The IgM and IgE Heavy Chain Domain 2 as Covalently Linked Homodimerization Modules for the Generation of Fusion Proteins with Dual Specificity

Abstract

The present invention provides a polypeptides comprising a heavy chain domain 2 (HD2) from IgM or IgE and at least one pharmaceutically active moiety, complexes thereof and their use for therapy and prophylaxis.

Description

[0001] The present invention provides polypeptides comprising a heavy chain domain 2 (HD2) from IgM or IgE and at least one pharmaceutically active moiety, complexes thereof and their use for therapy and prophylaxis.

BACKGROUND

[0002] Fusion of therapeutic proteins such as antibodies, cytokines and growth factors to homodimerization modules generates bivalent molecules generally displaying improved efficacy due to increased valency but also improved pharmacokinetic properties (Deyev & Lebedenkov, 2008; Cuesta et al., 2010; Kontermann, 2010). Furthermore, combining two different effector molecules allows for the generation of molecules with dual specificity. Such multivalent molecules can be applied for dual targeting approaches using bispecific antibodies or using bifunctional molecules composed of an effector molecule and an antibody moiety (Muller & Kontermann, 2010; Schrama et al., 2006; Deckert, 2009). Various homo-dimerization and multimerization modules have been established including the Fc region as well as C.sub.H3 and C.kappa. domains of immunoglobulins (Jazayeri & Carroll, 2008; Hu et al., 1996; Giersberg et al., 2011) but also other protein domains and peptides, for example derived from streptavidin, p53, uteroglobin, tenascin and collagen (Ventura et al., 2009; Diibel et al., 1995; Pack & Pluckthun, 1992; Rheinnecker et al., 1996; Wilest et al., 2002; Fan et al., 2008). However, many of these modules are either non-human and, thus can induce an immune response. Furthermore, many of these modules are not covalently connected by disulfide bonds, thus have a reduced stability, and might be difficult to be produced. Finally, some of these multimerization domains, e.g. the Fc region of IgG, trigger undesired ADCC (antibody dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity) effector functions causing unwanted side effects.

[0003] To overcome these disadvantages in the prior art present inventors investigated into the usage of alternative multimerization modules. Surprisingly, it was found that the IgM C.sub.H2 domain (MHD2) and the IgE C.sub.H2 domain (EHD2) which replace the hinge region connecting two heavy chains in other Ig molecules, allow for the production of stable dimers due to the formation of disulfide bonds between two MHD2 or between two EHD2. Because of the central location of the MHD2 and EHD2 within the heavy chain of IgM or IgE, respectively, containing further heavy chain sequences at both ends, both domains were found to be ideally suited for the generation of dimeric fusion proteins composed of proteins fused to the N- and/or C-terminus of either HD2. Thus, the usage of the MHD2 or EHD2 domain allows for the generation of multivalent and bifunctional molecules held together by the covalently linked dimerization domains MHD2 or EHD2. Such dimers proved to be particularly stable whilst retaining their flexibility. Furthermore, no ADCC or CDC effector function is associated with their usages as dimerization module. Because they are derived from natural plasma proteins, they are easy to produce at high yields. Natural occurring N-glycosylation of these domains further improve stability and solubility under physiological conditions.

SUMMARY OF THE INVENTION

[0004] In a first aspect the present invention provides a polypeptide comprising a heavy chain domain 2 (HD2) from IgM or IgE and at least one pharmaceutically active moiety, under the proviso that the pharmaceutically active moiety is not a Fab or Fc fragment from IgM or IgE.

[0005] In a second aspect the present invention provides a nucleic acid molecule comprising a sequence encoding the polypeptide of the first aspect.

[0006] In a third aspect the present invention provides a vector comprising the nucleic acid molecule of the second aspect.

[0007] In a fourth aspect the present invention provides a complex comprising at least two polypeptides of the first aspect.

[0008] In a fifth aspect the present invention provides a cell comprising the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, or the complex of the fourth aspect.

[0009] In a sixth aspect the present invention provides a composition comprising the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the complex of the fourth aspect, or the cell of the fifth aspect.

[0010] In a seventh aspect, the present invention provides the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the complex of the fourth aspect, or the cell of the fifth aspect for use as a medicament.

