Bispecific antibodies against CD3 and CD20

Abstract

Bispecific antibodies directed to CD3 and CD20 and uses of such bispecific antibodies, in particular use thereof in the treatment of diseases in which specific targeting and T cell-mediated killing of cells that express CD20 is desired.

Description

(19) *EP004678661A2* (11) EP 4 678 661 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 14.01.2026 Bulletin 2026 / 03 (21) Application number: 25189898.7 (22) Date of filing: 08.01.2016 (51) International Patent Classification (IPC): C07K 16 / 30 (2006.01) (52) Cooperative Patent Classification (CPC): C07K 16 / 2809; A61P 35 / 00; A61P 35 / 02; C07K 16 / 2887; C07K 16 / 30; A61K 2039 / 505; C07K 2317 / 24; C07K 2317 / 31; C07K 2317 / 567; C07K 2317 / 73; C07K 2317 / 732; C07K 2317 / 75; C07K 2317 / 90; C07K 2317 / 92; C07K 2317 / 94 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR Designated Extension States: BA ME (30) Priority: 08.01.2015 PCT / EP2015 / 050276 15.07.2015 DK PA201500412 15.07.2015 DK PA201500413 16.07.2015 DK PA201500416 16.07.2015 DK PA201500415 (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 16700133.8 / 3 242 682 (71) Applicant: Genmab A / S 2500 Valby (DK) (72) Inventors: • ENGELBERTS, Patrick 3584 CT Utrecht (NL) • BREIJ, Esther, C. W. 3972 DK Driebergen (NL) • RADEMAKER, Rik 3584 CT Utrecht (NL) • ALTINTAS, Isil 3584 CT Utrecht (NL) • SATIJN, David 3584 CT Utrecht (NL) • VERPLOEGEN, Sandra 3584 CT Utrecht (NL) • RADERSMA, Riemke Van Dijkhuizen 3584 CT Utrecht (NL) • VAN DEN BRINK, Edward, N. 3584 CT Utrecht (NL) • SCHUURMAN, Janine 1111 CH Diemen (NL) • PARREN, Paul 3984 PR Odijk (NL) (74) Representative: Genmab A / S Carl Jacobsens Vej 30 2500 Valby (DK) Remarks: •The complete document including Reference Table(s) and the Sequence Listing(s) can be downloaded from the EPO website •This application was filed on 16.07.2025 as a divisional application to the application mentioned under INID code 62. •Claims filed after the date of filing of the application / after the date of receipt of the divisional application (Rule 68(4) EPC). (54) BISPECIFIC ANTIBODIES AGAINST CD3 AND CD20 (57) Bispecific antibodies directed to CD3 and CD20 and uses of such bispecific antibodies, in particular use thereof in the treatment of diseases in which specific targeting and Tcell-mediated killing of cells that express CD20 is desired. EP 4 67 8 66 1 A 2 Processed by Luminess, 75001 PARIS (FR) Description FIELD OF THE INVENTION

[0001] The present invention relates to bispecific antibodies directed to CD3 and CD20 and to uses of such bispecific antibodies, in particular use thereof in the treatment of diseases in which specific targeting and Tcell-mediated killing of cells that express CD20 is desired. BACKGROUND OF THE INVENTION

[0002] CD3 has been known for many years and therefore has been subject of interest in many aspects. Specifically antibodies raisedagainstCD3or theT-cellReceptorComplex,whichCD3 is part of, are known.An in vitrocharacterization of five humanized OKT3 effector function variant antibodies has been described (Xu et al., 2000, Cell Immunol. 200(1):16‑26).

[0003] Treatment with the anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in improved C-peptide responses and clinical parameters for at least 2 years after onset of type 1 diabetes in absence of continued immuno- suppressive medications (Herold et al., 2005, Diabetes, 54(6):1763‑9).

[0004] CD3 antibodies cross-reactive to cynomolgus and / or rhesus monkey CD3 have been described (WO2012162067, WO2008119567).

[0005] A promising approach to improve targeted antibody therapy is by delivering cytotoxic cells specifically to the antigen-expressing cancer cells. This concept of using T-cells for efficient killing of tumor cells has been described in Staerz, et. al., 1985, Nature 314:628‑631). However, initial clinical studies were rather disappointing mainly due to low efficacy, severeadverseeffects (cytokine storm)and immunogenicity of thebispecificantibodies (Muller andKontermann, 2010, BioDrugs 24: 89‑98). Advances in the design and application of bispecific antibodies have partially overcome the initial barrier of cytokine stormand improved clinical effectivenesswithout dose-limiting toxicities (Garber, 2014, Nat. Rev. DrugDiscov. 13: 799‑801;LumandThakur, 2011, BioDrugs25: 365‑379). Critical to overcome the initial barrier of cytokine storm as described for catumaxomab (Berek et al. 2014, Int. J. Gynecol. Cancer 24(9): 1583‑1589; Mau-Sørensen et al. 2015, Cancer Chemother. Pharmacol. 75: 1065‑1073), was the absence or silencing of the Fc domain.

[0006] The CD20 molecule (also called human B-lymphocyte-restricted differentiation antigen or Bp35) is a hydro- phobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al. (1989) J. Biol. Chem. 264(19):11282‑11287; and Einfield et al., (1988) EMBO J. 7(3):711‑717). CD20 is found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs and isexpressedduringearlypre-Bcell developmentand remainsuntil plasmacell differentiation.CD20 ispresentonboth normal B cells as well as malignant B cells. In particular, CD20 is expressed on greater than 90% of B cell non-Hodgkin’s lymphomas (NHL) (Anderson et al. (1984) Blood 63(6):1424‑1433), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder et al. (1985) J. Immunol. 135(2):973‑979).

[0007] Methods for treatingcanceraswell asautoimmuneand immunediseasesby targetingCD20areknown in theart. For example, the chimeric CD20 antibody rituximab has been used for or suggested for use in treating cancers such as non-Hodgkin’s lymphoma (NHL), chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). The humanmonoclonal CD20 antibody ofatumumab has been used for or suggested for use in treating among others various CLL indications, follicular lymphoma (FL), neuromyelitis optica (NMO), diffuse and relapsing-remitting multiple sclerosis (RRMS). The human monoclonal CD20 antibody obinutuzumab has been used for or suggested for use in treating CLL. Furthermore, the humanized CD20 antibody ocrelizumab is being developed for RRMS.

[0008] Gall et al. (2005ExperimentalHematology33: 452)disclose theCD3xCD20bispecificantibodyCD20bi resulting from the chemical heteroconjugation of theCD20-specific chimeric antibodyRituximab (Rituxan) to anti-CD3 (Orthoclone OKT‑3).

[0009] Stanglmaier et al. (2008 Int. J. Cancer: 123, 1181) describe the trifunctional bispecific anti-CD3xanti-CD20 antibody Bi20 / FBTA05 combining a CD20-specific mouse IgG2a and a CD3-specific rat IgG2b.

[0010] Wuet al. (2007 Nat Biotechnol. 25: 1290‑1297) andWO2011014659 describe a dual-specific (CD3 and CD20), tetravalent immunoglobulin G (dual-variable-domain immunoglobulin, DVD-Ig).

[0011] WO2011090762 describes the generation of a CD3xCD20 polypeptide heterodimer.

[0012] WO2011028952 describes amongst others the generation of CD3xCD20 bispecific molecules using Xencor’s XmAb bispecific Fc domain technology.

[0013] WO2014047231 describesREGN1979andotherCD3xCD20bispecific antibodies generated using theFcΔAdp technology from Regeneron Pharmaceuticals.

[0014] Sun et al. (2015, Science Translational Medicine 7, 287ra70) describe a B cell-targeting anti-CD20 / CD3 Tcell- dependent bispecific antibody constructed using "knobs-into-holes" technology.

[0015] Bispecific antibodies that bind to both CD3 and CD20 may be useful in therapeutic settings in which specific 2 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 targeting and T cell-mediated killing of cells that express CD20 is desired, and there is still a need for further efficient CD3xCD20 bispecific antibodies. SUMMARY OF THE INVENTION

[0016] It is an object of the present invention to provide novel efficient bispecific antibodies comprising a first antigen- binding region derived from a CD3 antibody and a second antigen-binding region derived from a CD20 antibody.

[0017] ThenovelCD3xCD20bispecificantibodiesareuseful in therapeutic settings inwhichspecific targetingandTcell- mediated killing of cells that express CD20 is desired. The novel CD3xCD20 bispecific antibodies are highly efficient in killing CD20 expressing cells, including cells with low CD20 copy numbers, and have been shown to be highly potent in eradicating tumor cells in animalmodels. The novel CD3xCD20 bispecific antibodies are advantageous by inducing rapid and strong killing of cells at low dosing. The novel CD3xCD20 bispecific antibodies are furthermore capable of inducing cytotoxicity by both CD4+ Tcells andCD8+ Tcells whichmakes them suitable for engaging Tcells for killing CD20 positive tumors and other diseases involving CD20 positive cells. In addition, the CD3xCD20 bispecific antibodies are efficient in depleting B cells from lymphoid structures. Accordingly, it is an object of the present invention to provide a bispecific CD3xCD20 antibodywhich is capable of inducing cytotoxicity by bothCD4+ Tcells andCD8+ Tcells. It is a further object of the present invention to provide a bispecific CD3xCD20 antibody which is highly efficient in killing CD20 expressing cells such as CD20 expressing tumor cells. It is a further object of the present invention to provide a bispecific CD3xCD20 antibody which is highly efficient in killing CD20 expressing cancers.

[0018] These and other aspects of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: Binding of bispecific CD3xCD20 antibodies to Daudi (A-F) and Jurkat (G-I) cells. (A) Binding of bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR and IgG1‑7D8, (B) Binding of bsIgG1-huCD3-H1L1- FEALxCD20‑2F2-FEAR and IgG1‑2F2, (C) Binding of bsIgG1-huCD3-H1L1-FEALxCD20-RTX-FEAR and IgG1- RTX, (D) Binding of bsIgG1-huCD3-H1L1-FEALxCD20‑11B8-FEAR and IgG1‑11B8, (E) Binding of bsIgG1-huCD3- H1L1-FEALxCD20-GA101-FEAR and IgG1-GA101, (F) Binding of bsIgG1-huCD3-H1L1-FEALxCD20‑2C6-FEAR (and IgG1‑7D8). Data shown are geometric means of fluorescence intensity (geomean) (A-E) and median fluores- cence intensity (F) of binding toDaudi cells, as determinedby flowcytometry, for two representative experiments (A-E from one, F from the other). (G) Binding of bsIgG1-huCLB-T3 / 4-FEALxCD20‑7D8-FEAR (huCLB-T3 / 4x7D8), bsIgG1-huCLB-T3 / 4-FEALxCD20‑2F2-FEAR (huCLB-T3 / 4x2F2), bsIgG1-huCLB-T3 / 4-FEALxCD20-GA101-FEAR (huCLB-T3 / 4xGA101), bsIgG1-huCLB-T3 / 4-FEALxCD20‑11B8-FEAR (huCLB-T3 / 4x11B8) and monospecific biva- lent IgG1‑7D8-FEAR toDaudi cells, (H)Bindingof bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR, bsIgG1-huCD3-H 1L1-FEALxCD20‑2F2-FEAR, bsIgG1-huCD3-H1L1-FEALxCD20-GA101-FEAR, bsIgG1-huCD3-H1L1- FEALxCD20-RTX-FEAR, bsIgG1-huCD3-H1L1-FEALxCD20‑11B8-FEAR, bsIgG1-huCD3-H1L1- FEALxCD20‑2C6-FEAR, bsIgG1-huCD3-H1L1-FEALxb12-FEAR and monospecific, bivalent IgG1-huCD3-H1L1- FEAL to Jurkat cells (I) Binding of bsIgG1-huCLB- T3 / 4-FEALxCD20‑7D8-FEAR, bsIgG1-huCLB-T3 / 4- FEALxCD20‑2F2-FEAR. bsIgG1-huCLB-T3 / 4-FEALxCD20-GA101-FEAR, bsIgG1-huCLB-T3 / 4-FEALxCD20- RTX-FEAR, bsIgG1-huCLB-T3 / 4-FEALxCD20‑11B8-FEAR, bsIgG1-huCLB- T3 / 4-FEALxCD20‑2C6-FEAR, bsIgG1-huCLB-T3 / 4-FEALxb12-FEAR and monospecific, bivalent IgG1-huCLB-T3 / 4-FEAL to Jurkat cells. Data shown are median fluorescence intensity, as determined by flow cytometry, of one representative experiment. Figure 2: Concentration-dependent simultaneous binding of bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR (CD3xCD20) to T cells and B cells. Simultaneous binding of the bispecific antibody bsIgG1-huCD3-H1L1- FEALxCD20‑7D8-FEAR (CD3xCD20) to B and T cells in blood was analyzed by flow cytometry. Data shown are from one representative experiment. Data shown are the number of double-positive (CD19 andCD4 [A] or CD19 and CD8 [B])events, asdeterminedby thenumberofevents in theupper right quadrantof theCD4 / CD19or theCD8 / CD19 flow cytometry dot-plot. Closed and open symbols indicate data fromdifferent healthy donors. IgG1‑2F2 (2F2, CD20- specific) and bsIgG1-huCD3-H1L1-FEALxb12-FEAR (CD3xb12, CD3-specific) were included as negative control antibodies. Figure 3: Inductionof cytotoxicity in vitroby CD3xCD20 bispecific antibodies inhuman B-cell lymphoma and B cell leukemia cell lines. (A) Daudi cells were incubated with bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR (CD3x7D8), bsIgG1-huCD3-H1L1-FEALxCD20‑11B8-FEAR (CD3x11B8), the monospecific CD20 antibodies IgG1‑7D8 (7D8), IgG1‑7D8-FEAR (7D8-FEAR; with inactive Fc region), IgG1‑11B8-F405L or IgG1‑11B8-FEAR (11B8-FEAR; with inactive Fc region), and the bispecific control antibody bsIgG1-huCD3-H1L1-FEALxb12-FEAR (CD3xb12) PBMCs were used as effector cells. (B) Daudi cells were incubated with BsIgG1-huCD3-H1L1- 3 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 FEALxCD20‑7D8-FEAR(CD3x7D8), themonospecificCD20antibody IgG1‑7D8-FEAR(7D8-FEAR;with inactiveFc region) and bsIgG1-huCD3-H1L1-FEALxb12-FEAR (CD3xb12), purified T cells were used as effector cells. (C-F) Daudi cells were incubated with CD3xCD20 bispecific antibodies based on two different CD3 arms (huCD3-H1L1- FEAL and huCLB-T3 / 4-FEAL Fab arm, represented by open and closed symbols, respectively) and four different CD20 arms: 7D8 (BsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR and BsIgG1-huCLB-T3 / 4-FEALxCD20‑7D8- FEAR; [C]), 11B8 (BsIgG1-huCD3-H1L1-FEALxCD20‑11B8-FEAR and BsIgG1-huCLB-T3 / 4-FEALxCD20‑11B8- FEAR; [D]), GA101 (BsIgG1-huCD3-H1L1-FEALxCD20-GA101-FEAR and BsIgG1-huCLB-T3 / 4-FEALxCD20- GA101-FEAR; [E]) and 2F2 (BsIgG1-huCD3-H1L1-FEALxCD20‑2F2-FEAR and BsIgG1-huCLB-T3 / 4- FEALxCD20‑2F2-FEAR; [F]). (G-M) Different B-cell lines were used as target cells and incubated with antibodies as indicated. CD3xCD20 bispecific antibodies contained the huCD3-H1L1-FEAL Fab arm and different CD20 Fab arms (7D8, 11B8, 2F2,GA101orRTX)as indicated.CD3antibodyalonewas IgG1-huCD3-H1L1.PurifiedTcellswere used as effector cells. (N) Daudi cells were incubated with bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR (CD3x7D8), bsIgG1-huCD3-H1L1-FEALxCD20‑2C6-FEAR (CD3x2C6) and bsIgG1-huCD3-H1L1-FEALxb12- FEAR (CD3xb12), purified T cells were used as effector cells. Data shown are mean percentages specific lysis ± S.D of triplicate wells and data for each graph were obtained from one representative experiment. Figure 4: Dose-dependent induction of cytotoxicity in vitro by bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR using purified total T cells (CD3+), CD4+ T cells (CD3+CD8-) and CD8+ T cells (CD3+CD8+) as effector cells. Daudi cells were incubated with the different T-cell subsets, as indicated, and a dilution series of bsIgG1-huCD3- H1L1-FEALxCD20‑7D8-FEAR or the control antibodies IgG1-huCD3-H1L1-FEALxb12-FEAR and IgG1‑7D8-FEAR (data not shown). Data shown are mean percentages of tumor cell lysis ± S.E.M. of triplicate wells (A, B). Figure 5: Kinetics of bsIgG1-huCLB-T3 / 4-FEALxCD20‑7D8-FEAR-dependent cytoxicity in Daudi cells.Daudi cells were incubated with bsIgG1-huCLB-T3 / 4-FEALxCD20‑7D8-FEAR in presence of purified Tcells isolated from twodifferent donors (AandB).Cytotoxicitywasassessedafter4, 12,24,48and72hoursof incubation (A)orafter3,16 and 24 hours of incubation (B). Data shown are percentages cell kill ± S.D. of triplicate wells of cells incubated with bsIgG1-huCLB-T3 / 4-FEALxCD20‑7D8-FEAR for different incubation times. Data shown in A and B are from two independent experiments, using purified T cells isolated from two different donors. Figure 6: Efficacy of induction of cytotoxicity in vitro by CD3xCD20 bispecific antibodies at different effector to target ratios.Acytotoxicity assaywasperformedusingdifferentCD3xCD20bispecific antibodiesanddifferentE / T ratios. CD3xCD20 bispecific antibodies included bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR (A), bsIgG1- huCD3-H1L1-FEALxCD20‑11B8-FEAR (B), bsIgG1-huCD3-H1L1-FEALxCD20-GA101-FEAR (C), bsIgG1- huCD3-H1L1-FEALxCD20-RTX-FEAR (D) and bsIgG1-huCD3-H1L1-FEALxCD20‑2F2-FEAR (E). As a negative control, bsIG1-huCLB-T3 / 4-FEALxb12-FEARwas includedatanE / Tratioof 10:1.Datashownaremeanpercentages lysis ± S.D. of triplicate wells as determined in a cytotoxicity assay for one representative experiment. Each line represents a different E / T ratio (as indicated). Figure 7: Cytotoxic activivity of CD3xCD20 bispecific antibodies in the Raji-luc co-engraftment model in NOD-SCID mice. (A) Average tumor size in mice that were treated with vehicle (PBS) or bsIgG1-huCD3-H1L1- FEALxCD20‑7D8-FEAR at the indicated doses. Error bars indicate S.E.M. (B) Tumor size in individual mice after treatment with PBS or bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR on day 21 after tumor inoculation. Statistical analysis of dataat day21wasperformedusingKruskalWallis (Dunn’smultiple comparisonaspost-test). **p<0.01 (C) Kaplan-Meier plotswith tumorsize cut-offset at 600mm3.Statistical significanceof differences in survival between the bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR-treated groups and the PBS-treated control groupwere assessed by Mantel Cox analysis. *p<0.05, **p<0.01, n.s. not significant (D) Average tumor size in mice that were treated with vehicle (PBS) or bsIgG1-huCLB-T3 / 4-FEALxCD20‑7D8-FEAR at the indicated doses. Error bars indicate S.E.M. (E) Tumor size in individual mice in the different treatment groups on day 25 after tumor inoculation. Statistical analysis was performed using Kruskal Wallis test (Dunn’s multiple comparison as post-test) (F) Kaplan-Meier plots with tumorsize cut-off set at 500 mm3. Statistical significance of differences between treatment groups and the vehicle control groupwasanalysed byMantel Cox analysis. *p<0.05, ** p< 0.01, n.s. not significant. (G) Average tumor size in mice that were treated with vehicle (PBS), bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR or bsIgG1-huCD3-H1L1- FEALxCD20‑11B8-FEAR at the indicated doses. Error bars indicate S.E.M. (H) Tumor size in individual mice after treatment with PBS, or bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR or bsIgG1-huCD3-H1L1-FEALxCD20‑11B8- FEAR on day 20 after tumor inoculation. Statistical analysis of data at day 20 was performed using one-way ANOVA (Tukey’smultiple comparison as post-test). *p<0.05, **p<0.01 (I) Kaplan-Meier plots with tumorsize cut-off set at 500 mm3. Statistical significance of differences in survival between the bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR treated groups and thePBS-treated control groupwere assessed byMantel Cox analysis. *p<0.05, **p<0.01, n.s. not significant. Figure 8: Anti-tumor activity of bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR in a Daudi-luc xenograft model in HIS mice. (A) Average tumor size in the Daudi-luc xenograft model in BRGS-HIS mice after treatment with PBS (vehicle control), bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR (mAb2) or the control bispecific antibody bsIgG1- 4 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 huCD3-H1L1-FEALxb12-FEAR (mAb1) at the indicated dose levels. Tumor burden was assessed by biolumines- cence imaging. Error bars indicate S.E.M. (B) Statistical analysis was performed at day 21 (Kruskal Wallis test followed by Dunn’s multiple comparison post-test) ** p< 0.01 (C) Characterization of peripheral blood leukocyte populations in Daudi-luc xenograft-bearing BRGS-HIS mice after treatment with bsIgG1-huCD3-H1L1- FEALxCD20‑7D8-FEAR or the control bispecific antibody bsIgG1-huCD3-H1L1-FEALxb12-FEAR, as determined by flow cytometry at day 9. %hCD45+ cells represents the total percentage of human leukocytes in mouse peripheral blood. %hCD19+, %hCD3+ and %hCD3+FSChi represent the percentage of B cells, T cells and activated T cells, respectively, within the human leukocyte population. Figure 9: Study of effects of a single dose of bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR in cynomolgus monkeys.B-cell counts (CD19+CD21+ cells) (A) and T-cell counts (CD4+ plus CD8+ cells) (B) over time in peripheral blood of cynomolgus monkeys treated with different doses of bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR (0.01, 0.1, 1 or 10mg / kg). Days ‑18 and ‑11 show pre-dose B‑ and T-cell counts. B cells (C) and Tcells (D) as percentage of the total lymphocyte population over time in lymph node samples from cynomolgus monkeys treated with different doses of bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR (0.01, 0.1, 1 or 10 mg / kg). Plasma levels of IL‑2, IL‑6, IL‑8, IL‑10, IFN-γ and TNF-α in cynomolgus monkeys treated with different doses of bsIgG1-huCD3-H1L1- FEALxCD20‑7D8-FEAR (0.01, 0.1, 1 or 10 mg / kg) (E). Pharmacokinetic profile of bsIgG1-huCD3-H1L1- FEALxCD20‑7D8-FEAR in blood samples at pre-dose and at various time-points after dosing up to 70 days (F). The dotted line shows the predicted pharmacokinetic profile of IgG1, using a two-compartment model, with k10 (clearance constant) at 0.006 h‑1, Vc (plasma volume) 40 mL·kg‑1 and 3.5 kg bodyweight. Figure 10: Binding of bispecific CD3xCD20 antibodies to wild type CD20 and CD20-AxP expressed in HEK293F cells. Binding of CD3xCD20 bispecific antibodies (bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR [huCD3x7D8], bsIgG1-huCLB-T3 / 4-FEALxCD20‑7D8-FEAR [huCLB-T3 / 4x7D8], bsIgG1-huCD3-H1L1- FEALxCD20‑2F2-FEAR [huCD3x2F2], bsIgG1-huCLB-T3 / 4-FEALxCD20‑2F2-FEAR [huCLB-T3 / 4x2F2], bsIgG1- huCD3-H1L1-FEALxCD20‑11B8-FEAR [huCD3x11B8], bsIgG1-huCLB-T3 / 4-FEALxCD20‑11B8-FEAR [huCLB- T3 / 4x11B8], bsIgG1-huCD3-H1L1-FEALxCD20-GA101-FEAR [huCD3xGA101], bsIgG1-huCD3-H1L1- FEALxCD20-RTX-FEAR [huCD3xRTX], bsIgG1-huCD3-H1L1-FEALxCD20‑2C6-FEAR [huCD3x2C6]) to wild type CD20 (A) and CD20 mutant (CD20-AxP) (B) expressed in HEK293F cells was measured by flow cytometry. Data shown are mean fluorescence intensities of one representative experiment. Figure 11: T cell activation upon incubation of PBMC with bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR.

