A Recombinant Bispecific CD20×CD95 Antibody With Superior Activity Against Normal and Malignant B-cells

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Accepted Article Preview: Published ahead of advance online publication A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells

Kristina Nalivaiko, Martin Hofmann, Karina Kober,Nadine Teichweyde, Peter H. Krammer, Hans-Georg Rammensee, Ludger GrosseHovest,Gundram Jung

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Cite this article as: Kristina Nalivaiko, Martin Hofmann, Karina Kober,Nadine Teichweyde, Peter H. Krammer, Hans-Georg Rammensee, Ludger Grosse-Hovest,Gundram Jung, A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells, Molecular Therapy accepted article preview online 19 November 2015; doi:10.1038/mt.2015.209

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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

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Received 06 July 2015; accepted 30 October 2015; Accepted article preview online 19 November 2015

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A recombinant bispecific CD20XCD95 antibody with superior activity against normal and malignant B-cells Kristina Nalivaiko1, Martin Hofmann1,3, Karina Kober1, Nadine Teichweyde1, Peter H. Krammer2,4, Hans-Georg Rammensee1,4, Ludger Grosse-Hovest1,3, Gundram Jung1,4 Affiliations: 1 Department of Immunology, Eberhard Karls Universität Tübingen, Germany 2 Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Present address: Synimmune GmbH, Auf der Morgenstelle 15, Tübingen, Germany 4 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Contributions: K.N. performed most of the experiments, and contributed to data presentation and editing of the paper. M.H. developed the FACS method used for the cell depletion experiments and contributed to the analysis of the respective experiments. K.K. was involved in the initial construction of recombinant variants of BS9520. N.T. generated the recombinant CD20 antibodies (together with L.G.H.). P.H.K contributed the method used for measuring antibody production by PWM activated cells. H.G.R suggested some of the experiments. L.G.H. invented the Fabsc-format and was involved in the construction of BS9520 and BS95Mel. G.J. designed the concept of the study, most of the experiments (together with K.N.) and wrote the manuscript.

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Competing financial interests: LGH and GJ have filed patent applications covering the stimulation of CD95 with recombinant, bispecific antibodies. Patent rights have been licensed to Baliopharm AG, Jülich, Germany. GJ is a member of the scientific advisory board of Baliopharm. The remaining authors declare no competing financial interests.

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Acknowledgements: This work was supported by a fellowship from the Catholic Academic Student Exchange Service (Katholischer Akademischer Ausländer-Dienst, KAAD), Germany, to K.N. and, in part, by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, SFB773, project C4). We thank Dr. O. Planz for help with the animal experiments and B. Pömmerl for skillful technical assistance. Corresponding author: Gundram Jung Department of Immunology Eberhard Karls Universität Tübingen E-mail: [email protected] Phone: x49-7071-29-87621 FAX: x49-7071-29-5653

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Monoclonal antibodies directed to the B-cell specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here we compare the anti-B-cell activity of three different antibodies directed to CD20, (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof and (iii) a bispecific CD20XCD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant- as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from PBMC cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its non-optimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20XCD95-antibody might become a new, prototypical reagent for the treatment of Bcell mediated autoimmune disease.

