Dual antigen-induced bipartite functional complementation

Abstract

The present invention relates to a set of polypeptides and its uses. In particular, the present invention relates to a set of polypeptides whereby this set comprises two polypeptides each of which comprises a targeting moiety “T” binding to an antigen “Λ” and a fragment of “F” of a functional domain, wherein said two polypeptides are not associated with each other in absence of a substrate that has “A” at (on) its surface and wherein, upon dimerization of “F”, the resulting dimer becomes functional. Furthermore, medical and diagnostic uses of said set are described. Moreover, the present invention relates to nucleic acid molecule(s) encoding said set of polypeptides. The present invention also relates to a vector comprising the nucleotide sequence of nucleic acid molecule(s) encoding said set of polypeptides. Furthermore, the present invention relates to pharmaceutical compositions comprising said set of polypeptides. Moreover, the present invention relates to a kit comprising said set of polypeptides.

Claims

1. A method of treatment by dual antigen-induced bipartite functional complementation, said method comprising a step of administering to a subject in need thereof a pharmaceutically effective amount of a set of polypeptides comprising: a first polypeptide P1 comprising (i) a targeting moiety T1, wherein said targeting moiety T1 specifically binds to an antigen A1, and (ii) a fragment F1 of a functional domain F, wherein neither said fragment F1 by itself nor said polypeptide P1 by itself is functional with respect to the function of said functional domain F, and a second polypeptide P2 comprising (i) a targeting moiety T2, wherein said targeting moiety T2 specifically binds to an antigen A2, and (ii) a fragment F2 of said functional domain F, wherein neither said fragment F2 by itself nor said polypeptide P2 by itself is functional with respect to the function of said functional domain F, wherein said antigen A1 is different from said antigen A2, wherein said polypeptide P1 and said polypeptide P2 are not associated with each other in the absence of a cell that carries both antigens A1 and A2 at its surface, wherein, upon dimerization of said fragment F1 of said polypeptide P1 with said fragment F2 of said polypeptide P2, the resulting dimer is functional with respect to the function of said functional domain F, wherein said fragment F1 comprises a V.sub.H domain of an antibody and said fragment F2 comprises a V.sub.L domain of the same antibody, wherein said targeting moiety T1 comprises an immunoglobulin module, wherein said targeting moiety T2 comprises an immunoglobulin module, wherein said functional domain F specifically binds or is capable of specifically binding to an antigen, wherein said antigen is different from antigen A1 and antigen A2, and wherein said polypeptide P1 comprises the amino acid sequence SEQ ID NO: 124 and said polypeptide P2 comprises the amino acid sequence SEQ ID NO: 125.

2. The method according to claim 1, wherein said cell that carries both antigens A1 and A2 at its surface induces dimerization of the fragment F1 of said polypeptide P1 with the fragment F2 of said polypeptide P2, whereas a cell which does not carry both antigens A1 and A2 at its cell surface does not induce dimerization of the fragment F1 of said polypeptide P1 with the fragment F2 of said polypeptide P2.

3. The method according to claim 1, wherein said polypeptides P1 and P2 have, in the absence of said cell that carries both antigens A1 and A2 at its surface, a dissociation constant K.sub.D with each other in the range of 10.sup.−7 M to 10.sup.−3 M.

4. The method according to claim 1, wherein said antigen A1 and said antigen A2 are expressed on the surface of cells of a tumour or on the surface of progenitor/precursor cells of a tumour.

5. The method according to claim 1, wherein said antigen A1 is HLA-A2 and antigen A2 is CD45.

6. The method according to claim 1, wherein said targeting moiety T1 comprises an immunoglobulin module I1 comprising a V.sub.L domain linked to a V.sub.H domain; and wherein said targeting moiety T2 comprises an immunoglobulin module I2 comprising a V.sub.L domain linked to a V.sub.H domain.

7. The method according to claim 6, wherein said immunoglobulin module I1 comprises a single-chain variant fragment (scFv) of an antibody; and wherein said immunoglobulin module I2 comprises an scFv of an antibody.

8. The method according to claim 1, wherein said functional domain F is or comprises an immunoglobulin module.

9. The method according to claim 8, wherein said functional domain F is or comprises a variant fragment (Fv) of an antibody.

10. The method according to claim 1, wherein said fragment F1 comprises a V.sub.H domain of an anti-CD3 antibody and said fragment F2 comprises a V.sub.L domain of the same antibody.

11. The method of claim 1, wherein the method comprises treating a patient who is suffering from cancer and/or a tumour.

12. The method of claim 1, wherein said antigen specifically bound by said functional domain F is present on cells of the human immune system.

13. The method of claim 1, wherein said binding of said functional domain F to said antigen activates cells of the human immune system.

14. The method of claim 1, wherein said functional domain F comprises a T cell engaging domain.

15. The method of claim 1, wherein said functional domain F comprises a T cell engaging domain specifically binding to CD3; or a T cell engaging domain specifically binding to CD3ε.

16. The method of claim 1, wherein said polypeptide P1 and said polypeptide P2 have, in the absence of said cell that carries both antigens A1 and A2 at its surface, a dissociation constant K.sub.D of above 10.sup.−7 M.

Description

(1) In the following, reference is made to the figures:

(2) FIG. 1A to FIG. 1D show the principle of the invention. FIG. 1A: Antigens and design of polypeptides P1 and P2. FIG. 1B: If a cell expresses both antigens 1 and 2 at its cell surface, simultaneous binding of polypeptide P1 and polypeptide P2 to the surface of this cell brings P1 and P2 in close proximity, causes association of fragments F1 and F2 and restoration of the biological function of domain F by complementation. No restoration of biological function occurs if only antigen A1 (FIG. 1C) or antigen A2 (FIG. 1D) is present on the cell surface.

(3) FIG. 2 shows an exemplary embodiment of the invention in an allogeneic transplantation setting for haematopoietic neoplasias with mismatched HLA antigens. In this situation, the dual information of recipient HLA haplotype (HLA.sub.patient) and haematopoietic lineage origin (CD45) is displayed exclusively on leukemic blasts and other haematopoietic cells of the patient. The first polypeptide P1 comprises a single-chain variable fragment antibody construct directed against the HLA of the patient (targeting moiety T1) fused to the V.sub.L fragment of anti CD3 (fragment F1). The second polypeptide P2 comprises a single-chain variable fragment construct specific for the haematopoietic lineage marker CD45 (targeting moiety T2), fused to the V.sub.H split-fragment of anti CD3 Fv (fragment F2).

(4) CD45: antigen specific for haematopoietic cells. HLA.sub.patient: HLA-antigen specific for patient cells, i.e. an allelic variant of the human MEW that is present on the surface of patient cells (=cells of the recipient of cell transplantation), but absent from the surface of donor cells. αCD45 scFv: scFv with binding specificity for CD45. αHLA.sub.patient scFv: scFV with binding specificity for HLA.sub.patient. CD3(V.sub.H): variable region of an immunoglobulin heavy chain of an antibody with binding specificity for CD3. CD3(V.sub.L): variable region of an immunoglobulin light chain of an antibody with binding specificity for CD3.

(5) Upon binding of the two constructs through their αCD45 scFv and αHLA.sub.patient scFv, respectively, to a cell carrying both the CD45 and the HLA.sub.patient antigen, assembly of CD3(V.sub.H) with CD3(V.sub.L) leads to functional complementation of the antibody with binding specificity for CD3, thus allowing for specific recruitment and activation of T cells through the CD3 molecules at their cell surface.

(6) FIG. 3A-FIG. 3B show the constructs used in the experiments depicted in FIG. 4-FIG. 9. (Construct 85 differs from construct 71 by the fact that construct 85 has a Flag tag while construct 71 has a myc tag. Construct 75 differs from construct 82 by the fact that construct 75 has a Flag tag while construct 82 has a myc tag.) V.sub.HCD3: variable region of the heavy chain of an anti-CD3 antibody; V.sub.LCD3: variable region of the light chain of an anti-CD3 antibody; V.sub.HA2: variable region of the heavy chain of an anti-HLA-A2 antibody; V.sub.LA2: variable region of the light chain of an anti-HLA-A2 antibody; V.sub.L45: variable region of the heavy chain of an anti-CD45 antibody; V.sub.H45: variable region of the light chain of an anti-CD45 antibody; L18, L7, L15, L6, L19: linker of 18, 7, 15, 6, 19 amino acids, respectively.

(7) FIG. 4 shows conventional tandem bispecific single chain scFv constructs used to control the assay system. Briefly, bispecific antibody constructs with specificity for CD3 and HLA A2 were titrated as indicated to a co-culture of U266, a HLA A2 positive, CD45 positive myeloma cell line, and HLA A2 negative T cells (monocyte depleted peripheral blood mononuclear cells), and production of interleukin 2 by T cells was determined. Substantial T cell stimulatory capacity was detected for the two FvCD3−HLA-A2 constructs 85 and 71, which differ by their respective Flag or Myc-Tags (For domain structure of constructs see FIG. 3). Bispecific tandem Fv constructs in HLA-A2−CD3 configuration were less efficient and single chain constructs addressing either HLA-A2 or CD3 did not stimulate T cells at all. Positive control is conducted using unspecific PHA-L (phytohemagglutinin) stimulation.