FIGURES

[0011] FIG. 1: The IgM heavy chain domain 2 (MHD2) and IgE heavy chain domain 2 (EHD2) is used as covalently linked homodimerization module. Further modules can be fused to either the N- or C-terminus, or to both ends. Each of the modules X.sub.1, X.sub.2, . . . X.sub.m, as well as Y.sub.1, Y.sub.2, . . . Yn, can be identical or different. Numbers of fused modules is between 1 to n and does not have to be identical between N- and C-terminal fusion.

[0012] FIG. 2: Sequence analysis of the human MHD2 and EHD2 as well as alignment of the two sequences. Inter- and intradomain cysteine bonds as well as potential N-glycosylation sites (NIT, NAS) are marked.

[0013] FIG. 3: a) Assembly of bivalent or tetravalent scFv-MHD2 fusion proteins. Bivalent, monospecific scFv-MHD2 fusion proteins are generated by fusing a scFv either to the N-terminus or the C-terminus of the MHD2, respectively. Tetravalent, bispecific scFv-MHD2 fusion proteins are generated by fusing a scFv directed against antigen A to the N-terminus of the MHD2 and another scFv directed against antigen B to the C-terminus of the MHD2. b) Schematic arrangement of the variable domains of the scFvs and the MHD2. For secretion into the cell culture supernatant the fusion proteins contain an N-terminal leader sequence (L). For purification and detection the fusion proteins contain further a C-terminal hexahistidyl-tag (His6). c) SDS-PAGE analysis of the purified MHD2 fusion proteins directed against EGFR or HER2, or both antigens. Proteins were analyzed under reducing (1-3) or non-reducing (4-6) conditions (1 and 3, scFv.sub.EGFR-MHD2; 2 and 5, MHD2-scFv.sub.HER2; 3 and 6, scFv.sub.EGFR-MHD2-scFv.sub.HER2). The gel was stained with Coomassie brilliant blue G250. Dimeric assembly of the fusion proteins is indicated by the upper band visible under non-reducing conditions corresponding to disulfide-bond linked dimers.

[0014] FIG. 4: Bioactivity of scFv-MHD2 fusion proteins. a) Binding of the antibody-MHD2 fusion proteins to immobilized receptor-Fc fusion proteins in ELISA. The three scFv-MHD2 fusion proteins showed binding to the respective EGFR- and or HER2-Fc fusion proteins. No binding was seen with an HER3-Fc fusion protein included as negative control. b) Binding of the scFv-MHD2 fusion proteins to various cell lines as indicated analyzed by flow cytometry. Bound proteins were detected with a FITC-labelled anti-His-tag antibody.

[0015] FIG. 5: a) Assembly of the MHD2-scTNF, scFv-MHD2-scTNF, and scFv-MHD2 fusion proteins. Bivalent MHD2-scTNF fusion proteins are generated by fusing scTNF to the C-terminus of the MHD2. The scFv-MHD2-scTNF fusion proteins are generated by fusing a scFv to the N-terminus of the MHD2 and scTNF to the C-terminus of the MHD2. b) Schematic arrangement of the variable domains of the scFvs, the MHD2 and the scTNF in the fusion proteins. For secretion into the cell culture supernatant the fusion proteins contain an N-terminal leader sequence (L). For purification and detection the fusion proteins contain further a C-terminal hexahistidyl-tag (His6). c) SDS-PAGE analysis of the purified fusion proteins under reducing (1-4) or non-reducing (5-8) conditions (1 and 5, scTNF; 2 and 6, scFv.sub.EGFR-MHD2; 3 and 7, MHD2-scTNF; 4 and 8, scFv.sub.EGFR-MHD2-scTNF). The gel was stained with Coomassie brilliant blue G250. Dimeric assembly of the fusion proteins is indicated by the upper band visible under non-reducing conditions corresponding to disulfide-bond linked dimers (lanes 6-8). c) Size exclusion chromatography (SEC) of scTNF. d) SEC of MHD2-scTNF. e) SEC of scFv.sub.EGFR-MHD2-scTNF. This analysis further demonstrates dimeric assembly of the fusion proteins. f) Determination of the melting point of scFv.sub.EGFR-MHD2-scTNF by dynamic light scattering.