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Chimeric or humanized second generation antibodies recognizing CD20, a protein expressed on normal and malignant B-cells, have already been prototypical in several respects: Rituximab, a chimeric version of the parental mouse antibody 2B8, was the first antibody to demonstrate convincing anti-tumor effects in humans and has become a cornerstone of current lymphoma treatment1. In addition, given the activity of CD20 antibodies not only against malignant- but also against normal B-cells, those reagents have been used to treat B-cell associated autoimmune diseases2, such as rheumatoid arthritis, Wegeners granulomatosis, Sjögrens syndrome3 and, more recently, multiple sclerosis4,5. For immunosuppression, Rituximab as well as newly developed CD20 antibodies, such as Ocrelizumab and Ofatumumab, were used. The latter reagents are derived from the parental antibodies 2H76 and B-Ly1, respectively, and are directed against different but overlapping epitopes of the CD20 molecule. CD20 antibodies were also the first to benefit from Fc-optimization7, a promising strategy to further improve the therapeutic activity of antibodies, that largely depends on the interaction of antibody Fc parts and Fc-receptors (FcRs) expressed on immune cells. In principle, Fcoptimization of an IgG antibody may be achieved by genetic engineering of the glycosylation machinery8 or the amino acid sequence of the CH2 domain9, the region responsible for Fc-FcR interaction. Both of these techniques have been used by the industry to develop third generation antibodies up to the stage of clinical trials: Roche’s glyco-engineered CD20antibody GA101 is the successor of the prototypical Rituximab antibody10. Xencor introduced the amino acid modifications S239D and I332E (SDIE-modification) into the CH2 domain of XmAb 5574, an antibody directed to CD19, an alternative B-cell associated antigen11. GA10112, 13 as well as XmAb 557411, exerted remarkably enhanced antibody dependent cytotoxicity (ADCC) against malignant lymphoma cells if compared to the respective nonoptimized antibody variants. Recently, a clinical trial with more than 700 CLL patients demonstrated superior therapeutic activity of GA101 compared to that of Rituximab14, and a phase I trial with XmAB 5574 demonstrated safety and efficicacy in relapsed CLL15. As CD19 and CD20, the death receptor CD95 (Apo-1, Fas) is expressed on malignant- as well as on normal B-cells. Normal cells, however, express only marginal amounts of the protein, unless they undergo activation that is accompanied by a greatly enhanced CD95 expression16. Previously, we have demonstrated that bispecific CD20xCD95-Fab2-antibodies, generated by chemical hybridization, are capable of inducing CD95-mediated apoptosis selectively in malignant cells expressing CD2017. Later, we found that induction of apoptosis depends on

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mutual bi-cellular crosspresentation of bispecific antibodies by cells expressing the relevant target antigen18. For the work described here we have used the CD20 antibody 2H76, contained in Ocrelizumab, to construct a bispecific CD20xCD95 antibody (BS9520) in a newly developed, recombinant format, termed Fabsc19. For comparison, we have also generated a chimeric CD20 antibody (CH20) as well as an Fc-optimized version of this reagent carrying the SDIE modification described above (SDIE20, Fig.1). We have evaluated the cytolytic activity of these reagents against malignant lymphoma cells as well as normal resting and activated B-cells. We have also explored the consequences of this activity, that is, suppression of tumor growth in a SCID mouse model and inhibition of antibody production by normal B-cells, in order to assess and compare the anti-B cell activity of the different reagents.

RESULTS Construction and characterization of the antibodies Fig. 1 depicts the antibodies compared in this paper. All reagents contain the CD20 antibody 2H7, either as a chimeric version (CH20) or as an Fc-optimized, SDIE-modified variant thereof (SDIE20). For construction of the bispecific CD20XCD95 molecule (BS9520), we used the Fabsc-format consisting of an N-terminal Fab-part linked to a C-terminal single chain antibody by an Fc-attenuated CH2 domain (Fig. 1c)19. Fc-attenuation is required to prevent multimerization of the CD95 arm –and, thus, CD95 stimulation- on the surface of FcRexpressing cells 17, 18. We also produced a Fabsc-control antibody, designated BS95Mel, directed to the melanoma associated chondroitin sulfate proteoglycan CSPG4. Previously, we have compared bispecific antibodies with FLT3xCD3-specificity expressed in the Fabsc- as well as the widely used bispecific single chain (bssc)-format, also termed BiTE, and have found that the Fabsc-format is superior with respect to (i) increased production rates, (ii) preserved affinity of the N-terminal Fab-arm and (iii) diminished aggregation19. In fact, the latter advantage became strikingly evident during the development of BS9520: we did not succeed in constructing a functional bispecific antibody containing the CD95 binding part as a C-terminal single chain molecule due to extensive aggregation of this moiety. Only when we expressed the CD95 antibody as the N-terminal Fab-part within the Fabsc-format a monomeric and functional bispecific molecule was obtained (Supplementary Fig. 1). Not unexpectedly, expression of the CD20 antibody in the single chain format resulted in a significant loss of avidity if compared to the intact, monospecific and bivalent CD20 antibodies. In contrast, the avidity of BS9520 towards CD95 is higher and only moderately reduced if compared to the parental CD95 antibody Apo-1 (Supplementary Fig. 2) . In accordance with these findings we observed only weak binding of BS9520 to resting normal B cells due to the low avidity of its CD20 binding part. In contrast, binding to activated cells was considerably more pronounced because the CD95 expression on these cells allows binding of the high affinity CD95 binding part of the molecule (Supplementary Fig.3).

