NOVEL MEANS AND METHODS FOR TREATING HBV INFECTION AND ASSOCIATED CONDITIONS

Abstract

The present invention relates to a polypeptide comprising (a) a first set of six complementarity determining regions (CDRs) configured to bind a first antigen; and (b) (ba) a second set of six CDRs configured to bind a second antigen; or (bb) a ligand capable of binding to a second antigen; wherein (i) said first antigen is selected from Hepatitis B virus (HBV) small surface antigen; HBV medium surface antigen; and HBV large surface antigen; and (ii) said second antigen is selected from surface antigens presented by immune effector cells such as natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Also provided are compositions for use in a method of treating or preventing HBV infection and/or a condition caused by said HBV infection, said condition caused by said HBV infection being selected from liver cirrhosis and hepatocellular carcinoma.

Claims

1-15. (canceled)

16. A polypeptide comprising: (a) a first set of six complementarity determining regions (CDRs) that bind to a first antigen; wherein said first set of six CDRs has the sequences of SEQ ID NOs: 1 to 6; and (b) a second set of six CDRs that bind to a second antigen; wherein said second set of six CDRs has the sequences of SEQ ID NOs: 25 to 30 wherein (i) said first antigen is Hepatitis B virus (HBV) small surface antigen; and (ii) said second antigen is a CD28 surface antigen, and wherein said CDRs are part of immunoglobulin domains.

17. The polypeptide of claim 16, wherein (a) said first set of six CDRs is comprised in a first scFv fragment; and/or (b) said second set of six CDRs is comprised in a second scFv fragment.

18. The polypeptide of claim 16, wherein said first set of six CDRs binds an epitope of said first antigen which epitope is located in said HBV small surface antigen.

19. The polypeptide of claim 16, wherein said polypeptide further comprises a dimerization region, wherein said dimerization region provides for covalent and/or non-covalent dimerization.

20. The polypeptide of claim 17, wherein said polypeptide further comprises a spacer region, said spacer region comprising a CH2 domain and a CH3 domain, said spacer region being located between (i) said first scFv fragment and (ii) said second scFv fragment in the amino acid sequence of said polypeptide, and said CH2 domain and/or said CH3 domain being mutated in one or more positions to diminish or abolish the binding to F.sub.c receptors.

21. The polypeptide of claim 16, wherein within each set of six CDRs the order of CDRs is as follows: CDR1 of heavy chain, CDR2 of heavy chain, CDR3 of heavy chain, CDR1 of light chain, CDR2 of light chain, and CDR3 of light chain.

22. The polypeptide of claim 16, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence which exhibits at least 80% identity to SEQ ID NO: 44, provided that the CDRs of said amino acid sequence exhibiting at least 80% identity are identical to SEQ ID NOs: 1 to 6 and 25 to 30, respectively.

23. A nucleic acid molecule encoding the polypeptide of claim 16.

24. A complex comprising a first and a second polypeptide, said first polypeptide being as defined in claim 16, and the second polypeptide comprising: (A) a first set of six complementarity determining regions (CDRs) that bind to a first antigen; wherein said first set of six CDRs has the sequences of SEQ ID NOs: 1 to 6, 7 to 12, or 13 to 18; and (B) (ba) a second set of six CDRs that bind to a second antigen; wherein said second set of six CDRs has the sequences of SEQ ID NOs: 19 to 24, 25 to 30, 31 to 36, or 37 to 42, or (bb) a ligand capable of binding to a second antigen; wherein (i) said first antigen is selected from Hepatitis B virus (HBV) small surface antigen; HBV medium surface antigen; and HBV large surface antigen; and (ii) said second antigen is selected from surface antigens presented by immune effector cells, and wherein said CDRs are part of immunoglobulin domains; wherein (a) there is at least one covalent linkage between said first and said second polypeptide; or (b) said first and said second polypeptide are bound to each other non-covalently.

25. A composition comprising (A) a first polypeptide according to claim 16 and a second polypeptide comprising: (a) a first set of six complementarity determining regions (CDRs) that bind to a first antigen; wherein said first set of six CDRs has the sequences of SEQ ID NOs: 1 to 6, 7 to 12, or 13 to 18; and (b) (ba) a second set of six CDRs that bind to a second antigen; wherein said second set of six CDRs has the sequences of SEQ ID NOs: 19 to 24, 25 to 30, 31 to 36, or 37 to 42, or (bb) a ligand capable of binding to a second antigen; wherein (i) said first antigen is selected from Hepatitis B virus (HBV) small surface antigen; HBV medium surface antigen; and HBV large surface antigen; and (ii) said second antigen is selected from surface antigens presented by immune effector cells, and wherein said CDRs are part of immunoglobulin domains and/or (B) one or more complexes according to claim 24, wherein the first polypeptide and the second polypeptide are distinct from each other with regard to the first antigen and/or the second antigen to which they bind.

26. The composition of claim 25, wherein the first polypeptide is a polypeptide binding to HBV small surface antigen and CD28; and the second polypeptide is a polypeptide binding to HBV small or large surface antigen and CD3.

27. The complex of claim 24 or the composition of claim 25, wherein said complex and said composition comprise or consist of a tetravalent antibody which is bispecific, trispecific or tetraspecific.

28. A pharmaceutical composition comprising one or more polypeptides of claim 16, one or more complexes of claim 24 and/or one or more compositions of claim 25.

29. An in vitro or ex vivo immune effector cell comprising a polypeptide of claim 16 or a complex according to claim 24 bound to a surface antigen of said immune effector cell.

Description

[0096] The figures illustrate the invention.

[0097] FIG. 1:

[0098] scFv fragments are obtained by fusion of two variable domains. Fusion involves the use of a flexible peptide linker which does not or not substantially interfere with the structure of each variable domain.

