Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells

This information is current as Patrick J. Engelberts, Marleen Voorhorst, Janine Schuurman, of September 27, 2021. Tom van Meerten, Joost M. Bakker, Tom Vink, Wendy J. M. Mackus, Esther C. W. Breij, Stefanie Derer, Thomas Valerius, Jan G. J. van de Winkel, Paul W. H. I. Parren and Frank J. Beurskens

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 2, 2016, doi:10.4049/jimmunol.1600811 The Journal of Immunology

Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells

Patrick J. Engelberts,* Marleen Voorhorst,* Janine Schuurman,* Tom van Meerten,† Joost M. Bakker,* Tom Vink,* Wendy J. M. Mackus,* Esther C. W. Breij,* Stefanie Derer,‡ Thomas Valerius,‡ Jan G. J. van de Winkel,*,x Paul W. H. I. Parren,*,{,‖ and Frank J. Beurskens*

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity + (CDC) of CD20 B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab9)2 fragments, as well as C1q-binding–deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for

whole or unmodiﬁed IgG. Experiments using human serum depleted of speciﬁc complement components demonstrated that the Downloaded from observed lytic activity, which we termed “accessory CDC,” remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR2 B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into + + + + 2 CD20 cells: OFA-F(ab9)2 fragments were able to induce CDC in the CD20 BCR cell population, but not in the CD20 BCR 9 population. Importantly, OFA-F(ab )2 fragments were able to induce CDC ex vivo in malignant B cells isolated from patients http://www.jimmunol.org/ with mantle cell lymphoma and Waldenstro¨m macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab–induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic. The Journal of Immunology, 2016, 197: 000–000.

he type I CD20–specific mAbs such as rituximab (RTX) type II CD20 Abs is not due to differences in the intrinsic capacity to and ofatumumab (OFA) efficiently recruit executor compo- activate complement, as all Abs are of the IgG1 isotype. Type II nents of the innate immune system, leading to efficient de- CD20 Abs furthermore have been shown to induce CDC when pre-

T by guest on September 27, 2021 pletion of CD20+ tumor cells in B cell lymphomas and B cell chronic sent at high concentrations in whole blood (8). Therefore, the dif- lymphocytic leukemia (CLL) (1, 2). The mechanisms employed ferent capacity to induce CDC upon binding to CD20-expressing include Ab-dependent cell-mediated cytotoxicity (ADCC), Ab- cells instead seems related to a different density or orientation of dependent cell-mediated phagocytosis (ADCP), and, specifically, Ab Fc domains upon binding to CD20 (7–10). Related to this, type I complement-dependent cytotoxicity (CDC) (3–6). This is in contrast but not type II Abs induce redistribution of CD20 into lipid raft to type II CD20 Abs (e.g., obinutuzumab, 11B8), which are also domains. potent inducers of ADCC and ADCP but are inefficient inducers of Ab-mediated CDC is dependent on the classical pathway of CDC (4, 7). The difference in CDC capacity between these type I and complement activation, which starts when complement component C1q binds to the CH2 domain in the Fc region of complexed IgG or IgM (11). C1 binding and activation requires its binding to the *Genmab, 3584 CM Utrecht, the Netherlands; †Department of Hematology, Univer- sity Medical Center Groningen, 9713 GZ Groningen, the Netherlands; ‡Division of C1q binding site on an array of Fc domains. On IgM molecules, Stem Cell Transplantation and Immunotherapy, 2nd Department of Medicine, Christian the C1q binding site is presented after conformational changes Albrechts University, 24105 Kiel, Germany; xDepartment of Immunology, University Medical Center Utrecht, 3584 EA Utrecht, the Netherlands; {Department of Cancer following Ag binding (12, 13). Efficient C1 binding to IgG mol- and Inflammation Research, Institute of Molecular Medicine, University of Southern ecules is dependent on hexamerization of IgG Fc domains of Abs ‖ Denmark, 5000 Odense, Denmark; and Department of Hematology and Blood Trans- bound to cell surface Ag, which is mediated by extensive inter- fusion, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands molecular Fc–Fc contacts between neighboring Abs (11, 14–16). ORCIDs: 0000-0002-6274-8554 (P.J.E.); 0000-0002-9738-9926 (J.S.); 0000-0002-5581- 6950 (T.v.M.); 0000-0001-6205-1733 (T. Vink); 0000-0002-5368-6849 (E.C.W.B.); For our studies on the mechanism of complement activation by 0000-0002-6142-5577 (S.D.); 0000-0001-9181-8067 (T. Valerius); 0000-0002-4365- type I CD20 Abs, we generated F(ab9)2 fragments of OFA and 3859 (P.W.H.I.P.); 0000-0002-7165-5771 (F.J.B.). RTX. These F(ab9)2 fragments were intended to serve as negative Received for publication May 10, 2016. Accepted for publication October 5, 2016. controls in complement activation studies, because with the ab- Address correspondence and reprint requests to Prof. Paul W.H.I. Parren, Genmab, sence of an IgG Fc domain, they cannot bind C1q and activate the Yalelaan 60, 3584 CM Utrecht, the Netherlands. E-mail address: P.Parren@genmab. complement pathway. Remarkably, we observed complement- com dependent lysis of B cell lymphoma cells upon incubation with The online version of this article contains supplemental material. these type I CD20 Ab F(ab9)2 fragments. In this study, we investi- Abbreviations used in this article: ADCC, Ab-dependent cell-mediated cytotoxicity; ADCP, Ab-dependent cell-mediated phagocytosis; CDC, complement-dependent cy- gated the underlying mechanisms behind this seemingly IgG Fc- totoxicity; CLL, chronic lymphocytic leukemia; MCL, mantle cell lymphoma; NHS, independent complement activation. We show that type I CD20 Abs, normal human serum; OFA, ofatumumab; PI, propidium iodide; RTX, rituximab; in addition to the recruitment of complement components through WM, Waldenstro¨m macroglobulinemia. the Fc domain, are able to induce CDC by activation of the classical Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 complement pathway in a BCR-dependent manner.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600811 2 THE BCR CONTRIBUTES TO CD20 Ab–MEDIATED CDC