(8) FIG. 5 shows exquisite and highly specific T cell stimulatory capacity if a pair of complementing constructs according to the invention is used, but not if only one of the two constructs of a pair is used individually. Briefly, V.sub.LCD3−scFvHLA-A2 (construct 42), V.sub.HCD3−scFvCD45(V.sub.L−V.sub.H) (construct 45) and V.sub.HCD3−scFvCD45(V.sub.H−V.sub.L) (construct 55) were titrated separately or in the combinations of constructs 42 and 45, or 42 and 55 to co-cultures of U266 and T cells as described. High T cell stimulatory capacity was demonstrated for the combinations of 42/45 or 42/55 with minute activity, if only one of these constructs was given separately. These results show that the V.sub.L and V.sub.H domains of FvCD3 have to cooperate in order to reconstitute or complement T cell engaging function. Importantly, the scFvCD45 targeting moiety could be switched from (V.sub.L−V.sub.H) to the (V.sub.H−V.sub.L) configuration, clearly indicating that the modular character of the constructs allows replacement of a targeting moiety by another targeting moiety with desired specificity. The assay system was controlled by the use of single chain constructs CD45(V.sub.L−V.sub.H) and CD45(V.sub.H−V.sub.L) which did not stimulate T cells to produce IL2.

(9) FIG. 6 shows a first of three competitive blocking experiments. The bispecific tandem construct FvCD3−HLA-A2 (construct 71) was given to co-cultures of U266 and T cells as described and stimulatory function was determined through induced IL2 production by T lymphocytes. The T cell stimulating function was blocked by single chain constructs that occupy the targeted epitope on the HLA A2 molecule (construct 4, concentration*100). Intrinsic stimulation of T cells by the HLA A2 or CD3 specific single chain constructs (construct 4 (concentration*100) or construct 36 (concentration*9)) was ruled out. PHA-L was used as positive control.

(10) FIG. 7 shows that “tridomain constructs” (i.e. constructs according to the invention) first have to bind on the surface of a single cell to dimerize and complement T cell engaging functions the competitive epitope blocking experiments. Briefly, constructs 42 and 45 were given to co-cultures of U266 cells and HLA-A2 negative T lymphocytes and stimulatory capacity was determined by IL2 production of T cells. In experimental situations where the epitopes on HLA A2 or CD45 molecules were competitively blocked by constructs 4 or 46 (both concentrations*100), T cell stimulatory function was abrogated. These results clearly indicate that the two respective “tridomain constructs” have to bind simultaneously onto the surface of a cell in order to restore or to complement T cell engaging function. Intrinsic stimulatory activity of either construct (42, 45, 4, 46 and 36) was ruled out using different concentrations.

(11) FIG. 8 shows the analogous experiment to FIG. 7 for the combination of constructs 42 and 55. Again, T cell stimulatory capacity of the combination of the two “tridomain constructs” was abrogated by competitive blocking of antigenic epitopes on the HLA A2 or the CD45 molecule. Importantly, these results again show that the targeting module can be easily replaced by another module with appropriate specificity. More importantly, the V.sub.L-V.sub.H-V.sub.L configuration of construct 42 and the V.sub.H-V.sub.H-V.sub.L configuration of construct 55 impede homo- or hetero-dimerization or self-assembling of the constructs without prior binding to a substrate expressing both, HLA A2 and CD45 antigens.

(12) FIG. 9 shows lysis of U266 cells by HLA A2 negative T cells in a sample comprising both V.sub.LCD3−scFvHLA-A2 and V.sub.HCD3−scFvCD45(V.sub.H−V.sub.L) constructs (“both constructs”). No significant lysis was observed in control samples comprising only one of the two constructs.

(13) FIG. 10 shows the On-target restoration of the polypeptides. Binding of two separate polypeptides (P1 and P2) to their respective antigens on a target cell, each consisting of a specific single-chain variable antibody fragment (scFv, V.sub.H−V.sub.L) fused to the variable light (V.sub.L) or variable heavy chain domain (V.sub.H) of a CD3-specific antibody (Fragment F1 and F2), enables V.sub.H/V.sub.L heterodimerization and the formation of a functional CD3 binding site to engage T cells.

(14) FIG. 11A-FIG. 11D show that CD3 V.sub.H/V.sub.L dimerization engages T cells and is dual-antigen-restricted. U266 myeloma, primary T cell pro-lymphocytic leukemia (T-PLL), and THP-1 acute myeloid leukemia cells, all HLA-A2-positive and CD45-positive, were probed with HLA-A2-negative donor peripheral blood mononuclear cells (PBMC) and the polypeptides as indicated. T-cell engagement was assessed by reactive interleukin-2 (IL-2) production (FIG. 11A) and target cell lysis (FIG. 11B). The bispecific tandem scFv (CD3(V.sub.H−V.sub.L)−HLA-A2(V.sub.H−V.sub.L) antibody was used as a positive control. (FIG. 11C), Binding of the polypeptides on THP-1 cells is competitively blocked by an excess of scFvCD45 (left) and scFvHLA-A2 (right) inhibitors (blocking the individual antigen epitopes on the target cell), as indicated, and reactive IL2 production by donor PBMCs was investigated. (FIG. 11D), The single or double antigen negative cell lines RAJI and KMS-12-BM were probed with the polypeptides. PHA-L was used as a nonspecific stimulus control for PBMCs.

(15) FIG. 12A-FIG. 12B show targeted therapy by conditional CD3V.sub.H/V.sub.L complementation in vivo. (FIG. 12A), Survival of mice (n=6 per group) after intraperitoneal injection of 5×10.sup.6 THP-1 acute leukemic cells together with 1.25×10.sup.5 CMV-specific, HLA-A2-negative donor T cells and the polypeptides (0.5 μg) as indicated (tumor cells: T-cell ratio=40/1). (FIG. 12B), Caspase 3 activation was assessed in vitro by flow cytometry in HLA-A2/CD45 double-positive THP-1 and CD45-positive but HLA-A2-negative bystander cells after co-culture with donor T cells and the polypeptides (3 nM) as indicated. The bispecific tandem scFv (CD3(V.sub.H−V.sub.L)−HLA-A2(V.sub.H−V.sub.L)) antibody was used as a positive control.

(16) FIG. 13A-FIG. 13B show that EGFR- and EpCAM-directed polypeptides engage T cells for carcinoma cell destruction. EGFR and EpCAM double-positive human colon cancer cell line COLO-206F and melanoma cell line FM-55 (EGFR-positive but EpCAM-negative) were probed with PBMCs in the presence of polypeptides specific for EGFR (CD3(V.sub.H)-EGFR(V.sub.H−V.sub.L)) and EpCAM (CD3(V.sub.L)−EpCAM(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive interferon-γ (IFNγ) production (FIG. 13A) and activation of caspase 3 in target cells (FIG. 13B).

(17) FIG. 14 shows that HLA-A2 and CEA directed polypeptides redirect T cells for tumor cell destruction. Human colon cancer cell line COLO-206F, melanoma cell line FM-55 and ovarian cancer cell line OVCAR were probed with PBMCs in the presence of polypeptides specific for HLA-A2 (CD3(V.sub.L)−HLA-A2(V.sub.H−V.sub.L)) and CEA (CD3(V.sub.H)−CEA(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(18) FIG. 15 shows that HLA-A2 and EGFR directed polypeptides redirect T cells for tumor cell destruction. Human cell lines COLO-206F, FM-55 and OVCAR were probed with PBMCs in the presence of polypeptides specific for HLA-A2 (CD3(V.sub.L)−HLA-A2(V.sub.H−V.sub.L)) and EGFR (CD3(V.sub.H)−EGFR(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(19) FIG. 16 shows that HLA-A2 and Her2 directed polypeptides redirect T cells for tumor cell destruction. Human cell lines COLO-206F, FM-55 and OVCAR were probed with PBMCs in the presence of polypeptides specific for HLA-A2 (CD3(V.sub.L)−HLA-A2(V.sub.H−V.sub.L)) and Her2 (CD3(V.sub.H)−Her2(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(20) FIG. 17 shows that CD45 and HLA-A2 directed polypeptides redirect T cells for tumor cell destruction. In this experiment the split antiCD3 fragments (CD3(V.sub.H) and CD3(V.sub.L)) for the anti-CD45 and anti-HLA-A2 targeting moieties were exchanged, compared to the CD45 and HLA-A2 polypeptides used in FIG. 5,7-9, 11,12, 14-16. Human myeloma cell line U266 was probed with PBMCs in the presence of polypeptides specific for CD45 (CD3(V.sub.L)−CD45(V.sub.H−V.sub.L)) and HLA-A2 (CD3(V.sub.H)−HLA-A2(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(21) FIG. 18 shows that EGFR and EpCAM directed polypeptides redirect T cells for tumor cell destruction. Human colon cancer cell lines COLO-206F and CX-1 and ovarian cancer cell line OVCAR were probed with PBMCs in the presence of polypeptides specific for EpCAM (CD3(V.sub.L)−EpCAM(V.sub.H−V.sub.L)) and EGFR (CD3(V.sub.H)−EGFR(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(22) FIG. 19 shows that Her2 and EpCAM directed polypeptides redirect T cells for tumor cell destruction. Human ovarian cancer cell line OVCAR were probed with PBMCs in the presence of polypeptides specific for EpCAM (CD3(V.sub.L)−EpCAM(V.sub.H−V.sub.L)) and Her2 (CD3(V.sub.H)−Her2(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(23) FIG. 20 shows that CD45 and CD138 directed polypeptides redirect T cells for tumor cell destruction. Human myeloma cell line AMO-1 was probed with PBMCs in the presence of polypeptides specific for CD45 (CD3(V.sub.L)−CD45(V.sub.H−V.sub.L) upper panel, CD3(V.sub.H)−CD45(V.sub.H−V.sub.L) lower panel) and CD138 (CD3(V.sub.H)−CD138(V.sub.H−V.sub.L) upper panel, CD3(V.sub.L)−CD138(V.sub.H−V.sub.L) lower panel) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(24) FIG. 21 shows that targeting a single antigen (CD138) with CD138 directed polypeptides redirect T cells for tumor cell destruction. Human myeloma cell line AMO-1 was probed with PBMCs in the presence of polypeptides specific for CD138 (CD3(V.sub.L)−CD138(V.sub.H−V.sub.L) and (CD3(V.sub.H)−CD138(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(25) FIG. 22 shows that targeting a single antigen (CD45) with CD45 directed polypeptides redirect T cells for tumor cell destruction. Human myeloma cell lines AMO-1 and U266 were probed with PBMCs in the presence of polypeptides specific for CD45 (CD3(V.sub.L)−CD45(V.sub.H−V.sub.L) and (CD3(V.sub.H)−CD45(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(26) FIG. 23 shows the On-target restoration of two polypeptides directed against a single antigen on the cell surface, targeting two different epitopes (upper part) or the same epitope (lower part) on the antigen. Binding of two separate polypeptides (P1 and P2) to their respective epitope, on the same antigen, on a target cell. For targeting two different epitopes, the targeting moiety of each polypeptide consists of a specific single-chain variable antibody fragment (scFv). For targeting the same epitope, the targeting moiety of each polypeptide consists of the same single-chain variable antibody fragment (scFv). The targeting moieties are fused via peptide linkers to the variable light (V.sub.L) or variable heavy chain domain (V.sub.H) of a CD3-specific antibody (Fragment F1 and F2), enables V.sub.H/V.sub.L heterodimerization and the formation of a functional CD3 binding site (functional domain) to engage T cells.