[0099] FIG. 2:

[0100] Dimerization of two polypeptides of the invention by the formation of disulfide bonds. Each polypeptide comprises a bispecific bivalent antibody. Natural antibody dimerization in the endoplasmic reticulum of producer cells can result in formation of a bispecific tetravalent antibody, or a tri- or tetraspecific, tetravalent antibody if two bispecific bivalent antibodies are co-expressed (not shown).

[0101] FIG. 3:

[0102] Comparison of specific elimination of HBV surface antigen producing hepatoma target cells after administration of single bispecific antibodies and synergistic effects of simultaneous administration of two CTL-specific or two NK cell-specific bispecific antibodies. The CellTiter-Blue Cell Viability Assay is used.

[0103] FIG. 4:

[0104] A) Cytokine secretion as an indication of activation of immune effector cells in the presence of bispecific antibodies of the present invention. HBV-infected HepaRG cells were co-cultured with PBMC in the presence or absence of indicated bispecific antibodies.

[0105] B) Specific elimination of HBV-infected target cells in co-culture with immune effector cells and bispecific antibodies.

[0106] FIG. 5:

[0107] Viability of target cells co-cultured with PBMC In presence of Individual HBs-reactive bispecific antibodies. Single bispecific antibodies mediate lysis of target cells. A, C, E: Effect of stimulation with ?HBs??CD3 (A), ?HBs??CD28 (C) or summarized (E). B, D, F: Effect of stimulation with ?HBs??CD3 [Fc?ADCC] (B), ?HBs??CD28 [Fc?ADCC] (D) or summarized (F). The arrowhead indicates addition of PBMC and bispecific antibodies. Curves with dots represent HBs-transfected HuH7-S cells, curves with rhombuses represent HuH7 parental hepatoma cells. The xCELUgence real-time cytotoxicity assay is used. Normalization time of cell index: 0 h.

[0108] FIG. 6:

[0109] Viability of target cells co-cultured with PBMC in presence of HBs-reactive bispecific antibodies. Combination of bispecific antibodies mediate massive killing of target cells. A: Effect of stimulation with ?HBs??CD3 and ?HBs??CD28. B: Effect of stimulation with ?HBs??CD3 [Fc?ADCC] and ?HBs??CD28 [Fc?ADCC]. C, D: Effect of individual bispecific antibodies compared to combinations. The arrowhead indicates addition of PBMC and bispecific antibodies. Curves with dots represent HuH7-S cells, curves with rhombuses HuH7 cells. Normalization time of cell index: 0 h.

[0110] FIG. 7:

[0111] Viability of target cells co-cultured with PBMCs in the presence of different concentrations of bispecific antibodies. 50 ?l/50 ?l mixtures of antibody-containing supernatants of ?HBs??CD3/?HBs??CD28 (A), or ?HBs??CD3 [Fc?ADCC]/?HBs??CD28 [Fc?ADCC] (B), induced lysis of target cells earlier than 25 ?l/25 ?l mixtures, indicating dose-dependent effects. The arrowhead indicates addition of PBMCs and bispecific antibodies. Curves with dots represent HuH7-S cells, curves with rhombuses HuH7 cells. Normalization time of cell index: 0 h.

[0112] FIG. 8:

[0113] Viability of target cells co-cultured with different amounts of PBMC in the presence of a mixture of ?HBs??CD3 and ?HBs??CD28. 2?10.sup.5 PBMC mediate a significantly earlier elimination of HuH7-S cells than 1?10.sup.5 PBMC. The arrowhead indicates addition of PBMC and bispecific antibodies. Curves with dots represent HuH7-S cells, curves with rhombuses HuH7 cells. Normalization time of cell index: 0 h.

[0114] FIG. 9:

[0115] Viability of target cells co-cultured with PBMCs in presence of ?HBs??CD3/?HBs??CD28 mixtures for various time periods. Supernatants containing bispecific antibodies were removed after the indicated periods of stimulation. 4 h stimulation only led to a small decrease of target cell viability (78.5% endpoint viability). Stimulation of PBMC with bispecific antibodies for 8 h or longer induced elimination of target cells. After stimulation for 8 h and 12 h, killing of target cells was delayed as compared to 24 h or 48 h stimulation, suggesting continuous activation and re-targeting of effector cells. HuH7-S endpoint viabilities at 48 h were, however, comparable: 8 h stim.: 14.7%; 12 h stim.: 11.7%, 24 h stim.: 5.1%, 48 h stim.: 3.2%). The arrowhead indicates addition of PBMC and bispecific constructs. Viability kinetics for HuH7-S cells are shown. Normalization time of cell index: 0 h.

[0116] FIG. 10:

[0117] IL-2, IFN-? and TNF-? secretion of PBMC after co-culture with HuH7-S/HuH7 cells in presence of ?HBs??CD3/?HBs??CD28 at different time points. A: IL-2 concentration increased over time and reached a plateau at approximately 24 h with a concentration of about 1550 pg/ml. B: IFN-? secretion started between 8 h and 12 h and increased up to 12000 pg/ml (48 h). C: TNF-? production was detectable already after 4 h, increased continuously, reached its peak at 24 h (1700 pg/ml) and declined to 1400 pg/ml after 48 h. High background TNF-? secretion in the absence of HBs (HuH7 cells) could be detected, with the highest concentration after 4 h (?70 pg/ml) decreasing to 9 pg/ml after 48 h of co-culture.

[0118] FIG. 11:

[0119] LAMP-1 stainings after co-culture of PBMC with HuH7-S/HuH7 cells in presence of bispecific antibodies. Surface expression of the endosomal degranulation marker LAMP-1 is detected on CD4.sup.+ (A. B) and CD8.sup.+ (C, D) T cells after co-culture with HuH7-S (black line) or HuH7 (grey line) cells in the presence of either ?HBs??CD3/?HBs??CD28 (A, C) or ?HBs??CD3 [Fc?ADCC]/?HBs??CD28 [Fc?ADCC]. (B, D).