[0016] FIG. 6: a) Binding of the scFv.sub.EGFR-MHD2-scTNF, MHD2-scTNF and scFv.sub.EGFR-MHD2 to an EGFR-Fc fusion protein in ELISA. HER2-Fc and HER3-Fc were included as negative controls. An anti-human Fc antibody was used as coating control. b) Binding of the scFv.sub.EGFR-MHD2-scTNF fusion protein to different cells lines (A431, NCI-H460) in the presence or absence of monoclonal antibodies (anti-EGFR cetuximab, anti-HER trastuzumab) analyzed by flow cytometry (grey, cells alone, bold line; cells incubated with scFv.sub.EGFR-MHD2-scTNF; thin line, cells incubated with scFv.sub.EGFR-MHD2-scTNF and excess amounts of cetuximab or trastuzumab). c) Effect of the fusion proteins on killing of MEF-TNFR1 cells. d) Effect of the fusion proteins on killing of MEF-TNFR2 cells. Here, only the dimeric MHD2-scTNF fusion protein is capable of triggering TNFR2-mediated cell killing. e) IL-8 release from HT1080 cells induced by increasing concentrations of scFv.sub.EGFR-MHD-scTNF, MHD2-scTNF, and scTNF (n=4, +/-SD). scFv.sub.EGFR-MHD was included as negative control. f) Inhibition of scFv.sub.EGFR-MHD2-scTNF (20 pM) induced IL-8 secretion by excess amounts of anti-EGFR antibody Cetuximab (660 nM). No effects were seen for IL-8 release induced by MHD2-scTNF and scTNF, respectively. Trastuzumab (anti-HER2) was included as negative control.

[0017] FIG. 7: a) Assembly of the MHD2-scTRAIL and scFv-MHD2-scTRAIL fusion proteins. Bivalent MHD2-scTRAIL fusion proteins are generated by fusing scTRAIL to the C-terminus of the MHD2. The scFv-MHD2-scTRAIL fusion proteins are generated by fusing a scFv to the N-terminus of the MHD2 and scTRAIL to the C-terminus of the MHD2. b) Schematic arrangement of the variable domains of the scFvs, the MHD2 and the scTRAIL in the fusion proteins. For secretion into the cell culture supernatant the fusion proteins contain an N-terminal leader sequence (L). For purification and detection the fusion proteins contain further an N-terminal FLAG-tag (FLAG-tag). c) SDS-PAGE analysis of the purified fusion proteins under reducing (1-4) or non-reducing (5-8) conditions (1 and 4, scTRAIL; 2 and 5, MHD2-scTRAIL; 3 and 6, scFv.sub.EGFR-MHD2-scTRAIL). The gel was stained with Coomassie brilliant blue G250. Dimeric assembly of the fusion proteins is indicated by the upper band visible under non-reducing conditions corresponding to disulfide-bond linked dimers (lanes 4-6).

[0018] FIG. 8: Cytotoxic activity of scTRAIL and MHD2-scTRAIL on NCI-H460 (a) and Colo205 (b) cells in the presence of 250 ng/ml bortezomib. Cells were incubated for 24 h with the proteins and cytotoxicity was determined by crystal violet staining of remaining cells. MHD2-scTRAIL showed a 10- to 50-fold increased cytotoxic activity compared to scTRAIL.

