in this study and switching the fine specificity http://www.jimmunol.org/ Our further study of membrane-proximal targets revealed an of an Ab (i.e., the distance it binds from the cell surface), the most initial contradiction when targeting native CD52, as opposed to the effective effector mechanisms can be selected in each situation, C-flush construct. We identified that attaching the CAMPATH-1H potentially leading to more potent Ab therapeutics. epitope mimic to a GPI anchor (C-GPI construct) restored sensi- tivity of the target cell toward phagocytosis (Fig. 7C). GPI anchors Acknowledgments are a commonly found posttranslational modification; however, We thank all of the members of the Antibody and Vaccine Group (University there are still unknowns in terms of their structure, size, and of Southampton) for discussions relating to the experiments reported in this function. Resolved structures of GPI anchors are lacking; how- article and the Biomedical Imaging Unit (University of Southampton) for by guest on September 26, 2021 ever, computer modeling indicates that they may be far larger than assistance with confocal microscopy. The Rp3/Cp11 epitope construct was a anticipated (∼1.5 nm in size) (59). Assuming that CD52 uses a kind gift of Prof. M. Pule and Dr. B. Philip. The anti-idiotype for A20 was a similar anchor as modeled for CD59 (27, 59), then it is more kind gift of Prof. R. Levy. CAMPATH-1H was a kind gift of Drs. Steve equivalent in distance from the membrane to the C-1d construct, Cobbold and Geoff Hale. The domain-colored image of the TNFR1 struc- which is able to engage efficient ADCP, compared with the C-flush ture in Fig. 1 was produced by Emma Sutton, using the 1TNR data file sub- molecule, in support of our original hypothesis. mitted within the Protein Data Bank. In vivo experiments in syngeneic immune-competent mice revealed a clear depletion of A20 tumor cells expressing the R- Disclosures flush, but not R-8d, construct from the spleen of BALB/c mice M.S.C. is a retained consultant for BioInvent and has performed educational following treatment with RTX-m2a (Fig. 8C). This indicates that and advisory roles for Baxalta. He has received research funding from the smaller, more membrane-proximal epitope was more effective Roche, Gilead, and GlaxoSmithKline. M.J.G. is an advisor to BioInvent In- in depleting tumor cells and that ADCC and/or CDC were prin- ternational. The other authors have no financial conflicts of interest. cipally responsible. Although at odds with our recent findings identifying ADCP as the main effector mechanism (17, 60), it is reminiscent of our data using xenograft models (20) and likely References reflects the use of a highly selected in vitro cell line that has un- 1. Molina, A. 2008. A decade of rituximab: improving survival outcomes in non- Hodgkin’s . Annu. Rev. Med. 59: 237–250. dergone loss of complement defense and/or NK control. The lack 2. Weiner, L. M., J. C. Murray, and C. W. Shuptrine. 2012. Antibody-based im- of complement defense through CD55 and CD59 was subse- munotherapy of cancer. Cell 148: 1081–1084. quently confirmed (Supplemental Fig. 3). In contrast, no depletion 3. Ivanov, A., S. A. Beers, C. A. Walshe, J. Honeychurch, W. Alduaij, K. L. Cox, K. N. Potter, S. Murray, C. H. Chan, T. Klymenko, et al. 2009. Monoclonal was observed in the liver for either of the two tumor cell lines antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion fol- generated. lowed by lysosome-mediated cell death in human lymphoma and leukemia cells. J. Clin. Invest. 