[0020] Healthy donor PBMC were incubated with bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR or positive and negative control antibodies, andTcell activationwasassessed bymeasuringCD69expressionwithin theTcell population (CD28+ cells). Experiments were performed with PBMC isolated from five healthy donors. Results for two representative donors are shown. TABLE 1 SEQ ID NO: Clone name Sequence SEQ ID NO:1 huCD3 VH CDR1 GFTFNTYA SEQ ID NO:2 huCD3 VH CDR2 IRSKYNNYAT SEQ ID NO:3 huCD3 VH CDR3 VRHGNFGNSYVSWFAY SEQ ID NO:4 huCD3 VL CDR1 TGAVTTSNY huCD3 VL CDR2 GTN SEQ ID NO:5 huCD3 VL CDR3 ALWYSNLWV 5 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:6 huCD3 VH1 SEQ ID NO:7 huCD3 VH2 SEQ ID NO:8 huCD3 VH3 SEQ ID NO:9 huCD3 VH4 SEQ ID NO:10 huCD3 VL1 SEQ ID NO:11 huCD3 VL2 SEQ ID NO:12 huCD3 VL3 SEQ ID NO:13 Mature humanCD3ε (epsilon) 6 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:14 Human CD3δ (delta) SEQ ID NO:15 IgG1m(f) heavy chain con- stant region (amino acids po- sitions 118‑447 according to EU numbering) SEQ ID NO:16 IgG1m(f)‑LFLEDA heavy chain constant region (amino acids positions 118‑447 ac- cording to EU numbering) SEQ ID NO:17 VH huCLB-T3 / 4 SEQ ID NO:18 VL huCLB-T3 / 4 SEQ ID NO:19 Mature cyno CD3ε (epsilon) 7 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:20 Mature rhesus CD3ε (epsilon) SEQ ID NO:21 IgG1m(f)‑F405L (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:22 IgG1m(f)‑K409R (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:23 IgG1m(f)‑LFLEDA-F405L (FEAL) (amino acids posi- tions 118‑447 according to EU numbering) 8 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:24 IgG1m(f)‑LFLEDA-K409R (FEAR) (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:25 Parent murine VH of SP34 SEQ ID NO:26 Parent murine VL of SP34 SEQ ID NO:27 VH CD20 - 7D8 SEQ ID NO:28 VL CD20 - 7D8 SEQ ID NO:29 Human IgLC2 / IgLC3 constant domain SEQ ID NO:30 VL huCD3-LKNH SEQ ID NO:31 VL huCD3-T41K SEQ ID NO:32 VH CD20 - 7D8 CDR1 GFTFHDYA 9 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:33 VH CD20 - 7D8 CDR2 ISWNSGTI SEQ ID NO:34 VH CD20 - 7D8 CDR3 AKDIQYGNYYYGMDV SEQ ID NO:35 VL CD20 - 7D8 CDR1 QSVSSY VL CD20 - 7D8 CDR2 DAS SEQ ID NO:36 VL CD20 - 7D8 CDR3 QQRSNWPIT SEQ ID NO:37 VH CD20 - 2F2 SEQ ID NO:28 VL CD20 - 2F2 SEQ ID NO:38 VH CD20 - 2F2 CDR1 GFTFNDYA SEQ ID NO:39 VH CD20 - 2F2 CDR2 ISWNSGSI SEQ ID NO:34 VH CD20 - 2F2 CDR3 AKDIQYGNYYYGMDV SEQ ID NO:35 VL CD20 - 2F2 CDR1 QSVSSY VL CD20 - 2F2 CDR2 DAS SEQ ID NO:36 VL CD20 - 2F2 CDR3 QQRSNWPIT SEQ ID NO:40 VH CD20 - 11B8 10 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:41 VL CD20 - 11B8 SEQ ID NO:42 VH CD20 - 11B8 CDR1 GFTFSYHA SEQ ID NO:43 VH CD20 - 11B8 CDR2 IGTGGVT SEQ ID NO:44 VH CD20 - 11B8 CDR3 ARDYYGAGSFYDGLYGMDV SEQ ID NO:45 VL CD20 - 11B8 CDR1 QSVSSY VL CD20 - 11B8 CDR2 DAS SEQ ID NO:46 VL CD20 - 11B8 CDR3 QQRSDWPLT SEQ ID NO:47 VH CD20 - 2C6 SEQ ID NO:48 VL CD20 - 2C6 SEQ ID NO:49 VH CD20 - 2C6 CDR1 GFTFGDYT SEQ ID NO:50 VH CD20 - 2C6 CDR2 ISWNSGSI SEQ ID NO:51 VH CD20 - 2C6 CDR3 TKDNQYGSGSTYGLGV SEQ ID NO:52 VL CD20 - 2C6 CDR1 QSVSSY VL CD20 - 2C6 CDR2 DAS SEQ ID NO:53 VL CD20 - 2C6 CDR3 QQRSNWPLT 11 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:54 VL huCD3-CDR3 L97H ALWYSNHWV SEQ ID NO:55 huCLB-T3 / 4 VH CDR1 GFTFSSYG SEQ ID NO:56 huCLB-T3 / 4 VH CDR2 ISRYSRYI SEQ ID NO:57 huCLB-T3 / 4 VH CDR3 ARRPLYGSSPDY SEQ ID NO:58 huCLB-T3 / 4 VL CDR1 SSVTY huCLB-T3 / 4 VL CDR2 DTS SEQ ID NO:59 huCLB-T3 / 4 VL CDR3 FQGSGYPLT SEQ ID NO:60 IgG1m(a) CH3 region SEQ ID NO:61 IgG1m(f) CH3 region SEQ ID NO:62 IgG1m(ax) CH3 region SEQ ID NO:63 IgG1 heavy chain constant region - WT (amino acids positions 118‑447 according to EU numbering) 12 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:64 IgG1 heavy chain constant region - F405L (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:65 IgG1 heavy chain constant region - K409R (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:66 IgG1 heavy chain constant region - N297Q (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:67 IgG1 heavy chain constant region - LFLEDANQPS (amino acids positions 118‑447 according to EU numbering) 13 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:68 IgG1 heavy chain constant region - F405L N297Q (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:69 IgG1 heavy chain constant region - K409R N297Q (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:70 IgG1 heavy chain constant region - F405L LFLE- DANQPS (amino acids positions 118‑447 according to EU numbering) SEQ ID NO:71 IgG1 heavy chain constant region - K409R LFLE- DANQPS (amino acids posi- tions 118‑447 according to EU numbering) 14 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Clone name Sequence SEQ ID NO:72 huCD3 VH CDR1 affinity var- iant GFTFX1T YA, wherein X1 is selected from V, H, F, T, P, L, Q, D, K, W, S, G, A, C and R SEQ ID NO:73 huCD3 VH CDR1 affinity var- iant GFTFNX2YA, wherein X2 is selected from S, N, G, A, K, V, R, H, Q, P, I, F, M, Y, L, W, D, E and C SEQ ID NO:74 huCD3 VH CDR1 affinity var- iant GFTFNTX3A, wherein X3 is selected from F, H, N, M, W, G, Q, V, T, S, L, P, I, A, K, R and C SEQ ID NO:75 huCD3 VH CDR2 affinity var- iant IRSKYNX4YAT, wherein X4 is selected from S, Y, Q, W, L, A, I, M, D, T, K, R, G, F, E, V, C and P SEQ ID NO:76 huCD3 VH CDR2 affinity var- iant IRSKYNNYX5T, wherein X5 is selected from N, L, Y, W, H, M, G, F, K, S, V, R, Q, D, C, E, P and T SEQ ID NO:77 huCD3 VH CDR3 affinity var- iant VRX6GNFGNSYVSWFAY, wherein X6 is selected from A, S, V, N, K, L, T, I, P, Q, C, G, Y, W, F, and R SEQ ID NO:78 huCD3 VH CDR3 affinity var- iant VRHGNFX7NSYVSWFAY, wherein X7 is selected from P, Q, A, Y, H, I, N, V, E, L, F, W, M, R, C, S and T SEQ ID NO:79 huCD3 VH CDR3 affinity var- iant VRHGNFGNSYVX8WFAY, wherein X8 is selected from A, T, G, L, N, C, P, F, Q, H, R, K, E, W, and Y SEQ ID NO:80 huCD3 VH CDR3 affinity var- iant VRHGNFGNSYVSWFAX9, wherein X9 is selected from H, S, F, N, W, T, C, A, I, L, Q, V, E, M, K, R, G and P SEQ ID NO:81 huCD3 VL CDR1 affinity var- iant TGAVTX10SNY, wherein X10 is selected from S, A, G, R, V, F, I, E, M, H, N, Y, P, Q, D, K and L SEQ ID NO:82 huCD3 VL CDR3 affinity var- iant AX11WYSNLWV, wherein X11 is selected from C, F, Y, I, T, V, M, A, S, N, G, W, E, K, P, R and D SEQ ID NO:83 huCD3 VL CDR3 affinity var- iant ALWYSNX12WV, wherein X12 is selected from D, K, Q, R, G, V, E, T, N, Y, S, P, W, F and M

[0021] The CDR regions have been annotated according to the IMGT definitions. DETAILED DESCRIPTION OF THE INVENTION 15 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 Definitions

[0022] The term "human CD3" or "CD3" as used herein, refers to the human Cluster of Differentiation 3 protein which is part of the T-cell co-receptor protein complex and is composed of four distinct chains. CD3 is also found in other species, and thus, the term "CD3"maybeusedherein and isnot limited tohumanCD3unless contradictedby context. Inmammals, the complex contains a CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No P09693, or cynomolgus monkeyCD3γUniProtKB / Swiss-Prot NoQ95LI7), aCD3δ (delta) chain (humanCD3δUniProtKB / Swiss-Prot NoP04234, or cynomolgusmonkeyCD3δUniProtKB / Swiss-Prot NoQ95LI8), twoCD3ε (epsilon) chains (humanCD3εUniProtKB / S- wiss-Prot No P07766; cynomolgus CD3ε UniProtKB / Swiss-Prot No Q95LI5; or rhesus CD3ε UniProtKB / Swiss-Prot No G7NCB9), and a CD3ζ-chain (zeta) chain (human CD3ζ UniProtKB / Swiss-Prot No P20963, cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot No Q09TK0). These chains associate with a molecule known as the T-cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together comprise the TCR complex.

[0023] The term "humanCD20" or "CD20" refers to humanCD20 (UniProtKB / Swiss-Prot No P11836) and includes any variants, isoforms and species homologs of CD20 which are naturally expressed by cells, including tumor cells, or are expressed on cells transfected with the CD20 gene or cDNA. Species homologs include rhesus monkey CD20 (macaca mulatta; UniProtKB / Swiss-Prot No H9YXP1).

[0024] The term "chimeric antibody" as used herein, refers to an antibody wherein the variable region is derived from a non-humanspecies (e.g. derived from rodents) and theconstant region isderived fromadifferent species, suchashuman. Chimericantibodiesmaybegeneratedbyantibodyengineering. "Antibodyengineering" is a termusedgeneric for different kinds of modifications of antibodies, and which is a well-known process for the skilled person. In particular, a chimeric antibodymay be generated by using standardDNA techniques as described in Sambrook et al., 1989,Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, the chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody according to the present invention may be performed by other methods than described herein. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. They may typically contain non-human (e.g. murine) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms "variable region" or "variabledomains" asused in the context of chimericantibodies, refers toa regionwhich comprises the CDRs and framework regions of both the heavy and light chains of the immunoglobulin.

[0025] The term "humanized antibody" as used herein, refers to a genetically engineered non-human antibody, which containshumanantibodyconstant domainsandnon-humanvariabledomainsmodified tocontainahigh level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity- determining regions (CDRs),which together form theantigenbinding site, onto ahomologoushumanacceptor framework region (FR) (see WO92 / 22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-humanantibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibodymay comprise non-humanCDR sequences, primarily human framework regions optionally compris- ing one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

[0026] The term "human antibody" as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g.,mutations introducedby randomor site-specificmutagenesis in vitro or by somaticmutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producingmonoclonal antibody can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes.

[0027] A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system.Hybridoma production in themouse is a verywell established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0028] Human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system.

[0029] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of 16 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter- connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Funda- mental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein asCHorCH). Theheavy chain constant region typically is comprised of three domains,CH1,CH2, and CH3.Each light chain typically is comprisedof a light chainvariable region (abbreviatedhereinasVLorVL)anda light chain constant region (abbreviated herein asCL orCL). The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersedwith regions that aremore conserved, termed framework regions (FRs). Each VH andVL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901‑917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules (Brochet X., Nucl Acids Res. 2008;36:W503‑508andLefrancMP.,NucleicAcidsResearch1999;27:209‑212; seealso internet http addresshttp: / / www.imgt.org / ).

[0030] Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present invention is according to the EU-numbering (Edelman et al., ProcNatl Acad Sci USA. 1969May;63(1):78‑85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91‑3242).

[0031] The term "antibody" (Ab) in the context of thepresent invention refers to an immunoglobulinmolecule, a fragment of an immunoglobulinmolecule, or aderivativeofeither thereof,whichhas theability tospecificallybind toanantigenunder typical physiological conditionswith ahalf life of significant periodsof time, suchasat least about 30minutes, at least about 45minutes, at least about onehour, at least about twohours, at least about four hours, at least about 8hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or time sufficient for the antibody to recruit an effector activity). The variable regions of the heavy and light chains of the immunoglobulinmolecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibodymay be performedby fragments of a full-length antibody. Examples of antigen- binding fragments encompassed within the term "antibody" include (i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) F(ab’)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544‑546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; TrendsBiotechnol. 2003Nov;21(11):484‑90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan;.5(1):111‑24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, theymay be joined, using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain inwhich theVLandVH regionspair to formmonovalentmolecules (knownassingle chainantibodiesor single chain Fv (scFv), see for instance Bird et al., Science 242, 423‑426 (1988) and Huston et al., PNAS USA 85, 5879‑5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties andutility.Theseandotheruseful antibody fragments in thecontextof thepresent invention, aswell asbispecific formatsof such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.

[0032] The term "bispecific antibody" in the context of the present invention refers to an antibody having two different antigen-binding regions defined by different antibody sequences.

[0033] When used herein, unless contradicted by context, the term "Fab-arm" or "arm" refers to one heavy chain-light chain pair and is used interchangeably with "half molecules" herein.

[0034] Whenusedherein, unlesscontradictedbycontext, the term "Fc region" refers toanantibody regioncomprisingat least a hinge region, a CH2 domain, and a CH3 domain. 17 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0035] As used herein, the term "isotype" refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.

[0036] The term "monovalent antibody" means in the context of the present invention that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not capable of antigen crosslinking.

[0037] A "CD20 antibody" or "anti-CD20 antibody" is an antibody as described above, which binds specifically to the antigen CD20.

[0038] A "CD3 antibody" or "anti-CD3 antibody" is an antibody as described above, which binds specifically to the antigen CD3, in particular human CD3ε (epsilon).

[0039] A "CD3xCD20 antibody" or "anti-CD3xCD20 antibody" is a bispecific antibody, which comprises two different antigen-binding regions, one of which binds specifically to the antigen CD20 and one of which binds specifically to CD3.

[0040] Inapreferredembodiment, thebispecificantibodyof the invention is isolated.An "isolatedbispecificantibody,"as used herein, is intended to refer to a bispecific antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated bispecific antibody that specifically binds to CD20 andCD3 is substantially free of monospecific antibodies that specifically bind to CD20 or CD3).

[0041] The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three- dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in thebinding, suchasaminoacid residueswhichareeffectively blockedor coveredby the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).

[0042] The term "monoclonal antibody" as used herein refers to a preparation of antibodymolecules of singlemolecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "humanmonoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-humananimal, suchasa transgenicmouse, havingagenomecomprising a humanheavy chain transgeneanda light chain transgene, fused to an immortalized cell.

[0043] As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically isabindingwithanaffinity corresponding toaKDofabout10‑7Mor less, suchasabout10‑8Mor less, such as about 10‑9 M or less, about 10‑10 M or less, or about 10‑11 M or even less when determined by for instance surface plasmon resonance (SPR) technology inaBIAcore3000 instrumentusing theantigenas the ligandand theantibodyas the analyte, andbinds to thepredeterminedantigenwith anaffinity corresponding to aKD that is at least ten-fold lower, suchas at least 100 fold lower, for instanceat least 1,000 fold lower, suchasat least 10,000 fold lower, for instanceat least 100,000 fold lower than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than thepredeterminedantigenor a closely-related antigen. Theamountwithwhich theaffinity is lower is dependent on theKDof the antibody, so thatwhen the KD of the antibody is very low (that is, the antibody is highly specific), then the amount withwhich the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.

[0044] The term "kd" (sec‑1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.

[0045] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0046] When used herein the term "heterodimeric interaction between the first and second CH3 regions" refers to the interaction between the first CH3 region and the second CH3 region in a first-CH3 / second-CH3 heterodimeric protein.

[0047] When used herein the term "homodimeric interactions of the first and second CH3 regions" refers to the interaction between a first CH3 region and another first CH3 region in a first-CH3 / first-CH3 homodimeric protein and the interaction between a second CH3 region and another second CH3 region in a second-CH3 / second-CH3 homodimeric protein.

[0048] The term "reducing conditions" or "reducing environment" refers to a condition or an environment in which a substrate, here a cysteine residue in the hinge region of an antibody, is more likely to become reduced than oxidized.

[0049] The present invention also provides bispecific antibodies comprising functional variants of the VL regions, VH regions, or one ormoreCDRs of the bispecific antibodies of the examples. A functional variant of a VL, VH, or CDRused in the context of a bispecifc antibody still allows each arm of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or the specificity / selectivity of the parent bispecific antibody and in some cases such a bispecific antibodymay be associatedwith greater affinity, selectivity and / or specificity than the parent bispecific antibody.

[0050] Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The 18 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% homology=#of identical positions / total #of positionsx100), taking intoaccount thenumberofgaps,and the lengthofeach gap,which need to be introduced for optimal alignment of the two sequences. Thepercent identity between twonucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers andW.Miller, Comput. Appl. Biosci 4, 11‑17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12andagappenalty of 4. In addition, thepercent identity between twoaminoacid sequencesmaybe determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444‑453 (1970) algorithm.

[0051] Exemplary variants include those which differ from VH and / or VL and / or CDR regions of the parent bispecific antibody sequences mainly by conservative substitutions; for instance 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.

[0052] In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in the following table: Amino acid residue classes for conservative substitutions

[0053] Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

[0054] In the context of the present invention the following notations are, unless otherwise indicated, used to describe a mutation; i) substitution of an amino acid in a given position iswritten as e.g. K409Rwhichmeans a substitution of a Lysine in position 409 with an Arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of Lysine with Arginine in position 409 is designated as: K409R, and the substitution of Lysine with any amino acid residue in position 409 is designated as K409X. In case of deletion of Lysine in position 409 it is indicated by K409*.

[0055] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced, e.g. an expression vector encoding an antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK‑293F, Expi293F, PER.C6 or NS0 cells , and lymphocytic cells.

[0056] The term "treatment" refers to the administration of an effective amount of a therapeutically active bispecific antibody of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.

[0057] The term "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a bispecific antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the bispecifc antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0058] The term "anti-idiotypic antibody" refers to an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. Further aspects and embodiments of the invention

[0059] As described above, the invention relates to a bispecific antibody comprising two different antigen-binding regions, one which has a binding specificity for CD3 and one which has a binding specificity for CD20.

[0060] Thus, the invention relates to a bispecific antibody comprising (i) a first binding arm comprising a first antigen- binding region binding to human CD3ε (epsilon), wherein said first antigen-binding region comprises a) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, respectively; 19 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 b) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:54, respectively; c) heavy chain variable (VH) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:73, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X2 of SEQ ID NO:73 is selected from M and P, respectively; d) heavy chain variable (VH) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:74, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X3 of SEQ ID NO:74 is A, respectively; e) heavy chain variable (VH) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:1, 75, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X4 of SEQ ID NO:75 is E, respectively; f) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and77, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X6 of SEQ ID NO:77 is selected from F, G, I, K, L and N, respectively; g) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and78, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X7 of SEQ ID NO:78 is P, respectively; h) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and79, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X8 of SEQ ID NO:79 is selected from A and G, respectively; i) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and80, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X8 of SEQ ID NO:80 is selected from M, R and V, respectively; or j) heavy chain variable (VH) regionCDR1, CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:55, 56 and57, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:58, the sequence DTS, and the sequence as set forth in SEQ ID NO:59, respectively, and (ii) a second binding arm comprising a second antigen-binding region binding to human CD20.

[0061] Hereby bispecific antibodies with variying binding affinities for CD3 epsilon are provided. In one embodiment it is preferred that the bindning affinity for CD3 is lower than it is for the parent anti-CD3 binding arm. Experimental data have shown that bispescific CD3xCD20 antibodies as described above in c) to i) having an anti-CD3 binding arm with a substitutionwhich lower thebindingaffinity forCD3epsilonmaintains the tumor killing potencyof the parent bispecificanti- CD3xCD20 antibody.

[0062] In one embodiment, the invention relates to a bispecific antibody comprising (i) a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein said first antigen-binding region comprises (a) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NO:4, the sequenceGTN,and the sequenceasset forth inSEQ IDNO:5orSEQ IDNO:54, respectively, or (b) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:55, 56 and 57, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NO:58, the sequence DTS, and the sequence as set forth in SEQ ID NO:59, respectively, and (ii) a second binding arm comprising a second antigen-binding region binding to human CD20.

[0063] In one embodiment, the invention relates to a bispecific antibody comprising a first binding arm comprising a first antigen-binding region binding to humanCD3ε (epsilon), wherein said first antigen-binding region comprises heavy chain variable (VH) region CDR1, CDR2, andCDR3 having the sequences as set forth in SEQ IDNOs: 1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, respectively.

[0064] The term "binding arm comprising an antigen-binding region" means an antibody molecule or fragment that comprises an antigen-binding region. Thus, binding arm can be e.g. the six VH and VL CDR regions, the VH and VL sequences, the Fab fragment or a half-molecule antibody (i.e. comprising one heavy and one light chain). 20 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0065] In one embodiment, the first antigen-binding region comprises a first heavy chain variable sequence (VH), and a first light chain variable sequence (VL), and the second antigen-binding region comprises a second heavy chain variable sequence (VH), and a second light chain variable sequence (VL).

[0066] In one embodiment, the first binding arm comprises a first heavy chain comprising a first heavy chain variable sequence (VH) anda first heavy chain constant sequence (CH), and a first light chain comprising a first light chain variable sequence (VL) and a first light chain constant sequence (CL), and (ii) the second binding arm comprises a second heavy chain comprisinga secondheavy chain variable sequence (VH)andasecondheavy chain constant sequence (CH), anda second light chain comprising a second light chain variable sequence (VL) and a second light chain constant sequence (CL).

[0067] In one embodiment, the bispecific antibody is a full length antibody, such as a full length IgG1 antibody, e.g. a full length IgG1,λ (lambda),κ (kappa) antibody or IgG1,κ (kappa), κ (kappa) antibody. First binding arm

[0068] The first binding armcomprises a first antigen-binding region binding to humanCD3ε (epsilon), wherein said first antigen-binding region comprises a) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, respectively; b) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:54, respectively; c) heavy chain variable (VH) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:73, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X2 of SEQ ID NO:73 is selected from M and P, respectively; d) heavy chain variable (VH) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:74, 2, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X3 of SEQ ID NO:74 is A; e) heavy chain variable (VH) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:1, 75, and 3, respectively, and light chain variable (VL) regionCDR1,CDR2, andCDR3having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X4 of SEQ ID NO:75 is E; f) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and77, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X6 of SEQ ID NO:77 is selected from F, G, I, K, L and N, respectively; g) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and78, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X7 of SEQ ID NO:78 is P; h) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and79, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X8 of SEQ ID NO:79 is selected from A and G, respectively; i) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and80, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, wherein X8 of SEQ ID NO:80 is selected from M, R and V, respectively; or j) heavy chain variable (VH) regionCDR1, CDR2, andCDR3having the sequences as set forth in SEQ IDNOs:55, 56 and57, respectively, and light chainvariable (VL) regionCDR1,CDR2,andCDR3having thesequencesasset forth in SEQ ID NO:58, the sequence DTS, and the sequence as set forth in SEQ ID NO:59, respectively.

[0069] In one embodiment, the first binding arm comprises a first antigen-binding region binding to human CD3ε (epsilon), wherein said first antigen-binding region comprises (a) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) region CDR1,CDR2, andCDR3having the sequences as set forth in SEQ IDNO:4, the sequenceGTN, and the sequence as set forth in SEQ ID NO:5 or SEQ ID NO:54, respectively, or (b) heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:55, 56 and 57, respectively, and light chain variable (VL) region CDR1, 21 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 CDR2, andCDR3having the sequencesas set forth inSEQ IDNO:58, the sequenceDTS, and the sequenceasset forth in SEQ ID NO:59, respectively.

[0070] In one embodiment, the first binding arm comprises a first antigen-binding region binding to human CD3ε (epsilon), wherein said first antigen-binding region comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, andCDR3having the sequences as set forth in SEQ IDNO:4, the sequenceGTN, and the sequence as set forth in SEQ ID NO:5, respectively.

[0071] The six CDR sequences as defined in (a) above are derived from amouse antibody denoted SP34. Humanized versions of this antibody have been generated, and the humanized antibodies are denoted huCD3 herein and are further disclosed in WO2015001085 (Genmab).

[0072] The six CDR sequences as defined in (b) above are derived from huCLB-T3 / 4. huCLB-T3 / 4 is a humanized version of the murine CD3 antibody CLB-T3 / 4 (Parren et al., Res Immunol. 1991, 142(9):749‑63, hereby incorporated by reference in its entirety, including sequencedisclosures).Briefly, theCLB-T3 / 4murineVHandVLsequencesaspublished in Parren et al. (1991) were aligned to the human VH and VL repertoires using the IMGT’s V-QUEST. The closest human germlines that were found were IGHV3‑21*01 for the VH gene and IGKV3‑11*01(+IGKJ4*02) for the VL gene. All amino acid residues in the murine VH and VL sequences that differed were replaced by the human equivalent, except for those within the CDR regions of CLB-T3 / 4. As no related J-region was found for the VH sequence, the common WGQGTLVTVSS sequence was used for the FR4 region of the heavy chain. Both sequences were cloned into the relevant expression vectors and expressed by cotransfection in HEK293F cells. huCLB-T3 / 4 has a VH region comprising the sequence set forth in SEQ ID NO:17 (VH huCLB-T3 / 4) and a VL region comprising the sequence set forth in SEQ ID NO:18 (VLhuCLB-T3 / 4). huCLB-T3 / 4 has theVHCDR1,CDR2andCDR3sequences set forth inSEQ IDNOs:55, 56and 57, respectively, and the VL CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:58, the sequence DTS, and the sequence set forth in SEQ ID NO:59, respectively.

[0073] The various humanized huCD3 antibodies and huCLB-T3 / 4 bind to human CD3ε (epsilon).

[0074] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first heavy chain variable sequence (VH), wherein said VH sequence has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the VH sequences selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6; b) a VH sequence as set forth in SEQ ID NO:7; c) a VH sequence as set forth in SEQ ID NO:8; d) a VH sequence as set forth in SEQ ID NO:9; and e) a VH sequence as set forth in SEQ ID NO:17.

[0075] In one embodiment, the VH sequence of the first antigen-binding region is selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6; b) a VH sequence as set forth in SEQ ID NO:7; c) a VH sequence as set forth in SEQ ID NO:8; d) a VH sequence as set forth in SEQ ID NO:9; e) a VH sequence as set forth in SEQ ID NO:17.

[0076] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first VL sequence of the first antigen-binding region, wherein said VL sequence has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the VL sequences selected from the group consisting of: a) a VL sequence as set forth in SEQ ID NO:10; b) a VL sequence as set forth in SEQ ID NO:11; c) a VL sequence as set forth in SEQ ID NO:12; and d) a VL sequence as set forth in SEQ ID NO:18.

[0077] In one embodiment, the VL sequence of the first antigen-binding region is selected from the group consisting of: a VL sequence as set forth in SEQ ID NO:10; a) a VL sequence as set forth in SEQ ID NO:11; b) a VL sequence as set forth in SEQ ID NO:12; and 22 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 c) a VL sequence as set forth in SEQ ID NO:18.

[0078] Inoneembodiment, thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein comprisesfirst VH and VL sequences, wherein said VH andVL sequences of the first antigen-binding region are selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:10; b) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as set forth in SEQ ID NO:10; c) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as set forth in SEQ ID NO:10; d) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:11; e) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:12; f) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as set forth in SEQ ID NO:10; g) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as set forth in SEQ ID NO:11; h) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as set forth in SEQ ID NO:12; i) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as set forth in SEQ ID NO:11; j) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as set forth in SEQ ID NO:12; k) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as set forth in SEQ ID NO:11; l) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as set forth in SEQ ID NO:12; and m) a VH sequence as set forth in SEQ ID NO:17, and a VL sequence as set forth in SEQ ID NO:18.

[0079] Inoneembodiment, thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein comprisesfirst VH and VL sequences, wherein said VH andVL sequences of the first antigen-binding region are selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:10; b) a VH sequence as set forth in SEQ ID NO:17, and a VL sequence as set forth in SEQ ID NO:18

[0080] Inoneembodiment, thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein comprisesfirst VH and VL sequences as set forth in SEQ ID Nos: 17 and 18.

[0081] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed comprises a first antigen-binding region having at least 90% sequence identity to the VH sequence is as set forth in SEQ ID NO:6, and at least 90% sequence identity to the VL sequence is as set forth in SEQ ID NO:10. In one embodiment the sequence deviations are in the framework sequences andnot in theCDRsequences. Accordingly, in one embodiment, the invention relates to a bispecific antibody comprising a first antigen-binding region having at least 90% sequence identity to the VH sequence is as set forth in SEQ ID NO:6, and at least 90% sequence identity to the VL sequence is as set forth in SEQ ID NO:10wherein theCDRsequences are as set forth in SEQ IDNOs:1, 2, and 3, for the heavy chain and as set forth in SEQ IDNO:4, the sequenceGTN, and the sequence as set forth in SEQ IDNO:5 for the light chain so that the CDR sequences are unmutated.