[0120] FIG. 12:

[0121] FACS analysis of PBMC co-cultured with HuH7-S or HuH7 cells in the presence of ?HBs??CD3/?HBs??CD28 after 8 h, 12 h and 24 h. A, B; Percentages of IFN?.sup.+/IL-2.sup.+/TNF?.sup.+/CD154.sup.+ CD4.sup.+ T cells (A) or IFN?.sup.+/IL-2.sup.+/TNF?.sup.+/CD154.sup.+ CD8.sup.+ (B) T cells. C, D: Boolean combination gates of IFN?.sup.+, IL-2.sup.+ and/or TNF?.sup.+ CD4.sup.+ (C), or IFN?.sup.+, IL-2.sup.+ and/or TNF?.sup.+ CD8.sup.+ (D) T cells.

[0122] FIG. 13:

[0123] FACS analysis of PBMC co-cultured with immobilized or soluble HBsAg in the presence of ?HBs??CD3 [Fc?ADCC]/?HBs??CD28 [Fc?ADCC] after 24 h and 48 h. A, B; Percentages of IFN?.sup.+/IL-2.sup.+/TNF?.sup.+/CD154.sup.+ CD4.sup.+ T cells (A) or IFN?/IL-2.sup.+/TNF?.sup.+/CD154.sup.+ CD8.sup.+ (B) T cells. C, D: Boolean combination gates of IFN?.sup.+, IL-2.sup.+ and/or TNF?.sup.+ CD4 (C), or IFN?, IL-2.sup.+ and/or TNF?.sup.+ CD8.sup.+ (D) T cells.

[0124] FIG. 14:

[0125] HBsAg in the supernatant of HuH7-S cells (110.8 S/CO), HepG2.2.15 cells (41.7 S/CO) and HBV-infected HepaRG cells (16.5 S/CO).

[0126] FIG. 15:

[0127] Viability of HBV-infected/uninfected HepaRG cells co-cultured with PBMC in presence of bispecific antibodies. ?HBs??CD3 (A) and ?HBs??CD3/?HBs??CD28 (B) mediate significant target cell lysis. Endpoint viabilities of untreated cells are 65.9% (HBV+) and 62.9% (HBV?). The arrowhead indicates addition of PBMCs and bispecific constructs. Curves with dots represent HBV-infected HepaRG cells, curves with rhombuses uninfected HepaRG cells. Normalization time of cell index in xCELLigence assay: 0 h.

[0128] FIG. 16:

[0129] Reduction in tumor size in animal treated with bispecific antibodies. Mice bearing HBV-positive subcutaneous HepG2.2.15 tumors were treated with human PBMC and a mixture of ?HBs??CD3 and ?HBs??CD28 bispecific antibodies at four consecutive days. Mice were sacrificed and tumor size was analyzed.

[0130] The examples illustrate the invention.

EXAMPLE 1

Materials and Methods for Example 2

Cloning and Production of Bispecific Antibodies

[0131] Complementary DNAs coding for variable heavy and variable light chains of anti-CD3 (OKT3), anti-CD28 (9.3), anti-CD18 (A9) and anti-CD56 (NCAM29.2) were obtained by PCR amplification of reverse-transcribed mRNAs from the respective hybridoma using a set of primers covering all V.sub.H and V?/V? subtypes. PCR products were ligated into pCR2.1-TOPO (Invitrogen, Life Technologies) and sequenced. The anti-HBsAg scFv C8 was provided in a codon-optimized form in the plasmid pMP71-C8. Using primers containing appropriate restriction sites in the 5 and 3 flanks variable heavy and variable light chain cDNAs coding for the above mentioned antibodies were assembled with a glycine-serine linker into scFvs. The OKT3, 9.3, A9, and NCAM29.2 scFvs (N-terminally extended by (Gly).sub.4) were cloned at the 3 end of a cDNA present in pBluescript KS II+ (Stratagene) that codes for the Fc domain (hinge, CH2, CH3) of human IgG1 which was extended by glycine-serine linker GlyAsnSer(Gly.sub.4Ser).sub.3AlaSer at the 5 end and a StrepTag sequence (WSHPQFEK) and, in a second series of constructs, an additional glycine-serine linker (Gly.sub.4Ser).sub.3 at the 3 end. The C8 scFv coding sequence was cloned at the 5 end of the mentioned 5 glycine-serine linker. The complete scFv-linker-hIgG1Fc-linker-scFv sequence was subcloned into the mammalian expression vector pcDNA3.1(?) (Invitrogen). Maxi-prep plasmid DNA was used for transfection of HEK293 cells using the peqFECT transfection reagent (Peqab). Stable transfectants were selected using 0.8-1.0 mg/ml G418 and expanded. Supernatants from HEK transfectants were collected and analyzed by ELISA for the concentration of secreted, bispecific antibodies and by Western blot for the integrity of the secreted antibodies using goat anti-human IgG-Fc specific, peroxidase-labeled antibodies.

Cell Culture Conditions and HBV Infection

[0132] HuH7 hepatoma cells (Nakabayaski, et al. 1982. Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Res. 42: 3858-3863) and HEK293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 ?g/mL), and L-glutamine (2 mmol/L) (all from GIBCO, Life Technologies).

[0133] Peripheral blood mononuclear cells (PBMC) were isolated through density gradient centrifugation from heparinized whole blood using LSM 1077 Lymphocyte Separation Medium (PAA). 25 ml of blood was layered above 13 ml of LSM 1077. After centrifugation at 2000 rpm for 20 min (without break) at room temperature PBMC were harvested and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 ?g/ml), and L-glutamine (2 mmol/l) (all from GIBCO). After an overnight resting step PBMC or sorted NK cells were used for co-culture experiments.