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Activity against malignant B-cells Fig. 2 depicts the anti-proliferative activity of the three antibodies and -for comparison- of the prototypic CD20 antibody Rituximab towards various lymphoma cells in the presence of different amounts of PBMC as a source of effector cells. The antibody dependent cellular cytotoxicity (ADCC) of Rituximab and CH20 was comparable and clearly less pronounced than the activity of the Fc-optimized SDIE20 antibody. At concentrations <100 ng/ml and a PBMC : target ratio of 5 : 1 the activity of this antibody was higher than that of BS9520 well in accordance with the rather low avidity of the bispecific construct towards CD20. In contrast, at

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concentrations ≥100ng/ml BS9520 was superior. As expected, the cytotoxic activity of BS9520 does not depend on the presence of effector cells (Supplementary Fig. 5) and a control bispecific antibody directed to a melanoma associated antigen (BS95Mel) was only marginally effective. Whereas this pattern held true for three of the four cell lines tested, Raji cells were different: the cytotoxic activity of BS9520 against these cells was marginal, although they express sufficient amounts of CD20 as well as CD95 (data not shown), This indicated that Raji cells were largely resistant not only to CD95 mediated cell death but -to some extent- also to antibody mediated cytotoxicity. When we assessed depletion of SKW6.4 cells and Raji cells in a flow cytometry based assay, again at a PBMC:target ratio of 10:1, similar observations were made. After 48 hours almost complete depletion of the SKW6.4 cells by all CD20 targeting antibodies was observed. At low concentrations (<10ng/ml) the Fc-optimized CD20 antibody was most effective, whereas at concentrations ≥100 ng/ml BS9520 was more efficient (Fig. 3). If Raji cells were used as targets, the activity of BS9520 was again largely reduced, whereas depletion by monospecific antibodies appears to be comparable to that obtained when SKW6.4 cells were used as target cells. Variability of ADCC activity and cell susceptibility might explain this discrepancy. In any case, normal B cells remain unaffected by the BS9520 construct in all experiments. In contrast, upon incubation with monospecific antibodies, normal B cells expressing high amounts of CD19 are depleted and a small population of “CD19-low” cells remains (Supplementary Fig. 4). These findings demonstrate a specific activity of BS9520 against malignant- rather than normal, resting B cells. When we compared the anti-tumor activity of the antibodies in SCID mice inoculated with SKW6.4 cells, we found that BS9520 was the most effective reagent (Fig. 4a). 6 of 8 animals survived until day 120 when the experiment was terminated. To compensate for its lower serum half-life we applied 3x20µg of BS9520 on subsequent days or a single dose of 60µg of the monospecific antibodies. When we measured the serum half-life of the antibodies in C57BL/6NRj mice, however (Fig. 4b), it became obvious that this dosing scheme did probably not fully compensate for the lower half-life of BS9520: in the case of the monospecific antibodies a serum concentration of 10µg/ml is easily maintained for >24hrs, whereas the concentration of BS9510 at that time has fallen below 1 µg/ml, the detection limit of the assay. Thus, the superior activity of the bispecific reagent in this experimental setting is all the more remarkable.

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Depletion of normal B-cells

Next we evaluated the cytolytic activity of the antibodies against normal B-cells within PBMC cultures that contained either resting cells or cells that had been activated for 6 days with pokeweed mitogen (PWM), a T-cell dependent B cell mitogen 20. To measure B cell depletion, a flow cytometry (FACS) based assay was developed that allows determination of the absolute number of remaining B and T-cells in antibody treated PBMC cultures. To illustrate this assay FACS plots of one representative donor are presented in Figs. 5a and 5b. Figs. 5c-5f summarize results obtained with 4 resting and 5 PWM activated cell cultures. SDIE20 effectively depleted B-cells in resting as well as activated cultures. Again, the activity of CH20 as well as that of Rituxan was clearly less pronounced and highly variable between different donors, in particular, if resting cells were used. Most likely, this variability is due to the known variations in the ADCC activity of the PBMCs of different healthy donors. BS9520 is only marginally effective against resting cells, however, it kills B-cells in PWM activated cultures very effectively. Interestingly, we noted that BS9520 in contrast to the monospecific antibodies