119: 2143–2159. The differences in the clearance of R-flush–expressing cells 4. Alduaij, W., A. Ivanov, J. Honeychurch, E. J. Cheadle, S. Potluri, S. H. Lim, between the spleen and the liver reflect another factor that is not K. Shimada, C. H. Chan, A. Tutt, S. A. Beers, et al. 2011. Novel type II anti- clearly understood: the role of the tumor microenvironment in CD20 (GA101) evokes homotypic adhesion and actin- dependent, lysosome-mediated cell death in B-cell malignancies. Blood 117: regulating mAb efficacy. It is now evident that the tumor micro- 4519–4529. environment can promote infiltrating immune cells to adopt a 5. Oldham, R. J., K. L. Cleary, and M. S. Cragg. 2014. CD20 and its antibodies: past, present, and future. For. Immunopathol. Dis. Therap. 5: 7–23. more protumor/immune-suppressive phenotype (61); however, its 6. Borsos, T., and H. J. Rapp. 1965. Complement fixation on cell surfaces by 19S direct effect on mAb therapy once again is unclear. It is possible and 7S antibodies. Science 150: 505–506. that altering the epitope targeted within an Ag may allow for more 7. Diebolder, C. A., F. J. Beurskens, R. N. de Jong, R. I. Koning, K. Strumane, M. A. Lindorfer, M. Voorhorst, D. Ugurlar, S. Rosati, A. J. Heck, et al. 2014. efficacious Ab therapies to be developed that can overcome the Complement is activated by IgG hexamers assembled at the cell surface. Science specific suppressive elements present. In addition to direct anti- 343: 1260–1263. 12 EPITOPE DEFINES ANTIBODY EFFECTOR MECHANISMS

8. van Lookeren Campagne, M., C. Wiesmann, and E. J. Brown. 2007. Macrophage 35. Won, E. Y., K. Cha, J. S. Byun, D. U. Kim, S. Shin, B. Ahn, Y. H. Kim, complement receptors and pathogen clearance. Cell. Microbiol. 9: 2095–2102. A. J. Rice, T. Walz, B. S. Kwon, and H. S. Cho. 2010. The structure of the trimer 9. Muller-Eberhard,€ H. J. 1986. The membrane attack complex of complement. of human 4-1BB ligand is unique among members of the tumor necrosis factor Annu. Rev. Immunol. 4: 503–528. superfamily. J. Biol. Chem. 285: 9202–9210. 10. Bruhns, P. 2012. Properties of mouse and human IgG receptors and their con- 36. Beum, P. V., M. A. Lindorfer, and R. P. Taylor. 2008. Within peripheral blood tribution to disease models. Blood 119: 5640–5649. mononuclear cells, antibody-dependent cellular cytotoxicity of rituximab- 11. Cullen, S. P., and S. J. Martin. 2008. Mechanisms of granule-dependent killing. opsonized Daudi cells is promoted by NK cells and inhibited by monocytes Cell Death Differ. 15: 251–262. due to shaving. J. Immunol. 181: 2916–2924. 12. Freeman, S. A., J. Goyette, W. Furuya, E. C. Woods, C. R. Bertozzi, 37. Xia, M. Q., M. Tone, L. Packman, G. Hale, and H. Waldmann. 1991. Charac- W. Bergmeier, B. Hinz, P. A. van der Merwe, R. Das, and S. Grinstein. 2016. terization of the CAMPATH-1 (CDw52) antigen: biochemical analysis and Integrins form an expanding diffusional barrier that coordinates phagocytosis. cDNA cloning reveal an unusually small peptide backbone. Eur. J. Immunol. 21: Cell 164: 128–140. 1677–1684. 13. Bero´n, W., C. Alvarez-Dominguez, L. Mayorga, and P. D. Stahl. 1995. Mem- 38.Xia,M.Q.,G.Hale,M.R.Lifely,M.A.Ferguson,D.Campbell,L.Packman,and brane trafficking along the phagocytic pathway. Trends Cell Biol. 5: 100–104. H. Waldmann. 1993. Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol- 14. Clynes, R. A., T. L. Towers, L. G. Presta, and J. V. Ravetch. 2000. Inhibitory Fc anchored which is an exceptionally good target for complement lysis. receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6: 443–446. Biochem. J. 293: 633–640. 15. Lo´pez-Albaitero, A., S. C. Lee, S. Morgan, J. R. Grandis, W. E. Gooding, 39. Zent, C. S., C. R. Secreto, B. R. LaPlant, N. D. Bone, T. G. Call, T. D. Shanafelt, S. Ferrone, and R. L. Ferris. 