(27) FIG. 24 shows the possibility to use different effector ways to kill a target cell with a kit of polypeptide parts. To this end, the anti-CD3 module (F1 and F2) is replaced by an anti-HIS (hexa-histidine) module which, after simultaneous binding of polypeptide 1 and 2, complements a hexa-histidine binding site and thus binds histidine labeled payloads (eg. a HIS-tagged toxin). The targeting moiety T1 (V.sub.H−V.sub.L) of polypeptide P1 specifically binds to HLA-A2, the targeting moiety T2 (V.sub.H−V.sub.L) of polypeptide P2 specifically binds to CD45. The fragment F1 of polypeptide P1 comprises of a V.sub.H domain of an antibody against a hexahistidine-tag and fragment F2 of polypeptide P2 comprises a V.sub.L domain of the same antibody. Human myeloid leukemia cell line THP-1 was probed with a histidine (His) tagged Clostridium perfringens Iota toxin component Ia at 0.01 μg/ml in combination with indicated polypeptides. After 48 hours in culture the cell viability was measured using the ALAMARBLUE® assay. The results show a reduction of viability against the background of the assay for cells probed with the combination, but not with individual polypeptides. Control THP-1 cells were grown simultaneously in culture without toxin. Samples were run and analyzed as duplicates.

(28) FIG. 25 shows that HLA-A2 and CD45 directed polypeptides, comprising of a split antibody against a His-tag, kill tumor cells using a histidine (His) tagged Shiga toxin subunit A at a concentration of 0.01 μg/ml. The same experimental setup was used as in FIG. 24.

(29) FIG. 26 shows that HLA-A2 and CD45 directed polypeptides, comprising of a split antibody against a His-tag, kill tumor cells using a histidine (His) tagged Shiga toxin subunit A at a concentration of 0.1 μg/ml. The same experimental setup was used as in FIG. 24 and FIG. 25.

(30) FIG. 27 shows that EGFR and EpCAM directed polypeptides, comprising of a functional domain F with F1 and F2 are V.sub.H and H.sub.L of a antibody specific for digoxigenin (aDig), mark tumor cells using a digoxigenin labeled horse radish peroxidase (HRP) molecule. The targeting moiety T1 (V.sub.H−V.sub.L) of polypeptide P1 specifically binds to EGFR, the targeting moiety T2 (V.sub.H−V.sub.L) of polypeptide P2 specifically binds to EpCAM. The fragment F1 of polypeptide P1 comprises of a V.sub.H domain of an antibody against digoxigenin and fragment F2 of polypeptide P2 comprises a V.sub.L domain of the same antibody. Human colon cancer cell line Colo-206F was first probed with indicated polypeptides followed by probing with digoxigenin labeled HRP. The samples were analyzed using the (Invitrogen™, ELISA Kit) and the absorbance was read with a BioRAD-micro plate reader. For analysis the chromogen blank sample (no Digoxigenin-HRP) was set to 0. Samples were run and analyzed as duplicates.

(31) FIG. 28 shows that CD45 and HLA-CW6 directed polypeptides redirect T cells for patient cell destruction. Primary patient cells with known HLA-haplotypes were used. A51=cells of a patient with MDS (myelodysplastic syndrom), homozygous for the HLA-Cw6 haplotype. A49=cells of a patient after allogeneic bone marrow transplantation, heterozygous for the HLA-Cw6 haplotype. Patient cells were incubated with healthy PBMCs for 30 hours, in the presence of polypeptides specific for CD45 (CD3(V.sub.L)−CD45(V.sub.H−V.sub.L) and HLA-Cw6 (CD3(V.sub.H)−HLA-CW6(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(32) FIG. 29 shows that EGFR and EpCAM directed polypeptides redirect T cells for primary cancer patient cell destruction. A44 tumor cells were collected from the malignant ascites of a 48 years old male patient with metastatic pancreatic cancer. Patient tumor cells were incubated with patients own PBMCs (collected by phlebotomy) for 30 hours, in the presence of polypeptides specific for EpCAM (CD3(V.sub.L)−EpCAM(V.sub.H−V.sub.L) and EGFR (CD3(V.sub.H)−EGFR(V.sub.H−V.sub.L)) as indicated. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(33) FIG. 30 shows that CD45 and HLA-A2 directed polypeptides redirect CMV restricted CD8+ T cells for tumor cell destruction. Human tumor cells THP-1 and U266 were incubated with CMV restricted T-cells from a HLA-A2 negative healthy donor for 30 hours, in the presence of polypeptides specific for HLA-A2 (CD3(V.sub.L)−HLA-A2(V.sub.H−V.sub.L) and CD45 (CD3(V.sub.H)−CD45(V.sub.H−V.sub.L)) as indicated. The bispecific tandem scFv (CD3(V.sub.H−V.sub.L)×HLA-A2(V.sub.H−V.sub.L)-antibody was used as a positive control. T cell engagement was assessed by reactive IFNγ production. Samples were run and analyzed as duplicates.

(34) FIG. 31 shows the principle idea to eliminate autoimmune or hypersensitivity disorder causing B-cell clones with a kit of polypeptide parts, consisting of an allergen specific polypeptide and a cell type specific polypeptide. The first polypeptide P1 has at its targeting moiety an allergen (eg. Betv-1A, Der-f2, Conglutin-7, Can-f1, Feld-d1). The second polypeptide P2 has at its targeting moiety a specific single-chain variable antibody fragment (scFv, V.sub.H−V.sub.L) targeting a cell surface protein (eg. CD19, CD138, CD38). Both targeting moieties are fused to either the variable light (V.sub.L) or variable heavy chain domain (V.sub.H) of a CD3-specific antibody (Fragment F1 and F2).