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affected not only B-cells but also T-cells present in the PBMC cultures, albeit to a lesser extent. Again, bystander killing of T cells by BS9520 was observed only in PWM activated cultures. The pattern of cytolytic activity against activated B and T-cells described above was also observed when early apoptosis was measured by staining with annexin V and subsequent analysis by flow cytometry (Fig. 6): killing of activated B-cells by the Fc-optimized mono- and the bispecific antibody was comparable, but only the bispecific reagent causes significant apoptosis of activated T-cells. Suppression of antibody production Finally we wanted to evaluate to what extent the activity of the various antibodies against normal B and T cells affects antibody production. Fig. 7 shows the suppression of panclonal and specific antibody production in PBMC cultures stimulated with PWM and tetanus toxoid (TT), respectively. For TT stimulation PBMC of freshly immunized donors were used. In all experiments with stimulated PBMC from 6 (PWM-stimulation) and 4 (TT-stimulation) donors, significant suppression by BS9520 of panclonal as well as tetanus-specific antibody production was observed. SDIE20 induced a moderate and variable suppression in PWM activated cultures, its activity against TT-stimulated cells was marginal. CH20 as well as Rituximab did not exert significant suppressive effects in any of the experiments.

DISCUSSION

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The selective activation of the CD95 death receptor is one of several attractive applications for bispecific antibodies. However, the production of such reagents in pharmaceutical quality and quantity remains a formidable challenge despite considerable progress in recombinant antibody technology21, 22. The development of BS9520 strikingly illustrates this problem: As many other antibodies, the CD95 antibody used aggregates if expressed in a single chain format irrespective of VH-VL orientation (data not shown). This precluded the construction of antibodies in the widely used bispecific single chain format, also termed BiTE. Only when we expressed the CD95 antibody as the N-terminal Fab part within the Fabsc-format (Fig. 1) a functional, monomeric bispecific antibody was obtained. This confirms our previous findings that the Fabsc-format is particularly suited for the generation of bispecific antibodies19. Comparing the activity of BS9520 to that of the monospecific CD20 antibodies one has to consider the different cytolytic mechanisms used by these reagents as well as their markedly different serum half-life: killing by monospecific antibodies requires the presence of FcR expressing effector cells, whereas BS9520 acts via CD95-mediated apoptosis in the absence of effector cells. Notably, however, the activity of BS9520 at low doses seems to be somewhat improved if effector cells are present, possibly due to the binding of the reagent to normal B cells and a resulting bystander killing of lymphoma cells18 In any case, comparison of the reagents requires information about the target:effector ratios used. For SDIE20 a PBMC:target ratio of approx. 10:1 is required to “match” the activity of BS9520 against malignant lymphoma cells at antibody concentrations. At low doses (<10ng/ml) the Fc-optimized CD20 antibody

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SDIE20 appears to be more effective than BS9520. At doses (≥100ng/ml) both reagents reach maximal and comparable activity levels in two different cytolytic assays. In the case of the monospecific antibodies, concentrations ≥100 ng/ml appear to be easily achievable for several days after a single dose application. However, with regard to the bispecific antibody, the maintenance of such serum concentrations for longer periods of time may require repeated applications. When we measured the outgrowth of SKW6.4 cells in SCID mice, we applied 3x20 and 1x60 µg of the bi- and monospecific antibodies, respectively. Although this dosing