[0134] HepaRG cells were maintained in Williams E Medium (Invitrogen GmbH, Karlsruhe, Germany) supplemented with L-glutamine (5 mmol/l), glucose (0.06% [wt/vol]), HEPES (23 mmol/l, pH7.4), gentamycin (50 ?g/ml), penicillin (501 U/ml), streptomycin (50 ?g/ml), inosine (37 ?mol/l), hydrocortisone (4.8 ?g/ml), and insulin (1 ?g/ml). Prior to infection HepaRG cells were differentiated for 4 weeks using differentiation medium (Williams E Medium (as described above), supplemented with DMSO (1.75%). HepaRG cells were Infected using HBV stocks at a final m.o.i. of 200 and PEG (5%) in differentiation medium. Infection inoculum was removed after overnight incubation and replaced with differentiation medium and cultured for 6 days.

[0135] For co-cultures with redirected T cells, we changed from differentiation medium to hydrocortison-free medium 2 days before starting the co-culture to avoid immunosuppression mediated by the hydrocortison.

Transfection with HBV Surface Antigen Encoding Plasmids

[0136] HuH-7 cells were transfected with plasmids encoding the various surface antigens using FuGene transfection reagent (Promega). For 8 wells of a 96 well plate 3 ?l of FuGENE, 1 ?g of plasmid DNA were added to 100 ?l OptiMEM (Gibco). The transfection solution was Incubated for 15 min at room temperature in order for the FuGENE to bind the plasmid DNA. A final volume of 100 ?l was applied per well, after adding further OptiMEM and incubated for at least 24 h.

Magnetic Activated Cell Sorting (MACS) of NK Cells

[0137] NK cells were isolated from PBMC using a human CD56.sup.+CD16.sup.+ NK Cell Isolation Kit (Miltenyi) In a first negative selection step, all non-NK cells were removed by monoclonal antibodies directed against antigens not expressed on the surface of NK cells. In a second positive selection step, the NK cells were isolated by monoclonal CD16 antibodies conjugated to iron oxide microbeads and retained inside a magnetic field. After isolation NK cells were cultured in RPMI-1640 medium as described above.

Co-Culture of HBV-Positive Target Cells and Redirected Effector Cells

[0138] Target cells were cultured in a 96 well plate at full confluency. 1?10.sup.5 effector cells were added in a volume of 100 ?l medium per well. 100 ?l of the HEK supernatants containing the bispecific antibodies were applied per well. For determination of synergistic effects, 50 ?l of each bispecific antibody supernatant was added per well. Untreated target cells incubated with 200 ?l medium or with effector cells alone or with bispecific antibodies alone served as negative control.

Enzyme-Linked Immunosorbent Assay (ELISA) for Effector Cell Activation

[0139] Cytokine secretion resulting from activation of effector cells was detected by ELISA. Using the Human IFN-? ELISA MAX? (BioLegend). The absorbance at 450 nm was detected using the program Magellan6 and an InfiniteF200 (Tecan).

Target Cell Viability Assay

[0140] The target cell viability after co-culture was determined using the CellTiter-Blue Cell Viability Assay (Promega). This assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin) due to metabolic activity. Nonviable cells rapidly lose their metabolic capacity and thus do not generate a fluorescent signal. After removal of the supernatant 100 ?l of colorless DMEM containing 20% CellTiter-Blue Reagent was added per well to the co-cultures and incubated at 37? C. for 2 hours. The fluorescence signal was recorded at 560 nm using an InfiniteF200 (Tecan).

EXAMPLE 2

Results

[0141] In a first line of experiments we have evaluated the activity of the bispecific antibody constructs directed against CTL surface antigens CD3 and CD28 and against NK cell surface antigens CD16 and CD56. We employed plasmid-transfected hepatoma cell lines producing HBV surface antigens. After establishing the HBV protein expression, these target cells were co-cultured together with immune effector cells, namely PBMC and isolated NK cells, and bispecific antibody constructs. PBMC contain around 70% T cells but only 7% NK-cells. Therefore, we magnetically isolated CD16.sup.+ CD56.sup.+ NK cells. As negative controls we analyzed co-cultures with HBV-negative target cells, which had been preincubated with HBV? and subviral particle-containing supernatants. This control was employed to rule out activation of effector cells due to unspecific binding of HBV particles on the surface of HBV-negative target cells. Furthermore, we co-cultured HBV-positive target cells with immune effector cells in the absence of bispecific constructs to evaluate unspecific background cytotoxicity. To exclude a cytotoxic effect of the bispecific constructs, we prepared cultures of HBV-positive target cells without immune effector cells in the presence of bispecific constructs.

[0142] These experiments showed a specific activation of CTLs upon co-culture in the presence of the CD3- or CD28-specific constructs as determined by the secretion of the proinflammatory cytokine interferon gamma (IFN?) of up to 7000 pg/ml. This effect was further enhanced upon co-administration of CD3- and CD28-specific constructs demonstrating a synergistic effect.

[0143] Furthermore, the bispecific constructs mediated a specific cytotoxic elimination of HBsAg-producing HuH7 hepatoma cell lines (FIG. 3) of up to 90% reductions of target cell viability in comparison to controls. This cytotoxic response was observed for co-cultures of PBMC and HBV-positive target cells together with the bispecific constructs directed against CD3 and CD28 as well as for isolated NK-cells with constructs directed against CD16 and CD56. The co-administration of CTL- and NK-cell specific constructs further increased the cytotoxic effect synergistically to elimination rates above 95%. We observed unspecific background cytotoxicity of 15% to 40% for CTLs and NK cells, respectively.

[0144] In a second round of experiments we employed HBV-infected HepaRG hepatoma cells. This cell line allows for infection with HBV after a four week differentiation and mirrors the natural situation of HBV-infected tissues. Typically, Infection rates of HepaRG cells never reach 100% and this mixture of infected and non-infected cells mimics the situation in an HBV-infected individual under antiviral therapy, harboring both, infected and non-infected cells in the presence of free extracellular viral particles.

[0145] In co-cultures of immune effector cells and co-administered bispecific constructs, the HBV infected HepaRG cells mediated an efficient activation of both, CTLs and NK cells, with impressing amounts of IFN-? secretion of up 60,000 pg/ml (FIG. 4A). In this experiment we did not isolate or enrich NK cells prior to co-culture.