2009. Role of polymorphic Fc gamma receptor IIIa D. F. Jelinek, R. C. Tschumper, and N. E. Kay. 2008. Direct and complement and EGFR expression level in mediated, NK cell dependent in vitro dependent cytotoxicity in CLL cells from patients with high-risk early- cytotoxicity of head and neck squamous cell carcinoma cells. Cancer Immunol. intermediate stage chronic lymphocytic leukemia (CLL) treated with alemtu- Immunother. 58: 1853–1864. zumab and rituximab. Leuk. Res. 32: 1849–1856. € 16. Gul, N., L. Babes, K. Siegmund, R. Korthouwer, M. Bo¨gels, R. Braster, 40. Dyer, M. J., G. Hale, F. G. Hayhoe, and H. Waldmann. 1989. Effects of G. Vidarsson, T. L. ten Hagen, P. Kubes, and M. van Egmond. 2014. Macro- CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies: influ- phages eliminate circulating tumor cells after monoclonal antibody therapy. J. ence of antibody isotype. Blood 73: 1431–1439. Clin. Invest. 124: 812–823. 41. Hu, Y., M. J. Turner, J. Shields, M. S. Gale, E. Hutto, B. L. Roberts, Downloaded from 17. Tipton, T. R., A. Roghanian, R. J. Oldham, M. J. Carter, K. L. Cox, W. M. Siders, and J. M. Kaplan. 2009. Investigation of the mechanism of action C. I. Mockridge, R. R. French, L. N. Dahal, P. J. Duriez, P. G. Hargreaves, et al. of alemtuzumab in a human CD52 transgenic mouse model. Immunology 128: 2015. Antigenic modulation limits the effector cell mechanisms employed by 260–270. type I anti-CD20 monoclonal antibodies. Blood 125: 1901–1909. 42. Uchida, J., Y. Hamaguchi, J. A. Oliver, J. V. Ravetch, J. C. Poe, K. M. Haas, and 18. Shim, H. 2011. One target, different effects: a comparison of distinct therapeutic T. F. Tedder. 2004. The innate mononuclear phagocyte network depletes antibodies against the same targets. Exp. Mol. Med. 43: 539–549. B through Fc receptor-dependent mechanisms during anti-CD20 19. Cragg, M. S., S. M. Morgan, H. T. Chan, B. P. Morgan, A. V. Filatov, P. W. Johnson, antibody immunotherapy. J. Exp. Med. 199: 1659–1669. R. R. French, and M. J. Glennie. 2003. Complement-mediated lysis by anti-CD20 43. James, L. C., G. Hale, H. Waldmann, and A. C. Bloomer. 1999. 1.9 A˚ structure mAb correlates with segregation into lipid rafts. Blood 101: 1045–1052. of the therapeutic antibody CAMPATH-1H fab in complex with a synthetic http://www.jimmunol.org/ 20. Cragg, M. S., and M. J. Glennie. 2004. Antibody specificity controls in vivo peptide antigen. [Published erratum appears in 1999 J. Mol. Biol. 292: 1161.] J. effector mechanisms of anti-CD20 reagents. Blood 103: 2738–2743. Mol. Biol. 289: 293–301. 21. Teeling, J. L., W. J. Mackus, L. J. Wiegman, J. H. van den Brakel, S. A. Beers, 44. Chen, R., J. J. Knez, W. C. Merrick, and M. E. Medof. 2001. Comparative ef- R. R. French, T. van Meerten, S. Ebeling, T. Vink, J. W. Slootstra, et al. 2006. ficiencies of C-terminal signals of native glycophosphatidylinositol (GPI)- The biological activity of human CD20 monoclonal antibodies is linked to anchored proproteins in conferring GPI-anchoring. J. Cell. Biochem. 84: 68–83. unique epitopes on CD20. J. Immunol. 177: 362–371. 45. Bournazos, S., S. P. Hart, L. H. Chamberlain, M. J. Glennie, and I. Dransfield. 22. Beers, S. A., C. H. Chan, S. James, R. R. French, K. E. Attfield, C. M. Brennan, 2009. Association of FcgammaRIIa (CD32a) with lipid rafts regulates ligand A. Ahuja, M. J. Shlomchik, M. S. Cragg, and M. J. Glennie. 2008. Type II binding activity. J. Immunol. 182: 8026–8036. () anti-CD20 monoclonal antibody out performs type I (rituximab- 46. Bruhns, P., B. Iannascoli, P. England, D. A. Mancardi, N. Fernandez, S. Jorieux, like) reagents in B-cell depletion regardless of complement activation. Blood and M. Dae¨ron. 2009. Specificity and affinity of human Fcgamma receptors and 112: 4170–4177. their polymorphic variants for human IgG subclasses. Blood 113: 3716–3725. 