[0019] FIG. 9: a) Assembly of the EHD2-scTRAIL, scFv-EHD2-scTRAIL, and scFv-EHD2 fusion proteins. Bivalent scFv-EHD2 fusion proteins are generated by fusing an scFv directed against EGFR to the N-terminus of the EHD2. Bivalent EHD2-scTRAIL fusion proteins are generated by fusing scTRAIL to the C-terminus of the EHD2. The scFv-EHD2-scTRAIL fusion proteins are generated by fusing a scFv to the N-terminus of the EHD2 and scTRAIL to the C-terminus of the EHD2. b) Schematic arrangement of the variable domains of the scFvs, the EHD2 and the scTRAIL in the fusion proteins. For secretion into the cell culture supernatant the fusion proteins contain an N-terminal leader sequence (L). For purification and detection the fusion proteins contain further an N-terminal FLAG-tag (FLAG-tag). c) Immunoblot analysis of cell culture supernatant of HEK293 cells transiently transfected with an expression plasmid for scFv-EHD2 under non-reducing (1) or reducing (2) conditions. The fusion protein was detected with an anti-FLAG-tag antibody or anti-His-tag antibody. Dimeric assembly of the fusion protein is indicated by the upper band visible under non-reducing conditions corresponding to disulfide-bond linked dimers (lanes 1). d) Immunoblot analysis of cell culture supernatant of HEK293 cells transiently transfected with an expression plasmid for EHD2-scTRAIL under non-reducing (1) or reducing (2) conditions. The fusion protein was detected with an anti-FLAG-tag antibody. Dimeric assembly of the fusion protein is indicated by the upper band visible under non-reducing conditions corresponding to disulfide-bond linked dimers (lanes 1). e) Immunoblot analysis of cell culture supernatant of HEK293 cells transiently transfected with an expression plasmid for scFv-EHD2-scTRAIL under non-reducing (1) or reducing (2) conditions. The fusion protein was detected with an anti-FLAG-tag antibody. Dimeric assembly of the fusion protein is indicated by the upper band visible under non-reducing conditions corresponding to disulfide-bond linked dimers (lanes 1).

[0020] FIG. 10: Comparison of recombinant CH3 domain of human IgG1 (GHD3) (a), the human MHD2 domain (b) and the human EHD2 domain (c). SDS-PAGE analysis of the purified protein was performed under reducing (1) and non-reducing (2) conditions. Gels were stained with Coomassie brilliant blue G250. Dimeric assembly and linkage by disulfide-bonds was demonstrated for MHD2 and EHD2. Heterogeneity regarding N-glycosylation was also observed for MHD2 and EHD2 indicated by 2 to 3 bands under reducing and non-reducing conditions, respectively. Size-exclusion chromatography confirmed dimeric assembly of the domains, with the two N-glycosylated domains (MHD2, EHD2) migrating with an apparent size which is larger than that of the GHD3 domain. Determination of melting points by dynamic light scattering revealed for CH3 of IgG1 a first melting point at around 50.degree. C. (probably due to domain dissociation) and a second major melting point at 75.degree. C. (probably due to complete denaturation), while no sharp increase of signals but a gradual increase starting at around 56.degree. C. was observed for MHD2, indicating that the protein is rather stable up to a temperature of 92.degree. C. For EHD2, a single thermal melting point of approximately 80-82.degree. C. was determined.

[0021] FIG. 11: Nucleic acid and amino acid sequence of anti-HER2 scFvs (4D5). The leader peptide is underlined.

[0022] FIG. 12: Nucleic acid and amino acid sequence of anti-EGFR scFvs (hu225). The leader peptide is underlined.

[0023] FIG. 13: Nucleic acid and amino acid sequence of scTNF. Linkers connecting the individual TRAIL monomers are underlined.

[0024] FIG. 14: Nucleic acid and amino acid sequence of scTRAIL. Linkers connecting the individual TRAIL monomers are underlined.

[0025] FIG. 15: Nucleic acid and amino acid sequence of scFv.sub.EGFR-MHD2. The MHD2 is shown in grey. Leader peptide and linkers connecting the individual domains are underlined.

[0026] FIG. 16: Nucleic acid and amino acid sequence of MHD2-scFv.sub.HER2. The MHD2 is shown in grey. Leader peptide and linkers connecting the individual domains are underlined.

[0027] FIG. 17: Nucleic acid and amino acid sequence of scFv.sub.EGFR-MHD2-scFv.sub.HER2. The MHD2 is shown in grey. Leader peptide and linkers connecting the individual domains are underlined.

[0028] FIG. 18: Nucleic acid and amino acid sequence of MHD2-scTNF. The MHD2 is shown in grey. Leader peptide and linkers connecting the individual domains are underlined.

[0029] FIG. 19: Nucleic acid and amino acid sequence of scFv.sub.EGFR-MHD2-scTNF. The MHD2 is shown in grey. Leader peptide and linkers connecting the individual domains are underlined.