[0082] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed comprises a first antigen-binding region having at least 95% sequence identity to the VH sequence is as set forth in SEQ ID NO:6, and at least 95%sequence identity to the VL sequence is as set forth in SEQ IDNO:10. In one embodiment, the invention relates toabispecificantibody comprisingafirst antigen-binding regionhavingat least 95%sequence identity to theVHsequence is as set forth in SEQ ID NO:6, and at least 95% sequence identity to the VL sequence is as set forth in SEQ ID NO:10 wherein theCDRsequencesareasset forth inSEQIDNOs:1, 2, and3, for theheavychainandasset forth inSEQIDNO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5 for the light chain so that the CDR sequences are unmutated.

[0083] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed comprises a first antigen-binding region having at least 97% sequence identity to the VH sequence is as set forth in SEQ ID NO:6, and at least 97%sequence identity to the VL sequence is as set forth in SEQ IDNO:10. In one embodiment, the invention relates toabispecificantibody comprisingafirst antigen-binding regionhavingat least 97%sequence identity to theVHsequence is as set forth in SEQ ID NO:6, and at least 97% sequence identity to the VL sequence is as set forth in SEQ ID NO:10 wherein theCDRsequencesareasset forth inSEQIDNOs:1, 2, and3, for theheavychainandasset forth inSEQIDNO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5 for the light chain so that the CDR sequences are unmutated.

[0084] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed comprises a first antigen-binding region having at least 98% sequence identity to the VH sequence is as set forth in SEQ ID NO:6, and at least 98%sequence identity to the VL sequence is as set forth in SEQ IDNO:10. In one embodiment, the invention relates toabispecificantibody comprisingafirst antigen-binding regionhavingat least 98%sequence identity to theVHsequence 23 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 is as set forth in SEQ ID NO:6, and at least 98% sequence identity to the VL sequence is as set forth in SEQ ID NO:10 wherein theCDRsequencesareasset forth inSEQIDNOs:1, 2, and3, for theheavychainandasset forth inSEQIDNO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5 for the light chain so that the CDR sequences are unmutated.

[0085] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed comprises a first antigen-binding region having at least 99% sequence identity to the VH sequence is as set forth in SEQ ID NO:6, and at least 99%sequence identity to the VL sequence is as set forth in SEQ IDNO:10. In one embodiment, the invention relates toabispecificantibody comprisingafirst antigen-binding regionhavingat least 99%sequence identity to theVHsequence is as set forth in SEQ ID NO:6, and at least 99% sequence identity to the VL sequence is as set forth in SEQ ID NO:10 wherein theCDRsequencesareasset forth inSEQIDNOs:1, 2, and3, for theheavychainandasset forth inSEQIDNO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5 for the light chain so that the CDR sequences are unmutated.

[0086] Herebybispecicific antibodies of the inventionare providedwherein theVHandVLsequencesmayvarywithin at least 90% sequence identity to the parent sequences. It is preferred that the variants have the same properties as the parent antibodies. In certain embodiments the sequences only vary in the framework sequences so that the CDR sequences are identical to the parent CDR sequences.

[0087] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first antigen-binding region comprises the VH sequence is as set forth in SEQ ID NO:6, and the VL sequence is as set forth in SEQ ID NO:10.

[0088] In one embodiment, the first binding arm of the bispecific antibody as defined in any of the embodiments disclosed herein is derived from a mouse antibody.

[0089] In one embodiment, the first binding arm of the bispecific antibody as defined in any of the embodiments disclosed herein is derived from a humanized antibody.

[0090] In one embodiment, the first binding arm of the bispecific antibody as defined in any of the embodiments disclosed herein is derived from a full-length antibody antibody.

[0091] In one embodiment, the first binding arm of the bispecific antibody as defined in any of the embodiments disclosed herein is derived from a full-length IgG1,λ (lambda) or IgG1, κ (kappa) antibody. Second binding arm

[0092] SuitableCD20antibodies foruseas thesecondbindingarm in thebispecificantibodiesaccording to the invention are CD20 antibodies, which bind to an epitope on human CD20, which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172, but which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166. Examples of such antibodies are the antibodies denoted 2F2 and 7D8 as disclosed in WO2004035607 (Genmab) and the antibody denoted 2C6 as disclosed in WO2005103081 (Genmab). The CDR sequences of 2F2, 7D8 and 2C6 are disclosed in Table 1.

[0093] Further suitable CD20 antibodies for use as the second binding arm in the bispecific antibodies according to the invention are CD20 antibodies, which bind to an epitope on human CD20, which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172. An example of such an antibody is 11B8 as disclosed in WO2004035607 (Genmab). The CDR sequences of 11B8 are disclosed in Table 1.

[0094] Further suitable CD20 antibodies for use as the second antigen-binding region in the bispecific antibodies according to the invention are CD20 antibodies with a low functional off-rate meaning that the antibodies are slowly dissociated from CD20 upon binding.

[0095] The kd dissociation constant or koff rate may be determined by the method described under the heading "Dissociation rates of anti-CD20 F(ab)2 fragments" in Example 5 of WO2004035607. Thus, in one embodiment, the CD20antibodieshaveakddissociation constant of 1.0 x‑4 sec‑1 or below, suchasof 8.0 x10‑5 sec‑1 or below, suchas in the range of 8.0 x 10‑5 sec‑1 to 4.0 x 10‑5 sec‑1 as determined by the above method.

[0096] Further suitable CD20 antibodies for use as the second binding arm in the bispecific antibodies according to the invention are CD20 antibodies having the six CDR sequences of the antibody denoted 11B8 as disclosed in WO2004035607. The CDR sequences of 11B8 are disclosed in Table 1.

[0097] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a second antigen-binding regionwhich binds to humanCD20, which second antigen-binding region comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences selected from: (i) the VH CDR1 region of SEQ ID NO:32, the VH CDR2 region of SEQ ID NO:33, the VH CDR3 region of SEQ ID NO:34, (ii) the VH CDR1 region of SEQ ID NO:38, the VH CDR2 region of SEQ ID NO:39, the VH CDR3 region of SEQ ID NO:34, 24 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (iii)the VH CDR1 region of SEQ ID NO:42, the VH CDR2 region of SEQ ID NO:43, the VH CDR3 region of SEQ ID NO:44, or (iv)the VH CDR1 region of SEQ ID NO:49, the VH CDR2 region of SEQ ID NO:50, the VH CDR3 region of SEQ ID NO:51.

[0098] In a preferred embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a second antigen-binding region comprising the VH CDR1 region of SEQ ID NO:32, the VH CDR2 region of SEQ ID NO:33, and the VH CDR3 region of SEQ ID NO:34.

[0099] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a second antigen-binding regionwhich binds to humanCD20, which second antigen-binding region comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 and chain variable (LH) region CDR1, CDR2, and CDR3 sequences selected from: (i) the VH CDR1 region of SEQ ID NO:32, the VH CDR2 region of SEQ ID NO:33, the VH CDR3 region of SEQ ID NO:34, theVLCDR1 regionofSEQ IDNO:35, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:36, (ii) the VH CDR1 region of SEQ ID NO:38, the VH CDR2 region of SEQ ID NO:39, the VH CDR3 region of SEQ ID NO:34, theVLCDR1 regionofSEQ IDNO:35, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:36, (iii)the VH CDR1 region of SEQ ID NO:42, the VH CDR2 region of SEQ ID NO:43, the VH CDR3 region of SEQ ID NO:44, theVLCDR1 regionofSEQ IDNO:45, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:46, (iv)the VH CDR1 region of SEQ ID NO:49, the VH CDR2 region of SEQ ID NO:50, the VH CDR3 region of SEQ ID NO:51, theVLCDR1 regionofSEQ IDNO:52, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:53, (v) the VH CDR1 region of SEQ ID NO:32, the VH CDR2 region of SEQ ID NO:33, the VH CDR3 region of SEQ ID NO:34, theVLCDR1 regionofSEQ IDNO:45, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:46, (vi)the VH CDR1 region of SEQ ID NO:32, the VH CDR2 region of SEQ ID NO:33, the VH CDR3 region of SEQ ID NO:34, theVLCDR1 regionofSEQ IDNO:52, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:53, (vii) the VH CDR1 region of SEQ ID NO:38, the VH CDR2 region of SEQ ID NO:39, the VH CDR3 region of SEQ ID NO:34, theVLCDR1 regionofSEQ IDNO:45, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:46, (viii) the VH CDR1 region of SEQ ID NO:38, the VH CDR2 region of SEQ ID NO:39, the VH CDR3 region of SEQ ID NO:34, theVLCDR1 regionofSEQ IDNO:52, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:53, (ix)the VH CDR1 region of SEQ ID NO:42, the VH CDR2 region of SEQ ID NO:43, the VH CDR3 region of SEQ ID NO:44, theVLCDR1 regionofSEQ IDNO:35, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:36, (x) the VH CDR1 region of SEQ ID NO:42, the VH CDR2 region of SEQ ID NO:43, the VH CDR3 region of SEQ ID NO:44, theVLCDR1 regionofSEQ IDNO:52, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:53, (xi)the VH CDR1 region of SEQ ID NO:49, the VH CDR2 region of SEQ ID NO:50, the VH CDR3 region of SEQ ID NO:51, theVLCDR1 regionofSEQ IDNO:35, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:36, or (xii) the VH CDR1 region of SEQ ID NO:49, the VH CDR2 region of SEQ ID NO:50, the VH CDR3 region of SEQ ID NO:51, theVLCDR1 regionofSEQ IDNO:45, theVLCDR2 regionofDAS,and theVLCDR3 regionofSEQ IDNO:46.

[0100] In a preferred embodiment, the bispecific antibody as defined in any of the embodiments as disclosed herein comprises a second antigen-binding region comprising the VH CDR1 region of SEQ ID NO:32, the VH CDR2 region of SEQ IDNO:33, theVHCDR3 region ofSEQ IDNO:34, theVLCDR1 regionof SEQ IDNO:35, theVLCDR2 regionofDAS, and the VL CDR3 region of SEQ ID NO:36.

[0101] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VHsequencewhichhasat least 90%,at least 95%,at least 97%,or at least 99%aminoacid sequence identity to theamino acid sequence as set forth in SEQ IDNO:27, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:28.

[0102] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VHsequencewhichhasat least 90%,at least 95%,at least 97%,or at least 99%aminoacid sequence identity to theamino acid sequence as set forth in SEQ IDNO:37, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99%amino acid sequence identity to the amino acid sequence as set forth in SEQ IDNO:28. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:40, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:41.

[0103] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VHsequencewhichhasat least 90%,at least 95%,at least 97%,or at least 99%aminoacid sequence identity to theamino acid sequence as set forth in SEQ IDNO:47, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at 25 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 least 99%amino acid sequence identity to the amino acid sequence as set forth in SEQ IDNO:48. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:27, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequenceas set forth in SEQ IDNO:41. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%,or at least 99%aminoacid sequence identity to theaminoacid sequenceasset forth inSEQ IDNO:27, andaVL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:48. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99%aminoacid sequence identity to theaminoacid sequenceasset forth inSEQ IDNO:37, andaVLsequencewhichhas at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ IDNO:41. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprisesaVHsequencewhichhasat least 90%,at least 95%, at least 97%,or at least 99%aminoacid sequence identity to the amino acid sequence as set forth in SEQ ID NO:37, and a a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ IDNO:48. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ IDNO:40, and a VL sequencewhich has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:28. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:40, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequenceas set forth in SEQ IDNO:48. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%,or at least 99%aminoacid sequence identity to theaminoacid sequenceasset forth inSEQ IDNO:47, andaVL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:28. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:47, and a a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:41. In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprisessecondVHandVLsequences,wherein saidVHandVLsequencesof thesecondantigen-binding region are selected from the group consisting of: (i) the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:28, (ii) the VH sequence of SEQ ID NO:37, and the VL sequence of SEQ ID NO:28, (iii)the VH sequence of SEQ ID NO:40, and the VL sequence of SEQ ID NO:41, (iv)the VH sequence of SEQ ID NO:47, and the VL sequence of SEQ ID NO:48, (v) the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:41, (vi)the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:48, (vii) the VH sequence of SEQ ID NO:37, and the VL sequence of SEQ ID NO:41, (viii) the VH sequence of SEQ ID NO:37, and the VL sequence of SEQ ID NO:48, (ix)the VH sequence of SEQ ID NO:40, and the VL sequence of SEQ ID NO:28, (x) the VH sequence of SEQ ID NO:40, and the VL sequence of SEQ ID NO:48, (xi)the VH sequence of SEQ ID NO:47, and the VL sequence of SEQ ID NO:28, or (xii) the VH sequence of SEQ ID NO:47, and the VL sequence of SEQ ID NO:41.

[0104] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the second antigen-binding region comprises the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:28.

[0105] In one embodiment, the second binding arm of the bispecific antibody as defined in any of the embodiments disclosed herein is derived from a human antibody.

[0106] In one embodiment, the second binding arm of the bispecific antibody as defined in any of the embodiments disclosed herein is derived from a full-length antibody antibody.

[0107] In one embodiment, the second binding arm of the bispecific antibody as defined in any of the embodiments disclosed herein is derived from a IgG1, κ (kappa) antibody. 26 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 Bispecific antibody formats

[0108] The present invention provides bispecificCD3xCD20 antibodieswhich efficiently promote Tcell-mediated killing of CD20-expressing tumor cells. Depending on the desired functional properties for a particular use, particular antigen- binding regions can be selected from the set of antibodies or antigen-binding regions provided by the present invention. Many different formats and uses of bispecific antibodies are known in the art, and were reviewed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838‑47 and; MAbs, 2012 Mar-Apr;4(2):182‑97..

[0109] Abispecific antibody according to thepresent invention is not limited to any particular bispecific format ormethod of producing it.

[0110] Examples of bispecific antibody molecules which may be used in the present invention comprise (i) a single antibody that has two arms comprising different antigen-binding regions; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandemby an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig),whereeach light chainandheavychaincontains twovariabledomains in tandem throughashort peptide linkage (Wuet al.,GenerationandCharacterizationof aDualVariableDomain Immunoglobulin (DVD-Ig™)Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically-linked bispecific (Fab’)2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody,which isacombinationof scFvswithadiabody resulting inamultivalentmolecule; (vii) a so-called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied toFabs, can yield a trivalent bispecific bindingprotein consisting of two identical Fab fragments linked toa different Fab fragment; (viii) a so-called Scorpionmolecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.

[0111] In one embodiment, the bispecific antibody of the present invention is a diabody, a cross-body, or a bispecific antibody obtained via a controlled Fab-arm exchange (such as described in WO2011131746 (Genmab)).

[0112] Examples of different classes of bispecific antibodies include but are not limited to (i) IgG-like molecules with complementaryCH3domains to force heterodimerization; (ii) recombinant IgG-like dual targetingmolecules, wherein the two sides of themolecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment; (iv) Fc fusionmolecules, wherein single chain Fvmolecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; (v) Fab fusionmolecules, wherein different Fab-fragments are fused together, fused to heavy- chain constant-domains, Fc-regions or parts thereof; and (vi) ScFv- and diabody-based and heavy chain antibodies (e.g., domainantibodies, nanobodies)whereindifferent singlechainFvmoleculesordifferent diabodiesordifferent heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to heavy-chain constant-domains, Fc-regions or parts thereof.

[0113] Examples of IgG-like molecules with complementary CH3 domain molecules include but are not limited to the Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), the so-called Knobs-into- Holes molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) and the electrostatically-matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), the LUZ-Ymolecules (Genentech,Wranik et al. J. Biol. Chem. 2012, 287(52): 43331‑9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), the Strand Ex- change Engineered Domain body (SEEDbody) molecules (EMD Serono, WO2007110205), the Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecules (Xencor,WO2011028952), bivalent bispecific antibodies (WO2009080254) and theDuoBody®molecules (GenmabA / S, WO2011131746).

[0114] Examples of recombinant IgG-like dual targeting molecules include but are not limited to Dual Targeting (DT)‑Ig molecules (WO2009058383), Two-in-one Antibody (Genentech; Bostrom, et al 2009. Science 323, 1610‑1614.), Cross- linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208‑18), approaches with common light chain (Crucell / Merus, US7,262,028), κλBodies (NovImmune, WO2012023053) and CovX-body (CovX / Pfizer; Doppalapudi, V.R., et al 2007. Bioorg. Med. Chem. Lett. 17,501‑506.).

[0115] Examples of IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)‑Ig molecules (Abbott, US7,612,181), Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like Bispecific molecules (ImClone / Eli Lilly, Lewis etal. Nat Biotechnol. 2014 Feb;32(2):191‑8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009Oct 30;393(3):672‑92) and BsAbmolecules (Zymogenetics,WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0116] Examples of Fc fusionmolecules include but are not limited to ScFv / Fc Fusions (Pearce et al., BiochemMol Biol Int. 1997Sep;42(6):1179‑88), SCORPIONmolecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR100 27 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538) and Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine - China).

[0117] Examples of Fab fusion bispecific antibodies include but are not limited to F(ab)2molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677‑86.), Dual-Action or Bis-Fabmolecules (Genentech, Bostrom, et al 2009. Science 323, 1610‑1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), Bivalent Bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050‑7.) and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[0118] ExamplesofScFv‑, diabody-basedanddomainantibodies includebut arenot limited toBispecificTCell Engager (BiTE)molecules (Micromet,WO2005061547), TandemDiabodymolecules (TandAb) (Affimed) LeGall et al., ProteinEng Des Sel. 2004 Apr;17(4):357‑66.), Dual Affinity Retargeting Technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), Single-chain Diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479‑84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667‑75.), dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual targeting heavy chain only domain antibodies.

[0119] In one aspect, the bispecific antibody of the invention comprises a first Fc-region comprising a first CH3 region, and a second Fc-region comprising a secondCH3 region, wherein the sequences of the first and secondCH3 regions are different andaresuch that theheterodimeric interactionbetweensaidfirst andsecondCH3regions is stronger thaneachof the homodimeric interactions of said first and secondCH3 regions.More details on these interactions andhow they canbe achieved are provided in WO2011131746 andWO2013060867 (Genmab), which are hereby incorporated by reference.

[0120] As described further herein, a stable bispecific CD3xCD20 antibody can be obtained at high yield using a particular method on the basis of one homodimeric starting CD20 antibody and one homodimeric starting CD3 antibody containing only a few, fairly conservative, asymmetrical mutations in the CH3 regions. Asymmetrical mutationsmean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions.

[0121] In one aspect, the bispecific antibody as defined in any of the embodiments disclosed herein comprises first and secondheavy chains,wherein eachof said first andsecondheavy chain comprisesat least ahinge region, aCH2andCH3 region, wherein in said first heavy chain at least one of the amino acids in the positions corresponding to a positions selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, andwherein said first and said second heavy chains are not substituted in the samepositions.

[0122] Inoneembodiment, thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein comprisesfirst and second heavy chains,wherein (i) the amino acid in the position corresponding to F405 in a human IgG1heavy chain is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in said second heavy chain, or (ii) the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in said first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said second heavy chain.

[0123] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and the secondFc-region has anaminoacid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein the first and second Fc-regions are not substituted in the same positions.

[0124] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid substitution at position 366, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 368, 370, 399, 405, 407 and 409. In one embodiment the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.

[0125] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid substitution at position 368, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 370, 399, 405, 407 and 409.

[0126] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid substitution at position 370, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 399, 405, 407 and 409.

[0127] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid substitution at position 399, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 405, 407 and 409.

[0128] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid substitution at position 405, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407 and 409.

[0129] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- 28 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 region has an amino acid substitution at position 407, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 409.

[0130] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid substitution at position 409, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 407.

[0131] Accordingly, in oneembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, the sequences of said first and secondCH3 regions contain asymmetricalmutations, i.e.mutations at different positions in the two CH3 regions, e.g. a mutation at position 405 in one of the CH3 regions and amutation at position 409 in the other CH3 region.

[0132] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc- region has an amino acid other than Lys, Leu orMet, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino-acid substitution at a position selected from the groupconsistingof: 366, 368, 370, 399, 405and407. Inonesuchembodiment, said first Fc-regionhasanaminoacidother than Lys, Leu orMet, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino acid other than Phe, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or Leu, at position 405. In a further embodiment hereof, said first Fc-region has an amino acid other than Lys, LeuorMet, e.g.Gly, Ala, Val, Ile, Ser, Thr, Phe,Arg,His, Asp,Asn,Glu,Gln, Pro, Trp, Tyr, orCys, at position 409 and said second Fc-region has an amino acid other than Phe, Arg orGly, e.g. Leu, Ala, Val, Ile, Ser, Thr,Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405.

[0133] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-regioncomprisesaPheatposition405andanaminoacidother thanLys,LeuorMet, e.g.Gly,Ala,Val, Ile,Ser,Thr,Phe, Arg,His,Asp,Asn,Glu,Gln,Pro,Trp, Tyr, orCys, at position409andsaid secondFc-regioncomprisesanaminoacidother thanPhe,e.g.Gly,Ala,Val, Ile,Ser,Thr, Lys,Arg,His,Asp,Asn,Glu,Gln,Pro,Trp,Tyr, Leu,Met, orCys,at position405and a Lys at position 409. In a further embodiment hereof, said first Fc-region comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409and said secondFc-region comprisesanaminoacidother thanPhe,ArgorGly, e.g. Leu,Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and a Lys at position 409.

[0134] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-regioncomprisesaPheatposition405andanaminoacidother thanLys,LeuorMet, e.g.Gly,Ala,Val, Ile,Ser,Thr,Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises a Leu at position 405 and a Lys at position 409. In a further embodiment hereof, said first Fc-region comprises a Phe at position 405 and an Arg at position 409 and said second Fc-region comprises an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and a Lys at position 409. In another embodiment, said first Fc-region comprises Phe at position 405 and an Arg at position 409 and said second Fc-region comprises a Leu at position 405 and a Lys at position 409.

[0135] In a further embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises an amino acid other than Lys, Leu orMet, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405. In a further embodiment, said first Fc-region comprises an Arg at position 409 and said second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.

[0136] In an even further embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second Fc- region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.

[0137] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region comprises an amino acid other than Lys, Leu orMet, e.g.Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn,Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.

[0138] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region comprises an Arg at position 409 and said second Fc region comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.

[0139] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second Fc region comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.

[0140] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second Fc-region comprises an Ile at position 350, a Thr at position 370, a Leu at position 405 and a Lys at position 409.

[0141] In oneembodiment of the bispecificantibodyasdefined in anyof theembodimentsdisclosedherein, said first Fc- 29 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 regionhasanaminoacidother thanLys, LeuorMetat position409andsaidsecondFc-regionhasanaminoacidother than Pheat position405, suchasother thanPhe,ArgorGlyat position405; or said firstCH3 regionhasanaminoacidother than Lys, Leu orMet at position 409and said secondCH3 region has an amino acid other thanTyr, Asp,Glu, Phe, Lys,Gln, Arg, Ser or Thr at position 407.

[0142] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region having an amino acid other than Lys, Leu or Met at position 409 and a second Fc-region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.

[0143] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region having a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and a second Fc- region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.

[0144] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region having a Tyr at position 407 and an Arg at position 409 and a second Fc-region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.

[0145] In another embodiment, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407. In another embodiment, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has anAla, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.

[0146] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has a Gly, Leu, Met, Asn or Trp at position 407.

[0147] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino acid other than Tyr, Asp,Glu, Phe, Lys,Gln, Arg, Ser or Thr, e.g. Leu,Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and a Lys at position 409.

[0148] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an Ala, Gly, His, Ile, Leu,Met, Asn, Val or Trp at position 407 and a Lys at position 409.

[0149] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has aGly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.

[0150] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region has aTyr at position 407and anArg at position 409 and said secondFc-region has an amino acid other thanTyr, Asp,Glu, Phe, Lys,Gln, Arg, Ser or Thr, e.g. Leu,Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and a Lys at position 409.

[0151] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region has aTyr at position 407andanArgat position 409and said secondFc-region has anAla,Gly, His, Ile, Leu,Met, Asn, Val or Trp at position 407 and a Lys at position 409.

[0152] In anotherembodiment of thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein, said first Fc-region has a Tyr at position 407 and anArg at position 409 and said secondFc-region has aGly, Leu,Met, Asn or Trp at position 407 and a Lys at position 409.

[0153] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409, and the second Fc-region has (i) anaminoacidother thanPhe, LeuandMet, e.g.Gly,Ala,Val, Ile, Ser, Thr, Lys,Arg,His, Asp,Asn,Glu,Gln,Pro, Trp, Tyr, or Cys, at position 368, or (ii) a Trp at position 370, or (iii) anaminoacidother thanAsp,Cys,Pro,Glu orGln, e.g.Phe, Leu,Met,Gly,Ala,Val, Ile, Ser, Thr, Lys,Arg,His,Asn, Trp, Tyr, or Cys, at position 399 or (iv) anaminoacidother thanLys,Arg,Ser, Thr, orTrp, e.g.Phe, Leu,Met,Ala,Val,Gly, Ile,Asn,His,Asp,Glu,Gln,Pro, Tyr, or Cys, at position 366. 30 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0154] In one embodiment, the first Fc-region has an Arg, Ala, His or Gly at position 409, and the second Fc region has (i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) a Trp at position 370, or (iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399, or (iv) an Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366.

[0155] In one embodiment, the first Fc-region has an Arg at position 409, and the second Fc region has (i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) a Trp at position 370, or (iii) a Phe, His, Lys, Arg or Tyr at position 399, or (iv) an Ala, Asp, Glu, His, Asn, Val, Gln at position 366.

[0156] In addition to the above-specified amino-acid substitutions, said first and second Fc regionsmay contain further amino-acid substitutions, deletion or insertions relative to wild-type Fc sequences.

[0157] In a further embodiment, said first and second Fab-arms (or heavy chain constant domains) comprising the first and second Fc regions comprise, except for the specified mutations, a CH3 sequence independently selected from the following: (IgG1m(a)) (SEQ ID NO:60), (IgG1m(f)) (SEQ ID NO:61), and (IgG1m(ax) (SEQ ID NO:62).

[0158] In one embodiment, neither said first nor said second Fc-region comprises a Cys-Pro-Ser-Cys sequence in the (core) hinge region.

[0159] In a further embodiment, both said first and said second Fc-region comprise aCys-Pro-Pro-Cys sequence in the (core) hinge region.

[0160] In separate and specific embodiments, one or both Fab arms comprise a heavy-chain constant region sequence independently selected from SEQ ID NO:63, 64, 65, 66, 67, 68, 69, 70, and 71 (see Table 1).

[0161] In one embodiment of the bispecific antibody according to any of the embodiments as disclosed herein, (a) the first antigen-binding region comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, respectively, and (b) the second antigen-binding region which binds to human CD20 comprises the VH CDR1 region of SEQ IDNO:32, theVHCDR2 regionofSEQ IDNO:33, theVHCDR3 regionofSEQ IDNO:34, theVLCDR1 regionofSEQ ID NO:35, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:36, respectively.

[0162] In one embodiment of the bispecific antibody according to any of the embodiments as disclosed herein, (a) the first antigen-binding regioncomprises theVHsequenceasset forth inSEQIDNO:6,andaVLsequenceasset forth inSEQ IDNO:10, and (b) thesecondantigen-binding regioncomprises theVHsequenceasset forth inSEQ IDNO:27, and theVL sequence as set forth in SEQ ID NO:28.