Materials and Methods Rabbit anti-human C1q (Dako, Glostrup, Denmark), mouse anti-human Cell lines C4b (Brunschwig, Basel, Switzerland), and peroxidase-conjugated rabbit anti-mouse IgG (1:1000 dilution; Jackson ImmunoResearch Laboratories, The human lymphoma cell lines Daudi, Raji, WIL2-S, ARH-77, DOHH-2, Philadelphia, PA) were used for ELISA experiments. CA-46, and Ramos were obtained from the European Cell Culture Col- Mouse anti-CD55 (555696; BD Biosciences) and anti-IgM Ab HB-57– lection (Porton Down, U.K.), and the human lymphoma cell lines SU-DHL-4, allophycocyanin (a gift from Ron Taylor) were used for FACS experiments. SU-DHL-8, DB, JIYOYE, CL-1, EB-01, HAL-1, MEC-2, WSU-NHL, OCI- For transfection experiments an anti-human BCR (mouse anti-2F8; Ly7, RI-1, OCI-Ly19, and RC-K8 were obtained from the Deutsche Sammlung BioGenes, Berlin, Germany) followed by rat anti-mouse allophycocyanin von Mikroorganismen und Zellkulturen (Braunschweig, Germany). (BD Biosciences) and CD20 [OFA-FITC, OFA-F(ab9)2-FITC, or 11B8- Alemtuzumab-sensitive Wien 133 cells (Burkitt lymphoma cell line) were FITC; Genmab] were used. provided by Dr. Geoff Hale (BioAnaLab, Oxford, U.K.). Daudi, Raji, WIL2-S, ARH-77, DOHH-2, Ramos, WSU-NHL, CA-46, Complement activation and CDC and SU-DHL-4 cells were cultured in RPMI 1640 supplemented with 10% 9 m The capacity of immobilized Igs or F(ab )2 fragments to bind C1q or induce heat-inactivated FCS, 1 U/ml penicillin, 1 g/ml streptomycin, and 4 mM C4b deposition was assessed by ELISA as described previously (18). L-glutamine. CL-1 and SU-DHL-8 cells were cultured in RPMI 1640 CDC was assessed by either a standard chromium release assay utilizing supplemented with 20% heat-inactivated FCS. RC-K8, EB-01, HAL-1, 25% NHS or a propidium iodide (PI) exclusion assay as described previously OCI-Ly18, OCI-Ly19, RI-1, DB, and JIYOYE cells were cultured in (18, 19). To test the contribution of C1q in CDC experiments, 10% NHS (v/v) RPMI 1640 supplemented with 10% heat-inactivated FCS. MEC-2 and was pretreated with 50 mg/ml mouse anti-human C1q, 50 mg/ml isotype Wien 133 cells were cultured in IMDM supplemented with 10% heat- m control Ab, or buffer before addition to the cells. To determine the contri- inactivated FCS, 1 U/ml penicillin, and 1 g/ml streptomycin. OCI-Ly7 bution of endogenous Abs to CDC, IgM-deficient cord blood serum (a gift was cultured in IMDM supplemented with 20% heat-inactivated FCS, 1 U/ml m from Lucien Aarden) or IgG-depleted human serum that was obtained by penicillin, and 1 g/ml streptomycin. All media and supplements were ob- passing NHS over a protein G column (HiTrap; GE Healthcare) was used as Downloaded from tained from Lonza (Vervier, Belgium). complement source. After incubation for 45 min at 37˚C, cells were har- Isolation of peripheral blood from patients with B cell vested, PI was added, and lysis was detected by flow cytometry (FACSCanto II; BD Biosciences). malignancies Mononuclear cells were isolated from peripheral blood of CLL, mantle cell Flow cytometry lymphoma (MCL), or Waldenstro¨m macroglobulinemia (WM) patients as Surface expression of CD20, CD55, IgM-BCR, and/or IgG-BCR on B cell described previously (17). Blood was drawn after receiving the donors’ lymphoma cell lines and mononuclear cells isolated from patients was http://www.jimmunol.org/ written informed consents. Experiments reported in this study were ap- assessed by flow cytometry. One hundred thousand target cells were in- proved by the Ethics Committee of the Christian Albrechts University cubated with specific Abs at saturating concentrations for 30 min at 4˚C. (Kiel, Germany) in accordance with the Declaration of Helsinki. CLL, After washing, cells were analyzed using a FACSCanto II flow cytometer MCL, or WM cells were cultured in RPMI 1640 supplemented with 10% (BD Biosciences). heat-inactivated FCS, 1 U/ml penicillin, and 1 mg/ml streptomycin. To separately analyze normal peripheral blood B cell populations with high and low BCR expression, B cells were isolated from buffy coats Sera, complement-depleted sera, complement components, and obtained from healthy donors (Sanquin) using the untouched B cell depletion buffers kit (Dynabeads, 11351D; Thermo Fischer) according to the manufacturer’s instruction. The B cells were subsequently separated into an IgM BCRhigh Pooled normal human serum (NHS; AB positive) was obtained and an IgM BCRlow population using FITC-labeled Fab fragment targeting from Sanquin (Amsterdam, the Netherlands). C1q-, C5-, C8-, or factor Igm (PL-373; de Beer Medicals, Driessen, the Netherlands) and a FACSAria by guest on September 27, 2021 B–depleted sera, as well as purified complement components C5 and C8, III cell sorter (BD Biosciences). To assess CDC in the IgM BCRhigh and were obtained from Quidel (San Diego, CA). The procedure to deplete BCRlow populations, cells were exposed to OFA in the presence of NHS, sera of individual complement factors resulted sometimes in a modest and CDC was assessed by the PI exclusion assay as described above. reduced activity of these depleted sera. As a control, the activity of depleted serum was checked by assessing the CH50 or AP50 titer after ti- CDC in BCR+ and BCR2 CD20+ CEM T cells trating back the depleted component. C1q that had been purified from sera from multiple donors was a gift from Prof. M. Daha (Leiden University Human CD20+ CEM T cells were obtained by viral transduction as Medical Center). Heat-inactivated NHS was prepared by incubating NHS previously described (20). To generate CD20+BCR+ and CD20+BCR2 for 30 min at 56˚C. cells, CD20+ CEM T cells were cultured to log phase in RPMI 1640 Ca2+-deficient buffer was generated by supplementing PBS with 8 mM supplemented with 10% FCS and transfected with either IgG1 BCR (pG1f- 2+ 2+ 2+ EGTA and 2.5 mM MgCl2 (EGTA-Mg ). Ca Mg -deficient buffer was TM2F8, TM2F8 was cloned into pEE6.4; Lonza, Cologne, Germany) or prepared by adding 10 mM EDTA to PBS. empty vector (empty plasmid pEE6.4) with the Amaxa Nucleofector kit 9 (Lonza, Cologne, Germany), according to the manufacturer’s instructions. Abs, Fc variants, and F(ab )2 fragments Two days after transfection, cells were washed and incubated with Clinical OFA was obtained from GlaxoSmithKline. 7D8 and 11B8 are saturating concentrations of BCR-specific Abs as described above. Cell human, CD20-specific Abs of the IgG1 isotype and were recombinantly death was determined by PI exclusion as described above. produced at Genmab as described (18). Fc mutations to knock out C1q binding were introduced in mAb 7D8. mAb 7D8 only differs at only four Statistical analysis amino acid positions from OFA; it binds the same epitope on CD20 and Statistical analysis was performed using a one-way ANOVAwith a Dunnett has similar functional characteristics, including similarly efficient induc- multiple comparisons posttest. A p value #0.05 was considered significant. tion of CDC. To generate 7D8 variants with reduced or abrogated capacity to activate complement, amino acid mutations in the Fc domain were introduced in the expression plasmids encoding the Ab H chain using the Quik- Results Change XL kit (Stratagene, La Jolla, CA) according to the manufacturer’s C1q binding-deficient type I CD20 Abs retain the capacity to guidelines. Mutations were verified by DNA sequencing (LGC Genomics, Berlin, Germany). RTX was obtained from Roche (Grenzach-Whylen, induce CDC Germany). Alemtuzumab is a humanized CD52-specific IgG1 and was TheCD20Abs,OFA,RTX,and11B8,aswellasOFA-F(ab9)2 obtained from Genzyme (Cambridge, MA). fragments, were immobilized on ELISA plates and incubated with F(ab9)2 fragments of OFA, RTX, 7D8, and alemtuzumab were obtained by digestion of IgG with 2 mg/ml pepsin (Sigma-Aldrich) for 30–60 min. NHS to assess their capacity to bind C1q, the first step in the clas- Digested material was dialyzed with PBS and loaded on a protein A column sical pathway of complement activation. All intact Abs showed ef- 9 (Sigma-Aldrich) to separate the F(ab )2 fragments from the Fc tails. The ficient C1q binding (Fig. 1A). As expected, OFA-F(ab9)2 fragments 9 purity of the F(ab )2 fragments was confirmed by SDS-PAGE and ELISA. were unable to bind C1q. Similarly, surface-immobilized OFA, Mouse anti-human C1q (mIgG1 clone 85, directed to the globular head 9 region of C1q) was a gift from Prof. Lucien Aarden (Sanquin). A mouse RTX, and 11B8, but not OFA-F(ab )2 fragments, induced deposition IgG1 isotype control Ab was obtained from BD Biosciences (clone of C4b (Fig. 1B), an activation product of the classical complement MOPC-21). pathway that is generated upon C1-dependent cleavage of C4. The Journal of Immunology 3