[0146] Furthermore the bispecific antibody constructs resulted in a cytotoxic response of the activated immune effector cells leading to the specific elimination of HBV-infected target cells (FIG. 4B). We observed elimination rates of 50% to 70% for NK-cells and CTLs, respectively. Unspecific background cytotoxicity was absent in these experiments.

EXAMPLE 3

Methods for Example 4

[0147] To analyze the therapeutic potential of bispecific antibody constructs to successfully retarget T cells towards HBV-positive cells, in vitro co-culturing experiments were performed and analyzed in detail. We employed bispecific antibody constructs containing single chain binding domains directed against human CD3 (?CD3) and human CD28 (?CD28) and additionally, constructs containing directed mutations in their Fc spacer domain which should abrogate antibody dependent cellular cytotoxicity (?ADCC), by circumventing Fc? receptor binding. These were constructed as a safety measure to rule out unspecific activation of natural killer cells. On the other side, all bispecific antibody constructs harbored the HBV S-protein (HBsAg) specific binding domain C8. Peripheral blood mononucleated cells (PBMC) isolated from fresh venous blood of healthy donors were co-cultured with different human hepatoma cell lines as surrogate models for HBV-infection. We employed HuH7-S(HBV S-antigen transgenic) and as negative control the mother cell line HuH7 and HBV-infected or as control uninfected HepaRG cells. HepG2.2.15 (HBV genome transgenic) cell were used as controls for HBV? marker quantification. To provide bispecific antibody constructs, supernatant of producer cell lines containing bispecific antibodies was added. To visualize changes in target cell viability due to cytotoxicity mediated by bispecific antibodies over time, the xCELLigence system was employed. This technique allows for real-time monitoring of cell-viability over long time cultures. Therefore, target hepatoma cells were seeded on specially designed microtiter plates, which contain interdigitated gold microelectrodes to noninvasively monitor the viability of adherent target cells using electrical impedance as a readout. The cytotoxic elimination results in a change of the impedance, which can be converted into the so called cell index (CI) value, which is used to monitor cell viability.

Co-Culturing with Target Cells

[0148] At day zero, 3?10.sup.4 HuH7-S/HuH7 cells were seeded per well in a 96-well plate (E-Plate 96). At day 1, the supernatant was removed and 1?10.sup.5 primary human PBMC in 100 ?l PBMC medium or only 100 ?l medium for controls were added to the respective wells. Additionally, 100 ?l of supernatant containing bispecific antibodies, singly or in combinations were added. As negative control, 100 ?l DMEM medium were added to the wells, resulting in a total volume of 200 ?l. Co-cultures were monitored for 48 h or 72 h in the xCELLigence system.

[0149] HepaRG cells were grown to confluence, differentiated for 21 days and infected with HBV prior to immunotherapeutic experiments.

[0150] For the infection of HepaRG cells a virus stock was prepared in differentiation medium containing PEG and 50 ?l were added per well. The final concentration of PEG was 5% and the MOI of the virus stock was set to 200 (7.5?10.sup.6 virus particles/well). 16 h after addition of the infection master mix, cells were washed 3 times with PBS to remove residual virus. Differentiation medium was added, and medium was changed every 3 days for a total of 12 days. Before co-culturing experiments, medium was changed to co-culturing medium (depleted of the immunosuppressant hydrocortisone). Successful HBV infection of HepaRG cells was tested by measuring HBsAg (Axsym) and HBeAg (BEP III System) in the supernatant of infected cells.

PBMC Preparation

[0151] PBMC for co-culturing experiments were isolated from whole blood. Heparinized fresh blood was diluted 1:1 with RPMI wash-medium. 25 ml of diluted blood was over layered onto 15 ml Percoll and centrifuged at 960 g for 20 min without break in a swing-out centrifuge. The PBMC were isolated and transferred into 50 ml with RPMI medium. After washing, cells were resuspended in 10 ml PBMC medium and cell number was determined. The concentration was adjusted to 2?10.sup.6 cells/ml to ensure optimal conditions. PBMC were rested overnight at 37? C.

Fluorescence Activated Cell Sorting (FACS)

[0152] To examine effector functions of redirected PBMCs, FACS analysis was performed. Thereby, the secretion of the pro-inflammatory cytokines IFN-?, IL-2 and TNF-?, as well as the expression of the activation marker CD154 (CD40L) and the degranulation marker LAMP-1 (CD107a), respectively, where analyzed. The measurement of cytokine production was performed using intracellular cytokine staining. Therefore 0.2 ?g/ml Brefeldin A (BFA) was applied to cells and incubated for 4 hours at 37? C.

[0153] BFA blocks the forward transport between the endoplasmic reticulum and the Golgi apparatus and, as a consequence, exocytosis of cytokines is inhibited. In the case of simultaneous staining for LAMP-1, antibody was applied 1 h before adding BFA (to enable translocation of LAMP-1 to the cellular surface). Subsequently, cells were transferred to a 96-well plate (round bottom) and washed twice in 200 ?l FACS buffer. For staining of viable cells and exclusion of dead cells, the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was used. For fixation and permeabilization cells were resuspended in 100 ?l Cytofix/Cytoperm reagent and incubated on ice in the dark for 20 min. After washing, cells were resuspended in the prepared antibody mix or only stained with the respective single colors for systematic compensation. Staining took place on ice in the dark for 30 min. After washing, cells were resuspended in 200 ?l FACS buffer and transferred into FACS tubes for acquisition. Acquisition was performed using either a FACSCanto II or LSR Fortessa. FACS Diva software was used to record data, analysis was performed using FlowJo software.