[0163] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first antigen-binding region is a Fab arm derived from IgG1-huCD3-H1L1-FEAL, and the second antigen-binding region is a Fab arm derived from IgG1‑7D8-FEAR.

[0164] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first binding arm is a half-molecule antibody (i.e. comprising one heavy and one light chain) derived from IgG1-huCD3-H1L1- FEAL, and the second binding arm is a half-molecule antibody derived from IgG1‑7D8-FEAR.

[0165] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first binding arm is a half-molecule antibody derived from IgG1-huCD3-H1L1-FEAR, and the second binding arm is a half- molecule antibody derived from IgG1‑7D8-FEAL. Methods of preparing bispecific antibodies

[0166] Traditional methods such as the hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) ActaPharmacol Sin 26:649) can beused in the preparation of the bispecific antibodies of the invention.Co-expression in a host cell of twoantibodies, consisting of different heavy and light chains, leads to amixture of possible antibodyproducts in addition to the desired bispecific antibody, which can then be isolated by, e.g., affinity chromatography or similarmethods.

[0167] Strategies favoring the formation of a functional bispecific, product, upon co-expression of different antibody constructs can also be used, e.g., the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hydridomas producing different antibodies leads to a limited number of heterodimeric proteins because of preferential species-restricted heavy / light chain pairing. Another strategy to promote formation of heterodimers over homodimers is a "knob-into-hole" strategy in which a protuberance is introduced on a first heavy-chain polypeptide and a 31 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 corresponding cavity in a second heavy-chain polypeptide, such that the protuberance can be positioned in the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation. "Protuberances" are constructed by replacing small amino-acid side-chains from the interface of the first polypeptide with larger side chains. Compensatory "cavities" of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino-acid side-chains with smaller ones (US patent 5,731,168). EP1870459 (Chugai) andWO2009089004 (Amgen) describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In thesemethods, one ormore residues thatmakeup theCH3-CH3 interface in bothCH3domains are replacedwith a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. WO2007110205 (Merck) describe yet another strategy, wherein differences between IgA and IgG CH3 domains are exploited to promote heterodimerization.

[0168] Another in vitro method for producing bispecific antibodies has been described in WO2008119353 (Genmab), wherein a bispecific antibody is formed by "Fab-arm" or "half-molecule" exchange (swapping of a heavy chain and attached light chain) between twomonospecific IgG4‑ or IgG4-like antibodies upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.

[0169] A preferred method for preparing the bispecific CD3xCD20 antibodies of the present invention includes the methods described in WO2011131746 and WO13060867 (Genmab) comprising the following steps: a) providing a first antibody comprising an Fc region , said Fc region comprising a first CH3 region; b) providing a second antibody comprising a second Fc region , said Fc region comprising a second CH3 region, wherein the first antibody is a CD3 antibody and the second antibody is a CD20 antibody, or vice versa; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific CD3xCD20 antibody.

[0170] In one embodiment, the said first antibody together with said second antibody are incubated under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, wherein the heterodimeric interaction between said first and second antibodies in the resulting heterodimeric antibody is such that no Fab-arm exchange occurs at 0.5 mM GSH after 24 hours at 37° C.

[0171] Without being limited to theory, in step c), the heavy-chain disulfide bonds in the hinge regions of the parent antibodies are reduced and the resulting cysteines are then able to form inter heavy-chain disulfide bond with cysteine residues of another parent antibodymolecule (originally with a different specificity). In one embodiment of thismethod, the reducing conditions in step c) comprise the addition of a reducing agent, e.g. a reducing agent selected from the group consisting of: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl) phosphine (TCEP), L-cysteine and beta-mercapto-ethanol, preferably a reducing agent selected from the group con- sisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting.

[0172] For this method any of the CD3 and CD20 antibodes described above may be used including first and second CD3 andCD20 antibodies, respectively, comprising a first and / or second Fc region. Examples of such first and second Fc regions, including combination of such first and second Fc regions may include any of those described above. In a particular embodiment the first and second CD3 and CD20 antibodies, respectively, may be chosen so as to obtain a bispecific antibody as described herein.

[0173] In one embodiment of this method, said first and / or second antibodies are full-length antibodies.

[0174] The Fc regions of the first and second antibodiesmay be of any isotype, including, but not limited to, IgG1, IgG2, IgG3or IgG4. Inoneembodimentof thismethod, theFc regionsofbothsaidfirst andsaidsecondantibodiesareof the IgG1 isotype. In another embodiment, one of the Fc regions of said antibodies is of the IgG1 isotype and the other of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody comprises an Fc region of an IgG1 and an Fc region of IgG4 and may thus have interesting intermediate properties with respect to activation of effector functions.

[0175] In a further embodiment, one of the antibody starting proteins has been engineered to not bind Protein A, thus allowing to separate theheterodimericprotein fromsaidhomodimeric startingproteinbypassing theproductoveraprotein A column.

[0176] As described above, the sequences of the first and second CH3 regions of the homodimeric starting antibodies are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than eachof thehomodimeric interactionsof said first andsecondCH3regions.Moredetails on these interactionsandhow they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are hereby incorporated by reference in their entirety. 32 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0177] In particular, a stable bispecificCD3xCD20 antibody can be obtained at high yield using the abovemethod of the invention on the basis of two homodimeric starting antibodies which bind CD3 and CD20, respectively, and contain only a few, fairly conservative, asymmetricalmutations in theCH3 regions. Asymmetrical mutationsmean that the sequences of said first and second CH3 regions contain amino acid substitutions at nonidentical positions.

[0178] The bispecific antibodies of the inventionmay also be obtained by co-expression of constructs encoding the first and second polypeptides in a single cell. Thus, in a further aspect, the invention relates to a method for producing a bispecific antibody, said method comprising the following steps: a) providing a first nucleic-acid construct encoding a first polypeptide comprising a first Fc region and a first antigen- binding region of a first antibody heavy chain, said first Fc region comprising a first CH3 region, b) providing a second nucleic-acid construct encoding a second polypeptide comprising a second Fc region and a second antigen-binding region of a second antibody heavy chain, said second Fc region comprising a second CH3 region, wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions, and wherein said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino-acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407, optionally wherein said first and second nucleic acid constructs encode light chain sequences of said first and second antibodies c) co-expressing said first and second nucleic-acid constructs in a host cell, and d) obtaining said heterodimeric protein from the cell culture.

[0179] Thus, the present invention also relates to a recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody of the present invention.

[0180] In one embodiment of the present invention, the bispecific antibody is obtained by any of themethods according to the present invention.

[0181] Suitable expression vectors, including promoters, enhancers, etc., and suitable host cells for the production of antibodies arewell-known in the art. Examples of host cells include yeast, bacterial andmammalian cells, such asCHOor HEK cells.

[0182] Inoneembodiment, thebispecificantibodyasdefined inanyof theembodimentsdisclosedherein comprisesfirst and second CH3 regions, except for the specified mutations, comprising the sequence of SEQ ID NO:60 (IgG1m(a)).

[0183] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a secondFc-region, wherein neither said first nor said second Fc-region comprises aCys-Pro-Ser-Cys sequence in the hinge region.

[0184] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein both of said first and said second Fc-region comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

[0185] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second Fc-regions are human antibody Fc-regions.

[0186] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein said first and second Fc region, except for the specified mutations, comprise a sequence independently selected from the group consisting of SEQ IDNOS:63, 64, 65, 66, 67, 68, 69, 70, and 71.

[0187] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a firstFc-regionandasecondFc-region,wherein the first andsecondantigen-binding regionscomprisehumanantibodyVH sequences and, optionally, human antibody VL sequences.

[0188] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second antigen-binding regions are from heavy-chain antibodies.

[0189] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second antigen-binding regions comprise a first and second light chain.

[0190] In further embodiments, the co-expression method according to the invention comprises any of the further features described under the in vitro method above.

[0191] In a further aspect, the invention relates to an expression vector comprising the first and second nucleic-acid 33 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 constructs specified herein above. In a further embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of a light chain, a heavy chain or both light and heavy chains of an antibody, e.g. a human antibody.

[0192] An expression vector in the context of the present inventionmay be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In oneembodiment, aCD20oraCD3antibody-encodingnucleic acid is comprised in anakedDNAorRNAvector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355‑59 (1997)), a compacted nucleic acid vector (as described in for instance US 6,077, 835 and / or WO 00 / 70087), a plasmid vector such as pBR322, pUC 19 / 18, or pUC 118 / 119, a "midge"minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793‑800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (as described in for instance WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551‑55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance US 5,589,466 and US 5,973,972).

[0193] In one embodiment, the vector is suitable for expression of the CD20 antibody and / or the CD3 antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503‑5509 (1989), pET vectors (Novagen, Madison WI) and the like).

[0194] An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516‑544 (1987)).

[0195] An expression vector may also or alternatively be a vector suitable for expression in mammalian cells, e.g. a vector comprising glutamine synthetase as a selectable marker, such as the vectors described in Bebbington (1992) Biotechnology (NY) 10:169‑175.

[0196] A nucleic acid and / or vector may also comprises a nucleic acid sequence encoding a secretion / localization sequence,whichcan target apolypeptide, suchasanascent polypeptidechain, to theperiplasmic spaceor into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides.

[0197] The expression vector may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e. g., human CMV IE promoter / enhancer as well as RSV, SV40, SL3‑3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, anorigin of replication for plasmid product inE. coli, an antibiotic resistancegeneas selectablemarker, and / or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.

[0198] In one embodiment, the CD20 and / or CD3 antibody-encoding expression vector may be positioned in and / or delivered to the host cell or host animal via a viral vector.

[0199] In an even further aspect, the invention relates to a host cell comprising the first and second nucleic-acid constructs specified herein above.

[0200] Thus the present invention also relates to a recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody of the present invention, such as a transfectoma.

[0201] ThefirstCD20-specificantibodymaybeexpressed ina recombinant eukaryoticorprokaryotic host cell, suchasa transfectoma, which produces an antibody of the invention as defined herein or a bispecific antibody of the invention as definedherein. TheCD3-specific antibodymay likewise beexpressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody of the invention as defined herein or a bispecific antibody of the invention as defined herein.

[0202] Examples of host cells include yeast, bacterial, plant and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK‑293F, Expi293F, PER.C6 or NS0 cells or lymphocytic cells. For example, in one embodiment, the host cell may comprise a first and secondnucleic acid construct stably integrated into the cellular genome. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a first and second nucleic acid construct as specified above.

[0203] In an even further aspect, the invention relates to a transgenic non-human animal or plant comprising nucleic acids encoding one or two sets of a human heavy chain and a human light chain, wherein the animal or plant produces a bispecific antibody of the invention.

[0204] In a further aspect, the invention relates to a hybridoma which produces an antibody for use in a bispecific antibody of the invention as defined herein. In an even further aspect, the invention relates to a transgenic non-human animal or plant comprising nucleic acids encoding one or two sets of a human heavy chain and a human light chain, wherein the animal or plant produces an antibody for use in a bispecific antibody or a bispecific antibody of the invention. 34 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0205] In one aspect, the invention relates to a nucleic acid construct encoding one or more amino acid sequences set out in Table 1.

[0206] In one aspect, the invention relates to an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a first binding arm according to any one of the embodiments disclosed herein; (ii) a nucleic acid sequence encoding a light chain sequence of a first binding arm according to any one of the embodiments disclosed herein; (iii)a nucleic acid sequence encoding a heavy chain sequence of a second binding arm according to any one of the embodiments disclosed herein; (iv)a nucleic acid sequenceencodinga light chain sequenceof a secondbindingarmaccording to anyoneof theof the embodiments disclosed herein; (v) the nucleic acid set forth in (i) and the nucleic acid set forth in (ii); (vi)the nucleic acid set forth in (iii) and the nucleic acid set forth in (iv). (vii) the nucleic acid set forth in (i), (ii), (iii) and (iv).

[0207] In one aspect, the invention relates to a method for producing a bispecific antibody according to any one of the embodiments as disclosed herein, comprising the steps of a) culturing a host cell as disclosed herein comprising an expression vector as disclosed herein expressing the first antibody as disclosed herein and purifying said antibody from the culture media; b) culturingahost cell asdisclosedherein comprisinganexpressionvectorasdisclosedhereinexpressing thesecond antibody as disclosed herein and purifying said antibody from the culture media; c) incubating said first antibody together with said second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, and d) obtaining said bispecific antibody.

[0208] In one aspect, the invention relates to a host cell comprising an expression vector as defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell. Fc regions

[0209] In one aspect of the present invention, the bispecific CD3xCD20 antibody according to the present invention further comprises a first Fc region and a second Fc region whichmay be comprised in a first and a second Fab-armwhich respectively further comprise the first and second antigen-binding regions described above (or vice versa).

[0210] Inanotheraspectof thepresent invention, thebispecificCD3xCD20antibodycomprisesafirst andasecondFab- arm comprising a first and a second antigen-binding region, respectively. The bispecific CD3xCD20 antibody further comprises a first and a second Fc region.In one aspect of the present invention, the bispecific CD3xCD20 antibody comprises the first Fab-arm comprising the first antigen-binding region and the first Fc region, and the second Fab-arm comprising the second antigen-binding region and the second Fc region.

[0211] In another aspect of the present invention, the bispecific CD3xCD20 antibody comprises the second Fab-arm comprising the second antigen-binding region and the first Fc region, and the first Fab-arm comprising the first antigen- binding region and the second Fc region.

[0212] The first and second Fc-regions may each be of any isotype, including, but not limited to, IgG1, IgG2, IgG3 and IgG4, and may comprise one or more mutations or modifications. In one embodiment, each of the first and second Fc regions is of the IgG4 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, eachof thefirst andsecondFc regions isof the IgG1 isotypeor derived therefrom,optionallywithoneormore mutations or modifications. In another embodiment, one of the Fc regions is of the IgG1 isotype and the other of the IgG4 isotype, or is derived from such respective isotypes, optionally with one or more mutations or modifications.

[0213] In one embodiment, one or both of the Fc regions comprise amutation removing the acceptor site for Asn-linked glycosylation or is otherwise manipulated to change the glycosylation properties. For example, in an IgG1 Fc-region, an N297Q mutation can be used to remove an Asn-linked glycosylation site. Accordingly, in a specific embodiment, one or both Fc-regions comprise an IgG1 wildtype sequence with an N297Q mutation (SEQ ID NO:66, see Table 1).

[0214] In oneembodiment, oneor bothFc-regionsareeffector-function-deficient. For example, theFc-region(s)maybe of an IgG4 isotype, or a non-IgG4 type, e.g. IgG1, IgG2 or IgG3, which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall’Acqua WF et al., J Immunol. 177(2):1129‑1138 (2006) and Hezareh M, J Virol.; 75(24):12161‑12168 (2001). In one embodiment, one or both Fc-regions comprise an IgG1 wildtype sequence (SEQ ID NO:63, see Table 1). 35 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0215] The bispecific antibody according to the present invention may comprise modifications in the Fc region.When a bispecific antibody comprises such modifications it may become an inert, or non-activating, bispecific antibody. The term "inertness", "inert" or "non-activating" as used herein, refers to an Fc region which is at least not able to bind any Fcγ receptors, induce Fc-mediated cross-linking of FcRs, or induce FcR-mediated cross-linking of target antigens via two Fc regions of individual antibodies, or is not able to bind C1q. The inertness of an Fc region of a humanized or chimeric CD3 antibody is advantageously tested using the antibody in a monospecific format.

[0216] Several variants can be constructed to make the Fc region of an antibody inactive for interactions with Fcγ (gamma) receptors and C1q for therapeutic antibody development. Examples of such variants are described herein.

[0217] Thus, in one embodiment, the antibody comprises an Fc region which has been modified so that said antibody mediates reducedFc-mediated T-cell proliferation compared to awild-type antibody by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein said T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)‑based functional assay.

[0218] Thus,aminoacids in theFc region thatplayadominant role in the interactionswithC1qand theFcγ receptorsmay bemodified.Examplesofaminoacidpositions thatmaybemodified includepositionsL234,L235andP331.Combinations thereof, such as L234F / L235E / P331S, can cause a profound decrease in binding to human CD64, CD32A, CD16 and C1q.

[0219] Hence, in one embodiment, the amino acid in at least one position corresponding to L234, L235 and P331, may beA,AandS, respectively (Xuet al., 2000,Cell Immunol. 200(1):16‑26;Oganesyanet al., 2008,ActaCryst. (D64):700‑4). Also, L234F and L235E amino acid substitutions can result in Fc regions with abrogated interactions with Fcγ receptors and C1q (Canfield et al., 1991, J. Exp.Med. (173):1483‑91; Duncan et al., 1988, Nature (332):738‑40). Hence, in one embodiment, the amino acids in the positions corresponding to L234 and L235, may be F and E, respectively. A D265A amino acid substitution can decrease binding to all Fc gammaReceptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591‑604). Hence, in one embodiment, the amino acid in the position corresponding to D265 may be A. Binding to C1q can be abrogated by mutating positions D270, K322, P329, and P331. Mutating these positions to either D270Aor K322Aor P329Aor P331A canmake the antibody deficient in CDCactivity IdusogieEE, et al., 2000, J Immunol. 164: 4178‑84). Hence, in one embodiment, the amino acids in at least one position corresponding to D270, K322, P329 and P331, may be A, A, A, and A, respectively.

[0220] An alternative approach to minimize the interaction of the Fc region with Fcγ receptors and C1q is by removal of the glycosylation site of an antibody.Mutating position N297 to e.g. Q, A, or E removes a glycosylation site which is critical for IgG-Fc gammaReceptor interactions. Hence, in one embodiment, the amino acid in a position corresponding toN297, may be G, Q, A or E Leabman et al., 2013, MAbs; 5(6):896‑903). Another alternative approach to minimize interaction of the Fc region with Fcγ receptors may be obtained by the following mutations; P238A, A327Q, P329A or E233P / L234V / L235A / G236del (Shields et al., 2001, J. Biol. Chem. (276):6591‑604).

[0221] Alternatively, human IgG2 and IgG4 subclasses are considered naturally compromised in their interactions with C1q and Fc gammaReceptors although interactions with Fcγ receptors were reported (Parren et al., 1992, J. Clin Invest. 90: 1537‑1546;Bruhnset al., 2009, Blood113: 3716‑3725).Mutationsabrogating these residual interactions canbemade in both isotypes, resulting in reduction of unwanted side-effects associated with FcR binding. For IgG2, these include L234A and G237A, and for IgG4, L235E. Hence, in one embodiment, the amino acid in a position corresponding to L234 and G237 in a human IgG2 heavy chain, may be A and A, respectively. In one embodiment, the amino acid in a position corresponding to L235 in a human IgG4 heavy chain, may be E.

[0222] Other approaches to further minimize the interaction with Fc gamma Receptors and C1q in IgG2 antibodies include those described in WO2011066501 and Lightle, S., et al., 2010, Protein Science (19):753‑62.

[0223] The hinge region of the antibody can also be of importance with respect to interactions with Fcγ receptors and complement (Brekke et al., 2006, J Immunol 177:1129‑1138; Dall’Acqua WF, et al., 2006, J Immunol 177:1129‑1138). Accordingly, mutations in or deletion of the hinge region can influence effector functions of an antibody.

[0224] The term "cross-linking" as used herein, refers to the indirect bridging of antibody Fab arm(s) (monovalently or bivalently) bound to the target antigen by anFcR-bearing cell through binding to the antibody Fc region. Thus, an antibody which binds its target antigen on target antigen-bearing cells may cross-link that cell with another cell expressing FcRs.

[0225] The term "unspecific killing" as used herein, refers to the killing of cells by the cytotoxic function of Tcells or other effector cells, through tumor target antigen-independent activation of said cells. Thus, by unspecific killing is meant that effector cells, e.g. cytotoxic Tcells, are activated and induce cytotoxicity independent of tumor target binding, for example by binding of the antibody to CD3 and an FcγR.

[0226] Thus, in one embodiment, the bispecific antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of said first and second immunoglobulin heavy chains one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are not L, L, D, N, and P, respectively.

[0227] In one embodiment, in both the first and second heavy chains one or more amino acids in the position corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are not L, L, D, N, and 36 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 P, respectively.

[0228] In another embodiment, in at least one of the first and second heavy chains one or more amino acids in the positions corresponding to positions L234, L235 andD265 in a human IgG1 heavy chain, are not L, L and D, respectively, and the amino acids in the positions corresponding to N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0229] The term "amino acid corresponding to positions" as used herein refers to an amino acid position number in a human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Unless otherwise stated or contradicted by context, the amino acids of the constant region sequences are herein numbered according to the EU-index of numbering (described in Kabat, E.A. et al., 1991, Sequences of proteins of immunological interest. 5thEdition -USDepartment of Health andHumanServices,NIHpublicationNo. 91‑3242, pp 662, 680, 689). Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgG1 heavy chain. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention.

[0230] In the context of the present invention, the amino acid may be defined as described above.

[0231] The term "the amino acid is not" or similar wording when referring to amino acids in a heavy chain is to be understood tomean that the amino acid is any other amino acid than the specific amino acidmentioned. For example, the amino acid in the position corresponding to L234 in a human IgG1 heavy chain is not L,means that the amino acidmay be any of the other naturally or non-naturally occurring amino acids than L.

[0232] In one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain, is not D.

[0233] In one embodiment, in at least one of the first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain, is not D, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0234] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to position D265 in a human IgG1 heavy chain is hydrophobic or polar amino acids.

[0235] The term "hydrophobic" as used herein in relation to an amino acid residue, refers to an amino acid residue selected from thegroupconsistingof;A,C,F,G,H, I, L,M,R,T,V,W,andY.Thus, in oneembodiment, in at least oneof said first andsecondheavychains theaminoacid in thepositioncorresponding topositionD265 inahuman IgG1heavychain is selected from the group of amino acids consisting of; A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0236] The term "polar" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human heavy chain is selected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

[0237] In another embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is an aliphatic uncharged, aromatic or acidic amino acid.

[0238] The term "aliphatic uncharged" asusedherein in relation to aminoacid residues, refers to anyaminoacid residue selected from the group consisting of: A, G, I, L, and V. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, G, I, L, and V.

[0239] The term "aromatic" as used herein in relation to amino acid residues, refers to any amino acid residue selected from thegroup consisting of: F, T, andW.Thus, in oneembodiment, in at least oneof said first and secondheavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; F, T, and W.

[0240] The term "acidic" as used herein in relation to amino acid residues, refers to any amino acid residue chosen from the group consisting of: D and E. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in theposition corresponding to positionD265 inahuman IgG1heavy chain is selected from thegroupconsisting of;D and E.

[0241] In a particular embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, E, F, G, I, L, T, V, and W.

[0242] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain, is not D.

[0243] In one embodiment, in both the first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain, is not D, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively. 37 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0244] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is hydrophobic or polar amino acid.

[0245] Thus, in oneembodiment, in both said first andsecondheavychains theaminoacid in theposition corresponding to positionD265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of; A, C, F,G,H, I, L,M, R, T, V, W and Y.

[0246] Thus, in oneembodiment, in both said first andsecondheavychains theaminoacid in theposition corresponding to position D265 in a human heavy chain is selected from the group consisting of; C, E, H, K, N, Q, R, S, and T. In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of; A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0247] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to position D265 in a human heavy chain is selected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

[0248] Inanother embodiment, inbothsaidfirst andsecondheavychains theaminoacid in theposition corresponding to position D265 in a human IgG1 heavy chain is aliphatic uncharged, aromatic or acidic amino acids.

[0249] Thus, in oneembodiment, in both said first andsecondheavychains theaminoacid in theposition corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, G, I, L, and V.

[0250] Thus, in oneembodiment, in both said first andsecondheavychains theaminoacid in theposition corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; F, T, and W.

[0251] Thus, in oneembodiment, in both said first andsecondheavychains theaminoacid in theposition corresponding to position D265 in a human IgG1 heavy chain are selected from the group consisting of; D and E.

[0252] In a particular embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, E, F, G, I, L, T, V, and W.

[0253] In further embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain, is not N.

[0254] In one embodiment, in at least one of the first and second heavy chains the amino acid in the position corresponding to N297 in a human IgG1 heavy chain, is not N, and the amino acid in the position corresponding to position P331 in a human IgG1 heavy chain, is P.

[0255] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to positions N297 in a human IgG1 heavy chain, is not N.

[0256] In one embodiment, in both the first and second heavy chains the amino acid in the position corresponding to N297 in a human IgG1 heavy chain, is not N, and the amino acid in the position corresponding to positionP331 in a human IgG1 heavy chain, is P.

[0257] In further embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively.

[0258] In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0259] In one embodiment, in at least one of said first and second heavy chains the amino acids corresponding to positions L234andL235 in a human IgG1heavy chain are selected from the group consisting of; A,C,D,E, F,G,H, I, K,M, N, P, Q, R, S, T, Y, V.

[0260] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0261] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 andL235 in a human IgG1heavy chain are each selected from the group consisting of; A, C, F, G, H, I, M, R, T, V, W, and Y.

[0262] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group of amino acids consisting of; C, D, E, H, K, N, Q, R, S, and T.

[0263] In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 andL235 in a human IgG1heavy chain are each selected from the group consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0264] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively.

[0265] In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0266] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain are hydrophobic or polar amino acids. 38 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0267] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234andL235 in a human IgG1heavy chain areeach selected from thegroup consisting of; A,C, F,G,H, I,M,R, T, V, W, and Y.

[0268] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234andL235 in a human IgG1heavy chain are each selected from the groupof aminoacids consisting of; C,D, E, H, K, N, Q, R, S, and T.

[0269] In a particular embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0270] In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 andL235 in a human IgG1heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0271] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 andL235 in a human IgG1heavy chain are each selected from the group consisting of; A, G, I, and V.

[0272] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; F, T, and W.

[0273] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; D and E.

[0274] In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of; A, D, E, F, G, I, T, V, and W.

[0275] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively.

[0276] In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding toL234andL235 inahuman IgG1heavychain, areFandE;orAandA, respectively, and theaminoacids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0277] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively.

[0278] In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0279] In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E, respectively.

[0280] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E, respectively.

[0281] In one embodiment, in at least one of said first and second heavy chains at least the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are A and A, respectively.

[0282] In one embodiment, in both said first and second heavy chains at least the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are A and A, respectively.

[0283] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively.

[0284] In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, andD265 inahuman IgG1heavy chain, arenot L, L andD, respectively, and theaminoacids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0285] In one embodiment, in at least one of said first and second heavy chains the amino acids corresponding to positions L234andL235 in a human IgG1heavy chain are selected from the group consisting of; A,C,D,E, F,G,H, I, K,M, N, P,Q,R, S, T, Y, V, andW, and the amino acid corresponding to positionD265 is selected from the group consisting of; A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.

[0286] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0287] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to positionD265 in a human IgG1heavy chain is selected from the groupof amino acids consisting of; A, C, F,G, H, I, L,M, R, T, V,WandY, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, C, F, G, H, I, M, R, T, V, W, and Y.

[0288] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group of amino acids 39 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 consisting of; C, D, E, H, K, N, Q, R, S, and T, the amino acid in the position corresponding to position D265 in a human heavy chain is selected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

[0289] In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 andL235 in a human IgG1heavy chain are each selected from the group consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0290] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0291] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to positionD265 in a human IgG1heavy chain is selected from the group of amino acids consisting of; A,C, F,G,H, I, L,M,R, T, V,WandY, and the amino acids in the positions corresponding to positions L234and L235 in a human IgG1heavy chain are each selected from the group consisting of; A, C, F, G, H, I, M, R, T, V, W, and Y.