[0030] FIG. 20: Nucleic acid and amino acid sequence of MHD2-scTRAIL. The MHD2 is shown in grey. Leader peptide and Linkers connecting the individual domains are underlined.

[0031] FIG. 21: Nucleic acid and amino acid sequence of scFv.sub.EGFR-MHD2-scTRAIL. The MHD2 is shown in grey. Leader peptide and linkers connecting the individual domains are underlined.

[0032] FIG. 22: Nucleic acid and amino acid sequence of scDb.sub.EpCAMxEGFR-MHD2-scTRAIL. The MHD2 is shown in grey. Leader peptide and Linkers connecting the individual domains are underlined.

[0033] FIG. 23: Nucleic acid and amino acid sequence of scFv.sub.EGFR-EHD2. The EHD2 is shown in grey. Leader peptide and Linkers connecting the individual domains are underlined.

[0034] FIG. 24: Nucleic acid and amino acid sequence of EHD2-scTRAIL. The MHD2 is shown in grey. Leader peptide and Linkers connecting the individual domains are underlined.

[0035] FIG. 25: Nucleic acid and amino acid sequence of scFv.sub.EGFR-EHD2-scTRAIL. The MHD2 is shown in grey. Leader peptide and linkers connecting the individual domains are underlined.

[0036] FIG. 26: a) Schematic arrangement of scFv-EHD2 fusion proteins (L=leader peptide; His6=hexahistidyl tag). b) Schematic structure of scFv-EHD2 fusion proteins (the EHD2 domains are shown in white). c) Immunoblotting experiment showing the expression of anti-HER3 scFv-EHD2 fusion protein (lane 1). Proteins were separated on a 12% SDS-PAGE under reducing conditions and detected with a HRP-conjugated anti-His-tag antibody. The fusion protein has the expected size of approximately 40 to 45 kDa. The detected double band indicates glycosylated and non-glycosylated proteins.

[0037] FIG. 27: a) Schematic arrangement of bispecific scDb-EHD2 fusion protein (L=leader peptide; His6=hexahistidyl tag). b) Schematic structure of a scDb-EHD2 fusion protein (the EHD2 domains are shown in white). c) SDS-PAGE of purified fusion proteins under reducing or nonreducing conditions. Gel was stained with Coomassie. d) ELISA of binding of scFv-EHD2 and scDb-EHD2 to immobilized CEA (3 .mu.g/ml). Bound antibodies were detected with an HRP-conjugated anti-His-tag antibody.

[0038] FIG. 28: a) Schematic composition of the scFv-L3 fragment used to generate scFv-L3-EHD2 fusion proteins with the cysteine as position 3 of the linker sequence. b) Schematic structure of scFv-L3-EHD2 and scFv-L3-EHD2-scFv fusion proteins (the EHD2 domains are shown in white). c) Immunoblotting experiment showing the expression of anti-FAP scFv-L3 (lane 1), a diabody Db-Cys (lane 2), an scFv-L3-EHD2 (lane 3), and an scFv-L3-EHD3-scFv in the supernatant of transiently transfected HEK293 cells. Proteins were separated on a 12% SDS-PAGE under reducing conditions and detected with a HRP-conjugated anti-His-tag antibody. All proteins have the expected size.

[0039] FIG. 29: a) Schematic illustration of the various EHD2 fusion proteins. b) SDS-PAGE analysis of the purified fusion proteins under reducing (lanes 1-3) or non-reducing (lanes 4-6) conditions (scFv-EHD2, lanes 1, 4; EHD2-scTRAIL, lanes 2, 5; scFv-EHD2-scTRAIL, lanes 3, 6). c) SEC analysis of the purified fusion proteins. d) ELISA for binding of the fusion proteins to EGFR and TRAIL receptors 1 and 2 using recombinant receptor-Fc fusion proteins. HER2-Fc was included as negative control. e) Titration of scFv-EHD2 and scFv directed against EGFR in ELISA for binding to immobilized EGFR.

[0040] FIG. 30: Binding of EHD2 fusions proteins to EGFR-expressing cell lines analyzed by flow cytometry. a) Binding of EHD2-scTRAIL and scFv-EHD2-scTRAIL to Colo205, NCI-H460 and HEK293 using an anti-FLAG-tag antibody for detection. b) Binding of scFv-EHD2 to Colo205, NCI-H460 and HEK293 using an anti-His-tag antibody for detection.