Animal Experiments

[0154] For a first test of bispecific constructs in vivo, experiments with immunodeficient Rag2/IL2R?null-mice (international nomenclature: B10;B6-Rag2tm1Fwa II2rgtm1Wjl) were conducted. We injected 6 weeks old mice with 5?10.sup.8 cells of the HBV-transgenic human hepatoma cell line HepG2.2.15. Cells were injected subcutaneously into the flank of the animals. This resulted in tumor formation over a 14 day time period. HBV replication inside the tumor was monitored through determination of HBV viremia. Human PBMC were isolated from fresh human cord blood and stimulated on plates precoated with antibodies against human CD3 and CD28 at a cell concentration of 0.25?10.sup.6 PBMC per ml for 3 days. Subsequently cells were maintained in cell culture medium containing 300 U/ml of IL-2 for 7 days.

[0155] On day 14 after tumor induction, mice were injected with 2?10.sup.7 PBMCs per mouse intraperitoneally and received 100 ?l of ?CD3/?CD28 bispecific antibody constructs in supernatant of HEK producer cells into the tail vein per animal at four consecutive days. Mice were sacrificed on day 18 after tumor induction and analysed for tumor size. Subsequently, serum and tissue samples were stored for further analyses.

EXAMPLE 4

Bispecific Antibodies Mediate Specific Elimination of HBV Surface Protein Expressing Target Cells (HuH7-S)

[0156] To examine whether bispecific antibody constructs successfully retarget T cells towards HBsAg expressing target cells and induce target cell lysis, isolated PBMC were co-cultured with HuH7-S cells in the presence of bispecific antibody constructs. HuH7-S cells were stably transfected to express HBsAg and therefore mimicked HBV-infected hepatocytes. This results in the production and secretion of subviral particles into the supernatant and the incorporation of HBsAg into the cellular membrane. Untransfected HuH7 cells served as negative control.

Individual Bispecific Antibodies Provoke Killing of Target Cells

[0157] To analyze if the individual bispecific antibodies are able to stimulate T cell activation and mediate target cell lysis, PBMCs were co-cultured with HuH7-S/HuH7 cells in the presence of ?HBs??CD3, ?HBs??CD28, ?HBs??CD3 [Fc?ADCC] or ?HBs??CD28 [Fc?ADCC]bispecific tetravalent antibodies. The stimulation of effector cells by the single bispecific antibodies resulted in specific killing of HBsAg expressing target cells (FIG. 5). Bispecific antibodies directed against CD3 mediated elimination of target cells earlier and stronger than constructs directed against CD28, as the endpoint viability of HuH7-S cells treated with ?CD3 only accounted for 6.4% (?CD3?ADCC: 15.5%) compared to 44.42% for ?CD28 (?CD28?ADCC: 48.9%). Furthermore ?CD3?ADCC and ?CD28?ADCC required more time to induce lysis of target cells compared to ?CD3 and ?CD28, respectively. ?CD3?ADCC-mediated killing started approximately 35 h after starting the co-culture, whereas ?CD3 led to a decrease of target cell viability already after 12 h. A time shift of about 20 h could also be observed between ?CD28?ADCC and ?CD28-mediated target cell lysis. The stimulation with ?CD3 also led to detectable lysis of HBsAg-negative HuH7 cells with an endpoint viability of 78.1%, Indicating unspecific activation. The same was true for stimulation with ?CD3?ADCC in some experiments even if not seen here. The viability of HuH7 cells during co-culture in presence of the other bispecific constructs remained at 100%.

[0158] This data demonstrates that stimulation with each of the individual bispecific antibodies provokes elimination of target cells without further co-stimulation.

Bispecific Antibodies Mediate Target Cell Lysis in a Synergistic Manner

[0159] To further analyze whether combination of bispecific constructs leads to an enhanced activity and therefore cytotoxicity of effector cells, PBMCs were co-cultured with HuH7-S/HuH7 cells either in presence of the combinations of ?CD3/?CD28 or ?CD3?ADCC/?CD28?ADCC. As shown in FIG. 6, the combination of bispecific constructs led to massive killing of HBsAg expressing target cells with a remaining viability of 1.2% (?CD3/?CD28) and 4.4% (?CD3?ADCC/?CD28?ADCC), whereas nearly no HuH7 cells were eliminated (endpoint viability of HuH7 cells: ?CD3/?CD28: 92.4%; ?CD3?ADCC/?CD28?ADCC: 100.4%).

[0160] Again ?CD3/?CD28-mediated lysis of target cells was faster than the killing induced by constructs with mutated Fc region, even if killing of target cells started at approximately the same time after about 1 h (FIG. 6A, B). Combination of bispecific antibodies led to a faster elimination of target cells compared to lysis induced by individual bispecific constructs (FIG. 6C, D). This was expected, as T cells receive not only one signal as in the presence of individual constructs, but obtain both, activation and co-stimulatory signal if an antibodies directed against CD3 and CD28 are present.

[0161] Thus, combination of bispecific constructs mediate specific lysis of HBV surface protein expressing target cells in a synergistic manner.

Bispecific Antibodies Provoke Elimination of Target Cells in a Concentration Dependent Manner

[0162] To examine if the amount of bispecific antibodies had an effect on target cell lysis, two different amounts of bispecific constructs were used for co-culture. Therefore, the usual amount of antibodies (100 ?l supernatant in total?high) and the half of it (50 ?l supernatant in total?low) were used. The lower amount of bispecific antibodies could also induce lysis of target cells (endpoint viability of HuH7-S cells: ?CD3/?CD28: 12.6%; ?CD3?ADCC/?CD28?ADCC: 15.9%), whereas the higher amount caused elimination of target cells faster (FIG. 7) with only 1.5% (?CD3/?CD28) and 2.1% (?CD3?ADCC/?CD28?ADCC) of remaining viable cells. HuH7 cells were not affected in any case. Combination of either ?CD3/?CD28 or ?CD3?ADCC/?CD28?ADCC provoked killing of target cells in a concentration dependent manner.