[0292] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234andL235 in a human IgG1heavy chain are each selected from the groupof aminoacids consisting of; C,D, E,H, K,N,Q,R, S, andT, the amino acid in the position corresponding to positionD265 in a humanheavy chain is selected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

[0293] In a particular embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of;A,C,D,E,F,G,H, I,K,M,N,Q,R,S,T,V,W,andY,and theaminoacid in thepositioncorresponding topositionD265 ina human IgG1 heavy chain is selected from the group consisting of; A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0294] In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 andD265 in a human IgG1heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0295] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to positionD265 in a human IgG1heavy chain is selected from the group consisting of; A,G, I, L, andV, and theaminoacids in thepositions corresponding to positions L234andL235 in ahuman IgG1heavy chain are each selected from the group consisting of; A, G, I, and V.

[0296] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding topositionsL234,L235andD265 inahuman IgG1heavychainareeachselected from thegroupconsisting of; F, T, and W.

[0297] Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are each selected from the group consisting of; D and E.

[0298] In a particular embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, E, F, G, I, L, T, V, andW, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of; A, D, E, F, G, I, T, V, and W.

[0299] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively.

[0300] In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, andD265 in a human IgG1 heavy chain, are not L, L, andD, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0301] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0302] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to positionD265 inahuman IgG1heavychain is selected from thegroupconsistingof;A,G, I, L, andV, and theaminoacids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, G, I, and V.

[0303] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are each selected from the group consisting of; D and E.

[0304] In a particular embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, E, F, G, I, L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of; A, D, E, F, G, I, T, V, and W.

[0305] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding topositionsL234,L235,andD265 inahuman IgG1heavychain, areF,E, andA;orA,A,andA, respectively.

[0306] In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, andD265 in a human IgG1 heavy chain, are F, E, andA; or A, A, andA, respectively, and the 40 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0307] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively.

[0308] In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, andD265 inahuman IgG1heavy chain, areF,E, andA; orA,A, andA, respectively, and theaminoacids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0309] In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0310] In a particular preferred embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0311] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

[0312] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

[0313] In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S, respectively.

[0314] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S, respectively.

[0315] In a particular embodiment, the antibody according to the invention, comprises aVH sequence as set out in SEQ ID NO:8, a VL sequence as set out in SEQ ID NO: 10, and in at least one of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0316] In another embodiment, the antibody according to the invention, comprises a VH sequence as set out in SEQ ID NO:8, a VL sequence as set out in SEQ ID NO: 12, and in at least one of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0317] In another embodiment, the antibody according to the invention, comprises a VH sequence as set out in SEQ ID NO:6, a VL sequence as set out in SEQ ID NO: 10, and in at least one of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0318] In another embodiment, the antibody according to the invention, comprises a VH sequence as set out in SEQ ID NO:6, a VL sequence as set out in SEQ ID NO: 12, and in at least one of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0319] In another embodiment, the antibody according to the invention, comprises a VH sequence as set out in SEQ ID NO:9, a VL sequence as set out in SEQ ID NO: 10, and in at least one of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0320] In another embodiment, the antibody according to the invention, comprises a VH sequence as set out in SEQ ID NO:9, a VL sequence as set out in SEQ ID NO: 12, and in at least one of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0321] In one aspect, the bispecific antibody according to the invention comprises the human IgLC2 / IgLC3 constant domain lambda light chain of SEQ ID NO:29.

[0322] Several antibody variants were generated with one or more amino acid substitutions in the Fc region. A non- activating Fc region prevents the antibody from interactingwith Fcreceptors present on blood cells, such asmonocytes, or with C1q to activate the classical complement pathway. Reduction of the Fc activity was tested in antibody variants that contain different combinations of amino acid substitutions in the Fc region. Maximally five amino acid substitutions were introduced,which include themutationsN297Q,L234A,L235A,L234F,L235E,D265A,andP331S.Substitutions inoneor more of these five amino acid positions were introduced in the K409R and / or F405L IgG1 backbone. The following Fc region variants of the huCLB-T3 / 4 antibody were generated: N297Q (refers to the N297Q substitution, termed IgG1- huCLB-T3 / 4-N297Q), LFLE (refers to the L234F / L235E substitutions, termed IgG1-huCLB-T3 / 4-LFLE), LALA (refers to the L234A / L235A substitutions, termed IgG1-huCLB-T3 / 4-LALA), LFLENQ (refers to the L234F / L235E / N297Q substitu- tions, termed IgG1-huCLB-T3 / 4-LFLENQ), LFLEDA (refers to the L234F / L235E / D265A substitutions, termed IgG1- huCLB-T3 / 4-LFLEDA), DA (refers to the D265A substitution, termed IgG1-huCLB-T3 / 4-DA), DAPS (refers to the D265A / P331S substitutions, termed IgG1-huCLB-T3 / 4-DAPS), DANQ (refers to theD265A / N297Qsubstitutions, termed IgG1-huCLB-T3 / 4-DANQ), LFLEPS (refers to the L234F / L235E / P331S substitutions, termed IgG1-huCLB-T3 / 4- LFLEPS), and LFLEDANQPS (refers to the L234F / L235E / D265A / N297Q / P331S substitutions, termed IgG1-huCLB- T3 / 4-LFLEDANQPS).

[0323] In particular, in the IgG1-huCD3antibody variants a combination of three amino acid substitutions, which include the mutations L234F, L235E and D265A and is referred to as LFLEDA or FEA, were introduced in the K409R and F405L IgG1 backbones to generate antibodies with a non-activating Fc region. The resulting non-activating antibody variant is 41 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 termed with the suffix "FEAR" or "FEAL", respectively.

[0324] In one aspect, the bispecific antibodies according to the inventionmaybemodified in the light chain and / or heavy chain to increase the expression level and / or production yield. In one embodiment, the antibodies according to the invention may be modified in the light chain. Such modifications are known in the art and may be performed according to themethodsdescribed in e.g. Zheng, L.,Goddard, J.‑P.,Baumann,U.,&Reymond, J.‑L. (2004). Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3), 807‑14.

[0325] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first heavy chain and first light chain, wherein the amino acid in the position corresponding to position T41 in the lambda light chain of SEQ ID NO:10 of the first light chain is not T.

[0326] In oneembodiment thebispecificantibodyasdefined in anyof theembodimentsdisclosedherein, theaminoacid in the position corresponding to position T41 in the lambda light chain of SEQ IDNO:10 is selected fromH, I, K, L, Q,R and V.

[0327] In oneembodiment the bispecific antibody asdefined in anyof the embodiments disclosed herein,theaminoacid in the position corresponding to position T41 in the lambda light chain of SEQ ID NO: 10 is H, K or R.

[0328] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acid in the position corresponding to position T41 in the lambda light chain of SEQ ID NO: 10 of the first light chain is K.

[0329] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acid in the position corresponding to position F10 in the lambda light chain of SEQ IDNO:10 of the first light chain is not F, and one or more of the amino acid positions corresponding to the positions T41, K55, and L97 in the lambda light chain of SEQ ID NO: 10 of the first light chain are not T, K and L, respectively.

[0330] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acids in the positions corresponding to positions F10, T41, K55, and L97 in the lambda light chain of SEQ IDNO: 10 of the first light chain are not F, T, K and L, respectively.

[0331] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acids in the positions corresponding to positions F10, T41, K55, and L97 in the lambda light chain of SEQ IDNO: 10 of the first light chain are L, K, N, and H, respectively.

[0332] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acids in the positions corresponding to positions R23 and A35 in the lambda light chain of SEQ ID NO: 10 of the first light chain are not R and A, respectively.

[0333] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acids in the positions corresponding to positions R23 and A35 in the lambda light chain of SEQ ID NO: 10 of the first light chain are A and P, respectively.

[0334] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acids in the positions corresponding to positionsF10,R23,A35,R47,D71,A82,D83,S86, I87, andF89 in the lambda light chain of SEQ ID NO:10 of the first light chain are not F, R, A, R, D, A, D, S, I, and F, respectively.

[0335] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the amino acids in the positions corresponding to positionsF10,R23,A35,R47,D71,A82,D83,S86, I87, andF89 in the lambda light chain of SEQ ID NO:10 of the first light chain are L, A, P, T, G, P, E, A, E, and Y, respectively.

[0336] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, (i) the amino acid in the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 of the first light chain is not F, or (ii) the amino acid in the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 of the first light chain is not K, or (iii) the amino acid in the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 of the first light chain is not F, and theaminoacid in the position corresponding to positionK55 in the lambda light chain of SEQ ID NO:10 of the first light chain is not K.

[0337] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, (i) the amino acid in the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 of the first light chain is L, or (ii) the amino acid in the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 of the first light chain is N, or (iii) the amino acid in the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 of the first light chain is L, and the amino acid in the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 of the first light chain is N. 42 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0338] In one embodiment, the bispecific antibody according to the invention comprises a first light chain, wherein the aminoacid in theposition corresponding topositionT41 is selected fromH, I, K, L,Q,RorV, suchasselected fromH,Kand R, such asK. In one embodiment, the bispecific antibody according to the invention comprises a first light chain having the amino acids L, K, N, andH, respectively, in the positions corresponding to positions F10, T41, K55, and L97 in the lambda light chain of SEQ ID NO:10. In one embodiment, the bispecific antibody according to the invention comprises a first light chain,wherein theaminoacid in the position corresponding to positionR23 is selected fromA,G,H,K,Q,S, andT, suchas from A and G, and wherein the amino acid in the position corresponding to A35 is selected from I, L, M, P, V, G, F andW, such as from I, L, M, P, and V.

[0339] In one embodiment, the bispecific antibody according to the invention comprises a first light chain, wherein the amino acid in the position corresponding to position R23 is A or G, such as A, and the amino acid in the position corresponding to position A35 is P.

[0340] In one embodiment, the bispecific antibody according to the invention comprises a first light chain, wherein the amino acids in the positions corresponding to positions F10, R23, A35, R47, D71, A82, D83, S86, I87, and F89 in the lambda light chain of SEQ ID NO:10 are not F, R, A, R, D, A, D, S, I, and F, respectively.

[0341] In one embodiment, the bispecific antibody according to the invention comprises a first light chain, wherein the amino acid in the position corresponding to position R23 is selected from A, G, H, K, Q, S, and T, such as from A and G, wherein theaminoacid in theposition corresponding toA35 is selected from I, L,M,P,V,G,FandW,suchas from I, L,M,P, andwherein theamino acids in the positions corresponding to positions F10,R47,D71,A82,D83,S86, I87, andF89 in the lambda light chain of SEQ ID NO:10 are L, T, G, P, E, A, E, and Y, respectively.

[0342] In one embodiment, the bispecific antibody according to the invention comprises a first light chain, wherein the amino acid in the position corresponding to position R23 is A or G, and wherein the amino acids in the positions corresponding topositionsF10,A35,R47,D71,A82,D83,S86, I87, andF89 in the lambda light chainofSEQIDNO:10are L, P, T, G, P, E, A, E, and Y, respectively.

[0343] In one aspect, the bispecific antibodies according to the inventionmay bemodified in the first and / or second light chains to increase the affinity of the antibodies.

[0344] In one aspect, the bispecific antibodies according to the invention may be modified in the light chain of the first and / or second binding arm to reduce the affinity of the antibodies. Thismay be advantageous in some settings and lead to increased efficacy. In particular low affinity of the first binding arm (binding to human CD3ε (epsilon)) may have an impact on the motility of T cells in circulation and at tumor site thus leading to better engagement of T cells with tumor cells, cf. Mølhøj et al., Molecular Immunology 44 (2007). In particular this may be useful in bispecific formats, in which the CD3 antibodiesareusedasoneof thebindingarms.Modifications that lead to reducedantibodyaffinityare known in theart, see for example Webster et al. Int J Cancer Suppl. 1988;3:13‑6.

[0345] In one embodiment, the bispecific antibody according to the invention comprises a first light chain, wherein (i) the amino acid in the position corresponding to position F10 in the lambda light chain of SEQ ID NO: 10 is not F, or (ii) the amino acid in the position corresponding to position K55 in the lambda light chain of SEQ IDNO:10 is not K, or (iii) the aminoacid in the position corresponding to positionF10 in the lambda light chain ofSEQ IDNO:10 is not F, and the amino acid in the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 is not K.

[0346] In one embodiment, the antibody according to the invention comprises a constant light chain (LC), wherein (i) the amino acid in the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 is L, or (ii) the amino acid in the position corresponding to position K55 in the lambda light chain of SEQ ID NO: 10 is N, or (iii) theaminoacid in the position corresponding to positionF10 in the lambda light chain ofSEQ IDNO:10 is L, and the amino acid in the position corresponding to position K55 in the lambda light chain of SEQ ID NO: 10 is N.

[0347] In one embodiment, the bispecific antibody according to the invention comprises a light chain,wherein the amino acids in the positions corresponding to positions F10, T41, K55, and L97 in the lambda light chain of SEQ IDNO:10 are not F, T, K and L, respectively. Such modifications serve both to increase the expression level and to reduce the affinity.

[0348] In one embodiment, the bispecific antibody according to the invention comprises a first light chain, wherein the amino acids in the positions corresponding to positions F10, T41, K55, and L97 in the lambda light chain of SEQ IDNO:10 are L, K, N, and H, respectively. Such modifications serve both to increase the expression level and to reduce the affinity.

[0349] In a further aspect of the invention, mutations in the CDR regions of huCD3 have been made to optimize the binding affinity of the CD3 binding arm, such as to reduce the binding affinity of the CD3 arm.

[0350] Thus, in one embodiment, the CD3 binding arm of the bispecific antibody according to the invention comprises the six CDR sequences selected from the CDR sequences set forth in the the below Table 2. 43 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 TABLE 2 VH CDR1 (SEQ ID NO) VH CDR2 (SEQ ID NO) VH CDR3 (SEQ ID NO) VLCDR1 (SEQ ID NO) VL CDR2 VLCDR3 (SEQ ID NO) 72 2 3 4 GTN 5 72 2 3 81 GTN 5 72 2 3 4 GTN 82 72 2 3 4 GTN 83 73 2 3 4 GTN 5 73 2 3 81 GTN 5 73 2 3 4 GTN 82 73 2 3 4 GTN 83 74 2 3 4 GTN 5 74 2 3 81 GTN 5 74 2 3 4 GTN 82 74 2 3 4 GTN 83 1 75 3 4 GTN 5 1 75 3 81 GTN 5 1 75 3 4 GTN 82 1 75 3 4 GTN 83 1 76 3 4 GTN 5 1 76 3 81 GTN 5 1 76 3 4 GTN 82 1 76 3 4 GTN 83 1 2 77 4 GTN 5 1 2 77 81 GTN 5 1 2 77 4 GTN 82 1 2 77 4 GTN 83 1 2 78 4 GTN 5 1 2 78 81 GTN 5 1 2 78 4 GTN 82 1 2 78 4 GTN 83 1 2 79 4 GTN 5 1 2 79 81 GTN 5 1 2 79 4 GTN 82 1 2 79 4 GTN 83 1 2 80 4 GTN 5 1 2 80 81 GTN 5 1 2 80 4 GTN 82 1 2 80 4 GTN 83 1 2 3 81 GTN 5 1 2 3 4 GTN 82 44 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) VH CDR1 (SEQ ID NO) VH CDR2 (SEQ ID NO) VH CDR3 (SEQ ID NO) VLCDR1 (SEQ ID NO) VL CDR2 VLCDR3 (SEQ ID NO) 1 2 3 4 GTN 83

[0351] In one embodiment, the six CDR sequences may be inserted in any one of the huCD3 VH and VL framework sequencesVH1,VH2,VH3andVH4,andVL1,VL2,andVL3, respectively, replacing theCDRsequencesofhuCD3. Inone embodiment, the six CDR sequences are inserted in the huCD3 framework sequences VH1 and VL1.

[0352] In a further embodiment, the CD3 binding arm comprises the six CDR sequences selected from the Table 2, wherein X1 of SEQ ID NO:72 is selected from V, H, F, T, P, L, Q, D, K, W, G, A, C and R; X2 of SEQ ID NO:73 is selected from N, A, H, Q, P, F, M, Y, L, W, D, E and C; X4 of SEQ ID NO:75 is selected from Y, Q, W, L, A, I, M, D, T, K, R, G, F, E, V, C and P; X5 of SEQ ID NO:76 is selected from N, L, Y, W, H, M, G, F, K, S, V, R, Q, D, C, E and P; X10 of SEQ ID NO:81 is selected from A, G, R, V, F, E, M, H, N, Y, P, Q, D, K and L; X12 of SEQ ID NO:83 is selected from D, K, Q, G, V, E, T, N, Y, S, P, W, F and M.

[0353] Such huCD3 CDR variant sequences have reduced binding affinity compared to huCD3 wildtype CDR sequences. The six CDR sequences may be inserted in any of the huCD3 VH and VL framework sequences VH1, VH2,VH3andVH4, andVL1,VL2andVL3, respectively, replacing theCDRsequencesof huCD3. Inoneembodiment, the six CDRsequences are inserted in the huCD3 framework sequences VH1 andVL1. In a further embodiment, the six CDR sequences have been inserted in the huCD3 framework sequences VH1 and VL1, wherein the amino acid T in positon 41 of VL1 (SEQ ID NO:10) has been mutated to K.

[0354] In a further embodiment, the CD3 binding arm comprises the six CDR sequences selected from the Table 2, wherein X1 of SEQ ID NO:72 is selected from L, P, Q, D, K, W, S, G, A, C and R; X2 of SEQ ID NO:73 is selected from S, N, G, A, K, V, R, H, Q, P, I, F, M, Y, L, W, D, E and C; X3 of SEQ ID NO:74 is selected from M, W, G, Q, V, T, S, L, P, I, A, K, R and C; X4 of SEQ ID NO:75 is selected from W, L, A, I, M, D, T, K, R, G, F, E, V, C and P; X5 of SEQ ID NO:76 is selected from C, E, P and T: X6 of SEQ ID NO:77 is selected from A, S, V, N, K, L, T, I, P, Q, C, G, Y, W, F, and R; X7 of SEQ ID NO:78 is selected from P, C, S, and T; X8 of SEQ ID NO:79 is selected from A, T, G, L, N, C, P, F, Q, H, R, K, E, W, and Y ; X9 of SEQ ID NO:80 is selected from P, L, T, C, A, I, L, Q, V, E, M, K, R, G and P; X10 of SEQ ID NO:81 is selected from E, H, I, M, N, Y, P, Q, D, K and L; X11 of SEQ ID NO:82 is selected from F, Y, I, T, V, M, A, S, N, G, W, E, K, P, R and D; and X12 of SEQ ID NO:83 is selected from G, Y, V, N, T, S, H, E, P, W, F and M.

[0355] Such HuCD3 CDR variant sequences have reduced binding affinity compared to huCD3 wildtype CDR sequences. The six CDR sequences may be inserted in any of the huCD3 VH and VL framework sequences VH1, VH2,VH3andVH4, andVL1,VL2andVL3, respectively, replacing theCDRsequencesof huCD3. Inoneembodiment, the six CDRsequences are inserted in the huCD3 framework sequences VH1 andVL1. In a further embodiment, the six CDR sequences have been inserted in the huCD3 framework sequences VH1 and VL1, wherein the amino acid T in positon 41 of VL1 (SEQ ID NO:10) has been mutated to K.

[0356] In yet a further embodiment, the CD3 binding arm comprises the six CDR sequences selected from the CDR sequences set forth in the below Table 3, wherein X2 of SEQ ID NO:73 is selected from M and P; X3 of SEQ ID NO:74 is A; X4 of SEQ ID NO:75 is E; X6 of SEQ ID NO:77 is selected from F, G, I, K, L, and N; X7 of SEQ ID NO:78 is P; X8 of SEQ ID NO:79 is selected from A and G; and 45 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 X9 of SEQ ID NO:80 is selected from M, R and V. TABLE 3 VH CDR1 (SEQ ID NO) VH CDR2 (SEQ ID NO) VH CDR3 (SEQ ID NO) VLCDR1 (SEQ ID NO) VL CDR2 VLCDR3 (SEQ ID NO) 73 2 3 4 GTN 5 74 2 3 4 GTN 5 1 75 3 4 GTN 5 1 2 77 4 GTN 5 1 2 78 4 GTN 5 1 2 79 4 GTN 5 1 2 80 4 GTN 5

[0357] Such HuCD3 CDR variant sequences have reduced binding affinity compared to huCD3 wildtype CDR sequences. The six CDR sequences may be inserted in any of the huCD3 VH and VL framework sequences VH1, VH2,VH3andVH4, andVL1,VL2andVL3, respectively, replacing theCDRsequencesof huCD3. Inoneembodiment, the six CDRsequences are inserted in the huCD3 framework sequences VH1 andVL1. In a further embodiment, the six CDR sequences have been inserted in the huCD3 framework sequences VH1 and VL1, wherein the amino acid T in positon 41 of VL1 (SEQ ID NO:10) has been mutated to K.

[0358] In a further embodiment of the bispecific antibodyasdefined in anyof the embodiments disclosedherein, the first binding arm is derived fromaCD3antibody having a binding affinity value (KD) to humanCD3epsilon higher than 3.4x10‑8 M as determined by Bio-Layer Interferometry, such as from 3.5x10‑8 M to 9.9x10‑8 M, or from 1.0x10‑7 M to 9.9x10‑7 M as determined by Bio-Layer Interferometry.

[0359] In a further embodiment of the invention, one or both of the antibodies forming part of the bispecific antibody have been engineered to reduce or increase the binding to the neonatal Fc receptor (FcRn) in order to manipulate the serum half-life of the bispecific antibody. Techniques for increasing or reducing the serum half-life are well-known in the art. See for example Dall’Acqua et al. 2006, J. Biol. Chem., 281:23514‑24; Hinton et al. 2006,J. Immunol., 176:346‑56; and Zalevsky et al. 2010 Nat. Biotechnol., 28:157‑9.

[0360] In one aspect, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first constant heavy chain (HC) and a first constant light chain (LC), wherein the positions corresponding to positions L234, L235, andD265 in the human IgG1 heavy chain of SEQ IDNO:15 of both the first heavy chain and the second heavy chain are F, E, and A, respectively.

[0361] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first and second constant heavy chain (HC) and a first and second constant light chain (LC), wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain of SEQ ID NO:15 of both the first heavy chain and the second heavy chain are F and E, respectively.

[0362] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first binding arm is a Fab arm derived from IgG1-huCD3-H1L1-FEAR, and the second binding arm is a Fab arm derived from IgG1‑7D8-FEAL.

[0363] Herein, huCD3-H1L1 refers to thehumanizedSP34anti-CD3antibodyhavingVH1andVL1whichare set forth in table 1 as SEQ IDNos: 6 and 10. FEAL refers to L234F, L235E and D265A and F405Lmutations in the constant region of the antibody whereas FEAR refers to L234F, L235E and D265A and K409R mutations in the constant region of the antibodywherein the aminoacid positions corresponds to the aminoacid postions of human IgG1. "IgG1" refers to that the antibody constant regions are derived from the human IgG1 outside the specified mutations. 7D8 refers to the anti-CD20 antibody having the VH and VL sequence set forth in Table 1 as SEQ ID Nos: 27 and 28.

[0364] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first binding arm is a half-molecule antibody derived from IgG1-huCD3-H1L1-FEAR, and the second binding arm is a half- molecule antibody derived from IgG1‑7D8-FEAL.

[0365] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first binding arm is a Fab arm derived from IgG1-huCD3-H1L1-FEAL, and the second binding arm is a Fab arm derived from IgG1‑7D8-FEAR.

[0366] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein the first binding arm is a half-molecule antibody (i.e. comprising one heavy and one light chain) derived from IgG1-huCD3-H1L1- 46 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 FEAL, and the second binding arm is a half-molecule antibody (Fab arm and Fc arm) derived from IgG1‑7D8-FEAR.

[0367] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first and second constant heavy chain (HC) and a first and second constant light chain (LC), wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain of SEQ ID NO:15 of both the first constant heavy chain and the second constant heavy chain are F, E, and A, respectively, and wherein the position corresponding to F405 in the human IgG1 heavy chain of SEQ ID NO: 15 of the first constant heavy chain is L, and the position corresponding toK409 in the human IgG1heavy chain of SEQ IDNO: 15 of the second constant heavy chain isR, andwherein (i) the positions corresponding to positionsF10, T41, K55, andL97 in the lambda light chain of SEQ IDNO: 10 of the first constant light chain are L, K, N, and H, respectively, or (ii) the position corresponding to position T41 in the lambda light chain of SEQ ID NO: 10 of the first light constant chain is K. Further embodiments of the bispecific antibodies

[0368] Thebispecific antibody of the invention canbe of any isotype. The choice of isotype typicallywill be guided by the desired effector functions, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The effector function of the antibodies of the present invention may be changed by isotype switching to, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one embodiment, both Fc-regions of an antibody of the present invention are of the IgG1 isotype, for instance an IgG1,κ. In one embodiment, the two Fc-regions of a bispecific antibody are of the IgG1 and IgG4 isotypes, respectively. Optionally, the Fc-region may bemodified in the hinge and / or CH3 region as described elsewhere herein.

[0369] In one embodiment, the bispecific antibody of the invention is a full-length antibody, preferably an IgG1 antibody, in particular an IgG1,κ antibody or a variant thereof. In another embodiment, the bispecific antibody of the invention comprises an antibody fragment or a single-chain antibody. Antibody fragments may e.g. be obtained by fragmentation using conventional techniques, and the fragments screened for utility in the same manner as described herein for whole antibodies. For example, F(ab’)2 fragments may be generated by treating an antibody with pepsin. The resulting F(ab’)2 fragment may be treated to reduce disulfide bridges with a reducing agent, such as dithiothreitol, to produce Fab’ fragments. Fab fragmentsmay be obtained by treating an antibodywith papain. A F(ab’)2 fragmentmay also be produced by binding Fab’ fragments via a thioether bond or a disulfide bond. Antibody fragments may also be generated by expressionof nucleic acidsencoding such fragments in recombinant cells (see for instanceEvanset al., J. Immunol.Meth. 184, 123‑38 (1995)). For example, a chimeric gene encoding a portion of an F(ab’)2 fragment could include DNA sequencesencoding theCH1domainandhinge regionof theHchain, followedbya translational stop codon to yield sucha truncated antibody fragment molecule.

[0370] The bispecific CD3xCD20 antibodies of the inventionmay also be prepared from single chain antibodies. Single chain antibodies are peptides in which the heavy and light chain Fv regions are connected. In one embodiment, the bispecificantibodyof the present invention comprisesasingle-chainFv (scFv)wherein theheavy and light chains in theFv of a CD20 antibody of the present invention are joined with a flexible peptide linker (typically of about 10, 12, 15 or more amino acid residues) in a single peptide chain. Methods of producing such antibodies are described in for instance US 4,946,778, Pluckthun in ’The Pharmacology of Monoclonal Antibodies’, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269‑315 (1994), Bird et al., Science 242, 423‑426 (1988), Huston et al., PNASUSA 85, 5879‑5883 (1988) and McCafferty et al., Nature 348, 552‑554 (1990). A bispecific antibody can then be formed from two VH and VL from a single-chain CD20 antibody and a single-chain CD3 antibody, or a polyvalent antibody formed frommore than two VH and VL chains.