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schedule did not fully compensate for the markedly lower serum half-life of the bispecific antibody its activity was clearly superior. We speculate that it is the number of FcR expressing effector cells at the site of the tumor that is limiting for the monopecific antibodies at least in the SCID mouse model used. In principle, however, there is a limiting factor for the antitumor activity of BS9520 as well. It is implied by the data presented in Fig. 2 and Fig. 3: In these experiments killing of Raji cells was undetectable and low, respectively,, although these cells express sufficient amounts of CD20 as well as CD95. It is well established that malignant cells may readily aquire resistance towards CD95-mediated killing by downregulation of CD95-expression or –sensitivity23-25. In particular, it has been reported that CD95-sensitivity of malignant lymphoma cells isolated from biopsies is null or weak24, 26. In contrast, normal B- and T-cells acquire CD95 expression andsensitivity during activation16. We are not aware of data that convincingly establish a general link between an autoimmune phenotype and the CD95 resistance of B or T cells. Thus, induction of CD95 mediated cell death as a therapeutic strategy appears to be particularly promising if directed against normal (including “autoimmune cells”) rather than malignant B cells. In addition, the specific expression of CD95 on activated rather than resting B- and Tcells offers the perspective of a specific activity towards activated cells. Our observations confirm that BS9520 meets this expectation: it appears to be active primarily against activated B-cells whereas the Fc-optimized, monospecific SDIE20 antibody depletes resting cells as well. The variation of activity between different donors, particularly pronounced in the case of the monospecific antibodies, is likely to be due to the known variation of ADCC activity in PBMC cultures of normal healthy donors. What was unexpected, however, was the superior suppression of antibody production by BS9520 although the activity of the Fc-optimized CD20 antibody against activated B-cells was comparable. This discrepancy might be due to bystander killing of activated T-cells by BS9520, a phenomenon readily explained by the previously reported bi-cellular binding of bispecific antibodies stimulating CD95. This phenomenon results in the killing of CD95 single positive cells when these cells make contact with CD20-single positive positive target cells carrying a bispecific antibody as demonstrated by Herrmann et al 18. Given the well-established helper function of T-cells during antibody production, is tentative to speculate, that the killing of activated bystander T-cells enhances the anti-B cell activity of BS9520. Conceptually, this would constitute an additional advantage of the bispecific vs. the monospecific antibodies. Since this type of lytic activity is restricted to activated (rather than resting) T cells in the neighborhood of CD20 expressing B cells, we do not expect it to result in untolerable T cell depletion upon in vivo application of the reagent. An additional explanation for the superior suppressive effect of BS9520 on antibody production might be, that the susceptibility of B-cells towards CD95 mediated killing may change during the process of B cell activation that lasts 6 days in the experiments described here. In this regard, we have noticed in preliminary experiments that the sensitivity of B-cells towards CD95 mediated cell death is steadily increasing in PWM activated PBMC cultures from day 3 to day 6. Moreover, it has been reported that the small subpopulation of peripheral blood B cells in immunized human subjects, capable of producing specific antibody, is sensitive to CD95 mediated cell death27. Such cells might be killed by BS9520 induced bystander lysis18 even if they have lost CD20 expression during differentiation into antibody producing cells. In any case, the superior suppressive effects of BS9520 on antibody production implies that this reagent may be particularly suitable for the treatment of B cell mediated autoimmune disease.

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METHODS Cells and reagents Peripheral blood mononuclear cells (PBMCs), isolated from heparinized blood of healthy donors by density gradient centrifugation (Biocoll separating solution, Biochrom, Berlin, Germany), SKW6.4- Daudi-, Jurkat-, C1R-, JY-, and Raji-cell lines (ATCC, Manassas, USA) were kept in RPMI 1640 (Life Technologies, Darmstadt, Germany), mouse Sp2/0-Ag14 cells (ATCC) in IMDM (Lonza, Basel, Switzerland). All media were supplemented with 10% heatinactivated fetal calf serum (Biochrom), 100 U/ml penicillin, 100 µg/ml streptomycin (SigmaAldrich, Hamburg, Germany), 1 mM sodium-pyruvate (Biochrom), non-essential amino-acids (Biochrom), 2 mM L-glutamine (Lonza) and 50 µM beta-mercaptoethanol (Merck, Darmstadt, Germany). Human cells lines were cultured at 37°C a nd 5% CO2, the mouse myeloma cell line Sp2/0-Ag14 and transfected Sp2/0 cells were propagated at 7.5% CO2. Clinical grade material (Roche, Basel, Switzerland) diluted in PBS was used in all experiments utilizing the Rituximab® antibody.