Increased Concentrations of Effector Cells Enhance Lysis of Target Cells

[0163] It was of further interest if the number of effector cells had an impact on the elimination of target cells. Thus, the usual amount of PBMCs used for co-culture (1?10.sup.5) was compared to the double amount (2?10.sup.5). As it is demonstrated in FIG. 8, the higher number of PBMCs induced lysis of HuH7-S cells in the presence of ?CD3/?CD28 significantly faster with an endpoint viability of 4.5% compared to 11.7%, but also more HuH7 cells were killed, if the double amount of PBMCs was present (endpoint viability of HuH7 cells: 2?10.sup.5 PBMCs: 83.8%; 1?10.sup.5 PBMCs: 102.7%).

[0164] This data indicates that the elimination of target cells is dependent on the amount of effector cells.

Bispecific Antibodies Mediate Killing of Target Cells after Only 8 h of Co-Culture

[0165] To investigate the question, how long bispecific antibodies have to be present during co-culture to activate T cells and therefore induce cytotoxicity, the supernatant of co-cultures containing the bispecific antibodies was removed after different time periods and new DMEM standard medium was added. If supernatant containing ?CD3/?CD28 was removed after 4 h, PBMCs only induced a small decrease in target cell viability (78.5%), but were not able to provoke lysis of all target cells (FIG. 9). If supernatant containing bispecific antibodies was present for 8 h or longer, PBMCs were able to cause elimination of target cells. As it is illustrated in FIG. 10, PBMCs needed more time to induce target cell lysis if stimulation with ?CD3/?CD28 lasted for 8 h or 12 h compared to 24 h or 48 h, but the effect after 48 h was nearly similar (HuH7-S endpoint viability: 8 h: 14.7%; 12 h: 11.7%, 24 h: 5.1%, 48 h: 3.2%).

[0166] Bispecific antibodies mediate effector functions of T cells during co-culture with either HBsAg or HuH7-S cells

[0167] To investigate the activation and functionality of T cells during co-culture experiments, the secretion of cytokines was examined either by ELISA or FACS analysis.

Bispecific Constructs Mediate the Secretion of IFN-?, TNF-? and IL-2

[0168] In a time line experiment it was analyzed, when PBMCs start to secret cytokines upon contact with bispecific antibodies and how dynamics develop over time. Therefore, supernatant of co-cultures was removed 4 h, 8 h, 12 h, 24 h and 48 h after addition of PBMCs and ?CD3/?CD28. Cytokine production was measured by ELISAs for IL-2, IFN-? and TNF-?. The secretion of IL-2 increased over time, but after 4 h almost no IL-2 was detectable, after 8 h the concentration was already 316 pg/ml and during the following 4 hours, the concentration almost quadrupled (1119 pg/ml). There was no further rise between 24 h and 48 h and IL-2 concentration seemed to reach a plateau at about 1550 pg/ml (FIG. 10A). IFN-? secretion (FIG. 10B) needed more time, after 8 h still very low levels were detected. Between 8 h and 12 h, T cells started to secret IFN-?, because its concentration accounted already for 1800 pg/ml after 12 h. Subsequently (24 h) an increase in IFN-? production was observed, the concentration increased to around 10000 pg/ml. The highest amount was detected after 48 h (12000 pg/ml). For both, IL-2 and IFN?, the concentration on HBsAg negative cells increased over time, with the highest amount after 48 h (IL-2: 45 pg/ml; IFN?: 200 pg/ml) which also corresponds to observations concerning cell viability.

[0169] The secretion of TNF-? (FIG. 10C) increased up to 24 h, where it reached its peak concentration (1700 pg/ml). Then it declined and accounted for only 1400 pg/ml after 48 h. In contrast TNF-? secretion started earlier than the others, with around 100 pg/ml after 4 h followed by a steady rise up to 24 h. Interestingly, TNF-? production on HuH7 cells behaved in exactly the opposite way. With a relatively high background concentration compared to other cytokines, it showed the highest concentration after 4 h (?70 pg/ml) which declined over time and accounted for only 9 pg/ml after 48 h. PBMCs are induced to secret IL-2, IFN-? and TN-F? upon contact with ?CD3/?CD28 during co-culture with HBsAg-expressing cells, whereas the secretion dynamics differ among the individual cytokines.

Bispecific Constructs Activate CD8.sup.+ T Cells as Well as CD4.sup.+ T Cells

[0170] To analyze if PBMCs also show degranulation of cytotoxic vesicles, the translocation of LMAP-1 (CD107a), a degranulation marker, was investigated. After co-culture with HuH7-S/HuH7 cells in presence of ?CD3/?CD28 or ?CD3?ADCC/?CD28?ADCC, CD8.sup.+ T cells showed a clear shift in LAMP-1 staining, whereas the signal was stronger in samples stimulated with ?CD3?ADCC/?CD28?ADCC compared to ?CD3/?CD28 (FIG. 11 C, D). Interestingly, the same observation could be made for CD4.sup.+ T cells (FIG. 11 A, B). For ?CD3/?CD28 the translocation of LAMP-1 was more prominent in CD8 T cells, for ?CD3?ADCC/?CD28?ADCC exactly the opposite.

[0171] This data demonstrates, that not only CD8.sup.+ T cells, but also CD4.sup.+ are induced to secret cytotoxic granules upon contact with the bispecific antibodies and HBsAg.

[0172] To examine polyfunctionality of T cells after co-culture experiments, PBMC were stained for IFN-?, IL-2 and TNF-?, as well as for the activation marker CD154 (CD40L) which is predominantly expressed on CD4.sup.+ T cells, at 8 h, 12 h and 24 h after addition of PBMC and ?CD3/?CD28 (FIG. 12). CD4.sup.+ T cells showed a steady increase of IFN-?.sup.+ T cells (9.3% after 24 h), IL-2.sup.+ T cells (11.3% after 24 h), TNF-?.sup.+ T cells (14.7% after 24 h) and CD154.sup.+ T cells (28.0% after 24 h), whereas the major rise occurred between 12 h and 24 h (FIG. 12A).