(35) In the following, reference is made to certain (human) genes or proteins also referred to in the specification, the appended examples and figures as well as (partially) in the claims. Herein below, corresponding (exemplary) gene accession numbers are provided. Further accession numbers are also provided in the specification elsewhere herein as well as the appended examples. CD45: Gene ID: 5788, updated on 13 Jan. 2013, 3. Protein=P08575-1=Isoform 1, Last modified Jul. 19, 2003. Version 2 CD34: Protein: P28906-1/2 Last modified Jul. 15, 1998. Version 2. CD33: Gene ID: 945, updated on 30 Dec. 2012: Protein: P20138 [UniParc]. Last modified Oct. 17, 2006. Version 2. Checksum: 1C73E588240FBAD8 CD138: Gene ID: 6382, updated on 6 Jan. 2013, 4. Protein=P18827 [UniParc]. Last modified May 5, 2009. Version 3. CD15: Gene ID: 2526, updated on 5 Jan. 2013 CD1a: Gene ID: 909, updated on 30 Dec. 2012, P06126 [UniParc]. Last modified Feb. 9, 2010. Version 4. Checksum: C575C3C538F0AA29 CD2: Gene ID: 914, updated on 5 Jan. 2013; P06729 [UniParc]. Last modified Oct. 23, 2007. Version 2. Checksum: A03D853C3B618917 CD3e: Gene ID: 916, updated on 5 Jan. 2013, P07766 [UniParc]. Last modified Feb. 1, 1996. Version 2. Checksum: A1603D010E9957D7 CD4: Gene ID: 920, updated on 13 Jan. 2013; P01730 [UniParc]. Last modified Nov. 1, 1988. Version 1. Checksum: 20ED893F9E56D236 CD5: Gene ID: 921, updated on 30 Dec. 2012; P06127 [UniParc]. Last modified Nov. 30, 2010. Version 2. Checksum: 9131AEC9683EE1D3 CD8a: Gene ID: 925, updated on 30 Dec. 2012; Isoform 1/2 (membrane) P01732-1/2 (mCD8alpha) [UniParc]. Last modified Jul. 21, 1986. Version 1. Checksum: FCCA29BAA73726BB CD20: Gene ID: 931, updated on 6 Jan. 2013; P11836 [UniParc]. Last modified Oct. 1, 1989. Version 1. Checksum: AC5420F8B626BDD1 CD23: Gene ID: 2208, updated on 4 Jan. 2013; P06734 [UniParc]. Last modified Jan. 1, 1988. Version 1. Checksum: F86708C0E6515B87 CD31: Gene ID: 5175, updated on 13 Jan. 2013; Isoform Long [UniParc]. Last modified Apr. 1, 1990. Version 1. Checksum: C57BBFA200A407A6, P16284-1/2/3/4/5/6=Isoforms 1-6 CD43: Gene ID: 6693, updated on 30 Dec. 2012; P16150 [UniParc]. Last modified Apr. 1, 1990. Version 1. Checksum: C9C9AB8435D5E1FE CD56: Gene ID: 4684, updated on 30 Dec. 2012; Isoform 1 [UniParc]. Last modified Jul. 22, 2008. Version 3. Checksum: FD3B9DE80D802554, P13591-2/1/3/4/4/6, Isoforms 1-6 CD57: Gene ID: 27087, updated on 5 Jan. 2013 CD68: Gene ID: 968, updated on 6 Jan. 2013; Isoform Long (CD68.1) [UniParc]. Last modified May 15, 2007. Version 2. Checksum: 69E68D69EDE8EFBO, P34810-1/2, Isoform 1/2 CD79a: Gene ID: 973, updated on 5 Jan. 2013; Isoform 1 (Long) [UniParc].  Last modified Jun. 1, 1994. Version 2., Checksum: 6E5B837409969292, P11912-1/2, Isoform 1/2 CD146: Gene ID: 4162, updated on 30 Dec. 2012; Isoform 1 [UniParc]. Last modified Jan. 10, 2006. Version 2. Checksum: E46CB8AC7BA0738E, P43121-1/2, Isoform 1/2. surfactant proteins (A and B):  Gene ID: 6440, updated on 30 Dec. 2012 and Gene ID: 6439, updated on 30 Dec. 2012, P07988 [UniParc]. Last modified May 1, 1992. Version 3. Checksum: 9FD7F66678A35153, and Isoform 1 [UniParc]. Last modified Apr. 1, 1990. Version 2. Checksum: C26A21E33C60AA78, P11686-1/2, Isoform 1/2 synaptophysin:  Gene ID: 6855, updated on 30 Dec. 2012, P08247 [UniParc]. Last modified Aug. 1, 1991. Version 3. Checksum: 592289C43B12EFA7 nicotinic acetylcholine receptors:  Gene ID: 1138, updated on 30 Dec. 2012, Gene ID: 1136, updated on 6 Jan. 2013, Gene ID: 1139, updated on 13 Jan. 2013, Gene ID: 1137, updated on 30 Dec. 2012, Gene ID: 1141, updated on 5 Jan. 2013 muscle-specific kinase MUSK:  Gene ID: 4593, updated on 8 Jan. 2013, Isoform 1 [UniParc]. Last modified Jan. 1, 1998. Version 1. Checksum: 3DDC20E179FA010C, O15146-1/2, Isoform 1/2 voltage-gated calcium channel (P/Q-type):  Gene ID: 773, updated on 5 Jan. 2013; Isoform 1 (1A-1) (BI-1-GGCAG) [UniParc]. Last modified Jul. 15, 1999. Version 2. Checksum: 2F2F378ACE02FD56, O00555-1/2/3/4/5/6/7, Isoforms 1-7, Gene ID: 25398, updated on 11 Jan. 2013, J3KP41 [UniParc]. Last modified Oct. 3, 2012. Version 1. Checksum: AEDF4D2A5E49263F voltage-gated potassium channel (VGKC):  Gene ID: 3737, updated on 30 Dec. 2012, Gene ID: 3736, updated on 8 Jan. 2013, Gene ID: 3742, updated on 8 Jan. 2013 N-methyl-D-aspartate receptor (NMDA):  Gene ID: 2904, updated on 5 Jan. 2013, Q13224 [UniParc]. Last modified Jun. 20, 2001. Version 3. Checksum: 40AEB12BE6E50CEF; Gene ID: 2902, updated on 30 Dec. 2012, Isoform 3 (Long) (NR1-3) [UniParc]. Last modified Jun. 1, 1994. Version 1. Checksum: CDF5402769E530AB, Q05586-1/2/3/4/5, Isoforms 1-5 TSHR: Gene ID: 7253, updated on 4 Jan. 2013, Isoform Long [UniParc]. Last modified Mar. 29, 2005. Version 2. Checksum: D2EE9CEBFD64A65F, P16473-1/2/3, Isoforms 1-3 Amphiphysin:  Gene ID: 273, updated on 8 Jan. 2013, Isoform 1 (128 kDa) [UniParc].  Last modified Feb. 1, 1996. Version 1, Checksum: 78B4F75AB75BA357, P49418-1/2, Isoform 1-2 ganglioside GQ1B: Gene ID: 29906, updated on 30 Dec. 2012 GD3: Gene ID: 117189, updated on 22 Jun. 2012 Ca-125: Gene ID: 94025, updated on 30 Dec. 2012, Q8WXI7 [UniParc]. Last modified Mar. 1, 2003. Version 2. Checksum: B3E7BDF19997A440 Her-2/neu: Gene ID: 2064, updated on 13 Jan. 2013, 4. Protein=P04626-1/2/3/4=Isoform 1-4, Last modified Aug. 13, 1987. Version 1.  gross cystic disease fluid protein 15; Gene ID: 5304, updated on 30 Dec. 2012 CD117: Gene ID: 3815, updated on 6 Jan. 2013 CD30: Gene ID: 943, updated on 6 Jan. 2013; Isoform Long [UniParc]. Last modified Dec. 1, 1992. Version 1. Checksum: 7A407CC78A6E0BC8, P28908-1/2, Isoform 1/2 Platelet derived growth factor receptor PDGFR alpha:  Gene ID: 5159, updated on 13 Jan. 2013, Gene ID: 5156, updated on 13 Jan. 2013, Isoform 1 [UniParc]. Last modified Apr. 1, 1990. Version 1. Checksum: 5E3FB9940ACD1BE8, P16234-1/2/3, Isoforms 1-3; P09619 [UniParc]. Last modified Jul. 1, 1989. Version 1. Checksum: 038C15E531D6E89D Melanoma associated marker/Mart 1:  Gene ID: 2315, updated on 30 Dec. 2012; Q16655 [UniParc]. Last modified Nov. 1, 1996. Version 1. Checksum: B755BFF39CFCB16E CD133: Gene ID: 8842, updated on 13 Jan. 2013; Isoform 1 (AC133-1) (S2) [UniParc].  Last modified Jun. 1, 1998. Version 1. Checksum: D21CBC05ADB2DEDF, O43490-1/2/3/4/5/6/7, Isoforms 1-7

(36) In the following, reference is made to the examples which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1

Cloning of Recombinant Antibody Constructs

(37) DNA sequences derived from hybridoma cells and coding for the variable domains of anti-CD3, anti-CD45 and anti-HLA A2 antibodies, respectively, were used to generate the antibody constructs depicted in FIG. 3 by standard methods of molecular biology (see, e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001)). The constructs were designed to carry different affinity tags to facilitate identification and purification upon expression of recombinant proteins (Myc-, Flag, His-Tag). For details on domain arrangement, affinity tags and linkers of the constructs, see FIG. 3.

(38) pelB Leader codes for an amino acid sequence that directs a protein expressed in bacteria to the bacterial periplasm. The leader sequence is cleaved by bacterial enzymes and the protein can be isolated.

Example 2

Expression and Purification of Recombinant Antibodies

(39) Periplasmic Protein Expression:

(40) Recombinant antibody constructs were expressed in the periplasm of E. coli strain TG1 using an appropriate prokaryotic expression vector. Two litres of 2×TY medium including 0.1% glucose and 100 μg/ml ampicillin were inoculated with 20 ml of an overnight culture of transformed TG1 and grown to exponential phase (OD600 0.8-0.9) at 37° C. Since the antibody fragments are under control of the lactose promotor, protein expression was induced by addition of 1 mM IPTG followed by incubation at RT (room temperature) with shaking for additional 3 h. Cells were harvested by centrifugation for 10 min at 2,750×g and 4° C. and were resuspended in 100 ml or an appropriate buffer. Cell lysis was performed by adding 50 μg/ml freshly dissolved lysozyme [Roche Diagnostics] and incubating for 25 min on ice. Following, 10 mM MgSO.sub.4 were added to stabilise spheroblasts, and cells were centrifuged for 10 min at 6,200×g and 4° C. Finally, the supernatant obtained, containing the periplasmic protein, was dialysed against PBS overnight at 4° C. and was centrifuged again for 15 min as stated above. Afterwards, recombinant proteins were purified by Ni-NTA-IMAC (Nickel Nitrilo-triacetic acid Immobilised Metal Affinity Chromatography).

(41) Immobilised-Metal Affinity Chromatography (IMAC):

(42) For purification of recombinant proteins with a His.sub.6 tag, an IMAC was performed by means of immobilised nickel-nitrilotriacetic acid (NTA) agarose beads [Qiagen]. First, a column of 1 ml Ni-NTA agarose needed to be equilibrated with approximately 10 ml of sterile PBS or a sodium phosphate buffered solution with 20 mM imidazole. Then, crude protein, either precipitated from cytoplasmic expression or dialysed from periplasmic expression, was gradually applied to the column. After washing with about 20 ml of an appropriate IMAC wash buffer (sodium phosphate buffered solution containing 20-35 mM imidazole) until no more protein was detectable in the flow, bound protein was eluted from the column in 500 μl fractions with a sodium phosphate-buffered solution including 250 mM imidazole.

(43) All collected wash and elution fractions were tested for presence of protein by a qualitative Bradford assay by adding 10 μl of each sample to 90 μl of 1× Bradford solution. Verification of the purification process was performed by an SDS-PAGE analysis. For this purpose, eluted fractions were run in parallel with crude protein, flow, and wash fraction under reducing conditions. Finally, positive fractions determined by the colorimetric reaction were pooled into peak and minor fractions and dialysed against PBS overnight at 4° C. For usage in stimulation assays, purified proteins needed to be sterile filtrated, and their concentration has been determined. In addition, after protein quantification, 2 μg of further used fractions were also analysed by SDS-PAGE and Western blotting under reducing and non-reducing conditions.