[0371] In one embodiment, one or both Fc-regions of the CD3 and CD20 monoclonal antibodies for producing a bispecific antibody of the invention are effector-function-deficient. Conjugates

[0372] In a further aspect, the present inventionprovidesabispecificCD3xCD20antibody linkedor conjugated to oneor more therapeutic moieties, such as a cytokine, an immunesuppressant, an immune-stimulatory molecule and / or a radioisotope. Such conjugates are referred to herein as "immunoconjugates" or "drug conjugates". Immunoconjugates which include one or more cytotoxins are referred to as "immunotoxins".

[0373] In oneembodiment, the first and / or secondFc-region is conjugated to adrugor aprodrugor contains anacceptor group for the same. Such acceptor group may e.g. be an unnatural amino acid. Compositions

[0374] In a further aspect, the invention relates toa composition comprisingabispecificantibodyaccording to anyoneof 47 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 the embodiments disclosed herein.

[0375] In a further aspect, the invention relates to a pharmaceutical composition comprising: - a bispecific CD3xCD20 antibody as defined in any of the embodimets disclosed herein, and - a pharmaceutically acceptable carrier.

[0376] The pharmaceutical composition of the present invention may contain one bispecific antibody of the present invention or a combination of different bispecific antibodies of the present invention.

[0377] Thepharmaceutical compositionsmaybe formulated in accordancewith conventional techniques suchas those disclosed inRemington:TheScienceandPracticeofPharmacy, 19thEdition,Gennaro,Ed.,MackPublishingCo.,Easton, PA, 1995. A pharmaceutical composition of the present invention may e.g. include diluents, fillers, salts, buffers, detergents (e. g., a nonionic detergent, such as Tween‑20 or Tween‑80), stabilizers (e. g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0378] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, anti- bacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a bispecific antibody of the present invention. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, suchasethyl oleate, and / or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0379] Pharmaceutical bispecific antibodies of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0380] Pharmaceutical bispecific antibodies of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.

[0381] The pharmaceutical bispecific antibodies of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservativesor buffers,whichmayenhance theshelf life or effectivenessof thepharmaceutical composition. The bispecific antibodies of the present inventionmay be preparedwith carriers that will protect the bispecific antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocom- patible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art.

[0382] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0383] The actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or theamide thereof, the route of administration, the timeof administration, the rate of excretionof the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0384] The pharmaceutical composition may be administered by any suitable route and mode.

[0385] In one embodiment, a pharmaceutical composition of the present invention is administered parenterally. "Administered parenterally" as used herein meansmodes of administration other than enteral and topical administration, 48 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 usually by injection, and includeepidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, sub- capsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0386] Inoneembodiment thatpharmaceutical composition isadministeredby intravenousor subcutaneous injectionor infusion. Uses

[0387] In one aspect, the invention relates to the bispecific antibody according to any one of the embodiments disclosed herein, the composition as disclosed herein, or the pharmaceutical composition as disclosed herein for use as a medicament.

[0388] In one aspect, the invention relates to the bispecific antibody according to any one of the embodiments disclosed herein, the composition as disclosed herein, or the pharmaceutical composition as disclosed herein for use in the treatment of a disease.

[0389] In one aspect, the invention relates to amethod of treatment of a disease comprising administering the bispecific antibody according to any one of the embodiments disclosed herein, the composition as disclosed herein, or the pharmaceutical composition as disclosed herein to a subject in need thereof.

[0390] In one embodiment, the disease is mature B-cell malignancy.

[0391] In one embodiment, the disease is cancer, such as NHL or B cell leukemia.

[0392] The bispecific antibodies of the invention may be used for a number of purposes. In particular, the bispecific antibodies of the invention may be used for the treatment of various forms of cancer, including metastatic cancer and refractory cancer.

[0393] In particular, the bispecific antibodies according to the invention may be useful in therapeutic settings in which specific targeting and T cell-mediated killing of cells that express CD20 is desired, and they may be more efficient compared to a regular CD20 antibody in certain such indications and settings.

[0394] Thebispecificantibodiesof the inventionalsohaveadditional utility in therapyanddiagnosis of a variety ofCD20- related diseases. For example, the bispecific antibodies can be used to elicit in vivo or in vitro one or more of the following biological activities: to inhibit thegrowthof and / or differentiationof a cell expressingCD20; to kill a cell expressingCD20; to mediate phagocytosis or ADCCof a cell expressingCD20 in thepresenceof humaneffector cells; tomediateCDCof a cell expressing CD20 in the presence of complement; to mediate apoptosis of a cell expressing CD20; and / or to induce translocation into lipid rafts upon binding CD20.

[0395] In another embodiment, the bispecific antibodies of the invention can be used to effect Tcell-mediated immune responses, inflammation and microenvironment re-modelling.

[0396] In a particular embodiment, the bispecific antibodies are used in vivo to treat, prevent or diagnose a variety of CD20-related diseases. Examples of CD20-related diseases include, among others, B cell lymphoma, e.g., non- Hodgkin’s lymphoma (NHL), B cell leukemia and immune diseases, e.g., autoimmune diseases, such as those listed below.

[0397] In oneembodiment the bispecific antibodies according to the invention are used for the treatment ofNHLorB cell leukemia.

[0398] In one embodiment, the bispecific antibodies according to the invention are used for the treatment of CD20 antibody-resistant NHL or B cell leukemia, such as rituximab‑ or ofatumumab-resistant NHL or B cell leukemia, e.g. rituximab-resistant non-aggressive B-cell lymphoma.

[0399] In one embodiment, the bispecific antibodies according to the invention are used for the treatment of Acute Lymphoblastic Leukemia (ALL), such as relapsed or refractory ALL.

[0400] Inoneembodiment, thebispecificantibodiesaccording to the inventionareused for the treatmentofCLL,suchas relapsed or refractory CLL.

[0401] In one embodiment, the bispecific antibodies according to the invention are used for the treatment of FL, such as or relapsed or refractory FL.

[0402] In one embodiment, the bispecific antibodies according to the invention are used for the treatment of AdultGrade III LymphomatoidGranulomatosis; Adult Nasal Type Extranodal NK / T-cell Lymphoma; Anaplastic LargeCell Lymphoma; Angioimmunoblastic T-cell Lymphoma; Contiguous Stage II Adult Burkitt Lymphoma; Contiguous Stage II Adult Diffuse Large Cell Lymphoma; Contiguous Stage II Adult Diffuse Mixed Cell Lymphoma; Contiguous Stage II Adult Diffuse Small Cleaved Cell Lymphoma; Contiguous Stage II Adult Immunoblastic Large Cell Lymphoma; Contiguous Stage II Adult Lymphoblastic Lymphoma; Contiguous Stage II Grade 1 Follicular Lymphoma; Contiguous Stage II Grade 2 Follicular Lymphoma; Contiguous Stage II Grade 3 Follicular Lymphoma; Contiguous Stage II Mantle Cell Lymphoma; Contiguous Stage II Marginal Zone Lymphoma; Contiguous Stage II Small Lymphocytic Lymphoma; Cutaneous B-cell Non-Hodgkin Lymphoma; Epstein-Barr Virus Infection; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue;Hepatosplenic T-cell Lymphoma; Intraocular Lymphoma;NodalMarginal ZoneB-cell Lymphoma;Noncontiguous 49 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 Stage II Adult Burkitt Lymphoma; Noncontiguous Stage II Adult Diffuse Large Cell Lymphoma; Noncontiguous Stage II AdultDiffuseMixedCell Lymphoma;NoncontiguousStage IIAdultDiffuseSmallCleavedCell Lymphoma;Noncontiguous Stage II Adult Immunoblastic Large Cell Lymphoma; Noncontiguous Stage II Adult Lymphoblastic Lymphoma; Non- contiguousStage II Grade 1Follicular Lymphoma;NoncontiguousStage II Grade 2Follicular Lymphoma;Noncontiguous Stage II Grade 3 Follicular Lymphoma; Noncontiguous Stage II Mantle Cell Lymphoma; Noncontiguous Stage II Marginal Zone Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Noncutaneous Extranodal Lymphoma; Peripheral T-cell Lymphoma; Post-transplant Lymphoproliferative Disorder; Progressive Hairy Cell Leukemia, Initial Treatment; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Hodgkin Lymphoma;RecurrentAdult ImmunoblasticLargeCell Lymphoma;RecurrentAdult LymphoblasticLymphoma;Recurrent Adult T-cell Leukemia / Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides / Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma;RefractoryHairyCell Leukemia; Small IntestineLymphoma;SplenicMarginal ZoneLymphoma; Stage I Adult Burkitt Lymphoma; Stage I Adult Diffuse LargeCell Lymphoma; Stage I Adult DiffuseMixedCell Lymphoma; Stage IAdultDiffuseSmallCleavedCell Lymphoma;Stage IAdultHodgkinLymphoma;Stage IAdult ImmunoblasticLarge Cell Lymphoma;Stage IAdult Lymphoblastic Lymphoma;Stage IAdult T-cell Leukemia / Lymphoma;Stage ICutaneousT- cell Non-Hodgkin Lymphoma; Stage IGrade 1 Follicular Lymphoma; Stage IGrade 2 Follicular Lymphoma; Stage IGrade 3 Follicular Lymphoma; Stage I Mantle Cell Lymphoma; Stage I Marginal Zone Lymphoma; Stage I Small Lymphocytic Lymphoma; Stage IA Mycosis Fungoides / Sezary Syndrome; Stage IB Mycosis Fungoides / Sezary Syndrome; Stage II Adult Hodgkin’s Lymphoma; Stage II Adult T-cell Leukemia / Lymphoma; Stage II Cutaneous T-cell Non-Hodgkin Lym- phoma; Stage IIAMycosis Fungoides / Sezary Syndrome; Stage IIBMycosis Fungoides / Sezary Syndrome; Stage III Adult Burkitt Lymphoma; Stage III Adult Diffuse Large Cell Lymphoma; Stage III Adult Diffuse Mixed Cell Lymphoma; Stage III AdultDiffuseSmallCleavedCell Lymphoma;Stage III Adult Hodgkin Lymphoma;Stage III Adult Immunoblastic LargeCell Lymphoma; Stage III Adult Lymphoblastic Lymphoma; Stage III Adult T-cell Leukemia / Lymphoma; Stage III Cutaneous T- cell Non-Hodgkin Lymphoma; Stage III Grade 1 Follicular Lymphoma; Stage III Grade 2 Follicular Lymphoma; Stage III Grade 3 Follicular Lymphoma; Stage III Mantle Cell Lymphoma; Stage III Marginal Zone Lymphoma; Stage III Small Lymphocytic Lymphoma; Stage IIIA Mycosis Fungoides / Sezary Syndrome; Stage IIIB Mycosis Fungoides / Sezary Syndrome; Stage IVAdult Burkitt Lymphoma; Stage IVAdult Diffuse Large Cell Lymphoma; Stage IVAdult Diffuse Mixed Cell Lymphoma;Stage IVAdult DiffuseSmall CleavedCell Lymphoma;Stage IVAdult Hodgkin Lymphoma;Stage IVAdult Immunoblastic Large Cell Lymphoma; Stage IV Adult Lymphoblastic Lymphoma; Stage IV Adult T-cell Leukemia / Lym- phoma; Stage IV Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IV Grade 1 Follicular Lymphoma; Stage IV Grade 2 Follicular Lymphoma; Stage IV Grade 3 Follicular Lymphoma; Stage IV Mantle Cell Lymphoma; Stage IV Marginal Zone Lymphoma; Stage IV Small Lymphocytic Lymphoma; Stage IVA Mycosis Fungoides / Sezary Syndrome; Stage IVB Mycosis Fungoides / Sezary Syndrome; T-cell Large Granular Lymphocyte Leukemia; Testicular Lymphoma; Untreated Hairy Cell Leukemia; or Waldenström Macroglobulinemia.

[0403] In a particular embodiment, the antibodies of the invention are used to treat or to prevent NHL, as the antibodies deplete the CD20 bearing tumor cells.

[0404] NHL is a type of B cell lymphoma. Lymphomas, e.g., B cell lymphomas, are a group of related cancers that arise when a lymphocyte (a blood cell) becomes malignant. The normal function of lymphocytes is to defend the body against invaders: germs, viruses, fungi, even cancer. There are many subtypes and maturation stages of lymphocytes and, therefore, there are many kinds of lymphomas. Like normal cells, malignant lymphocytes can move to many parts of the body. Typically, lymphoma cells form tumors in the lymphatic system: bone marrow, lymph nodes, spleen, and blood. However, these cells can migrate to other organs. Certain types of lymphoma will tend to grow in locations in which the normal version of the cell resides. For example, it is common for follicular NHL tumors to develop in the lymph nodes.

[0405] CD20 is usually expressed at elevated levels on neoplastic (i.e., tumorigenic) B cells associated with NHL. Accordingly, CD20 binding antibodies of the invention can be used to depleteCD20 bearing tumor cells which lead toNHL and, thus, can be used to prevent or treat this disease.

[0406] The bispecific antibodies of the present invention also can be used to block or inhibit other effects of CD20. For example, it is known that CD20 is expressed on B lymphocytes and is involved in the proliferation and / or differentiation of these cells. Since B lymphocytes function as immunomodulators, CD20 is an important target for antibody mediated therapy to target B lymphocytes, e.g., to inactivate or kill B lymphocytes, involved in autoimmune disorders. Such autoimmune disorders include, for example, the above listed diseases

[0407] Similarly, the invention relates to amethod for killing a tumor cell expressingCD20, comprising administration, to an individual in need thereof, of an effective amount of a bispecific antibody of the invention.

[0408] Thepresent invention also relates to amethod for inhibiting growth and / or proliferation of one ormore tumor cells expressing CD20, comprising administration, to an indicidual in need thereof, of a bispecific antibody of the present invention. 50 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55

[0409] The present invention alto relates to a method for treating cancer, comprising a) selecting a subject suffering from a cancer comprising tumor cells expressing CD20, and b) administering to the subject the bispecific antibody of the present invention or a pharmaceutical composition of the present invention.

[0410] Also, the invention relates to the use of a bispecific antibody that binds to human CD3 and human CD20 for the preparation of amedicament for the treatment of cancer, such as one of the specific cancer indicationsmentioned herein.

[0411] The invention further relates to a bispecific antibody for use in the treatment of cancer, such as one of the cancer indications mentioned above.

[0412] In one embodiment the bispecific antibody is for use in the treatment of mature B-cell malignancies. In one embodiment the bispecific antibody is for use in the treatment of tumors expressing CD20. In one embodiment the bispecificantibody is for use in the treatment ofBcell lymphoma. Inoneembodiment thebispecificantibody is for use in the treatment of B cell lymphoma such as NHL. In one embodiment the bispecific antibody is for use in the treatment of precursor B cell lymphoblastic leukemia. In one embodiment the bispecific antibody is for use in the treatment of B cell chronic lymhocytic leukemia (CLL).

[0413] In one embodiment the bispecific antibody is for use in the treatment of small lymphocytic lymphoma (SLL).

[0414] In one embodiment the bispecific antibody is for use in the treatment of B cell prolymphocytic leukemia.

[0415] In one embodiment the bispecific antibody is for use in the treatment of lymphoplasmacytic lymphoma.

[0416] In one embodiment the bispecific antibody is for use in the treatment of mantle cell lymphoma (MCL).

[0417] In one embodiment the bispecific antibody is for use in the treatment of follicular lymphoma (FL), including low- grade, intermediate-grade and high-grade FL.

[0418] In one embodiment the bispecific antibody is for use in the treatment of B cell Hodgkin’s lymphoma.

[0419] In one embodiment the bispecific antibody is for use in the treatment of immune disorders in which CD20 expressing B cells are involved.

[0420] In one embodiment the bispecific antibody is for use in the treatment of psoriasis.

[0421] In one embodiment the bispecific antibody is for use in the treatment of sclerosis.

[0422] In one embodiment the bispecific antibody is for use in the treatment of inflammatory bowel disease.

[0423] For the abovementioned uses it is preferred that the antibody is bsIgG1-huCD3-H1L1-FEALxCD20‑7D8-FEAR, however it may be any of the bispecific CD3xCD20 antibodies disclosed herein.

[0424] The bispecific antibodies of the present invention have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of disorders involving cells expressing CD20. For example, the antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and non-human animals which respond to the bispecific antibodies against CD3 and CD20. Preferred subjects include human patients having disorders that can be corrected or ameliorated by inhibiting or controlling B cells (normal or malignant).

[0425] In oneaspect, the invention relates to adiagnostic composition comprising abispecific antibody according to any one of the embodiments as disclosed herein.

[0426] In one embodiment, the diagnostic composition is a companion diagnostic which is used to screen and select those patients who will benefit from treatment with the bispecific antibody.

[0427] In one embodiment, the bispecific antibodies of the present invention can be used to treat a subject with a tumorigenic disorder, e.g., a disorder characterized by the presence of tumor cells expressing CD20 including, for example, B cell lymphoma, e.g., NHL. Examples of tumorigenic diseaseswhich canbe treated and / or prevented includeB cell lymphoma, e.g., NHL, including precursor B cell lymphoblastic leukemia / lymphoma and mature B cell neoplasms, such as B cell chronic lymhocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma,mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate- grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse largeBcell lymphoma (DLBCL), Burkitt’s lymphoma, plasmacytoma, plasmacell myeloma, post-transplant lymphoproliferative disorder, Waldenström’s macroglobulinemia, malignant melanoma and anaplastic large-cell lymphoma (ALCL).

[0428] Further examples of B cell non-Hodgkin’s lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B cell lymphoma,mediastinal large B cell lymphoma, heavy chain diseases (including γ,µ, and a disease), lymphomas induced by therapy with immunosuppressive agents, such as cyclosporine-induced lym- phoma, and methotrexate-induced lymphoma.

[0429] In a further embodiment, the bispecific antibodies of the present invention can be used to treat B cell Hodgkin’s lymphoma.

[0430] Examples of immune disorders in which CD20 expressing B cells are involved which can be treated and / or prevented include autoimmune disorders, such as psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and 51 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 sclerosis, inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, respiratory distress syndrome, menin- gitis, encephalitis (incuding chronic fatigue syndrome / myalgic encephalitis (CFS / ME) and chronic fatigue syndrome / - myalgic encephalitis (CFS / ME), uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud’s syndrome, Sjögren’s syndrome, juvenile onset diabetes, Reiter’s disease, Behçet’s disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathies, immune-mediated thrombocyto- penias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves’ disease, Hashimoto’s thyroiditis, Wegener’s granulomatosis, Omenn’s syndrome, chronic renal failure, acute infectious mononucleosis, HIV, and herpes virus associated diseases. Further examples are severe acute respiratory distress syndrome and choreoretinitis. Furthermore, other diseases and disorders include those caused by or mediated by infection of B-cells with virus, such as Epstein-Barr virus (EBV).

[0431] Further examples of inflammatory, immune and / or autoimmune disorders in which autoantibodies and / or excessive B lymphocyte activity are prominent and which can be treated and / or prevented, include the following: vasculitidesandother vessel disorders,suchasmicroscopicpolyangiitis,Churg-Strausssyndrome,andotherANCA- associated vasculitides, polyarteritis nodosa, essential cryoglobulinaemic vasculitis, cutaneous leukocytoclastic angiitis, Kawasaki disease, Takayasu arteritis, giant cell arthritis, Henoch-Schönlein purpura, primary or isolated cerebral angiitis, erythema nodosum, thrombangiitis obliterans, thrombotic thrombocytopenic purpura (including hemolytic uremic syndrome), and secondary vasculitides, including cutaneous leukocytoclastic vasculitis (e.g., secondary to hepatitis B, hepatitis C, Waldenström’s macroglobulinemia, B-cell neoplasias, rheumatoid arthritis, Sjögren’s syndrome, or systemic lupus erythematosus); further examples are erythema nodosum, allergic vasculitis, panniculitis, Weber-Christian disease, purpura hyperglobulinaemica, and Buerger’s disease; skin disorders, such as contact dermatitis, linear IgA dermatosis, vitiligo, pyoderma gangrenosum, epidermolysis bullosa acquisita, pemphigus vulgaris (including cicatricial pemphigoid and bullous pemphigoid), alopecia areata (including alopecia universalis and alopecia totalis), dermatitis herpetiformis, erythema multiforme, and chronic autoimmune urticaria (including angioneurotic edema and urticarial vasculitis); immune-mediated cytopenias, such as autoimmune neutropenia, and pure red cell aplasia; connective tissue disorders, such as CNS lupus, discoid lupus erythematosus, CRESTsyndrome, mixed connective tissue disease, polymyositis / dermatomyositis, inclusion body myositis, secondary amyloidosis, cryoglobulinemia type I and type II, fibromyalgia, phospholipid antibody syndrome, secondary hemophilia, relapsing polychondritis, sarcoidosis, stiffman syndrome, and rheumatic fever; further examples are eosinophil fasciitis,myositis, and juvenile dermatomyositis; arthritides, such as ankylosing spondylitis, juvenile chronic arthritis, adult Still’s disease, and SAPHO syndrome; further examples are sacroileitis, reactive arthritis, Still’s disease, and gout; hematologic disorders, such as aplastic anemia, primary hemolytic anemia (including cold agglutinin syndrome), hemolytic anemia secondary to CLL or systemic lupus erythematosus; POEMS syndrome, pernicious anemia, and Waldenström’s purpura hyperglobulinaemica; further examples are agranulocytosis, autoimmune neutropenia, Franklin’s disease, Seligmann’s disease, µ-chain disease, paraneoplastic syndrome secondary to thymoma and lymphomas, and factor VIII inhibitor formation; endocrinopathies, such as polyendocrinopathy, and Addison’s disease; further examples are autoimmune hypogly- cemia, autoimmune hypothyroidism, autoimmune insulin syndrome, de Quervain’s thyroiditis, and insulin receptor antibody-mediated insulin resistance; hepato-gastrointestinal disorders, such as celiac disease,Whipple’s disease, primary biliary cirrhosis, chronic active hepatitis, and primary sclerosing cholangiitis; a further example is autoimmune gastritis; nephropathies, such as rapid progressive glomerulonephritis, post-streptococcal nephritis, Goodpasture’s syn- drome, membranous glomerulonephritis, and cryoglobulinemic nephritis; a further example is minimal change disease; neurological disorders, such as autoimmune neuropathies, mononeuritis multiplex, Lambert-Eaton’s myasthenic syndrome, Sydenham’s chorea, tabes dorsalis, and Guillain-Barré’s syndrome; further examples are myelopathy / - tropical spastic paraparesis, myasthenia gravis, acute inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy; cardiac and pulmonary disorders, such as fibrosing alveolitis, bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis, Löffler’s syndrome, myocarditis, and pericarditis; further examples are hypersensitivity pneumonitis, and paraneoplastic syndrome secondary to lung cancer; allergic disorders, such as bronchial asthma and hyper-IgE syndrome; a further example is amaurosis fugax; ophthalmologic disorders, such as idiopathic chorioretinitis; infectious diseases, such as parvovirus B infection (including hands-and-socks syndrome); and gynecological-obstretical disorders, such as recurrent abortion, recurrent fetal loss, and intrauterine growth retarda- 52 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 tion; a further example is paraneoplastic syndrome secondary to gynaecological neoplasms; male reproductive disorders, such as paraneoplastic syndrome secondary to testicular neoplasms; and transplantation-derived disorders, such as allograft and xenograft rejection, and graft-versus-host disease (including chronic graft-versus-host disease).

[0432] In one embodiment, the disease is an inflammatory, immune and / or autoimmune disorder selected from ulcerative colitis, Crohn’s disease, juvenile onset diabetes, multiple sclerosis, immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia (including autoimmune hemolytic anemia), myasthenia gravis, systemic sclerosis, and pemphigus vulgaris.

[0433] In a further embodiment of themethodsof treatment of thepresent invention, theefficacyof the treatment is being monitored during the therapy, e.g. at predefined points in time, by determining tumor burden orCD20 expression levels on the relevant tumor cells.

[0434] Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolusmay be administered, several divided dosesmay be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0435] The efficient dosages and the dosage regimens for the bispecific antibodies depend on the disease or condition tobe treatedandmaybedeterminedby thepersons skilled in theart. Anexemplary, non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.001‑10 mg / kg, such as about 0.001‑5 mg / kg, for example about 0.001‑2 mg / kg, such as about 0.001‑1 mg / kg, for instance about 0.001, about 0.01, about 0.1, about 1 or about 10mg / kg.Another exemplary, non-limiting range for a therapeutically effective amount of a bispecificantibodyof the present invention is about 0.1‑100 mg / kg, such as about 0.1‑50 mg / kg, for example about 0.1‑20 mg / kg, such as about 0.1‑10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[0436] A physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the bispecific antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect andgradually increase thedosageuntil thedesiredeffect is achieved. Ingeneral, a suitabledaily doseof abispecificantibodyof thepresent inventionwill be that amount of the compoundwhich is the lowest doseeffective to produce a therapeutic effect. Administration may e.g. be parenteral, such as intravenous, intramuscular or subcuta- neous. In one embodiment, the bispecific antibodiesmay be administered by infusion in a weekly dosage of calculated by mg / m2. Such dosages can, for example, be based on themg / kg dosages provided above according to the following: dose (mg / kg) x 70: 1.8. Such administrationmay be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administrationmay be performedby continuous infusionover aperiodof from2 to24hours, suchasof from2 to12hours. In oneembodiment, the bispecific antibodies may be administered by slow continuous infusion over a long period, such asmore than 24 hours, in order to reduce toxic side effects.

[0437] In one embodiment the bispecific antibodies may be administered in a weekly dosage of calculated as a fixed dose for up to8 times, suchas from4 to6 timeswhengivenonceaweek.Such regimenmaybe repeatedoneormore times as necessary, for example, after 6 months or 12 months. Such fixed dosages can, for example, be based on the mg / kg dosages provided above, with a body weight estimate of 70 kg. The dosagemay be determined or adjusted bymeasuring the amount of bispecific antibody of the present invention in the blood upon administration by for instance taking out a biological sampleandusinganti-idiotypicantibodieswhich target theCD20antigenantigen-binding regionof thebispecific antibodies of the present invention.

[0438] In oneembodiment, thebispecific antibodiesmaybeadministeredasmaintenance therapy, suchas, e.g., oncea week for a period of 6 months or more.

[0439] A bispecific antibodymay also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and / or reduce the risk of recurrencewhen a cancer is in remission.

[0440] The bispecific antibodies of the invention may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the bispecific antibody-containing medicament is for combination with one or more further therapeutic agents, such as a cytotoxic, chemotherapeutic or anti-angiogenic agent.

[0441] Such combined administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The present invention thus also provides methods for treating a disorder involving cells expressing CD20 as described above, which methods comprise administration of a bispecific antibody of the present invention combinedwith one ormore additional therapeutic agents as described below.

[0442] In one embodiment, the present invention provides a method for treating a disorder involving cells expressing 53 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 CD20 in a subject, whichmethod comprises administration of a therapeutically effective amount of a bispecific antibody of the present invention, and optionally at least one additional therapeutic agent, or an antibody binding to a different CD20 epitope than said antibody, to a subject in need thereof.