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Generation and purification of recombinant antibodies The variable domains of the 2H7 antibody (GenBank no.: M17954 and M17953) were synthesized de novo using the ligase chain reaction with overlapping oligonucleotides. For the generation of chimerized and Fc-optimized antibodies (amino-acid exchanges at S239D and I332E), the VJ and VDJ elements were amplified and cloned into a eukaryotic expression vector containing regulatory elements of the IgG locus, a human constant γ heavy- and κ light chain as described previously28. Heavy and light chain plasmids of the chimeric and optimized antibody constructs were linearized with AhdI and SfiI, respectively, and transfected into Sp2/0-Ag14 cells by electroporation. Antibodies were purified from culture supernatants of transfected Sp2/0 cells using protein A affinity chromatography (GE Healthcare, Munich, Germany). For construction of bispecific antibodies, the variable domains of the antibodies APO-1 (anti-CD95) and 9.2.27 (anti-chondroitin sulphate proteoglycan, CSPG4) were cloned from the respective hybridoma cells as previously described18 28. At the C-terminus of the Fab fragment of the APO-1 antibody a modified CH2 domain of human Igγ1 and the respective scFv-fragments of 2H7 or 9.2.27 were added. To abrogate FcR-binding, glycosylation sites and the formation of disulfide bonds the following modifications were introduced into the hinge region and the CH2 domain (EU-index): C226S; C229S; E233P; L234V; L235A; ∆G236; D265G; N297Q; A327Q; A330S. Bispecific Fabsc antibodies were purified from culture supernatants of transfected Sp2/0 cells by affinity chromatography on a KappaSelect column (GE Healthcare). The antibodies were analyzed by size exclusion chromatography on Superdex 200 using a SMART system equipped with a PC3.2/30 column (GE Healthcare). ADCC assay For the determination of antibody-dependent cellular cytotoxicity (ADCC) lymphoma target cells (SKW6.4, JY, C1R and Raji) were incubated with PBMC and varying concentrations of different antibodies for 24 h in 96 well plates and then pulsed with 0.5 µCi/well [methyl-3H]thymidine (Hartmann Analytics, Braunschweig, Germany). After 20 hrs cells were harvested on filter mats (Perkin Elmer, Waltham, MA, USA) and precipitated raioactivity was determined in a liquid scintillation counter (MicroBeta, Perkin Elmer). %inhibition of proliferation was calculated according to the formula: 100-(x/x0*100), where x and x0 are counts (cpm) measured in experimental wells (x) and in wells without antibodies (x0). Each data point represents the mean value of triplicate samples.

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Animal experiments All animal experiments were performed in accordance with the German animal protection law. Immunocompetent male C57BL/6NRj- and immunodeficient male C.B.-17 SCID-mice (CB17/lcr-PrkdcSCID/CRL) were purchased from Janvier (Le Genest-Saint-Isle, France) and Charles River (Sulzfeld, Germany), respectively. SCID mice were kept under specific pathogen free conditions. To determine survival after challenge with tumor cells groups of 8 C.B.-17 SCID mice were injected at day 0 with 1 x 107 SKW 6.4 cells i.v. Mice treated with CH20 and SDIE20 were injected i.p. with 60 µg of respective antibody at day 1, animals treated with BS9520 and BS95Mel with 20 µg at days 1, 2 and 3. Animals developing visible tumors, sense disturbances or paralysis of the hind legs were sacrificed and tumor growth was confirmed by necroscopy. After termination of the experiment at day 120 surviving mice were killed and subjected to necroscopy and histological examination. For determination of serum half-life C57BL/6 mice (3 animals per antibody) were injected i.v. with 50 µg of the respective antibodies. After 0.5, 1, 2 and 4 hrs (Fabsc) or 0.5, 3, 6 and 24 hrs (IgG-antibodies) mice were bled and serum was obtained. Serum concentrations were measured by incubating SKW 6.4 cells with prediluted serum samples. The amount of antibody bound to the cells was determined by flow cytometry and concentration values were calculated using a calibration curve obtained with samples that contained defined amounts of purified antibodies diluted in mouse serum (C57BL/6). Flow cytometry To measure binding avidity, antibodies were incubated in 96 well plates (1,5x105/well) for 30 minutes at 4°C with Daudi-(CD20+), Jurkat- (CD95+) ,SKMel-cells (CSPG4+) as well as resting and activated B cells . The cells were then stained with PE-conjugated goat-antihuman F(ab)2-fragments (Jackson ImmunoResearch, West Grove, USA), incubated for additional 30 minutes and analyzed by flow cytometry using a FACSCalibur™ (BD Biosciences). To determine antibody mediated killing of resting and activated B-cells, unstimulated and stimulated PBMC (6 days, 1 µg/ml pokeweed mitogen, Sigma-Aldrich) were adjusted to 2x106 cells/ml, incubated with antibodies (0.1 µg/ml) for 2 days in a 24 well plate and analyzed by flow cytometry. The lymphocyte population was defined by light scatter characteristics and the percentage of remaining B- and T-cells after Ab treatment was assessed after staining with CD4-FITC (clone HP2/6), CD8-APC (clone HIT8a, Biolegend, San Diego, CA, USA) and CD19-PacificBlue (clone HIB19, Biolegend). Dead cells were excluded by staining with 7Aminoactinomycin D (7-AAD BioLegend). All antibodies were incubated with cells for 30 minutes at 4°. By the acquisition of equal numbers of compensation particles (BD Biosciences) in each sample, absolute cell counts could be determined and used to calculate the percentage of remaining B- and T-cells. B-cell and T-cell gates were determined using FMO (‘fluorescence minus one’) controls 29, 30 with corresponding isotype controls purchased from Biolegend. Each data point represents the mean value of technical duplicates. To determine antibody activity against SKW6.4 or Raji cells, these cells (2x104/well) were incubated with PBMC from healthy donors (2x105/well ) in 96 well plates. After 48 hours cells were stained with CD19-PE and the absolute numbers of remaining target cells was determined as described above and related to control cultures without antibodies. In some experiments induction of apoptosis in activated B and T cells within PBMC cultures was measured after 4 hrs of antibody treatment (1 µg/ml). To this end, the cells were incubated with labeled antibodies (CD4-FITC, CD19 PacificBlue) washed, stained with PE-Annexin V (BioLegend) and analyzed by flow cytometry.