To assess CDC, we incubated OFA, RTX, 11B8, and OFA-F(ab9)2 heat-inactivated human serum (Fig. 1E and data not shown), fragments with Daudi Burkitt’s lymphoma cells in the presence indicating that active complement was required. In support of of NHS. To our surprise, not only the full-length Abs, but also the this, Daudi cells that survived exposure to OFA-F(ab9)2 frag- OFA-F(ab9)2 fragments, induced Daudi cell lysis (Fig. 1C), albeit the ments showed significantly higherexpressionofthecomplement OFA-F(ab9)2 fragments induced lysis more slowly and with lower regulatory protein CD55, in comparison with the whole pop- maximal kill. Nevertheless, OFA-F(ab9)2 fragments reproducibly ulation before OFA-F(ab9)2 fragment treatment (Supplemental killed at least 50% of the tumor cells. Similarly, RTX-F(ab9)2 frag- Fig. 2A). Similar data were previously obtained in B cell lym- ments induced lysis in Daudi cells in the presence of NHS, although phoma cells that survived exposure to RTX-mediated CDC in the not as efficiently as did RTX (Supplemental Fig. 1A). As previously presence of active complement, presumably due to CDC resis- reported, 11B8 was unable to induce CDC, confirming that type II tance in the cell population with the highest CD55 expression CD20 Abs are poor inducers of CDC (18). To confirm that CDC (22). inducedbytheCD20AbF(ab9) fragments is a general phenomenon 2 9 that may also be mediated by whole Abs lacking a C1q binding site, OFA-F(ab )2 fragment–mediated cytotoxicity is dependent on we generated a set of IgG1 mutants in the CD20 Ab 7D8. Indeed, an the classical pathway of complement activation IgG4 variant of 7D8 comprising a lysine to alanine mutation in the To study the role of the different complement activation pathways Fc fragment (K322A), which completed lacking C1q binding and in OFA-F(ab9)2 fragment–induced cytotoxicity, a set of experi- complement activation when immobilized in ELISA (Supplemental ments was performed in presence of NHS that had been depleted Fig. 1C, 1D), induced CDC of Daudi cells in human serum (Fig. of Ca2+ and Mg2+, resulting in complete abrogation of all com-