[0443] In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of a bispecific antibody of the present invention and at least one additional therapeutic agent to a subject in need thereof.

[0444] In one embodiment, such an additional therapeutic agent may be selected from a tyrosine kinase inhibitor (TKI), such as imatinib (Glivec, Gleevec STI571), ibrutinib (PCI‑32765, Imbruvica) or lapatinib (PTK787 / ZK222584).

[0445] In one embodiment, such an additional therapeutic agent may be selected from a Bruton tyrosine kinase (BTK) inhibitor, such as ibrutinib.

[0446] In one embodiment, such an additional therapeutic agentmay be selected fromaproteasome inhibitor (PI), such as carfilzomib.

[0447] In one embodiment, such an additional therapeutic agent may be selected from a immunomodulatory agent (IMID), such as pomalidomide, thalidomide, or lenalidomide.

[0448] In one embodiment, such an additional therapeutic agent may be selected from a phosphoinositide 3-kinase inhibitor, such as idelalisib or duvelisib.

[0449] In oneembodiment, suchanadditional therapeutic agentmaybeselected fromanauroraAkinase inhibitor, such as alisertib.

[0450] In one embodiment, such an additional therapeutic may be selected from a B-cell lymphoma‑2 (Bcl‑2) inhibitor, such as venetoclax.

[0451] In oneembodiment, suchanadditional therapeuticmaybeselected fromhistonedeacytelase (HDAC) inhibitors, such as panobinostat.

[0452] Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. In one embodiment, such therapeutic agents include one ormore chemotherapeutics, from the class of alkylating agents, antimetabolites, mitotic inhibitors, anti-tumor antibiotics, topoisomerase inhibitors or platinum analogs. Examples of such chemotherapeutic agents are doxorubicin (Adriamycin), cisplatin (Platinol), bleomycin (Blenoxane), carmustine (Gliadel), cyclophosphamide (Cytoxan, Procytox, Neosar), bendamustine, and chlorambucil (Leukeran).

[0453] In another embodiment, bispecific antibodies of the present invention may be administered in combination with chlorambucil; CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone or prednisolone); cyclophospha- mide andprednisolone; cyclophosphamide, vincristine, and prednisone; cyclophosphamide, vincristine, doxorubicin, and prednisone; fludarabine and an alkylating agent; dose-adjusted EPOCH (etoposide, prednisolone, vincristine, cyclopho- sphamide and doxorubicin); GemOx (gemcitabine and oxaliplatin); GDP (gemcitabine, dexamethasone and cisplatin) or in combination with other commonmulti-drugs regimens for NHL, such as disclosed, e.g., in Non-Hodgkin’s Lymphomas: Making sense of Diagnosis, Treatment, and Options, Lorraine Johnston, 1999, O’Reilly and Associates, Inc.

[0454] In one embodiment, such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, as- paraginase, gemcitabine or cladribine.

[0455] In another embodiment, such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, bu- sulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin.

[0456] In another embodiment, suchanadditional therapeutic agentmaybeselected fromananti-mitotic agent, suchas taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.

[0457] In another embodiment, such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.

[0458] Inanother embodiment, thepresent inventionprovidesamethod for treatingadisorder involvingcellsexpressing CD20 in a subject, whichmethod comprises administration of a therapeutically effective amount of a bispecific antibody of the present invention and at least one inhibitor of angiogenesis, neovascularization, and / or other vascularization to a subject in need thereof

[0459] Examples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY 12‑9566, AG 3340, BMS‑275291 and similar agents), inhibitors of endothelial cell migration and proliferation (such as TNP‑470, squalamine, 2-methoxyestradiol, combretastatins, endostatin, angiostatin, penicil- lamine, SCH66336 (Schering-Plough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) and similar agents), antagonists of angiogenic growth factors (such as such as ZD6474, SU6668, antibodies against angiogenic agents and / or their receptors (such as VEGF (e.g. bevacizumab), bFGF, and angiopoietin‑1), thalidomide, thalidomide analogs (such asCC‑5013), Sugen 5416, SU5402, antiangiogenic ribozyme (such as angiozyme), interferon a (such as interferon a2a), suramin and similar agents), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine 54 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 kinase inhibitors (such as SU011248), inhibitors of endothelial-specific integrin / survival signaling (such as vitaxin and similar agents), copper antagonists / chelators (such as tetrathiomolybdate, captopril and similar agents), carboxyamido- triazole (CAI), ABT‑627, CM101, interleukin‑12 (IL‑12), IM862, PNU145156E as well as nucleotide molecules inhibiting angiogenesis (such as antisense-VEGF-cDNA, cDNA coding for angiostatin, cDNA coding for p53 and cDNA coding for deficient VEGF receptor‑2).

[0460] Other examples of such inhibitors of angiogenesis, neovascularization, and / or other vascularization are anti- angiogenic heparin derivatives (e.g., heperinase III), temozolomide, NK4, macrophage migration inhibitory factor, cyclooxygenase‑2 inhibitors, inhibitors of hypoxia-inducible factor 1, anti-angiogenic soy isoflavones, oltipraz, fumagillin and analogs thereof, somatostatin analogues, pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin, thrombos- pondin, NM‑3, combrestatin, canstatin, avastatin, antibodies against other targets, such as anti-alpha-v / beta‑3 integrin and anti-kininostatin antibodies.

[0461] In oneembodiment, a therapeuticagent for use in combinationwithabispecificantibody for treating thedisorders as described above may be an anti-cancer immunogen, such as a cancer antigen / tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM / TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA), tumor- associated glycoprotein 72 (TAG‑72), gp100, Melan-A, MART‑1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., human papillomavirus vaccines) or tumor-derived heat shock proteins,

[0462] In oneembodiment, a therapeuticagent for use in combinationwithabispecificantibody for treating thedisorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNγ, IL‑2, IL‑4, IL‑6, IL‑7, IL‑10, IL‑12, IL‑13, IL‑15, IL‑18, IL‑23, IL‑24, IL‑27, IL‑28a, IL‑28b, IL‑29, KGF, IFNα (e.g., INFa2b), IFNβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα. Suitable chemokines may include Glu-Leu-Arg (ELR)‑negative chemokines such as IP‑10, MCP‑3, MIG, and SDF‑1α from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0463] In oneembodiment, a therapeuticagent for use in combinationwithabispecificantibody for treating thedisorders as described above may be a cell cycle control / apoptosis regulator (or "regulating agent"). A cell cycle control / apoptosis regulator may includemolecules that target andmodulate cell cycle control / apoptosis regulators such as (i) cdc‑25 (such asNSC663284), (ii) cyclin-dependent kinases thatoverstimulate thecell cycle (suchasflavopiridol (L868275,HMR1275), 7-hydroxystaurosporine (UCN‑01, KW‑2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerasemodulators (such as BIBR1532, SOT‑095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055). Non-limiting examples ofmolecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis‑2 ligand (Apo‑2L), antibodies that activate TRAIL receptors, interferon-γ (IFN-γ) and anti-sense Bcl‑2.

[0464] In oneembodiment, a therapeuticagent for use in combinationwithabispecificantibody for treating thedisorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / estinyl, an antiandrogene (such as flutaminde / eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy-progesterone / provera, megestrol acepate / megace), an adrenocorti- costeroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole / arimidex, aminoglutethi- mide / cytraden, exemestane) or a hormone inhibitor (such as octreotide / sandostatin).

[0465] In oneembodiment, a therapeuticagent for use in combinationwithabispecificantibody for treating thedisorders as described above may be an immune check-point inhibitor, such as molecules that block the activity of CTLA‑4, e.g. ipilimumab, PD‑1, e.g. pembrolizumab, PD-L1, TIM3, TIGIT, BTLA, VISTA or LAG‑3.

[0466] In oneembodiment, a therapeuticagent for use in combinationwithabispecificantibody for treating thedisorders as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0467] Examples of other anti-cancer agents, whichmay be relevant as therapeutic agents for use in combinationwith a bispecific antibody according to the invention for treating the disorders as described above are differentiation inducing agents, retinoicacidanalogues (suchasall trans retinoicacid, 13-cis retinoicacidandsimilar agents), vitaminDanalogues (suchas seocalcitol and similar agents), inhibitors of ErbB3,ErbB4, IGF-IR, insulin receptor, PDGFRa,PDGFRbeta, Flk2, Flt4, FGFR1,FGFR2,FGFR3,FGFR4,TRKA,TRKC,RON(suchasananti-RONantibody),Sea,Tie,Tie2,Eph,Ret,Ros, Alk, LTK, PTK7 and similar agents.

[0468] Examples of other anti-cancer agents, whichmay be relevant as therapeutic agents for use in combinationwith a bispecific antibody according to the invention for treating the disorders as described above are estramustine and epirubicin.

[0469] Examples of other anti-cancer agents, whichmay be relevant as therapeutic agents for use in combinationwith a bispecific antibody according to the invention for treating the disorders as described above are a HSP90 inhibitor like 17- allyl amino geld-anamycin, antibodies directed against a tumor antigen such as PSA,CA125, KSA, integrins, e.g. integrin β1, or inhibitors of VCAM.

[0470] Examples of other anti-cancer agents, whichmay be relevant as therapeutic agents for use in combinationwith a 55 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 bispecific antibody for treating the disorders as described above are calcineurin-inhibitors (such as valspodar, PSC 833 and other MDR‑1 or p-glycoprotein inhibitors), TOR-inhibitors (such as sirolimus, everolimus and rapamcyin), and inhibitors of "lymphocyte homing" mechanisms (such as FTY720), and agents with effects on cell signaling such as adhesion molecule inhibitors (for instance anti-LFA).

[0471] In yet another embodiment, the bispecific antibodies may be administered in conjunction with radiotherapy and / or autologous or allogeneic peripheral stem cell or bone marrow transplantation.

[0472] In still another embodiment, the bispecific antibodies may be administered in combination with one or more antibodies selected from anti-CD25 antibodies, anti-CD19 antibodies, anti-CD20 antibodies (e.g. ofatumumab or rituximab), anti-CD21 antibodies, anti-CD22 antibodies, anti-CD37 antibodies, anti-CD38 antibodies, anti-IL6R antibo- dies, anti-IL8 antibodies, anti-IL15 antibodies, anti-IL15R antibodies, anti-CD4 antibodies, anti-CD11a antibodies (e.g., efalizumab), anti-alpha‑4 / beta‑1 integrin (VLA4) antibodies (e.g., natalizumab), and CTLA4-Ig.

[0473] In a further embodiment, the bispecific antibodies may be administered in combination with one or more antibodies that block immune checkpoints, such as anti-CTLA‑4 (CD152) antibodies, anti-PD‑1 (CD279) antibodies, anti-PD-L1 (CD274) antibodies, anti-LAG‑3 (CD223) antibodies, anti-TIM3 antibodies, anti-CEACAM1 (CD66a) anti- bodies, anti-VISTA antibodies, anti-TIGIT antibodies, anti-BTLA (CD272) antibodies.

[0474] In a further embodiment, thebispecificantibodiesmaybeadministered in combinationwithoneormoreagonistic antibodies which are specific for costimulatory receptors on immune cells, such as anti‑4‑1BB (CD137) antibodies (e.g. urelumab), anti-OX40 (CD134) antibodies, anti-CD40 antibodies, anti-CD27 antibodies.

[0475] In a further embodiment, the bispecific antibodies may be administered in combination with one or more type II macrophage depleting or polarizing antibodies, such as anti-CSF‑1R (CD115) antibodies.

[0476] In a further embodiment, the bispecific antibodies may be administered in combination with one or more antibodies that bind to molecules involved in regulation of the innate immune system, such as anti-CD47 antibodies, anti-CD200 antibodies, anti-CD200R antibodies, antibodies against killer cell inhibitory receptors (KIRs), antibodies against CD94 / NKG2 receptors, anti-CD305 (LAIR1).

[0477] In another particular embodiment, the bispecifc antibodies are administered in combination with one or more antibodies selected from anti-CD19 antibodies, anti-CD21 antibodies, anti-CD22 antibodies, anti-CD37 antibodies, and anti-CD38 antibodies for the treatment of malignant diseases.

[0478] In another particular embodiment, the bispecific antibodies are administered in combination with an anti-CD20 antibody, such as ofatumumab.

[0479] In still another particular embodiment, the bispecific antibodies are administered in combinationwith oneormore antibodies selected from anti-IL6R antibodies, anti-IL8 antibodies, anti-IL15 antibodies, anti-IL15R antibodies, anti-CD4 antibodies, anti-CD11a antibodies (e.g., efalizumab), anti-alpha‑4 / beta‑1 integrin (VLA4) antibodies (e.g natalizumab), and CTLA4-Ig for the treatment of inflammatory diseases.

[0480] In one embodiment, the bispecific antibody of the invention is for use in combination with one or more other therapeutic antibodies, such as zanolimumab, daratumumab (Darzalex), ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), and / or efalizumab (Raptiva).

[0481] In another embodiment the bispecific antibody of the invention is for use in combination with one or more antibody-drug conjugates (ADCs), for example brentuximabvedotin (Adcetris), inotuzumab ozogamicin (CMC‑544), polatuzumab vedotin (RG7593), coltuximab ravtansine (SAR3419), indatuximab ravtansine (BT‑062), inotuzumab ozogamicin (CMC‑544), denintuzumab mafodotin (SGN-CD19), polatuzumab vedotin (RG7596) or a CD37-specific antibody drug conjugated (for example IMGN529 or AGS67E).

[0482] In another embodiment, the bispecific antibody of the invention can be used in combination with an anti- inflammatory or an immunosuppressive agent. For example, the combination therapy can include a composition of the present inventionwith at least one anti-inflammatory agent or at least one immunosuppressive agent. In one embodiment such therapeutic agents include one ormore anti-inflammatory agents, such as a steroidal drug or a NSAID (nonsteroidal anti-inflammatory drug). Preferred agents include, for example, aspirin and other salicylates, Cox‑2 inhibitors, such as celecoxib (Celebrex), NSAIDs such as ibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen), etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin).

[0483] In another embodiment, such therapeutic agents include one or more DMARDs, such as methotrexate (Rheumatrex), hydroxychloroquine (Plaquenil), sulfasalazine (Asulfidine), pyrimidine synthesis inhibitors, e.g., lefluno- mide (Arava), IL‑1 receptor blocking agents, e.g., anakinra (Kineret), and TNF-α blocking agents, e.g., etanercept (Enbrel), infliximab (Remicade) and adalimumab.

[0484] In another embodiment, such therapeutic agents include one or more immunosuppressive agents, such as cyclosporine (Sandimmune, Neoral) and azathioprine (Imural).

[0485] In aparticular embodiment, thebispecificantibodiesareadministered in combinationwith ananti-CD25antibody for the treatment of bullous pemphigoid, e.g., in patients with graft-versus-host disease. 56 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 Radiotherapy - surgery

[0486] In one embodiment, the present invention provides a method for treating a disorder involving cells expressing CD20 in a subject, whichmethod comprises administration of a therapeutically effective amount of aCD3xCD20 bispecifc antibody of the present invention, and radiotherapy to a subject in need thereof.

[0487] In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprisesadministrationofa therapeutically effectiveamountof aCD3xCD20bispecificantibodyof thepresent invention, and radiotherapy to a subject in need thereof.

[0488] In one embodiment, the present invention provides the use of a bispecific antibody of the present invention, for the preparation of a pharmaceutical composition for treating cancer to be administered in combination with radiotherapy.

[0489] Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient. The source of radiationmay be either external or internal to the patient being treated (radiation treatmentmay, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing suchmethods include, e.g., radium, cesium‑137, iridium‑192, americium‑241, gold‑198, cobalt‑57, copper‑67, technetium‑99, iodide‑123, iodide‑131, and indium‑111.

[0490] Ina further embodiment, thepresent inventionprovidesamethod for treatingor preventingcancer,whichmethod comprises administration to a subject in need thereof of a therapeutically effective amount of a bispecific antibody of the present invention, in combination with surgery. Diagnostic uses

[0491] Thus, in one aspect, the invention relates to a diagnostic composition comprising a bispecific CD3xCD20 antibody as defined herein, and to its use.

[0492] In another aspect, the invention relates to a kit for detecting cross-linking between CD3‑ and CD20-expressing cells, in a sample derived from a patient such as a blood sample, lymph node sample or bonemarrow sample, comprising i) a bispecific antibody according to any one of the embodiments as disclosed herein; and ii) instructions for use of said kit.

[0493] In one embodiment, the present invention provides a kit for diagnosis of cancer comprising a container comprising a bispecific CD3xCD20 antibody, and one or more reagents for detecting cross-linking of CD20 expressing cells andCD3expressing cells. Reagentsmay include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that may be visualized.

[0494] In a further aspect, the invention relates to a method for detecting whether cross-linking between CD3‑ and CD20-expressing cells occurs in a sample derived from a patient, such as a blood sample, lymph node sample or bone marrowsample, uponadministrationofabispecificantibodyaccording toanyoneof theembodimentsasdisclosedherein, comprising the steps of: (i) contacting thesamplewithabispecificantibodyaccording toanyoneof theembodimentsasdisclosedhereinunder conditions that allow for formation of a complex between said bispecific antibody and theCD3‑ andCD20-expressing cells; and (ii) analyzing whether a complex has been formed.

[0495] Detection of the complex can be done bymethods known in the art, such as by themethod disclosed in Example 5.

[0496] Ina furtheraspect, the invention relates toananti-idiotypicantibodywhichbinds to thefirst antigen-binding region asdefined inanyoneof theembodimentsdisclosedherein, orwhichbinds to thesecondantigen-binding regionasdefined in any one of the embodiments disclosed herein.