(44) In an alternative of Example 2, DNA coding for (V.sub.H)CD3−EGFR(V.sub.H−V.sub.L), (V.sub.H)CD3−CEA(V.sub.H−V.sub.L), (V.sub.H)CD3−Her2(V.sub.H−V.sub.L), (V.sub.H)CD3−HLA-A2(V.sub.H−V.sub.L), (V.sub.H)CD3−HLA-CW6(V.sub.H−V.sub.L) (V.sub.H)CD3−CD138(V.sub.H−V.sub.L), (V.sub.H)antiDig−EGFR(V.sub.H−V.sub.L), (V.sub.H)antiHis−HLA-A2(V.sub.H−V.sub.L), (V.sub.L)CD3−CEA(V.sub.H−V.sub.L), (V.sub.L)CD3−EpCAM(V.sub.H−V.sub.L), (V.sub.L)antiDig−EpCAM(V.sub.H−V.sub.L), (V.sub.L)CD3−CD45(V.sub.H−V.sub.L) were synthesised and proteins were produced and isolated by GenScript (Piscataway, N.J., USA). The DNA was codon optimized for E. coli expression (vector E3), expression optimized, grown in 2 litres standard LB-medium, protein was obtained from inclusion bodies or periplasm (pelB leader) in one step by Ni-HiTrap column. Bacterial endotoxins were removed by dialysis against 5 litres 1× phosphate buffered saline (PBS). The concentration was measured by Bradford protein assay with bovine serum albumin (BSA) as standard. The purity was estimated by densitometric analysis of a Coomassie Blue-stained SDS-PAGE gel. Aliquots were stored at −80° C. or +4° C. Storage buffer was used 1×PBS, 5% Glycerol, 0.5% sodium lauroyl sarcosine, pH 7.4.

Example 3

Cell Culture Techniques

(45) Cell Cultivation:

(46) Mammalian cells were cultivated in T75 tissue culture flasks in 20 ml of the appropriate culture medium at 37° C. with 5% CO.sub.2. Cells were split every 2-3 days. Adherent cells first needed to be detached with 1× trypsin-EDTA. Cells were counted using a vital stain, eosin or trypan blue. For storage, cells of 60-80% confluence were harvested by centrifugation for 5 min at 450×g, resuspended in FCS with 10% DMSO, aliquoted in cryovials, and gradually frozen to a temperature of −80° C. Cells were thawed quickly at 37° C. in a water bath and cautiously added to 5 ml medium. In order to remove DMSO, cells were centrifuged again, resuspended in fresh medium and transferred into a tissue culture flask.

(47) Preparation of Peripheral Blood Mononuclear Cells (PBMC):

(48) PBMC, comprising lymphocytes and monocytes, were previously isolated from the buffy coat of a healthy human donor by density centrifugation using the Ficoll based lymphocyte separation solution LSM 1077 (PAA Laboratories, Pasching, Austria). Since, during usage, these PBMC nevertheless appeared as an inhomogeneous cell population, the separation from remaining erythrocytes, granulocytes, and thrombocytes was repeated as follows. Thawed PBMC, resuspended in 30 ml RPMI 1640 medium containing 10% FCS and Pen-Strep, were cautiously layered onto 10 ml of LSM 1077 and centrifuged for 5 min at 800×g without braking. After discarding the upper phase, PBMC concentrated in the interphase were transferred into a fresh tube, resuspended in 30 ml of medium, and centrifuged for 5 min at 450×g. Monocytes were removed by cultivating PBMC in a Ø10 cm tissue culture plate overnight, allowing adherence of monocytes to the plate. Finally, PBMC, remaining in solution, were harvested.

(49) In an alternative of Example 3, Primary human cancer cells from a patient with metastatic pancreatic cancer were extracted from the ascites bags of the patient (FIG. 29). 4 litres with fresh collected malignant ascites were stored in 2 litres glass bottles at 4° C. over night. The next day the cell pellet from the glass bottom was washed in 1×PBS and resuspended in culture medium (DMED supplemented with 200 μM l-glutamine, 10% heat inactivated FBS, penicillin (200 U/mL), streptomycin (200 μg/mL) and sodium pyruvate (1 mM) (GIBCO®)). Adherend cells were cultured in incubator 36° C., 5% CO.sub.2, 90% humidity. The same day the ascites was collected from the patient, 20 ml peripheral blood for PBMC extraction was collected. Primary leukemic cells were obtained from a 71 year old male patient with T-cell-prolymphocytic leukemia (T-PLL) (FIG. 11A) relapsing 32 days after matched allogeneic stem cell transplantation. The leukemic T-PLL cells were extracted as PBMCs from the peripheral blood of the patients. At the time the sample was drawn the patient had >90% leukemic blast in his blood count in routine clinic diagnostic. From all patients an informed consent, approved by the University hospital of Würzburg ethical committee, was signed.

(50) In an alternative of Example 3, generation of cytomegalievirus (CMV)-specific human T-cells: Briefly, dendritic cells (DC) were generated from plastic adherent monocytes from PBMC of HLA-A0201 negative, B0702+ donor. After 72 h of culture in GM-CSF/IL4-containing DC medium (Cellgenix), DC were matured in medium containing IL4 (100 ng/ml), GM-CSF (800 IU/ml), LPS (10 ng/ml) and IFNγ (100 U/ml) plus 2.5 ug/ml CMV pp65 derived peptide TPRVTGGG (SEQ ID NO:201). After 16 h, DC were irradiated (30Gy) and co-incubated with CD45RO.sup.−, CD57.sup.− naïve CD8.sup.+ T-cells at a 1:4 ratio in medium containing 5% AB serum and IL21 (long/ml). Fresh medium, IL7 and IL15 was added on days 3, 5 and 7 of culture, before evaluation on day 10-12. Cells were cultured in Cellgenix DC medium. Human AB serum was used from PAA. One single batch was used throughout all experiments. IL4, IL7, IL15, IL21 were either purchased from Peprotech or Cellgenix (with identical results). GM-CSF was purchased from Gentaur. LPS (E. coli O:15) was purchased from Sigma. The HLA-B0702-restricted CMV-specific peptide TPRVTGGG (SEQ ID NO:201) was purchased from jpt. For in vivo experiments, CMV-specific T-cells were further purified using APC-labelled MHC-multimers (Immudex). MHC multimer staining was performed at room temperature, followed by isolation of MHC-multimer+ T-cells with anti-APC-beads (Miltenyi).

Example 4

Functional Assays

(51) Flow Cytometry:

(52) Binding of antibody fusion proteins to antigen-presenting tumour cells and/or T lymphocytes was tested by flow cytometry. For this purpose, 2.5-5×10.sup.5 cells were incubated with 10 μg/ml of scFv or 0.004-4 μg/ml of titrated fusion proteins in 100 μl of a suitable buffer solution (such as PBS+bovine serum albumin, or other acceptable buffer solution) per well on a 96-well V-shaped plate at 4° C. for 2 h. After washing three times with 150 μl of a suitable buffer solution, cells were incubated with FITC-conjugated anti-His.sub.6 tag or anti-Flag Tag or anti-myc Tag antibody at RT for 30 min and washed again two times. For gating and testing for background staining, additionally two samples of each cell type were prepared, one of unstained cells and one stained with FITC-conjugated anti-His.sub.6 tag antibody without any protein. Finally, cells were resuspended in 500 μl of a suitable buffer solution, transferred into FACS tubes, and analysed by flow cytometry.

(53) PBMC Stimulation Assay:

(54) Stimulatory properties of recombinant proteins were tested in a cell-based stimulation assay. Thereby, T-cell activation mediated by bispecific antibodies and “tridomain constructs” was determined by measuring PBMC stimulation in terms of the IL-2 release induced.

(55) Measurement of stimulatory Activity of Constructs:

(56) CD45 pos/HLA A2 myeloma cell line U266 were seeded in a flat-bottomed 96-well cell culture plate at a density of 105 cells per well in 100 μl of culture medium. Titrated stimulatory proteins were added as indicated in 100 μl medium per well and were preincubated for 1 h at 37° C. and 5% CO2 to ensure sufficient binding. Unstimulated PBMC, thawed and isolated the day before, were then added at indicated density and incubated for 24 h at 37° C. and 5% CO.sub.2. Finally, plates were centrifuged for 5 min at 450×g to harvest cell-free supernatants for IL-2 quantification in ELISA.

(57) IL-2 Sandwich ELISA:

(58) As an indicator for the stimulatory activity, T-cell activation induced by bispecific antibodies was measured in terms of the IL-2 release. Upon PBMC stimulation, concentration of secreted IL-2 in the supernatant was determined by an IL-2 sandwich ELISA.

(59) First, a 96-well ELISA plate was coated with 400 ng/100 μl per well of mouse anti-human IL-2 antibody overnight at 4° C., followed by saturation of nonspecific binding sites with a suitable blocking buffer for 2 h at RT. In the meantime, serial 1:2 dilutions of an IL-2 standard were prepared in duplicate in reagent diluent starting with a maximum IL-2 concentration of 1,000 pg/ml. Then, supernatants containing IL-2 were 1:3 diluted in RPMI 1640 medium containing 10% FCS and Pen-Strep (Penicillin-Streptomycine). Both diluted supernatants and standards were transferred into the ELISA plate and incubated for 2 h at RT. Following, IL-2 was detected by incubation with 17.5 ng/100 μl per well of biotinylated goat anti-human IL-2 antibody for 2 h at RT. Finally, 100 μl of HRP-conjugated streptavidin, 1:200 diluted in reagent diluent, was added per well and incubated for 20 min at RT. Each plate was developed using a TMB substrate solution. In order to achieve a background signal, at least 2 wells on each plate were incubated with reagent diluent or medium only and the detecting antibody plus TMB. Between each incubation step, the plate was washed three times with PBS containing 0.05% Tween-20 and once with PBS only.

(60) A seven point standard curve was created by plotting the absorbance signals of each standard sample against the IL-2 concentration. Thus, the amount of IL-2 of each supernatant could be determined by interpolation of the standard curve fitted with the nonlinear regression equation for one phase exponential association using GraphPad Prism®.