1D). Similar results were obtained with other 7D8 mutants lacking plement activity, or of Ca2+ alone, resulting in inactivation of the Downloaded from C1q binding (Supplemental Fig. 1C, 1D). Our observations appeared classical and lectin complement pathways, but not the alterna- to be specific for CD20 Abs, as F(ab9)2 fragments or C1q binding- tive pathway. Under these conditions, both OFA-F(ab9)2 fragments deficient mutants of the potent CDC-inducing CD52 Ab alemtuzu- and intact OFA were unable to induce cytotoxicity in Daudi cells mab (21) were unable to induce cytotoxicity (data not shown). (Fig. 2A). This confirmed that active complement was required OFA-F(ab9)2 fragment– and 7D8-IgG4-K322A–mediated cy- and demonstrated that complement activation did not occur

totoxicity was observed in the presence of human serum, but not through the alternative pathway. http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Type I CD20 Abs or Ab fragments that lack the capacity to bind C1q are able to elicit lysis of Daudi cells in the presence of NHS. (A and B)

OFA, OFA-F(ab9)2 fragments, RTX, and 11B8 were immobilized on ELISA plates in the presence of NHS. C1q binding (A) and C4b deposition (B) were assessed by ELISA. (C) OFA, OFA-F(ab9)2 fragments, RTX, and 11B8 were incubated with Daudi cells in presence of NHS, and tumor cell lysis was assessed by quantifying PI+ cells using flow cytometry. (D) 7D8 and 7D8-IgG4-K322A were incubated with Daudi cells in presence of NHS, and CDC + was assessed by quantifying PI cells using flow cytometry. (E) OFA and OFA-F(ab9)2 fragments were incubated with Daudi cells in presence of NHS or heat-inactivated NHS (HI-NHS), and CDC was assessed by quantifying PI+ cells using flow cytometry. Data shown are representative of at least three replicate experiments. 4 THE BCR CONTRIBUTES TO CD20 Ab–MEDIATED CDC