[0173] The same was true for CD8 T cells, whereas the percentage of IFN-?.sup.+ and IL-2.sup.+ cells with 18.4% and 11.3% outnumbered CD4 T cells. The amount of TNF-?.sup.+ and CD154.sup.+ CD8.sup.+ T cells was decreased with 10.1% and 6.25% compared to CD4.sup.+ T cells (FIG. 12B). PBMC on HuH7 cells showed no activation in any sample. Boolean combination gates were used for further analysis of T cells secreting cytokines (FIG. 12C, D). After 24 h 3.1% of CD4.sup.+ T cells and 2.1% of CD8.sup.+ T cells were IFN?, IL-2.sup.+ and TNF?.sup.+, indicating polyfunctionality of T cells. Therefore, ?CD3/?CD28 mediates activation of PBMCs during co-culture with HuH7-S/HuH7 cells resulting in polyfunctional CD4.sup.+ and CD8.sup.+ T cells.

[0174] To exclude the possibility that false positive signals were detected due to unspecific binding of antibodies to dead target cells during FACS analysis, PBMC were cultured in the presence of Immobilized HBsAg. Additionally the effect of soluble HBsAg was examined, as HBV infected patients exhibit high amounts of HBsAg in their blood. PBMCs were again stained for IFN-?, IL-2 and TNF-?, as well as for CD154, but only 24 h and 48 h after addition of PBMC and ?CD3?ADCC/?CD28?ADCC (FIG. 13). Again CD4.sup.+ T cells showed an increase of IFN-?.sup.+ T cells (3.4% after 24 h, 6.8% after 48 h), and CD154.sup.+ T cells (17.2% after 24 h, 19.9% after 48 h).

[0175] There were less IL-2.sup.+ T cells after 48 h (4.9%) compared to 24 h (5.5%), TNF-?.sup.+ T cells also decreased (14.9% after 24 h, 8.1% after 48 h) (FIG. 13A). CD8.sup.+ T cells only showed a decrease in TNF-?.sup.+ T cells (12.6% after 24 h, 7.4% after 48 h), whereby this reduction was also observed in ELISA (FIG. 10). The percentage of IFN-?.sup.+, IL-2.sup.+ and CD154.sup.+ CD8.sup.+ T cells increased between 24 h and 48 h (IFN?.sup.+: 4.7% after 24 h, 8.5% after 48 h, IL-2.sup.+: 5.1% after 24 h, 7.2% after 48 h, CD154.sup.+: 8.3% after 24 h, 10.4% after 48 h) (FIG. 13B). Again the percentage of IFN-?.sup.+ and IL-2.sup.+ CD8.sup.+ T cells outnumbered CD4.sup.+ T cells and the amount of TNF-?.sup.+ and CD154.sup.+ CD8.sup.+ T cells was decreased compared to CD4.sup.+ T cells. After 48 h also some T cells seemed to be activated by the soluble HBsAg, as TNF?.sup.+ T cells reached 1.1% (CD4.sup.+ T cells) and 1.0% (CD8.sup.+ T cells), CD154.sup.+ T cells 2.7% (CD4.sup.+ T cells) and 3.1% (CD8 T cells), IFN?.sup.+ CD8.sup.+ T cells 1.2% and IL-2.sup.+ CD8.sup.+ T cells 1.4%. Again boolean gates were used for further analysis of T cells secreting cytokines (FIG. 13C, D). 0.35% (after 24 h) and 0.63% (after 48 h) of CD4.sup.+ T cells, 0.3% (after 24 h) and 1.0% (after 48 h) of CD8.sup.+ T cells were IFN-?.sup.+, IL-2.sup.+ and TNF-?.sup.+, indicating polyfunctionality of T cells. ?CD3ADCC/?CD28?ADCC mediates activation of PBMCs during co-culture with immobilized HBsAg cells resulting in polyfunctional CD4.sup.+ and CD8.sup.+ T cells. The activation due to soluble HBsAg remains poor.

Bispecific Antibodies Mediate IFN?Secretion and Killing of HBV Infected HepaRG Cells

[0176] Finally, it was of interest, if bispecific antibodies are able to retarget T cells towards HBV infected HepaRG cells. Success of infection was tested by the measurement of HBsAg in the supernatant of infected cells. Compared to results of HuH7-S or HepG2.2.15 cells, the concentration of HBsAg produced by HBV infected HepaRG cells was very low. Additionally the values in different wells varied a lot, indicated by the relatively high standard deviation (FIG. 14).

[0177] Nevertheless, the infection was successful and co-culture of PBMCs with HepaRG cells in presence of bispecific antibodies was performed. As can be seen in FIG. 15, viability of untreated cells decreased over time, with a remaining viability of 65.9% (HBV+) and 62.9% (HBV?) after 56 h. In comparison, ?CD3 and the combination of ?CD3/?CD28 mediated specific lysis of HBV infected HepaRG cells. ?CD28 alone could not induce specific elimination of target cells. If ?CD3 was present during co-culture, the viability of HBV infected cells decreased to 25.3%, while non-infected HepaRG cells remained at 53.5% (FIG. 15A). The stimulation of effector cells by ?CD3/?CD28 also led to significant killing of HBV infected HepaRG cells (FIG. 15B), whereby 37.5% of target cells remained viable (not infected HepaRG cells: 62.4%).

[0178] Therefore, ?CD3 or ?CD3/?CD28 Induce specific lysis of HBV infected HepaRG cells.

Bispecific Antibodies Mediate Reduction of HBV-Positive Tumors In Vivo

[0179] Immunodeficient mice injected with human HBV-transgenic hepatoma cell line HepG2.2.15 to develop subcutaneous HBV-positive tumors were injected with human PBMC and bispecific constructs directed against CD3 and CD28 (FIG. 16). The treatment resulted in a marked reduction in tumor size in comparison to not-treated or mock treated (animals receiving PBMC and PBS) animals. The tumor size was reduced by about fifty percent in treated animals.