(61) IFN-γ ELISA (Alternative of Example 4):

(62) In 100 μl cell culture supernatant the IFN-γ concentration was measured using the human IFN-γ ELISA Kit (Invitrogen™) after manufacturer's protocol. Briefly 50 μL of Incubation Buffer was added to each well of a precoated 96-well plat. 50 μL of the Standard Diluent Buffer to zero wells. 50 μL of standards and samples to each well. 50 μL of biotinylated Hu IFN-γ Biotin Conjugate solution into each well. Taped gently on the side of the plate to mix. Covered plate with plate cover and incubate for 1 hour and 30 minutes at room temperature. Thoroughly aspirated solution from wells and discarded the liquid. Washed wells 4 times. Added 100 μL Streptavidin-HRP Working Solution to each well. Covered plate with the plate cover and incubated for 45 minutes at room temperature. Thoroughly aspirated solution from wells and discarded the liquid. Added 100 μL of Stabilized Chromogen to each well. The liquid in wells turned blue. We incubated for 15-30 minutes at room temperature and in the dark. Added 100 μL of Stop Solution to each well. Taped side of plate gently to mix. The solution in the wells changed from blue to yellow. The absorbance of each well was read with a BioRad plate reader at 450 nm.

(63) Cytotoxicity Assay:

(64) The HLA-A2/CD45 positive cell line U266 or myeloma cell line U266 was labelled with 10 μM CFSE (Invitrogen Vybrant CFDA SE Cell Tracer Kit) in 350 μl PBS for 10 min at room temperature (RT) in the dark. The labelling reaction was stopped by the addition of 5 ml fetal calf serum (FCS), followed by a 1-minute incubation at RT. After 2 washes, the CFSE-labelled target cells were resuspended in assay medium and co-incubated with Peripheral Blood Mononuclear Cells (PBMC) from a HLA-A2 negative healthy donor at a ration of 1:10 (5*10.sup.5 U266 and 5*10.sup.6 PBMCs in 2 ml) and 27 nM of antibody constructs as indicated. A sample treated with Triton was used as positive control (100% lysis) and a sample without antibody construct as negative control (0% lysis). After 24 h, apoptotic cells were visualized by 7AAD stain (Biozol, 10 min at RT) and % specific Lysis of CFSE labelled U266 cells was calculated employing flow cytometry techniques.

(65) Caspase-3 Assay (Alternative of Example 4):

(66) Staining was performed after co-incubating of the target cells with T-cells (tumor cells: T-cells ratio 2:1) with or without the specific polypeptides for 4 h. Surface staining for HLA-A2 and CD45 was performed first, followed by fixation and permeabilization (Fix+Perm, BD Biosciences). Activated Caspase-3 antibody was then added for 30 min. (BD Biosciences). Cells were washed with 1×PBS+5% human serum (HS, PAA Laboratories) and analyzed on a BD-FACS Canto-II. % specific apoptosis was calculated as (% experimental value−spontaneous release)/(100%−% spontaneous release)*100.

(67) ALAMARBLUE® Assay (Alternative of Example 4):

(68) The ALAMARBLUE® assay (Abd Serotec) was used to measure proliferation and viability of cells after exposure to toxins. Briefly, cells were grown in 100 μl cell culture medium per well (96 well plate). For analysis 10 μl ALAMARBLUE® was added per well and incubated in the incubator for 30-120 minutes. The absorbance was read with a BioRad plate reader at 570 nM and 600 nM. For blank media only was used. The percent difference in reduction of cell proliferation between the different polypeptide groups was calculated as indicated by the manufacturer, using cells growing in culture without toxin as control.

(69) Digoxigenin Assay (Alternative of Example 4):

(70) First peroxidase from horseradish (HRP, Sigma-Aldrich Chemie gmbH) was labelled with digoxigenin NETS-ester (Sigma-Aldrich Chemie gmbH) in a 1/3 molar ratio. Dig-HRP was cleaned up with micro Bio-Spin™ chromatography columns (BioRad and and stored at 4° C. in the dark. Colo-206F cells were first incubated with indicated polypeptides at various concentrations for 90 minutes. Cells were washed with PBS and resuspended in cell culture medium with Dig-HRP and incubated for 30 minutes. Afterward cells were washed twice with PBS and resuspended in 50 μl PBS. 50 μL of Stabilized Chromogen (Invitrogen™) was added for 15-30 minutes at room temperature in the dark. 50 μL of Stop Solution was added and the absorbance was read with a BioRad plate reader at 450 nm.

(71) Mice (Alternative of Example 4):

(72) The HLA.A2 transgenic, immunodeficient mice (NodScid IL-2rg−/−HLA.A2/B2m tg; Stock number 14570, The Jackson Laboratory, Bar Harbor, Me., USA) for the in vivo experiment (FIG. 12A) were maintained in our certified animal facility (ZEMM, Center for experimental molecular medicine, University hospital Würzburg) in accordance with European guidelines. Female Mice, 6-10 weeks old, were divided into five groups, six mice per group (n=30). 5×10.sup.6 THP-1 cells, 1.25×10.sup.5CMV specific CD8+ T-cells (tumour cell: T-cell ratio 40/1) and the 0.5 μg of the polypeptides were injected intraperitoneally (i.p.) as indicated. After injection, mice were monitored by daily inspection. A second injection of 1.16×10.sup.5 CMV-specific CD8+ T-cells/mouse was given at day 13 and injections of the polypeptides were repeated every three days a week. The animals were sacrificed when the increase in body weight was greater 80% or if they appeared moribund according to institutional guidelines.

(73) Domain structure, affinity tags and linkers of the constructs or polypeptides used in Examples 5-9 or FIG. 4-FIG. 11 are shown in FIG. 3. These constructs and all constructs or polypeptides used in FIG. 4-FIG. 30 were prepared as described in Examples 1 and 2. Cell culture and functional assays in Examples 5-9 and culture, functional assays and in vivo work as to FIG. 4-FIG. 30 were carried out as described in Examples 3 and 4.

Example 5

(74) The CD45 and HLA A2 positive myeloma target cell line U266 was co-incubated with HLA A2 negative T cells (monocyte depleted PBMCs (peripheral blood mononuclear cells) from a healthy donor and varying amounts of HLA A2 and CD3 bispecific antibody constructs as indicated (Numbers 85, 82, 75 and 71). PHA-L (phytohemagglutinin, a lectin that causes unspecific stimulation of T cells; 1 μg/ml final concentration) was used as positive control and single chain scFv constructs with specificity for HLA A2 (Number 4) or CD3 (Number 36) were investigated. IL2 (Interleukin-2) production by T cells was measured by ELISA techniques. No IL2 production was found in experimental situations without any constructs. Data obtained is depicted in FIG. 4.

Example 6

(75) The CD45 and HLA A2 positive myeloma target cell line U266 was co-incubated with HLA A2 negative T cells (monocyte depleted PBMCs) from a healthy donor and varying amounts of “tridomain constructs” added either separately (Numbers 42, 45, 55; numbers referring to constructs as depicted in FIG. 3) or in combinations (42+45 or 42+55). PHA-L and single chain scFv constructs with specificity for CD45 (Numbers 46 and 17) were given as controls. IL2 production by T cells was measured by ELISA techniques. No IL production was found in experimental situations without any constructs. Data obtained is depicted in FIG. 5.

Example 7

(76) The CD45 and HLA A2 positive myeloma target cell line U266 was co-incubated with HLA A2 negative T cells (monocyte depleted PBMCs) from a healthy donor and the HLA A2 and CD3 bispecific antibody construct alone (number 71, 27 nM) or in combination with single chain scFv constructs that block the antigenic epitopes on HLA A2 (Number 4, hundredfold excess compared to the concentration of construct 71, i.e. 2700 nM) or CD3 (Number 36, ninefold excess compared to the concentration of construct 71, i.e. 243 nM). IL2 production by T cells was measured by ELISA techniques and PHA-L is given as control. Data obtained is depicted in FIG. 6.

Example 8

(77) The CD45 and HLA A2 positive myeloma target cell line U266 was co-incubated with HLA A2 negative T cells (monocyte depleted PBMCs) from a healthy donor and the combination of constructs 42 and 45. T cell stimulatory function was blocked by single chain constructs specific for HLA A2 (number 4) or CD45 (number 46). Complementation of T cell stimulatory function was tested by assaying constructs 42 and 45 separately or the single chain scFv construct directed against CD3 (number 36). IL2 production by T cells was measured by ELISA techniques and PHA-L is given as control. Concentration of constructs was 27 nM, unless indicated otherwise. (“9×” indicates a concentration of 243 nM, “100×” a concentration of 2700 nM.) Data obtained is depicted in FIG. 7.

Example 9

(78) The CD45 and HLA A2 positive myeloma target cell line U266 was co-incubated with HLA A2 negative T cells (monocyte depleted PBMCs) from a healthy donor and the combination of constructs 42 and 55. T cell stimulatory function was blocked by single chain constructs specific for HLA A2 (number 4) or CD45 (number 46). Complementation of T cell stimulatory function was tested by assaying constructs 42 and 55 separately or the single chain scFv construct directed against CD3 (number 36). IL2 production by T cells was measured by ELISA techniques and PHA-L is given as control. Concentration of constructs was 27 nM, unless indicated otherwise. (“9×” indicates a concentration of 243 nM, “100×” a concentration of 2700 nM.) Data obtained is depicted in FIG. 8.

(79) The results of the preceding Examples clearly demonstrate that two constructs (42+45) or (42+55) first have to bind their ligands on the surface of a single cell in order to subsequently complement T cell engaging function.