To study which components of complement were required for the in serum devoid of IgM (Supplemental Fig. 3A) or depleted for IgG cytotoxicity of OFA-F(ab9)2 fragments, we performed experiments (Supplemental Fig. 3B), serum amyloid P, or C-reactive protein (data in serum that had been depleted of specific complement compo- not shown). nents. Neither OFA-F(ab9)2 fragments nor OFA showed CDC Taken together, these results show that type I CD20 F(ab9)2 activity in sera depleted of C5 or C8 (Fig. 2B), components that fragments or Ab variants that are unable to bind C1, retain the are essential for the initiation of terminal complement activation capacity to induce complement-mediated lysis via the classical and formation of the membrane attack complex, respectively (23). pathway, with, paradoxically, a critical role for C1q. In contrast, OFA-F(ab9) fragments were able to induce lysis of 2 9 Daudi cells in the presence of NHS that had been depleted of OFA-F(ab )2 fragment–mediated CDC is dependent on BCR factor B (Fig. 2C). This confirmed that the alternative pathway expression was not involved in OFA-F(ab9)2 fragment–dependent CDC, as We hypothesized that an alternative docking site for C1q must be factor B is an essential intermediate in the alternative pathway, but present on the plasma membrane, in or near the CD20-OFA-F(ab9)2 not the classical or lectin pathways. fragment immune complexes. The BCR complex is expressed in Finally, OFA-F(ab9)2 fragment–mediated cytotoxicity was close proximity to CD20 on B cells (27) and contains a cell surface– shown to be mediated through the classical pathway and not the bound Ab (surface Ig) that could provide such a C1q docking lectin pathway. OFA-F(ab9)2 fragments were unable to induce site. We addressed the potential role of the BCR in OFA-F(ab9)2 cytotoxicity in C1q-depleted serum, whereas reconstitution with fragment–mediated complement activation by measuring induc- C1q restored cytotoxic activity (Fig. 2D). Furthermore, an Ab specific tion of CDC in a panel of human B cell lymphoma cell lines that for the globular head of C1q was able to completely inhibit the displayed variable levels of IgM- or IgG-BCRs on the cell surface. Downloaded from cytotoxic activity of 7D8, 7D8-F(ab9)2 fragments, and 7D8-IgG4- OFA-F(ab9)2 fragments were able to induce CDC in seven out of K322A in NHS (Fig. 2E). eight cell lines with an IgM BCR (Fig. 3A, Table I), indicating that Activation of the classical pathway may also be induced by other our observations in Daudi cells seem to represent a common serum factors such as naturally occurring autoantibodies (e.g., anti- phenomenon for B cell lines expressing a BCR of the IgM sub- hinge Abs) or the pentraxins serum amyloid P (24) and C-reactive class. In contrast, OFA-F(ab9)2 fragment–mediated cytotoxicity protein (25, 26). However, a role for these factors in OFA-F(ab9)2 was observed in only one out of seven IgG-BCR–expressing cell http://www.jimmunol.org/ fragment–mediated CDC was excluded by performing experiments lines, whereas all of these cell lines were susceptible to CDC by guest on September 27, 2021

FIGURE 2. OFA-F(ab9)2 fragment–mediated cytotoxicity is dependent on the classical pathway of complement activation. (A–D) OFA and

OFA-F(ab9)2 fragments were incubated with Daudi cells in the presence of normal NHS or NHS that had been depleted of Ca2+ and Mg2+ or Ca2+ alone (A), complement components C5 or C8 (B), factor B (C), or C1q (D). Cytotoxicity of OFA and OFA-F(ab9)2 fragments was assessed by ﬂow cytometric quantiﬁcation of PI+ cells. (E)

Cytotoxicity of OFA, 7D8, 7D8-F(ab9)2 fragments, and 7D8-IgG4-K322A in NHS that had been preincubated with a C1q Ab or an isotype control Ab. Cytotoxicity was assessed as described above. All experiments were performed in triplicate; representative experiments are shown. The Journal of Immunology 5 Downloaded from

FIGURE 3. OFA-F(ab9)2 fragments induces CDC in B cells with different BCR subtypes. (A) CDC activity of OFA and OFA-F(ab9)2 fragments was assessed in the IgM-BCR–expressing OCI-Ly7 cell line (left panel), the IgG-BCR–expressing SU-DHL-4 cell line (middle panel), and the BCR2 Raji cell line. Error bars indicate the SD of three replicates samples per experiment. Representative examples of at least triplicate experiments are shown. Statistical http://www.jimmunol.org/ analysis was done with a one-way ANOVA followed by a Dunnett multiple comparisons posttest to compare the mean of each column to that of the no Ab control. OFA induced lysis of all three cell lines (one-way ANOVA, p , 0.05), whereas OFA-F(ab9)2 fragments only induced signiﬁcant lysis of OCI-Ly7 cells expressing an IgM BCR and SU-DHL-4 cells expressing an IgG BCR (one-way ANOVA, p , 0.05) but not of BCR2 Raji cells. *p , 0.05, ***p ,

0.0005, ****p , 0.0001. (B) CDC activity of OFA, OFA-F(ab9)2 fragments, and anti-IgM-F(ab9)2 fragments in the OCI-Ly7 DLBCL cell line. CDC was assessed by ﬂow cytometric evaluation of PI+ cells. The ﬁgure shows a representative example of at least three experiments.

induced by intact OFA (Fig. 3A, Table I). OFA-F(ab9)2 fragment– cells (Supplemental Fig. 2B). This again suggests that suscepti- mediated CDC was not observed in any of the six B cell lines bility to OFA-F(ab9)2 fragment–mediated CDC may be linked to devoid of BCR expression (Fig. 3A, Table I). The different sen- its BCR expression. by guest on September 27, 2021 sitivity of IgM- and IgG-BCR–expressing cell lines for OFA-F In OCI-Ly7 cells, a cell line with high IgM-BCR surface ex- (ab9)2 fragment–mediated CDC could not be attributed to differ- pression, CDC was induced not only by OFA and OFA-F(ab9)2 ences in BCR or CD20 expression levels, although analysis of a fragments, but also by polyclonal IgM-specific F(ab9)2 fragments larger number of cell lines would be required to draw firm con- (Fig. 3B). This indicates that, under certain conditions, crosslinking clusions. When examining the lysis of Daudi cells more closely, of the BCR in itself is sufficient to initiate complement activation we observed that the average BCR expression level of cells that and complement-mediated lysis. survived exposure to OFA-F(ab9)2 fragments in the presence of A direct relationship between BCR expression and OFA-F(ab9)2 NHS was lower than the average BCR expression in untreated fragment–mediated CDC was established by transfection of an