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In all FACS experiments binding to Fc receptors was blocked with Flebogamma (50 µg/ml, Grifols, Germany). Acquisitions were performed using the FACSCanto™ II cell analyzer. For data analysis the FlowJo software (TreeStar, USA) was used. Inhibition of antibody production by PWM- or antigen-stimulated PBMC Freshly isolated PBMC from normal donors or from individuals recently immunized with Tetanus toxoid (TT) were stimulated for 6 days with 1µg/ml PWM 16 and 25 ng/mL TT (Statens Serum Institute, Herredsvejen, Denmark), respectively, as described by Lum et al 31. Cells were then washed twice, adjusted to 3,2x106 cells/ml in 24 well plates and incubated with antibodies for 2 days. To prevent detection of the antibodies as newly produced human IgG in PWM stimulated cultures, antibody concentrations used were 0,1 µg/ml and 1 µg/ml in cultures stimulated with PWM and TT, respectively. This was appropriate, since we had noticed in preliminary experiments that (i) IgG production in PWM-stimulated PBMC cutures varied between 0.5 and 5 µg/ml under the experimental conditions used and (ii) a concentration of 0.1 µg/ml was almost as effective as 1 µg/ml in the depletion of B cells as well as in the suppression of antibody production. Panclonal human IgG and TT specific antibodies were measured in the cell culture supernatants by ELISA. To this end, an adapted sandwich procedure was performed in Maxisorp 96 well plates (Nunc, Roskilde, Denmark) that consisted of incubation steps with the following reagents, separated by extensive washing: (i) 1:1000 diluted goat anti-human IgG (Jackson ImmunoResearch laboratories, USA) and 5 µg/mL TT, respectively, in PBS. (ii) blocking solution containing 10% BSA in PBS, (iii) sample dilutions of PBMC supernatants, (iv) 1:10000 diluted biotinylated mouse anti-human IgG F(ab’)2 (Jackson Immunoresearch Laboratories, Bar Harbor, USA), (v) 1:5000 diluted poly-HRP Streptavidin (Immunotools, Friesoythe, Germany) (vi) TMB Microwell Peroxidase Substrate System (KPL, Gaithesburg, USA). Optical density at 450 nm was determined using a Spectra Max 340 ELISA reader (Molecular devices, Sunnyvale, USA). Antibody concentration were calculated using standard curves obtained by diluting human IgG (Flebogamma, Grifols, Germany) and Tetagam P (CSL Behring, Marburg, Germany), respectively, in culture medium.