Example 10

(80) Lysis of the CD45 and HLA A2 positive myeloma target cell line U266 by HLA A2 negative T cells (monocyte depleted PBMCs) in the presence of V.sub.LCD3−scFvHLA A2 (27 nMol) or V.sub.H−scFvCD45 (27 nMol) or the combination of both of these constructs (27 nMol each) was determined using flow cytometry based techniques. Percent lysis was calculated by apoptotic U266 cells divided through total U266 cells and background apoptosis was subtracted. Data obtained is depicted in FIG. 9.

Example 11

(81) As parts of the final bipartite construct, two polypeptides were designed, each composed of an antigen-binding single-chain variable fragment (scFv) and either the variable light (V.sub.L) or variable heavy chain (V.sub.H) domain of a T cell-activating anti-CD3 antibody (FIG. 10). When these two polypeptides bind their respective antigens on the surface of a single cell, the V.sub.L and V.sub.H domains interact with each other to reconstitute the original anti-CD3 binding site. The thus on-target formed trispecific heterodimer engages and stimulates T cells for tumor cell destruction.

(82) This scenario is fully validated in vitro when T lymphocytes are confronted with target cells that have been incubated with the two different polypeptides. As proof of principle, major histocompatibility antigen HLA-A2 and the hematopoetic lineage marker CD45 were targeted as first and second antigens, which both are expressed on U266 myeloma cells, primary cells from a patient with pro-lymphocytic leukemia of the T cell lineage (T-PLL), and THP-1 acute myeloid leukemic blasts (FIG. 11). Due to the described V.sub.L/V.sub.H interaction, the now trispecific heterodimer potently stimulates T cells to secrete interleukin-2 (IL-2) (FIG. 11A) and to lyse the labeled tumor cells at nanomolar concentration (FIG. 11B), the cytotoxic efficacy being quite similar to that of a bispecific T cell-activating antibody, which was employed as a positive control (FIG. 11A, left panel), Mack, 1995, Proc Natl Acad Sci 92, 7021-7025. When the polypeptides were added separately from each other, they did not induce T lymphocytes to lyse target cells. These results are in line with structural data indicating that both, V.sub.H and V.sub.L domains are required to confer sufficient affinity to the target antigen (FIG. 11A, FIG. 11B), Colman, 1987, Nature 326, 358-363; Amit, 1986, Science 233, 747-753. Moreover, the results reveal that possible homodimerization of either V.sub.H or V.sub.L arms results in a negligible measurable biological effect.

(83) To demonstrate that the two molecules must first bind their antigens on the surface of the target cell for V.sub.H/V.sub.L heterodimerization to occur, single-chain variable fragments specific for HLA-A2 and CD45 were used to block the respective epitopes on the target. As shown in FIG. 11C, when present in great excess, these inhibitors prevented the two polypeptides from triggering T cells in a dose-dependent manner. Furthermore, T cells were not stimulated when the target cells were omitted (data not shown) or when target cells were probed that express CD45 only (RAJI cells, FIG. 11D) or neither target molecule (KMS-12-BM, FIG. 11D).

Example 12

(84) For in vivo proof of concept, a model of allogeneic mismatch stem cell transplantation was resorted in which a patient's residual leukemic and hematopoietic cells, all HLA-A2 and CD45-positive, must be eliminated to give the allogeneic donor stem cells (HLA-A2-negative, CD45-positive) a chance to engraft and to reconstitute hematopoiesis (see FIG. 2). To put the specificity of the bipartite construct to the test, immunodeficient mice expressing the human HLA-A2 transgene on virtually all nucleated cells were used, the question being whether HLA-A2-positive but CD45-negative murine tissues would suffer collateral damage. THP-1 cells were injected intraperitoneally with or without CD8 T lymphocytes from an HLA-A2-negative donor, which had been selected for specificity to cytomegalovirus (CMV) to avoid human anti-murine immune reactivity. Intraperitoneal tumors developed rapidly in mice that did not receive the polypeptides, and in mice treated either with single molecule types or with the combination of both polypeptides but without T cells. In all instances, fatal disseminated disease developed within 3 to 4 weeks (FIG. 12A). In stark contrast, all tumor-bearing mice treated with T cells and repeated injections of both polypeptides survived the end of the experiment on day 31, albeit with palpable tumors at the injection site. These results clearly show that the bipartite construct truly redirects T cells irrespective of their specificity at tumor cells that simultaneously express both target molecules (HLA-A2 and CD45) in vivo. As an aside, a T cell recruiting bispecific antibodies against HLA-A2 would wreak havoc by redirecting T cells against all HLA-A2 positive murine tissues. Likewise, a CD45-binding bispecific antibody would have mediated lysis of all hematopoietic cells, including THP-1 leukemic blasts and T cells from the donor. In our set-up, however, injection of HLA-A2-specific polypeptide into the HLA-A2 transgenic animals caused no apparent toxicity.

(85) To further examine possible toxicity to bystanders, we employed a highly sensitive apoptosis assay on THP-1 cells and HLA-A2-negative but CD45-positive monocytes, the latter representing the healthy bystander compartment. As depicted in FIG. 12B, we observed caspase-3 activation in THP-1 cells but not in monocytes treated in the same well with the combination of the polypeptides or the bispecific positive control and donor T cells. THP-1 cells cultured with T cells and individual polypeptides were unaffected. These observations again clearly show initiation of apoptosis exclusively in the double antigen positive target population, while the HLA-A2-negative bystander cells are spared. These experiments model quite accurately the dire clinical situation of leukemia patients with a HLA-mismatched stem cell transplant. The combinatorial approach of using a distinctive HLA molecule and CD45 aims at enhancing the desired graft versus leukemia effects by retargeting the donor's T cells against leukemic blasts of both, myeloid and lymphoid origin.

Example 13

(86) To venture into solid tumors, we targeted the combinatorial approach to epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) antigens. Both antigens are over-expressed in various carcinomas and have been extensively studied in clinical phase II and III trials. The expression of EGFR is closely associated with cell proliferation, while EpCAM is present at the basolateral surface of virtually all simple epithelia and was recently found to act like a signaling protein in the Wnt pathway, Maetzel, 2009, Nat Cell Biol 11, 162-171. As FIG. 13A illustrates, the two polypeptides trigger the release of interferon-γ (IFNγ) from co-incubated donor lymphocytes and mediate apoptosis of the double-positive cancer cell line COLO-206F at nanomolar concentrations (FIG. 13a, b), but only when given in combination and not with either part alone. As a descendant of neuroepithelial tissue, the melanoma cell line FM-55 lacks EpCAM, and therefore was completely resistant to the polypeptides (FIG. 13a, b). Though the expression of EGFR and EpCAM overlaps broadly on proliferating carcinoma cells, non-proliferating epithelial cells, e.g., of liver and pancreas solely expressing EGFR or EpCAM antigens, respectively, should be less susceptible to or protected from the two-pronged attack. Notably, hepatic and pancreatic toxicities have been dose-limiting for high-affinity monoclonal EpCAM antibodies in clinical trials (for review see, Munz, 2010, Cancer Cell Int 10:44).

Example 14

(87) The further validation of the bipartite functional complementation strategy was performed by extensive in vitro experiments, using a combination of different polypeptides, targeting various cell surface antigens on different human cell lines.

(88) The HLA A2 positive human tumor cell lines FM-55 (myeloma), Colo-206F (colon cancer) and OVCAR (ovarian cancer) were co-incubated with HLA-A2 negative PBMCs from a healthy donor, polypeptide against HLA-A2 (CD3(V.sub.L)−HLA-A2(V.sub.H−V.sub.L)) and with a second polypeptide targeting either CEA (CD3(V.sub.H)−CEA(V.sub.H−V.sub.L)), EGFR (CD3(V.sub.H)−EGFR(V.sub.H−V.sub.L)) or Her2 (CD3(V.sub.H)−Her2(V.sub.H−V.sub.L)). IL2 or IFN-γ production by lymphocytes was measured by ELISA techniques. These data demonstrate that (i) a specific combination of antigens, an antigen signature, can be expressed on carcinomas of various origin (skin, neuroepithelial, gut and ovary tissue), (ii) the antigen signature is approachable with our bipartite functional complementation strategy using a set of polypeptides specific for the antigen signature. Data obtained are depicted in FIG. 14, FIG. 15 and FIG. 16.

Example 15

(89) To demonstrate the exchangeability of the functional domain, the fragments F1 and F2 of a set of polypeptides were exchanged with each other, retaining their specific complementation ability for on target restoration of their original antibody domain to engage T cells. Therefore the set of polypeptides against the CD45 and HLA-A2 target antigen was used. The polypeptide against CD45 had CD3(V.sub.L) as fragment F1 and the polypeptide against HLA-A2 had CD3(V.sub.H) as fragment F2. The CD45 and HLA-A2 positive myeloma cell line U266 was co-incubated with HLA-A2 negative T cells from a healthy donor and polypeptides against CD45 (CD3(V.sub.L)−CD45(V.sub.H−V.sub.L)) and HLA-A2 (CD3(V.sub.H)−HLA-A2(V.sub.H−V.sub.L)) in varying amounts. T cell engagement was assessed by reactive IFNγ production, measured by ELISA techniques. No IFNγ production was found in experimental situations without any polypeptides. Data obtained is depicted in FIG. 17.

Example 16

(90) The bipartite functional complementation strategy was further tested by targeting a set of antigens, already used as targets for antibody therapy of cancer (EGFR, EpCAM and Her2) (Her2 is a target for Trastuzumab in breast cancer, EGFR is a target for Cetuximab in colorectal cancer and EpCAM is a target for Catumazumab for the treatment of neoplastic ascites). The EGFR, EpCAM and Her2 positive cells (Colo-206F, CX-1 and OVCAR) were co-incubated with PBMCs from a healthy donor and the combination of polypeptides against EGFR (CD3(V.sub.H)−EGFR(V.sub.H+V.sub.L)), EpCAM (CD3(V.sub.L)−EpCAM(V.sub.H+V.sub.L)) and Her2 (CD3(V.sub.H)−Her2(V.sub.H+V.sub.L)). Complementation of lymphocyte stimulatory function was assessed by reactive IFNγ production, measured by ELISA techniques. No IFNγ production was found in experimental situations without any polypeptides. Data obtained is depicted in FIG. 18 and FIG. 19.