Table I. Sensitivity of various B cell lymphoma cell lines to OFA F(ab9)2 fragment–mediated CDC

BCR Expression

Cell Line OFA-F(ab9)2–Mediated CDC Subtype Molecules/Cell (sABC) CD20 Expression (Molecules/Cell) (sABC) Wien 133 + IgM 52,400 102,000 MEC-2 2 IgM 30,500 168,000 Daudi ++ IgM 150,000 133,000 OCI-Ly7 ++ IgM 342,000 392,000 Ramos ++ IgM 171,000 222,000 ARH-77 2 IgG 20,400 223,000 WSU-NHL 2 IgG 117,000 339,000 DOHH-2 2 IgG 23,900 249,000 SU-DHL-4 + IgG 77,500 241,000 OCI-Ly19 2 IgG 22,300 47,900 SU-DHL-8 2 IgG 26,000 83,900 DB 2 IgG 233,000 307,000 HAL-01 2 Not expressed ,7,000 100,000 Raji 2 Not expressed ,7,000 174,000 JIYOYE 2 Not expressed ,7,000 40,000 RC-K8 2 Not expressed ,7,000 150,000

Surface expression of CD20 and the BCR was assessed by flow cytometry. To assess CDC, cells were incubated with OFA-F(ab9)2 fragments in the presence of 20% NHS. Specific lysis was assessed by quantifying PI+ cells using flow cytometry. 2, no lysis; +, percentage specific lysis $2-fold the background level; ++, percentage specific lysis $3-fold the background level; sABC, specific Ab-binding capacity. 6 THE BCR CONTRIBUTES TO CD20 Ab–MEDIATED CDC

IgG1-BCR into CD20+ CEM T cells. Flow cytometry confirmed a type II CD20 Ab 11B8 did not induce CDC in either CD20+BCR+ or population of BCR-expressing (BCR+) cells in the BCR-transfected CD20+BCR2 cells. The findings demonstrate that the BCR contrib- CD20+ CEM T cells, whereas BCR expression was absent in cells utes to type I CD20 Ab–mediated complement activation. The BCR- + + that had been transfected with empty vector (Fig. 4A). CD20 BCR dependent CDC activity in the presence of OFA-F(ab9)2 fragments CEM T cells were exposed to OFA and OFA-F(ab9)2 fragments in the was named “accessory CDC.” presence of NHS, and complement-mediated lysis was assessed by quantifying the percentage of PI+ cells within the CD20+BCR+ and Accessory CDC in patient-derived tumor material CD20+BCR2 populations. Whereas CD20+BCR+ cells were suscep- Finally, to assess whether accessory CDC might contribute to the tible to complement-mediated lysis after opsonization with OFA-F antitumor activity of type I CD20 Abs in the clinic, we performed a + 2 (ab9)2 fragments, the CD20 BCR cell population in the same set of experiments using PBMCs obtained from three CLL patients, sample was resistant to CDC mediated by OFA-F(ab9)2 fragments but three MCL patients, and one WM patient. Expression of CD20 and not OFA. The cells in lower panel of Fig. 4A were mock transfected the BCR (IgM) was confirmed for all patients (Fig. 5A). Interestingly, and therefore only contain a CD20-expressing population. As ex- OFA-F(ab9)2 fragments were able to induce ex vivo killing of tumor pected, these cells were only lysed in the presence of OFA and not cells isolated from MCL and WM patients, in which OFA-F(ab9)2 OFA F(ab9)2 fragments (Fig. 4B). For cells treated with OFA, CDC fragment–mediated tumor cell killing was particularly efficient in was more efficient in CD20+BCR+ than in CD20+BCR2 (mock- the WM patient, that showed high BCR expression (Fig. 5B). OFA-F transfected) cells (p = 0.0397), even though CD20 expression was (ab9)2 fragments showed statistically significant tumor cell lysis comparable between the BCR+ and BCR2 populations. Similarly, compared with the no Ab control for MCL (p = 0.0214) and WM

OFA-mediated CDC was higher in peripheral blood B cells with high (p = 0.0062) (Fig. 5C), but not for CLL (p = 0.4129). Downloaded from BCR expression than in the population with low BCR expression These data indicate that, also for clinically relevant samples, (Supplemental Fig. 4). This demonstrates that the BCR indeed also accessory CDC can contribute to the antitumor activity of type I contributes to the CDC activity of IgG1 CD20 Abs. As expected, the CD20 Abs (Fig. 6). http://www.jimmunol.org/

FIGURE 4. Transfection of CD20+ CEM T cells with BCR sensitizes cells for OFA-F

(ab9)2 fragment–mediated CDC. (A)BCR and CD20 expression in CD20+ CEM T cells that had been transfected with an