23. Reichert, J. M. 2017. Therapeutic monoclonal antibodies approved or in review 47. Bruggemann,€ M., G. T. Williams, C. I. Bindon, M. R. Clark, M. R. Walker, by guest on September 26, 2021 in the European Union or the United States. The Antibody Society. Available at: R. Jefferis, H. Waldmann, and M. S. Neuberger. 1987. Comparison of the ef- http://www.antibodysociety.org/news/approved-antibodies/. Accessed: January fector functions of human immunoglobulins using a matched set of chimeric 23, 2017. antibodies. J. Exp. Med. 166: 1351–1361. 24. Golay, J., M. Lazzari, V. Facchinetti, S. Bernasconi, G. Borleri, T. Barbui, 48. Niederfellner, G., A. Lammens, O. Mundigl, G. J. Georges, W. Schaefer, A. Rambaldi, and M. Introna. 2001. CD20 levels determine the in vitro sus- M. Schwaiger, A. Franke, K. Wiechmann, S. Jenewein, J. W. Slootstra, et al. ceptibility to rituximab and complement of B-cell chronic lymphocytic leuke- 2011. Epitope characterization and crystal structure of GA101 provide insights mia: further regulation by CD55 and CD59. Blood 98: 3383–3389. into the molecular basis for type I/II distinction of CD20 antibodies. Blood 118: 25. Derer, S., P. Bauer, S. Lohse, A. H. Scheel, S. Berger, C. Kellner, M. Peipp, and 358–367. T. Valerius. 2012. Impact of epidermal growth factor receptor (EGFR) cell 49. Banner, D. W., A. D’Arcy, W. Janes, R. Gentz, H. J. Schoenfeld, C. Broger, surface expression levels on effector mechanisms of EGFR antibodies. J. H. Loetscher, and W. Lesslauer. 1993. Crystal structure of the soluble human 55 Immunol. 189: 5230–5239. kd TNF receptor-human TNF beta complex: implications for TNF receptor ac- 26. Bindon, C. I., G. Hale, and H. Waldmann. 1988. Importance of antigen speci- tivation. Cell 73: 431–445. ficity for complement-mediated lysis by monoclonal antibodies. Eur. J. Immunol. 50. Shuford, W. W., K. Klussman, D. D. Tritchler, D. T. Loo, J. Chalupny, 18: 1507–1514. A. W. Siadak, T. J. Brown, J. Emswiler, H. Raecho, C. P. Larsen, et al. 1997. 4- 27. Treumann, A., M. R. Lifely, P. Schneider, and M. A. Ferguson. 1995. Primary 1BB costimulatory signals preferentially induce CD8+ T cell proliferation and structure of CD52. J. Biol. Chem. 270: 6088–6099. lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186: 28. Cho, H. S., K. Mason, K. X. Ramyar, A. M. Stanley, S. B. Gabelli, 47–55. D. W. Denney, Jr., and D. J. Leahy. 2003. Structure of the extracellular region of 51. Wyzgol, A., N. Muller,€ A. Fick, S. Munkel, G. U. Grigoleit, K. Pfizenmaier, and HER2 alone and in complex with the Herceptin Fab. Nature 421: 756–760. H. Wajant. 2009. Trimer stabilization, oligomerization, and antibody-mediated 29. Haso, W., D. W. Lee, N. N. Shah, M. Stetler-Stevenson, C. M. Yuan, I. H. Pastan, cell surface immobilization improve the activity of soluble trimers of CD27L, D. S. Dimitrov, R. A. Morgan, D. J. FitzGerald, D. M. Barrett, et al. 2013. Anti- CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand. J. Immunol. CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic 183: 1851–1861. leukemia. Blood 121: 1165–1174. 52. Nam, K. O., H. Kang, S. M. Shin, K. H. Cho, B. Kwon, B. S. Kwon, S. J. Kim, 30. Varghese, B., A. Widman, J. Do, B. Taidi, D. K. Czerwinski, J. Timmerman, and H. W. Lee. 2005. Cross-linking of 4-1BB activates TCR-signaling pathways S. Levy, and R. Levy. 2009. Generation of CD8+ T cell-mediated immunity in CD8+ T lymphocytes. J. Immunol. 174: 1898–1905. against idiotype-negative lymphoma escapees. Blood 114: 4477–4485. 53. Wallis, R., D. A. Mitchell, R. Schmid, W. J. Schwaeble, and A. H. Keeble. 