Example 17

(91) To test an antigen combination with close clinical correlation, the combination CD45 and CD138 was used to target human multiple myeloma (MM) cells. The majority of human MM cells are positive for CD45 and CD138. A T cell recruiting bispecific antibodies against CD45 would kill all hematopoetic cells of a patient and against CD138 would cause severe side effects because of its expression on various normal tissues (epithelial cells, endothelia, trophoblastic cells and glandular cells of the GI tract, The Human Protein Atlas, Version: 10.0, Atlas updated: 2012 Sep. 12). In contrast the combination of CD45 and CD138 is found exclusively on plasma cells and MM cells and is therefore a good antigen signature for the targeted therapy approach. The CD45 and CD138 positive human multiple myeloma cell line AMO-1 was co-incubated with PBMCs from a healthy donor and the combination of polypeptides against CD45 (CD3(V.sub.L)−CD45(V.sub.H+V.sub.L)) and CD138 (CD3(V.sub.H)−CD138(V.sub.H+V.sub.L)). Complementation of lymphocyte stimulatory function was assessed by reactive IFNγ production, measured by ELISA techniques. No IFNγ production was found in experimental situations with single polypeptides or without any polypeptides. Data obtained is depicted in FIG. 20.

Example 18

(92) A further application of the bipartite functional complementation strategy is to target single antigens on the cell surface and to kill single antigen positive tumor cells. One major drawback for T cell recruiting bispecific antibodies with functional antiCD3 binding sides are severe side effects caused by unspecific T-cell activation and cytokine release (Linke, R. et al. Catumaxomab: clinical development and future directions. MAbs 2, 129-136 (2010)). The advantage of this bipartite functional complementation strategy is the fact, antibodies that the T-cell activating antiCD3 functional domain is exclusively restored on the target cell. Without the target cell, no T-cell activating domain is present. The CD45 and CD138 positive human multiple myeloma cells AMO-1 and U266 were co-incubated with PBMCs from a healthy donor and the combination of polypeptides against a single target antigen, either CD138 (CD3(V.sub.H)−CD138(V.sub.H+V.sub.L)+CD3(V.sub.L)−CD138(V.sub.H+V.sub.L)) or CD45 (CD3(V.sub.H)−CD45(V.sub.H+V.sub.L)+CD3(V.sub.L)−CD45(V.sub.H+V.sub.L)). Complementation of lymphocyte stimulatory function was assessed by reactive IFNγ production, measured by ELISA techniques. No IFNγ production was found in experimental situations with single polypeptides or without any polypeptides. Data obtained are depicted in FIG. 21 and FIG. 22. In FIG. 23 the single antigen approach is illustrated, by using a set of polypeptides targeting two different epitopes (upper part) or the same epitope (lower part) on the target antigen A1.

Example 19

(93) This is an example to demonstrate that the functional complementation strategy can be further elaborated for targeted payload delivery and that different effector ways are possible to kill a target cell. By complementing the F1 and F2 fragments of a set of bound polypeptides on target, the newly formed antibody binding site can bind any molecule it is specific for. In order to direct a HIS-tagged payload precisely to a target cell, the V.sub.H and V.sub.L fragments of an anti-HIS(hexa-histidine)-antibody were used. After simultaneous binding of polypeptide 1 (antiHis(V.sub.L)−CD45(V.sub.H−V.sub.L) and polypeptide 2 (antiHis(V.sub.H)−HLA-A2(V.sub.H−V.sub.L) to their specific target antigens CD45 and HLA-A2, a hexa-histidine binding site is complemented on target that binds histidine labeled payloads with high high affinity. The payload be a HIS-tagged toxin as given in this example here. The CD45 and HLA-A2 positive cells THP-1 were co-incubated with a histidine(His)-tagged Clostridium perfringens Iota toxin component Ia (FIG. 24) or a histidine(His)-tagged Shiga toxin subunit A (FIG. 25, FIG. 26) in combination with polypeptides against CD45 (antiHis(V.sub.L)−CD45(V.sub.H−V.sub.L)) and HLA-A2 (antiHis(V.sub.H)−HLA-A2(V.sub.H−V.sub.L)). Complementation of his-tagged toxin binding and subsequent target cell killing was assessed by measuring cell viability using an ALAMARBLUE® assay. At the highest concentration of polypeptides used (80 nM), a clear difference in target cell killing, measured as reduction in cell viability, was found in experimental situations with a combination of both polypeptides compared to single polypeptides.

Example 20

(94) To further demonstrate the versatility, flexibility and the exchangeability of the bipartite functional complementation strategy, the V.sub.H and V.sub.L fragments of an anti-Digoxigenin antibody were used to identify and mark double antigen positive cells with Digoxigenin-labeled HRP (horse raddish peroxidase). EGFR and EpCAM positive Colo-206F cells were co-incubated with polypeptides against EGFR (antiDig(V.sub.H)−EGFR(V.sub.H+V.sub.L)) and EpCAM (antiDig(V.sub.L)−EpCAM(V.sub.H+V.sub.L)). On target complementation of the functional domain anti-Digoxigenin, indicated by Digoxigenin-HRP labelling of Colo-206F cells, was assessed by measuring the peroxidase activity, using a standard ELISA Kit (Invitrogen™). A clear difference in Dig-HRP labeled target cells was found in experimental situation with a combination of both polypeptides compared to single polypeptides. Data obtained are depicted in FIG. 27.

Example 21

(95) Using the human leukocytic antigens (HLA) as one arm for dual antigen restricted bipartite functional complementation, this haplotype strategy was further validated by exchanging the functional domains of the polypeptides with V.sub.H and V.sub.L fragments of an anti-HLA-Cw6 antibody. HLA-Cw6 positive primary patient PBMCs were co-incubated with HLA-Cw6 negative PBMCs from a healthy donor, polypeptide against CD45 (CD3(V.sub.L)−CD45(V.sub.H−V.sub.L)) and HLA-Cw6 (CD3(V.sub.H)−HLA-Cw6(V.sub.H−V.sub.L)). IFNγ production by lymphocytes was measured by ELISA techniques. These data demonstrate that hematopoietic cells of patients with other haplotypes than HLA-A2 can be targeted simply by exchanging one targeting domain (anti HLA-A2, FIG. 5, FIG. 7-FIG. 9, FIG. 11-FIG. 12) by another (anti HLA-Cw6). Data obtained are depicted in FIG. 28.

Example 22

(96) The dual antigen-induced bipartite functional complementation strategy was further validated in an in vitro patient assay, using freshly isolated primary patient cancer cells and antigen targets already used for cancer therapy in clinic or clinical trials (EGFR, EpCAM, CEA and Her2). Malignant cells of a 48 years old male patient with metastatic pancreatic cancer were co-incubated with the patients own peripheral blood lymphocytes and the combination of polypeptides against EGFR (CD3(V.sub.H)−EGFR(V.sub.H+V.sub.L)), EpCAM (CD3(V.sub.L)−EpCAM(V.sub.H+V.sub.L)), Her2 (CD3(V.sub.H)−Her2(V.sub.H+V.sub.L)), CEA (CD3(V.sub.H)−CEA(V.sub.H−V.sub.L)) and HLA-A2 (CD3(V.sub.L)−HLA-A2(V.sub.H−V.sub.L)). Complementation of lymphocyte stimulatory function was assessed by reactive IFNγ production, measured by ELISA techniques. No IFNγ production was found in experimental situations without any polypeptides. These data demonstrate the potential of this strategy to use patients own immune cells to target and kill his malignant transformed cells. Data obtained are depicted in FIG. 29.

Example 23

(97) A highly enriched CD3/CD8 positive CMV restricted T-cell population was used to show that any T cell, irrespective of its specificity, can serve as effector cell an kill double antigen positive tumor cells by this complementation strategy. The CD45 and HLA-A2 positive U266 and THP-1 cells were co-incubated with cytomegalievirus (CMV) specific T-cells from a HLA-A2 negative healthy donor and polypeptides against CD45 (CD3(V.sub.H)−CD45(V.sub.H−V.sub.L)) and HLA-A2 (CD3(V.sub.L)−HLA-A2(V.sub.H−V.sub.L)) in varying amounts. The bispecific tandem scFv (CD3(V.sub.H−V.sub.L)×HLA-A2(V.sub.H−V.sub.L))-antibody was used as a positive control. T cell engagement was assessed by reactive IFNγ production, measured by ELISA techniques. No IFNγ production was found in experimental situations with single polypeptides or without any polypeptides. Data obtained are depicted in FIG. 30. Cells from the same frozen aliquot batch, CMV specific T-cells and THP-1 cells, were used for the in vivo murine model (FIG. 12A).

Example 24

(98) This illustration depicts the potential to target allergen/autoimmune specific B-cell clones with the bipartite functional complementation strategy. By using a synthetic allergen as targeting moiety, the allergen linked polypeptide will bind specifically to its clonotypic B-cell receptor expressed on the surface of the allergen specific B-cell clone. The second arm of the bipartite strategy will use a B-cell specific polypeptide (CD19, CD20, CD38, CD138), restricting the followed complementation of the effector domain with subsequent target cell killing to the allergen specific B-cell clone. The ultimate goal of this strategy is to eliminate the B cell clone that causes and allergic or autoimmune disease (upper part of FIG. 31) whilst sparing B cells with other specificities or cells other than B cells (eg. mast cells or basophilic cells) which bind the antibody responsible for the disease via Fc-receptors (lower part of FIG. 31).

(99) The features of the present invention disclosed in the specification, the claims, and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.