IgG-BCR (upper panels) or empty vector by guest on September 27, 2021 (lower panels). Expression of BCR and CD20 was assessed by ﬂow cytometry, using allophycocyanin-labeled IgG-speciﬁc mouse Fc fragments or FITC-labeled OFA, respectively. P2 indicates the population of BCR2 cells; P3 indicates the population of BCR+ cells. (B) CDC activity of OFA, OFA-F(ab9)2 fragments, and 11B8 in CD20+BCR+ and CD20+BCR2 cell populations within the BCR-transfected cultures (orange and blue bars, respectively) and in untransfected cells (gray bars). Bars are means of triplicate experiments; error bars indicate the SD. Statistical analysis was done with a one- way ANOVA followed by a Dunnett multiple comparisons posttest to compare the mean of each column to that of the mean of the control (CD20+Mock). OFA ANOVA, p = 0.0467. Dunnett multiple comparison: CD20+ mock-transfected cells (CD20+Mock) versus CD20+BCR+, p = 0.0397. CD20+Mock versus CD20+ + IgG , p = 0.8387. OFA-F(Ab9)2 ANOVA, p = 0.0208. Dunnett multiple comparison: CD20+Mock versus CD20+BCR+, p = 0.0220. CD20+Mock versus CD20+IgG+, p =0.9790.*p , 0.05. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Accessory CDC in primary tumor cells. (A) Coexpression of CD20 and the IgM-BCR in tumor material isolated from a patient with CLL (upper panel), MCL (middle panel), and WM (lower panel). Expression of CD20, IgG-BCR, and IgM-BCR was assessed by flow cytometry. (B) Dose-dependent cytotoxicity of OFA and OFA-F(ab9)2 fragments in CLL (left panel), MCL (middle panel), and WM (right panel) patient–derived tumor cells ex vivo. (C) CDC activity of OFA and OFA-F(ab9)2 fragments in tumor material isolated from three CLL patients, three MCL patients, and one WM patient. CDC was assessed using the classical chromium release assay. Error bars indicate the SEM of three replicate samples. Statistical analysis was done with a one-way ANOVA followedbya Dunnett multiple comparisons posttest to compare the means of each column to that of the no Ab control per indication. OFA induced significant lysis in all indications (one-way ANOVA, p , 0.05), whereas OFA-F(ab9)2 fragments only induced significant lysis in patient-derived MCL and WM samples (one-way ANOVA, p , 0.05) but not of patient-derived CLL samples. *p , 0.05, **p , 0.005. 8 THE BCR CONTRIBUTES TO CD20 Ab–MEDIATED CDC

Discussion Recent data suggest that BCRs of the IgM and IgD subclass are All IgG1 Abs have an intrinsic capacity to bind C1q and activate organized in preformed oligomeric structures in distinct protein complement. However, the ability of specific IgG1 Abs to induce islands on the plasma membrane of resting B cells (31, 32), and that CDC upon target binding is strongly dependent on the characteristics B cell activation induces remodeling of such BCR protein islands of both the Ab as well as the target and epitope recognized (11). CDC into smaller structures, with a different protein and lipid compo- occurs upon formation of ordered IgG hexamers on the plasma sition (32). We hypothesize that binding of type I CD20 Abs or membrane of target cells, the agglomerate of IgG Fc parts, thus F(ab9)2 fragments to the plasma membrane induces remodeling of providing the optimal docking site for C1q (14). In the present study, BCR protein islands, in such a way that the BCR clusters provide we demonstrate that, at least for typeICD20Abs,CDCcanalsobe sufficient avidity to allow C1q binding and complement activation. initiated by F(ab9)2 fragments and other Ab variants that are unable to Based on this idea, we propose a model for type I CD20 Ab- bind C1q, and that CDC in this case appears dependent on C1q dependent complement activation in BCR-expressing malignant binding by the BCR. This is an intriguing observation, as it is gen- B cells (Fig. 6). Upon binding to CD20 on the plasma membrane, erally assumed that Abs that are able to induce CDC upon binding to type I CD20 IgG Abs form hexamers that provide a docking site a cellular target provide the C1q docking site. Importantly, this phe- for C1q and induce CDC (14, 15). Hexamer formation by the nomenon, which we named accessory CDC, was observed in almost CD20 Abs is dependent on the IgG Fc domain and independent all B cell lines that expressed an IgM BCR, as well as in malignant of the BCR (Fig. 6A). Ab binding results in clustering of CD20 B cells from patients with MCL and WM. The absence of significant and the BCR in lipid raft domains, which potentially also facil- lysis of CLL patient material may be due to low and heterogeneous itates C1q binding by the BCR cluster (Fig. 6B). As illustrated Downloaded from CD20 expression on these cells. in Fig. 6C, the combination of these two mechanisms leads to CD20 and the BCR have been shown to colocalize on the plasma maximal CDC activity of type I CD20 Abs. The detailed molec- membrane within lipid raft domains (27). Type I but not type II ular organization of such CD20–BCR complexes requires further CD20 Abs caused direct association of CD20 with the BCR and investigation. enhanced the formation of CD20- and BCR-enriched lipid rafts C1q binding and complement activation by the BCR was pre- (27–30). Furthermore, type I CD20 mAbs and the BCR were viously observed by Rossbacher and Shlomchik (33) who showed shown to induce similar intracellular signaling pathways, and that crosslinking of an IgM-BCR using a highly multivalent Ag http://www.jimmunol.org/ CD20 mAbs were shown to induce phosphorylation of BCR- induced deposition of C3d on B cells, an effect that was abolished specific adaptor proteins, which was dependent on BCR expres- in cells with a mutated BCR that was unable to bind C1q (33). In sion (30). Collectively, these data indicate that CD20 and the BCR line with this, we were able to induce CDC of OCI-Ly7 cells by can reside together in lipid-rich domains, and that cross-talk exists ligation of the IgM-BCR with polyclonal IgM-specific F(ab9)2 between the two molecules. fragments. by guest on September 27, 2021