2010. 31. Kearney, J. F., A. Radbruch, B. Liesegang, and K. Rajewsky. 1979. A new mouse Paths reunited: initiation of the classical and lectin pathways of complement myeloma cell line that has lost immunoglobulin expression but permits the activation. Immunobiology 215: 1–11. construction of antibody-secreting hybrid cell lines. J. Immunol. 123: 1548– 54. de Jong, R. N., F. J. Beurskens, S. Verploegen, K. Strumane, M. D. van Kampen, 1550. M. Voorhorst, W. Horstman, P. J. Engelberts, S. C. Oostindie, G. Wang, et al. 32. Horton, R. M., H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease. 1989. En- 2016. A novel platform for the potentiation of therapeutic antibodies based on gineering hybrid without the use of restriction enzymes: gene splicing by antigen-dependent formation of IgG Hexamers at the cell surface. PLoS Biol. 14: overlap extension. Gene 77: 61–68. e1002344. 33. Sanchez, A. B., T. Nguyen, R. Dema-Ala, A. C. Kummel, T. J. Kipps, and 55. Vidarsson, G., G. Dekkers, and T. Rispens. 2014. IgG subclasses and allotypes: B. T. Messmer. 2010. A general process for the development of peptide-based from structure to effector functions. Front. Immunol. 5: 520. immunoassays for monoclonal antibodies. Cancer Chemother. Pharmacol. 66: 56. Natsume, A., M. In, H. Takamura, T. Nakagawa, Y. Shimizu, K. Kitajima, 919–925. M. Wakitani, S. Ohta, M. Satoh, K. Shitara, and R. Niwa. 2008. Engineered 34. Perosa, F., E. Favoino, M. A. Caragnano, and F. Dammacco. 2006. Generation of antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities. biologically active linear and cyclic has revealed a unique fine speci- Cancer Res. 68: 3863–3872. ficity of rituximab and its possible cross-reactivity with acid sphingomyelinase- 57. Davis, S. J., and P. A. van der Merwe. 2006. The kinetic-segregation model: TCR like phosphodiesterase 3b precursor. Blood 107: 1070–1077. triggering and beyond. Nat. Immunol. 7: 803–809. The Journal of Immunology 13

58. Choudhuri, K., D. Wiseman, M. H. Brown, K. Gould, and P. A. van der Merwe. 62. Simpson, T. R., F. Li, W. Montalvo-Ortiz, M. A. Sepulveda, K. Bergerhoff, 2005. T-cell receptor triggering is critically dependent on the dimensions of its F.Arce,C.Roddie,J.Y.Henry,H.Yagita,J.D.Wolchok,etal.2013.Fc- peptide-MHC ligand. Nature 436: 578–582. dependent depletion of tumor-infiltrating regulatory T cells co-defines the 59. Rudd, P. M., B. P. Morgan, M. R. Wormald, D. J. Harvey, C. W. van den Berg, efficacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. 210: 1695– S. J. Davis, M. A. Ferguson, and R. A. Dwek. 1997. The glycosylation of the 1710. complement regulatory protein, human erythrocyte CD59. J. Biol. Chem. 272: 63. Marson, A., K. Kretschmer, G. M. Frampton, E. S. Jacobsen, J. K. Polansky, 7229–7244. K. D. MacIsaac, S. S. Levine, E. Fraenkel, H. von Boehmer, and R. A. Young. 60. Beers, S. A., R. R. French, H. T. Chan, S. H. Lim, T. C. Jarrett, R. M. Vidal, 2007. Foxp3 occupancy and regulation of key target genes during T-cell stim- S. S. Wijayaweera, S. V. Dixon, H. Kim, K. L. Cox, et al. 2010. Antigenic ulation. Nature 445: 931–935. modulation limits the efficacy of anti-CD20 antibodies: implications for antibody 64. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, selection. Blood 115: 5191–5201. M. Collins, and M. C. Byrne. 2002. CD4(+)CD25(+) immunoregulatory T cells: 61. Ruffell, B., N. I. Affara, and L. M. Coussens. 2012. Differential macrophage gene expression analysis reveals a functional role for the glucocorticoid-induced programming in the tumor microenvironment. Trends Immunol. 33: 119–126. TNF receptor. Immunity 16: 311–323. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021