[0497] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. EMBODIMENTS

[0498] 1. A bispecific antibody comprising (i) a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein said first antigen-binding region comprises (a) heavy chain variable (VH) region CDR1, 57 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 CDR2, and CDR3 having the sequences as set forth in SEQ IDNOs:1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ IDNO:4, the sequenceGTN, and the sequence as set forth in SEQ IDNO:5, respectively, or (b) heavy chain variable (VH) regionCDR1, CDR2, andCDR3 having the sequences as set forth in SEQ ID NOs:55, 56 and 57, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ IDNO:58, the sequence DTS, and the sequence as set forth in SEQ IDNO:59, respectively, and (ii) a second binding arm comprising a second antigen-binding region binding to human CD20. 2. A bispecific antibody according to embodiment 1 comprising a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon), wherein said first antigen-binding region comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ IDNOs:1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, respectively. 3. The bispecific antibody according to embodiment 1 or 2, wherein said first antigen-binding region comprises a first heavy chain variable sequence (VH), and a first light chain variable sequence (VL), and said second antigen-binding region comprisesa secondheavy chain variable sequence (VH), andasecond light chain variable sequence (VL) and wherein said variable sequences each comprises threeCDR sequences, CDR1, CDR2 andCDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively. 4. The bispecific antibody according to any one of the preceding embodiments, wherein (i) said first binding arm comprises a first heavy chain comprising a first heavy chain variable sequence (VH) and a first heavy chain constant sequence (CH), and a first light chain comprising a first light chain variable sequence (VL) and a first light chain constant sequence (CL), and (ii) said second binding arm comprises a second heavy chain comprising a second heavy chain variable sequence (VH) and a second heavy chain constant sequence (CH), and a second light chain comprising a second light chain variable sequence (VL) and a second light chain constant sequence (CL). 5. The bispecific antibody according to any one of the preceding embodiments, wherein said VH sequence of the first antigen-binding regionhasat least 90%,at least 95%,at least 97%,orat least 99%aminoacidsequence identity to the amino acid sequence as set forth in the VH sequences selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6; b) a VH sequence as set forth in SEQ ID NO:7; c) a VH sequence as set forth in SEQ ID NO:8; d) a VH sequence as set forth in SEQ ID NO:9; and e) a VH sequence as set forth in SEQ ID NO:17. 6. The bispecific antibody according to any one of the preceding embodiments, wherein said VL sequence of the first antigen-binding regionhasat least 90%,at least 95%,at least 97%,orat least 99%aminoacidsequence identity to the amino acid sequence as set forth in the VL sequences selected from the group consisting of: a) a VL sequence as set forth in SEQ ID NO:10; b) a VL sequence as set forth in SEQ ID NO:11; c) a VL sequence as set forth in SEQ ID NO:12; and d) a VL sequence as set forth in SEQ ID NO:18. 7. The bispecific antibody according to any one of the preceding embodiments, wherein the framework sequences of saidVHsequenceof thefirst antigen-binding regionhasat least 90%,at least 95%,at least 97%,or at least 99%amino acid sequence identity to the framework amino acid sequence as set forth in the VH sequences selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6; b) a VH sequence as set forth in SEQ ID NO:7; c) a VH sequence as set forth in SEQ ID NO:8; d) a VH sequence as set forth in SEQ ID NO:9; and e) a VH sequence as set forth in SEQ ID NO:17. 8. The bispecific antibody according to any one of the preceding embodiments, wherein the framework sequences of 58 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 saidVLsequenceof the first antigen-binding regionhasat least 90%,at least 95%, at least 97%,or at least 99%amino acidsequence identity to the frameworkaminoacidsequenceasset forth in theVLsequencesselected from thegroup consisting of: a) a VL sequence as set forth in SEQ ID NO: 10; b) a VL sequence as set forth in SEQ ID NO: 11; c) a VL sequence as set forth in SEQ ID NO:12; and d) a VL sequence as set forth in SEQ ID NO: 18. 9. The bispecific antibody according to any of embodiments 5 to 8 wherein the CDR sequences of said VH and VL sequences are not mutated but are as set forth in table 1. 10. The bispecific antibody according to any of embodiments 5 to 7wherein the VHandVL sequences only deviate in the framework sequences. 11.Thebispecificantibodyaccording toanyof embodiments5 to8wherein the the respectiveFR1,FR2,FR3andFR4 frameworksequencesof theVHandVLsequencesof thefirst antigen-binding regionhasat least 90%,at least 95%,at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of said VH and VL sequences and wherein the CDR sequences are not mutated. 12. Thebispecific antibodyaccording to anyoneof theprecedingembodiments,wherein saidVHsequenceof the first antigen-binding region is selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6; b) a VH sequence as set forth in SEQ ID NO:7; c) a VH sequence as set forth in SEQ ID NO:8; d) a VH sequence as set forth in SEQ ID NO:9; e) a VH sequence as set forth in SEQ ID NO:17. 13.The bispecific antibody according to any one of the preceding embodiments, wherein said VL sequence of the first antigen-binding region is selected from the group consisting of: a) a VL sequence as set forth in SEQ ID NO: 10; b) a VL sequence as set forth in SEQ ID NO: 11; c) a VL sequence as set forth in SEQ ID NO: 12; and d) a VL sequence as set forth in SEQ ID NO: 18. 14. The bispecific antibody according to any one of the preceding embodiments, wherein said VH and VL sequences of the first antigen-binding region are selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:10; b) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as set forth in SEQ ID NO:10; c) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as set forth in SEQ ID NO:10; d) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:11; e) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:12; f) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as set forth in SEQ ID NO:10; g) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as set forth in SEQ ID NO:11; h) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as set forth in SEQ ID NO:12; i) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as set forth in SEQ ID NO:11; j) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as set forth in SEQ ID NO:12; k) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as set forth in SEQ ID NO:11; l) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as set forth in SEQ ID NO:12; and m) a VH sequence as set forth in SEQ ID NO:17, and a VL sequence as set forth in SEQ ID NO:18. 15. The bispecific antibody according to any one of the preceding embodiments, wherein said first antigen-binding region comprises a VH sequence and a VL sequence selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ ID NO:10; 59 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 b) a VH sequence as set forth in SEQ ID NO:17, and a VL sequence as set forth in SEQ ID NO:18. 16. The bispecific antibody according to any one of the preceding embodiments, wherein said first antigen-binding region comprises the VH sequence having at least 90% sequence identity to the VH sequence set forth in SEQ ID NO:6, and the VL sequence having at least 90% sequence identity to the VL sequence as set forth in SEQ IDNO:10. 17. The bispecific antibody according to any one of the preceding embodiments, wherein said first antigen-binding region comprises the VH sequence having at least 95% sequence identity to the VH sequence set forth in SEQ ID NO:6, and the VL sequence having at least 95% sequence identity to the VL sequence as set forth in SEQ IDNO:10. 18. The bispecific antibody according to any one of the preceding embodiments, wherein said first antigen-binding region comprises the VH sequence having at least 97% sequence identity to the VH sequence set forth in SEQ ID NO:6, and the VL sequence having at least 97% sequence identity to the VL sequence as set forth in SEQ IDNO: 10. 19. The bispecific antibody according to any one of the preceding embodiments, wherein said first antigen-binding region comprises the VH sequence as set forth in SEQ ID NO:6, and the VL sequence as set forth in SEQ ID NO: 10. 20. The bispecific antibody according to any one of the preceding embodiments, wherein the first binding arm is derived from a mouse antibody. 21. The bispecific antibody according to any one of the preceding embodiments, wherein the first binding arm is derived from a humanized antibody. 22.The bispecific antibody according to any one of the preceding embodiments, wherein the first binding arm is derived from a full-length antibody. 23.The bispecific antibody according to any one of the preceding embodiments, wherein the first binding arm is derived from a full-length IgG1,λ (lambda) or IgG1, κ (kappa) antibody. 24. The bispecific antibody according to any of the preceding embodiments, wherein the second antigen-binding region is derived froman antibodywhich binds to an epitope on humanCD20, which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172. 25. The bispecific antibody according to embodiments 21, wherein the second antigen-binding region is derived from an antibody which binds to an epitope on human CD20, which further comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166. 26. The bispecific antibody according to any of the preceding embodiments, wherein the second antigen-binding region which binds to human CD20 comprises: (i) the VHCDR1 region of SEQ IDNO:32, the VHCDR2 region of SEQ IDNO:33, the VHCDR3 region of SEQ ID NO:34, (ii) the VHCDR1 region of SEQ IDNO:38, theVHCDR2 region of SEQ IDNO:39, theVHCDR3 region of SEQ ID NO:34, (iii)the VHCDR1 region of SEQ IDNO:42, the VHCDR2 region of SEQ IDNO:43, theVHCDR3 region of SEQ ID NO:44, or (iv)theVHCDR1 region of SEQ IDNO:49, theVHCDR2 region of SEQ IDNO:50, theVHCDR3 region of SEQ ID NO:51. 27. The bispecific antibody according to any of the preceding embodiments, wherein the second antigen-binding region which binds to human CD20 comprises: (i) the VL CDR1 region of SEQ ID NO:35, the VL CDR2 region DAS, the VL CDR3 region of SEQ ID NO:36, (ii) the VL CDR1 region of SEQ ID NO:45, the VL CDR2 region DAS, the VL CDR3 region of SEQ ID NO:46, (iii)the VL CDR1 region of SEQ ID NO:52, the VL CDR2 region DAS, the VL CDR3 region of SEQ ID NO:53. 28. The bispecific antibody according to any of the preceding embodiments, wherein the second antigen-binding region which binds to human CD20 comprises: 60 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 (i) the VHCDR1 region of SEQ IDNO:32, the VHCDR2 region of SEQ IDNO:33, the VHCDR3 region of SEQ ID NO:34, the VL CDR1 region of SEQ ID NO:35, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:36, (ii) the VHCDR1 region of SEQ IDNO:38, theVHCDR2 region of SEQ IDNO:39, theVHCDR3 region of SEQ ID NO:34, the VL CDR1 region of SEQ ID NO:35, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:36, (iii)the VHCDR1 region of SEQ IDNO:42, the VHCDR2 region of SEQ IDNO:43, theVHCDR3 region of SEQ ID NO:44, the VL CDR1 region of SEQ ID NO:45, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:46, (iv)theVHCDR1 region of SEQ IDNO:49, theVHCDR2 region of SEQ IDNO:50, theVHCDR3 region of SEQ ID NO:51, the VL CDR1 region of SEQ ID NO:52, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:53, (v) theVHCDR1 region of SEQ IDNO:32, theVHCDR2 region of SEQ IDNO:33, theVHCDR3 region of SEQ ID NO:34, the VL CDR1 region of SEQ ID NO:45, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:46, (vi)theVHCDR1 region of SEQ IDNO:32, theVHCDR2 region of SEQ IDNO:33, theVHCDR3 region of SEQ ID NO:34, the VL CDR1 region of SEQ ID NO:52, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:53, (vii) theVHCDR1 regionofSEQ IDNO:38, theVHCDR2 regionofSEQ IDNO:39, theVHCDR3 regionofSEQ ID NO:34, the VL CDR1 region of SEQ ID NO:45, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:46, (viii) theVHCDR1 regionofSEQ IDNO:38, theVHCDR2regionofSEQIDNO:39, theVHCDR3regionofSEQID NO:34, the VL CDR1 region of SEQ ID NO:52, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:53, (ix)theVHCDR1 region of SEQ IDNO:42, theVHCDR2 region of SEQ IDNO:43, theVHCDR3 region of SEQ ID NO:44, the VL CDR1 region of SEQ ID NO:35, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:36, (x) theVHCDR1 region of SEQ IDNO:42, theVHCDR2 region of SEQ IDNO:43, theVHCDR3 region of SEQ ID NO:44, the VL CDR1 region of SEQ ID NO:52, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:53, (xi)theVHCDR1 region of SEQ IDNO:49, theVHCDR2 region of SEQ IDNO:50, theVHCDR3 region of SEQ ID NO:51, the VL CDR1 region of SEQ ID NO:35, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:36, or (xii) theVHCDR1 regionofSEQ IDNO:49, theVHCDR2 regionofSEQ IDNO:50, theVHCDR3 regionofSEQ ID NO:51, the VL CDR1 region of SEQ ID NO:45, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:46. 29. The bispecific antibody according to any of the preceding embodiments, wherein the second antigen-binding regionwhich binds to humanCD20 comprises the VHCDR1 region of SEQ IDNO:32, the VHCDR2 region of SEQ ID NO:33, theVHCDR3regionofSEQIDNO:34, theVLCDR1regionofSEQIDNO:35, theVLCDR2regionofDAS,and the VL CDR3 region of SEQ ID NO:36. 30. The bispecific antibody according to any one of the preceding embodiments, wherein the second antigen-binding region which binds human CD20 comprises: (i) aVHsequencewhichhasat least 90%,at least 95%,at least 97%,or at least 99%aminoacid sequence identity to the amino acid sequence as set forth in SEQ ID NO:27, and a VL sequence which has at least 90%, at least 95%,at least 97%,or at least 99%aminoacid sequence identity to theaminoacid sequenceasset forth inSEQ ID NO:28, (ii) a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:37, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:28, (iii)a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:40, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:41, (iv)a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence 61 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 identity to the amino acid sequence as set forth in SEQ ID NO:47, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:48, (v) a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:27, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:41, (vi)a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:27, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:48, (vii) a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:37, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:41, (viii) a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ IDNO:37, and a aVL sequencewhich has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:48, (ix)a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:40, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:28, (x) a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:40, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:48, (xi)a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:47, and a VL sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:28, or (xii) a VH sequence which has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ IDNO:47, and a aVL sequencewhich has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:41. 31. The bispecific antibody according to any one of the preceding embodiments, wherein the second antigen-binding region which binds to human CD20 comprises: (i) the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:28, (ii) the VH sequence of SEQ ID NO:37, and the VL sequence of SEQ ID NO:28, (iii)the VH sequence of SEQ ID NO:40, and the VL sequence of SEQ ID NO:41, (iv)the VH sequence of SEQ ID NO:47, and the VL sequence of SEQ ID NO:48, (v) the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:41, (vi)the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:48, (vii) the VH sequence of SEQ ID NO:37, and the VL sequence of SEQ ID NO:41, (viii) the VH sequence of SEQ ID NO:37, and the VL sequence of SEQ ID NO:48, (ix)the VH sequence of SEQ ID NO:40, and the VL sequence of SEQ ID NO:28, (x) the VH sequence of SEQ ID NO:40, and the VL sequence of SEQ ID NO:48, (xi)the VH sequence of SEQ ID NO:47, and the VL sequence of SEQ ID NO:28, or (xii) the VH sequence of SEQ ID NO:47, and the VL sequence of SEQ ID NO:41. 32. The bispecific antibody according to any one of the preceding embodiments, wherein the second antigen-binding region which binds to human CD20 comprises the VH sequence of SEQ ID NO:27, and the VL sequence of SEQ ID NO:28. 33. The bispecific antibody according to any one of the preceding embodiments, wherein the second binding arm is derived from a human antibody. 62 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 34. The bispecific antibody according to any one of the preceding embodiments, wherein the second binding arm is derived from a full-length antibody. 35. The bispecific antibody according to any one of the preceding embodiments, wherein the second binding arm is derived from a full-length IgG1,κ (kappa) antibody. 36. The bispecific antibody according to any one of the preceding embodiments, wherein (a) the first antigen-binding region comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NO:4, the sequence GTN, and the sequence as set forth in SEQ ID NO:5, respectively, and (b) thesecondantigen-binding regionwhichbinds tohumanCD20comprises theVHCDR1regionof SEQ IDNO:32, the VHCDR2 region of SEQ IDNO:33, the VHCDR3 region of SEQ IDNO:34, the VLCDR1 region of SEQ ID NO:35, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:36, respectively. 37. The bispecific antibody according to any one of the preceding embodiments, wherein (a) the first antigen-binding region comprises theVHsequence as set forth in SEQ IDNO:6, and aVL sequence as set forth in SEQ IDNO:10, and (b) the secondantigen-binding regioncomprises theVHsequenceasset forth inSEQ IDNO:27, and theVLsequence as set forth in SEQ ID NO:28. 38. The bispecific antibody according to any one of the preceding embodiments, wherein the first antigen-binding region is a half-molecule antibody derived from IgG1-huCD3-H1L1-FEAL, and the second antigen-binding region is a half-molecule antibody derived from IgG1‑7D8-FEAR. 39. Thebispecific antibodyaccording to anyoneof the preceding embodiments,wherein the first bindingarm is a half- molecule antibody derived from IgG1-huCD3-H1L1-FEAR, and the second binding arm is a half-molecule antibody derived from IgG1‑7D8-FEAL. 40. The bispecific antibody according to any one of the preceding embodiments 1‑37, wherein each of said first and secondheavy chain comprisesat least a hinge region, aCH2andCH3 region,wherein in said first heavy chain at least one of the amino acids in the positions corresponding to a positions selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368,K370,D399, F405,Y407, andK409 ina human IgG1heavy chain hasbeensubstituted, andwherein said first and said second heavy chains are not substituted in the same positions. 41. The bispecific antibody according to embodiment 40, wherein (i) the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1heavy chain isR in said secondheavy chain, or (ii) the aminoacid in the position corresponding toK409 in a human IgG1heavy chain is R in said first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said second heavy chain. 42. The bispecific antibody according to any one of the preceding embodiments 1‑41, wherein the bispecific antibody comprises a first constant heavy chain (HC) and a first constant light chain (LC), wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain of SEQ IDNO:15 of both the first heavy chain and the second heavy chain are F, E, and A, respectively. 43.The bispecific antibody according to any one of the preceding embodiments 1‑41, wherein the bispecific antibody comprises a first and second constant heavy chain (HC) and a first and second constant light chain (LC), wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain of SEQ IDNO:15 of both the first heavy chain and the second heavy chain are F and E, respectively. 44. Thebispecific antibodyaccording to anyoneof the precedingembodiments 1‑43,wherein the first bindingarm is a half-molecule antibody derived from IgG1-huCD3-H1L1-FEAL, and the second binding arm is a half-molecule antibody derived from IgG1‑7D8-FEAR. 45. Thebispecific antibodyaccording to anyoneof the precedingembodiments 1‑43,wherein the first bindingarm is a half-molecule antibody derived from IgG1-huCD3-H1L1-FEAR, and the second binding arm is a half-molecule antibody derived from IgG1‑7D8-FEAL. 63 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 46. The bispecific antibody according to any one of the preceding embodiments 1‑45, wherein the bispecific antibody comprises a first and second constant heavy chain (HC) and a first and second constant light chain (LC), wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain of SEQ ID NO:15 of both the first constant heavy chain and the second constant heavy chain are F, E, and A, respectively, and wherein the position corresponding to F405 in the human IgG1 heavy chain of SEQ IDNO:15 of the first constant heavy chain is L, and thepositioncorresponding toK409 in thehuman IgG1heavychainofSEQ IDNO:15of thesecondconstant heavy chain is R. 47.The bispecific antibody according to any one of the preceding embodiments 1‑45, wherein the bispecific antibody comprises a constant heavy chain (HC) and a constant light chain (LC), wherein the positions corresponding to positions L234andL235 in the human IgG1heavy chain of SEQ IDNO:15 of both the first heavy chain and the second heavy chain areFandE, respectively, andwherein theposition corresponding toF405 in thehuman IgG1heavy chain ofSEQ IDNO:15of the first heavy chain is L, and theposition corresponding toK409 in thehuman IgG1heavy chain of SEQ ID NO:15 of the second heavy chain is R. 48. The bispecific antibody according to any one of the preceding embodiments, wherein said antibody comprises an Fc regionwhichhasbeenmodifiedso that bindingofC1q to said antibody is reducedcompared toawild-typeantibody by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%,whereinC1qbinding is determinedby ELISA. 49.A nucleic acid construct encoding one or more amino acid sequences of embodiments 1‑48. 50. A nucleic acid construct encoding a bispecific antibody as defined in any og embodiments 1‑48. 51. An expression vector comprising: (i) a nucleic acid sequence encoding a heavy chain sequence of a first binding arm as defined in any one of the preceding embodiments 1‑23; (ii) a nucleic acid sequence encoding a light chain sequence of a first binding arm as defined in any one of the preceding embodiments 1‑23; (iii) a nucleic acid sequence encoding a heavy chain sequence of a second first binding armas defined in any one of the preceding embodiments 24‑32; (iv) a nucleic acid sequenceencoding a light chain sequenceof a second first bindingarmasdefined in anyoneof the preceding embodiments 24‑32; (v) the nucleic acid set forth in (i) and the nucleic acid set forth in (ii); (vi) the nucleic acid set forth in (iii) and the nucleic acid set forth in (iv). (vii) the nucleic acid set forth in (i), (ii), (iii) and (iv). 52. A host cell comprising an expression vector of embodiment 51. 53. The host cell according to embodiment 52, wherein said host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell. 54. A composition comprising a bispecific antibody according to any one of the preceding embodiments 1‑48. 55. A pharmaceutical composition comprising the bispecific antibody according to any one of the preceding embodiments 1‑48 and a pharmaceutically acceptable carrier. 56. The bispecific antibody according to any one of the preceding embodiments 1‑48, the composition according to embodiment 54, or the pharmaceutical composition according to embodiment 55 for use as a medicament. 57.The bispecific antibody according to any one of the preceding embodiments 1‑48, the composition according to embodiment 54, or the pharmaceutical composition according to embodiment 55 for use in the treatment of a disease. 58. The bispecific antibody for use according to embodiment 57 wherein the use is for treating cancer. 59. The bispecific antibody for use according to embodiment 58 wherein the use is for treating B cell malignancies. 64 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 60. The bispecific antibody for use according to embodiment 58 or 59 wherein the use is for treating NHL or B cell leukemia. 61.A method of treatment of a disease comprising administering the bispecific antibody according to any one of the preceding embodiments 1‑48, the composition according to embodiment 54, or the pharmaceutical composition according to embodiment 55 to a subject in need thereof. 62. Use of a bispecific antibody according to any one of the preceding embodiments 1‑48 for the manufacture of a medicament. 63. Use of a bispecific antibody according to any one of the preceding embodiments 1‑48 for the manufacture of a medicament for the treatment of cancer. 64. The use according to embodiment 63 wherein the use is for treating B cell malignancies. 65. The use according to embodiments 63 or 64 wherein the use is for treating NHL or B cell leukemia. 66. Themethod or use according to any of embodiments 56‑65wherein themethod or use of the bispecific antibody of any of embodiments 1‑48 is used in combination with one or more further therapeutic agent, such as a chemother- apeutic agent. 67.Amethod for producingabispecificantibodyaccording to anyoneof theprecedingembodiments 1‑48, comprising the steps of a) culturing a host cell comprising an expression vector according to embodiment 51(v) and purifying said antibody from the culture media; b) culturing a host cell comprising an expression vector according to embodiment 51(vi) and purifying said antibody from the culture media; c) incubating said first antibody together with said second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, and d) obtaining said bispecific antibody. 68. A diagnostic composition comprising a bispecific antibody according to any one of the preceding embodiments 1‑48. 69. A method for detecting whether cross-linking between CD3‑ and CD20-expressing cells occurs in a sample derived from a patient such as a blood sample, lymph node sample or bonemarrow sample, upon administration of a bispecific antibody according to any one of the preceding embodiments 1‑48, comprising the steps of: (i) contacting the sample with a bispecific antibody according to any one of the preceding embodiments 1‑48, under conditions that allow for formation of a complex between said bispecific antibody and the CD3‑ andCD20- expressing cells; and (ii) analyzing whether a complex has been formed. 70. A kit for detecting cross-linking betweenCD3‑andCD20-expressing cells, in a sample derived fromapatient such as a blood sample, lymph node sample or bone marrow sample, comprising i) a bispecific antibody according to any one of the preceding embodiments 1‑48; and ii) instructions for use of said kit. 71. An anti-idiotypic antibody which binds to the first antigen-binding region as defined in any one of the preceding embodiments 1‑23, or which binds to the second antigen-binding region as defined in any one of the preceding embodiments 24‑32. EXAMPLES Example 1 ‑ Generation of humanized CD3 antibodies and non-activating antibody variants 65 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 Humanization of CD3 antibodies

[0499] Humanization of amurine CD3 antibody SP34 (US8236,308, described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK) using their improved version of the germline humanization (CDR-grafting) technology (EP0629240). Using this technology, 4 different VH chains (SEQ ID NOs:6, 7, 8, and 9) and 3 different VL chains (SEQ ID NOs:10, 11, and 12) were designed. By combining these 4 VHwith the 3 VL chains, 12 different antibodies were generated. The humanized variants are described herein as huCD3. Thus, humanized variants comprising aVHand aVL according to the invention, are described as, e.g., IgG1-huCD3-H1L1 meaning that said specific variant is of the IgG1 isotype, is a humanized SP34 CD3-specific antibody and comprises the VH amino acid sequence termed "H1" and is defined according to SEQ ID NO:6, and the VL amino acid sequence termed "L1" and is defined according to SEQ ID NO:10.Thus,H1 refers to thevariableheavychain regionVH1,L1 refers to thevariable light chain regionVL1,andso forth.

[0500] In particular, the variants IgG1-huCD3-H1L1 (humanizedCD3 comprising the VH1 sequence set forth in SEQ ID NO:6 and the VL1 sequence set forth in SEQ ID NO:10), IgG1-huCD3-H1L2 (humanized CD3 comprising the VH1 sequenceset forth inSEQIDNO:6and theVL2sequenceset forth inSEQIDNO:11), IgG1-huCD3-H1L3 (humanizedCD3 comprising the VH1 sequence set forth in SEQ ID NO:6 and the VL3 sequence set forth in SEQ ID NO:12), IgG1-huCD3- H3L3 (humanizedCD3 comprising the VH3 sequence set forth in SEQ IDNO:8 and the VL3 sequence set forth in SEQ ID NO:12), IgG1-huCD3-H4L1 (humanized CD3 comprising the VH4 sequence set forth in SEQ ID NO:9 and the VL1 sequenceset forth inSEQ IDNO:10), IgG1-huCD3-H3L1 (humanizedCD3comprising theVH3sequenceset forth inSEQ ID NO:8 and the VL1 sequence set forth in SEQ ID NO:10), IgG1-huCD3-H3L3 (humanized CD3 comprising the VH3 sequenceset forth inSEQ IDNO:8and theVL3sequenceset forth inSEQ IDNO:12), and IgG1-huCD3-H4L3 (humanized CD3 comprising the VH4 sequence set forth in SEQ IDNO:9 and the VL3 sequence set forth in SEQ IDNO:12) have been usedas the first antigen-binding regionof the bispecific antibodies according to the invention.Herein, "IgG1-huCD3", if not further defined refers to IgG1-huCD3-H1L1.

[0501] In some examples the CD3 antibody comprising the heavy and light chain variable region sequences of huCLB- T3 / 4 (SEQ ID NOs:17 and 18, respectively) were used as the first antigen-binding region of the bispecific antibodies according to the invention. huCLB-T3 / 4 is a humanized version of the murine CD3 antibody CLB-T3 / 4 (Parren et al., Res Immunol. 1991, 142(9):749‑63). Both sequences (SEQ ID NOs:17 and 18) were cloned into the relevant pcDNA3.3 (Invitrogen) expression vectors and expressed by cotransfection in HEK293F cells. The resulting CD3 antibody is described as IgG1-huCLB-T3 / 4.

[0502] The humanized CD3 antibodies are further disclosed in WO2015001085. CD20 antibodies

[0503] TheCD20antibodies usedas the secondbinding armof the present bispecific antibodies are further disclosed in WO2004035607 (Genmab) and WO2005103081 (Genmab). Control antibodies

[0504] The following antibodies were used as control antibodies in the examles: CD3 antibodies

[0505] IgG1-CD3 (the parental CD3 antibody SP34 having the VH andVL sequences set forth in SEQ IDNO:25 and SEQ ID NO:26, respectively) IgG1-huCD3 (H1L1) (having the VH and VL sequences set forth in SEQ ID NO:6 and SEQ ID NO:10, respectively) IgG1-huCLB-T3 / 4 bsIgG1-huCD3-H1L1-FEALxb12-FEAR (bispecific antibody using as the second arm the antibody b12 which is a gp120 specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812‑23). IgG1-huCD3-H1L1-FEAL IgG1-huCLB-T3 / 4-FEAL CD20 antibodies

[0506] IgG1‑7D8 (having the VH and VL sequences set forth in SEQ ID NO:27 and SEQ ID NO:28, respectively) 66 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 IgG1‑11B8 (having the VH and VL sequences set forth in SEQ ID NO:40 and SEQ ID NO:41, respectively) IgG1‑2F2 (having the VH and VL sequences set forth in SEQ ID NO:37 and SEQ ID NO:28, respectively) IgG1-RTX (having the VH and VL sequences of rituximab) IgG1-GA101 (having the VH and VL sequences of obinutuzumab, CHEMBL1743048, US8883980, with a wild type human IgG1 Fc domain) IgG1‑2C6 (having the VH and VL sequences set forth in SEQ ID NO:47 and SEQ ID NO:48, respectively). IgG1‑7D8-FEAR IgG1‑11B8-FEAR IgG1‑2F2-FEAR IgG1-GA101-FEAR IgG1‑2C6-FEAR TABLE 4: Quantification of 7D8 CD20 antibody binding on different B cell lines Quantitative flow cytometry (QIFIKIT®, Dako; cat. no K0078) was performed as described (Poncelet and Carayon, 1985, J. Immunol. Meth. 85: 65‑74), to quantify the binding of 7D8 CD20 antibody on different human B-cell lines used in the examples, as an indication of CD20 expression. To this end, human B-cell lines were incubated with a saturating concentration of 7D8, and the number of bound 7D8 molecules was determined using quantitative flow cytometry. The anti-human CD20 antibody 7D8, that was engineered to express a murine Fc domain (10 µg / mL, mmIgG1‑7D8 (Overdijk et al. 2012, J. Immunol. 189: 3430‑3438), was used in this assay. B-ALL: B cell acute lymphoblastic leukemia, ABC-DLBCL: activated B cell diffuse large B-cell lymphoma, GC-DLBCL: germinal center diffuse large B-cell-lymphoma, FL: follicular lymphoma. Lymp homa type Cell line 50,000‑100,000 ABC* / cell 100,000‑200,000 ABC / cell > 200,000 ABC / cell Burkitt’s lymphoma Daudi X Burkitt’s lymphoma Raji X B-ALL Nalm‑16 X ABC-DLBCL OCI-Ly7 X GC-DLBCL SU-DHL‑4 X FL WSU-NHL X *ABC: antibody-binding capacity Example 2 ‑ Generation of bispecific antibodies by 2-MEA-induced Fab-arm exchange

[0507] An in vitromethod for producing bispecific antibodies is described inWO2008119353 (Genmab) and reported by van der Neut-Kolfschoten et al. (Science. 2007Sep 14; 317(5844):1554‑7). Herein, the bispecific antibodieswere formed by "Fab-arm"or "half-molecule"exchange (swappingofaheavychainandattached light chain)between twomonospecific IgG4‑ or IgG4-like antibodies upon incubation under mildly reducing conditions. Without being limited to theory, this Fab- arm exchange reaction was the result of a disulfide-bond isomerization reaction wherein the inter heavy-chain disulfide bonds in the hinge regions of monospecific antibodies were reduced and the resulting free cysteines form a new inter heavy-chain disulfide bond with cysteine residues of another antibody molecule with a different specificity. The resulting products were bispecific antibodies having two Fab arms with different sequences.

[0508] The knowledge of this natural IgG4 Fab-arm exchange was adapted to generate a method to produce stable IgG1-based bispecific antibodies (WO2011131746 (Genmab)). The bispecific antibody product generated by thismethod described below will no longer participate in IgG4 Fab-arm exchange. The basis for this method was the use of complimentary CH3 domains, which promote the formation of heterodimers under specific assay conditions. To enable the production of bispecific antibodies by thismethod, IgG1molecules carrying certainmutations in theCH3domainwere generated: in one of the parental IgG1 antibody T350I, K370T and F405L mutations (or minimally F405L) in the other parental IgG1 antibody the K409R mutation.

[0509] To generate bispecific antibodies, these two parental antibodies, each antibody at a final concentration of 0.5 mg / mL (equimolar concentration), were incubated with 25 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100µLTris-EDTA (TE) at 37°C for 90min. The reduction reaction is stopped when the reducing agent 2-MEA is removed 67 EP 4 678 661 A2 5 10 15 20 25 30 35 40 45 50 55 by using spin columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer’s protocol. Example 3 ‑ Generation of mutants to optimize the production of the humanized CD3 antibodies Generation of huCD3-L1 mutant plasmids

[0510] Several IgG1-huCD3-H1L1 variants with mutations in the L1 light chainwere generated in order to improve the expression levels of IgG1-huCD3-H1L1 in transient transfection assays, cf. Table 5. The selection of residues was based on comparisons with germline sequences or screening for the presence of rare residues in the huCD3-L1 sequence in combination with crystal structures from homologous antibodies. The selected sequences were synthesized at GeneArt (Life Technologies, Germany). p33L encodes the constant domain of the human IgLC2 / IgLC3 lambda light chain of SEQ ID NO:29. p33G1f encodes the IgG1m(f) heavy chain constant region of SEQ ID NO:15. TABLE 5 LC constructs LC Mutants Antibody name after co-expression with HC VH1 encoding plasmid p33L-huCD3-VL1 - IgG1-huCD3-H1L1 p33L-huCD3-VL1-F10L F10L IgG1-huCD3-H1L1-LF10L p33L-huCD3-VL1-R23A R23A IgG1-huCD3-H1L1-LR23A p33L-huCD3-VL1-A35P A35P IgG1-huCD3-H1L1-LA35P p33L-huCD3-VL1-T41K T41K IgG1-huCD3-H1L1-LT41K p33L-huCD3-VL1-K55N K55N IgG1-huCD3-H1L1-LK55N p33L-huCD3-VL1-L97H L97H IgG1-huCD3-H1L1-LL97H p33L-huCD3-VL1-LKNH F10L, T41K, K55N, L97H IgG1-huCD3-H1L1-LLKNH p33L-huCD3-VL1- LTGPEAEY F10L, R47T, D71G, A82P, D83E, S86A, I87E, F89Y IgG1-huCD3-H1L1-LLTGPEAEY p33L-huCD3-VL1-LAPTG- PEAEY F10L, R23A, A35P, R47T, D71G, A82P, D83E, S86A, I87E, F89Y IgG1-huCD3-H1L1-LLAPTGPEAEY Transient expression in Expi293F cells

[0511] Forasingleantibody, theplasmidsencodingheavychain (HC)and light chain (LC)were transiently transfected in Freestyle Expi293F cells (Life technologies, USA) using ExpiFectamine 293 (Life technologies). In total 1.5 µg HC encoding plasmid and 1.5 µg LC encoding plasmid (Table 5) were diluted in 150 µL Opti-MEM (Gibco, USA). To prepare the transfection mix, 8 µL ExpiFectamine 293 was diluted in 150 µL Opti-MEM and incubated for 5 minutes at room temperature. Next, the DNA / Opti-MEM and ExpiFectamine 293 / Opti-MEM solutions were mixed, incubated for 20 minutes at room temperature and added to 2.55 mL Expi293 Expression Medium containing 7.5x106 Expi293F cells and 50 U / mL Pen-Strep. The cells were incubated at 37°C, 8% CO2 and shaken at 200 rpm. To enhance expression, 21 hoursafter transfection, 15µLenhancermix1and150µLenhancermix2wereadded.Thecellswere incubated for 4days followed by the harvest of the supernatant. Supernatantswere spun at 3,000xg and filter sterilized over a 0.2µmfilter. The IgG expression levels were measured on the Octet RED (ForteBio, US) using anti-human IgG sensors (ForteBio, USA). IgG concentration analysis

[0512] The IgG1-huC...

Claims

1. A bispecific antibody comprising a first binding arm comprising a first antigen-binding region binding to human CD3ε (epsilon) and a second binding arm comprising a second antigen-binding region binding to human CD20, wherein: (i) said first binding arm comprises a first heavy chain comprising a first heavy chain variable sequence (VH) as set forth in SEQ ID NO:6 and a first heavy chain constant sequence (CH), and a first light chain comprising a first light chain variable sequence (VL) as set forth in SEQ ID NO:10 and a first light chain constant sequence (CL), and said second binding arm comprises a second heavy chain comprising a second heavy chain variable sequence (VH) as set forth in SEQ ID NO:27 and a second heavy chain constant sequence (CH), and a second light chain comprising a second light chain variable sequence (VL) as set forth in SEQ ID NO:28 and a second light chain constant sequence (CL); (ii) said first and said second heavy chain constant sequences are IgG1 constant sequences, wherein: in the first and second heavy chains the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in said second heavy chain, the numbering of said amino acid positions being in accordance with Eu-numbering; and (iii) wherein the bispecific antibody comprises a light chain constant sequence (CL) as set forth in SEQ ID NO:29.

2. The bispecific antibody according to claim 1, which is a full-length antibody.

3. The bispecific antibody according to claim 1 or 2, which is a full-length IgG1 antibody.

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