FIGURE 6. Proposed model for type I CD20 Ab–mediated CDC in BCR-expressing tumor cells. (A) Upon binding to CD20 on the cell membrane, type I CD20 Abs form hexameric structures that are dependent on intermolecular interactions between Fc domains of bound IgG molecules. These IgG hexamers provide an optimal docking site for C1q. (B) Binding of type I CD20 Abs induces clustering of CD20 and BCR molecules in lipid raft domains, which appears to facilitate complement activation via a process that is independent of the CD20 Ab’s Fc domain. (C) The combination of these two mechanisms of complement activation leads to maximal CDC activity of type I CD20 Abs. The Journal of Immunology 9

The concentration of OFA-F(ab9)2 fragments required to observe 14. 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. accessory CDC in cell lines and tumor samples was well within the Complement is activated by IgG hexamers assembled at the cell surface. Science expected plasma levels of patients that are undergoing treatment 343: 1260–1263. with type I CD20 Abs (34). This suggests that accessory CDC may 15. de Jong, R. N., F. J. Beurskens, S. Verploegen, K. Strumane, M. D. van Kampen, M. Voorhorst, W. Horstman, P. J. Engelberts, S. C. Oostindie, G. Wang, et al. contribute to the antitumor activity of type I CD20 Abs in the clinic. 2016. A novel platform for the potentiation of therapeutic antibodies based on Whether accessory CDC has a role in normal physiology remains antigen-dependent formation of IgG hexamers at the cell surface. PLoS Biol. 14: an open question. One could speculate that recognition of a multi- e1002344. 16. Pawluczkowycz, A. W., F. J. Beurskens, P. V. Beum, M. A. Lindorfer, J. G. van valent Ag by the BCR could induce sufﬁcient crosslinking to allow de Winkel, P. W. Parren, and R. P. Taylor. 2009. Binding of submaximal C1q CDC, similar to the CDC we and others observed after crosslinking promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher the BCR with polyclonal anti-IgM. However, we are not aware of levels of CDC are induced by OFA than by RTX. J. Immunol. 183: 749–758. studies addressing this subject. 17. Peipp, M., J. J. Lammerts van Bueren, T. Schneider-Merck, W. W. Bleeker, In summary, accessory CDC is a novel effector mechanism of type I M. Dechant, T. Beyer, R. Repp, P. H. van Berkel, T. Vink, J. G. van de Winkel, et al. 2008. Antibody fucosylation differentially impacts cytotoxicity mediated CD20 Abs. Accessory CDC occurred through the classical pathway of by NK and PMN effector cells. Blood 112: 2390–2399. complement activation, but it was independent of the CD20 Ab Fc 18. Teeling, J. L., R. R. French, M. S. Cragg, J. van den Brakel, M. Pluyter, domain. Instead, accessory CDC was facilitated by the BCR. Together H. Huang, C. Chan, P. W. Parren, C. E. Hack, M. Dechant, et al. 2004. Char- acterization of new human CD20 monoclonal antibodies with potent cytolytic with known effector mechanisms of type I CDC Abs, such as ADCC, activity against non-Hodgkin lymphomas. Blood 104: 1793–1800. ADCP, and “classical” CDC, accessory CDC may contribute to 19. Ro¨sner, T., S. Lohse, M. Peipp, T. Valerius, and S. Derer. 2014. Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of therapeutic activity of CD20 Abs and potentially other Abs that induce decay-accelerating factor expressing tumor cells through the alternative pathway BCR clustering. ampliﬁcation loop. J. Immunol. 193: 1485–1495. Downloaded from 20. van Meerten, T., R. S. van Rijn, S. Hol, A. Hagenbeek, and S. B. Ebeling. 2006. Complement-induced cell death by rituximab depends on CD20 expression level Disclosures and acts complementary to antibody-dependent cellular cytotoxicity. Clin. P.J.E., M.V., J.S., T. Vink, E.C.W.B, J.G.J.W., P.W.H.I.P., and F.J.B. are Cancer Res. 12: 4027–4035. all employees of Genmab. J.M.B and W.J.M.M are former employees of 21. de Weers, M., Y. T. Tai, M. S. van der Veer, J. M. Bakker, T. Vink, D. C. Jacobs, L. A. Oomen, M. Peipp, T. Valerius, J. W. Slootstra, et al. 2011. Daratumumab, a Genmab. P.J.E., M.V., J.S., T. Vink, E.C.W.B, J.G.J.W., P.W.H.I.P., F.J.B., novel therapeutic human CD38 monoclonal antibody, induces killing of multiple

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