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Figure legends

Figure 1 The three CD20-antibodies compared in this paper. (a) a chimeric antibody with variable regions derived from the 2H7 antibody and human constant domains, (b) the chimeric molecule with an optimized Fc-part, (c) a bispecific molecule in the Fabsc-format with an Nterminal CD95-specificity and a C-terminal single chain binding CD20. To prevent homodimerization and binding to Fc-receptors cysteins (x) and amino acid residues mediating FcR-binding (o), respectively, were replaced as described in the Materials and Methods section. As a control a bispecific Fabsc-antibody was produced that is directed to the melanoma associated antigen CSPG4 (BS95Mel). Figure 2 Antibody dependent cellular cytotoxicity against various lymphoma cell lines. PBMCs and various target cells were incubated with the indicated antibodies at PBMC:target ratios of 5:1 or 10:1 and pulsed after 24 hrs with 3H-thymidine. Mean values and standard deviations of triplicate samples are indicated.

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Figure 3 Depletion of (a) SKW 6.4 and (b) Raji lymphoma cells in the presence of PBMC. Cells were incubated with the indicated antibodies at PBMC:target ratios of 10:1 and analyzed after 48 hrs by flow cytometry as described in the Materials and Methods section. The mean values of technical duplicates are indicated. Representative results with one (of three) different healthy donors are shown

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Figure 4 Anti-tumor activity and serum elimination of the various anti CD20 antibodies. In (a) groups of eight C.B.-17 SCID mice were inoculated with 1x107 SKW6.4 lymphoma cells and treated after 24 hrs with 1x60 µg (day 1) and 3x20 µg (day 1,2,3, i.p.) of mono- and bispecific antibodies, respectively. Mice were killed when signs of lymphoma growth were present and tumor growth was confirmed by necroscopy. All long term surviving animals were sacrificed at day 120 and were subjected to a histological examination. In one animal of the BS9520 group local tumor growth in the thymus was detected. In (b) C57BL/6 mice were injected with 50 µg of the respective antibody i.v. and serum concentrations were determined at the indicated time points as described in the Materials and Methods section. Mean values and standard deviations obtained with three mice per time point are indicated.

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Figure 5 Depletion of B and T cells in resting and PWM-activated PBMC cultures. PBMC, either untreated (resting) or activated with 1 µg/ml PWM for six days, were incubated for two days with the indicated antibodies (0,1 µg/ml) and were then analyzed by flow cytometry as described in the Materials and Methods section. In (a) and (b) FACS analysis of B-cell depletion within PBMC cultures of one donor, marked with an open square in (c)-(f), is shown. In (c)-(f) results obtained with 4 resting (c,e) and 5 activated (d,f) PBMC cultures from different healthy donors are summarized. Symbols indicate the mean values of technical duplicates. Figure 6 Annexin V staining of CD19+ and CD4+ cells in activated PBMC cultures of one donor (marked with filled circles in Fig. 4) were incubated with the respective antibodies (1 µg/mL) and stained with Annexin V. After 4h cells were analyzed by flow cytometry as described in the Methods section. One representative experiment of three is shown.

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Figure 7 Suppression of panclonal and specific IgG production in PBMC cultures by various antibodies. PBMC of different healthy donors were stimulated for six days with PWM (a) and tetanus toxoid (b). Cells were then washed and incubated for two days with the indicated antibodies. Antibody production in the culture supernatants was estimated by ELISA as described in the Materials and Methods section. Supplementary Figure 1 Size exclusion chromatography of the recombinant BS9520 antibody. ~10 µg of purified antibody were separated using a Superdex 200 column (PC3.2/30). The peak retention volume of the following calibration proteins is indicated: catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa) and ribonuclease A (13,7 kDa). Supplementary Figure 2 Binding of antibodies to CD20+ Daudi (a) CD95+ Jurkat cells (b). Supplementary Figure 3 Binding of antibodies to (a) resting and (b) PWM activated B-cells.

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Supplementary Figure 4 Depletion of SKW 6.4 lymphoma cells in the presence of PBMC. Cells were co-cultured in the presence of the indicated antibodies at different concentrations (indicated on the right) and analyzed after 48 hrs by flow cytometry. SKW 6.4 were defined as FSC-A high and CD19+ (large gate), B-cells as FSC-A low and CD19+ (small gate).

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Supplementary Figure 5 Inhibition of proliferation of different lymphoma cells in the absence of effector cells. Cells were incubated with the indicated antibodies and pulsed after 24 hrs with 3 H-thymidine for additional 20 hrs. Mean values and standard deviations of triplicate samples are indicated.

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