[0041] FIG. 31: In vitro cytotoxicity of EHD2-scTRAIL and scFv-EHD2-scTRAIL in comparison to scTRAIL. Cytotoxicity was analyzed with two cell lines (NCI-H460, Colo205) in the absence or presence of bortezomib (250 ng/ml). Cells were incubated for 16 h with the fusion proteins and viable cells were determined by crystal violet staining.

[0042] FIG. 32: In vitro cytotoxicity of EHD2-scTRAIL and scFv-EHD2-scTRAIL in the absence or presence of cetuximab. Cytotoxicity was analyzed with two cell lines (NCI-H460, Colo205) in presence of bortezomib (250 ng/ml) with or without an 100-fold molar excess of cetuximab (Cet.), which blocks binding of the anti-EGFR scFv to cells. Cells were incubated for 16 h with the fusion proteins and viable cells were determined by crystal violet staining.

[0043] FIG. 33: In vitro cytotoxicity of scFv-EHD2-scTRAIL in comparison to scFv-scTRAIL. Cytotoxicity was analyzed with two cell lines (NCI-H460, Colo205) in the absence or presence of bortezomib (250 ng/ml). Cells were incubated for 16 h with the fusion proteins and viable cells were determined by crystal violet staining.

[0044] FIG. 34: Pharmacokinetics of EHD2 fusion proteins in mice. scTRAIL and the EHD2 fusion proteins (25 .mu.g per animal) were injected i.v. into CD1 mice (n=3) and serum concentrations were determined by ELISA.

[0045] FIG. 35: a) In vivo antitumor activity of EHD2-scTRAIL and scFv-EHD2-scTRAIL in comparison to scTRAIL. NMRI nude mice bearing s.c. Colo205 xenograft tumors received four i.v. injections of the proteins (0.35 nmol of the fusion proteins and 0.7 nmol of scTRAIL) every four days as well as eight i.p. injection of bortezomib (Brt, 5 .mu.g/injection) every second day. a) tumor growth. b) Tumor volumes at day 13. c) In vivo antitumor activity of scFv-EHD2-scTRAIL in comparison with scFv-EHD2 and bortezomib treatment. NMRI nude mice bearing s.c. Colo205 xenograft tumors received four i.v. injections of the proteins (1 nmol of the fusion proteins) as well as four i.p. injection of bortezomib (Brt, 5 .mu.g/injection) every second day. b) Tumor volumes at day 21. e) An ALT-assay was performed 4 or 24 hours after injection of scFv-EHD2-scTRAIL (1 nmol, i.v.) together with bortezomib (5 i.p.) into CD1 mice. No statistically significant differences were observed in comparison to a PBS control. All values were below the threshold of 50 U/L.

[0046] FIG. 36: a-c) Purified TNF variants (a: MHD2-scTNF.sub.R2, b: EHD2-scTNF.sub.R2 with a 16 aa linker between EHD2 and scTNFR2 as well as c: EHD2-scTNF.sub.R2 with a 28 aa linker between EHD2 and scTNFR2) were analyzed by 8% SDS-PAGE under reducing (+2-ME) or non-reducing (-2-ME) conditions and stained with Coomassie. d) Binding studies with TNFR2-Fc fusion protein. Plates were coated with 1 .mu.g/ml TNFR2-Fc fusion protein (Enbrel). TNF fusion proteins were incubated for 1 h at RT and bound TNF fusion proteins were detected using HRP-conjugated anti-TNF antibodies and TMB substrate (n=2).

[0047] FIG. 37: Nucleic acid and amino acid sequence of Anti-CEA scFv-EHD2

[0048] FIG. 38: Nucleic acid and amino acid sequence of Anti-HER2 scFv-EHD2

[0049] FIG. 39: Nucleic acid and amino acid sequence of Anti-HER3 scFv-EHD2

[0050] FIG. 40: Nucleic acid and amino acid sequence of Anti-CEAxCD3 scDb-EHD2

[0051] FIG. 41: Nucleic acid and amino acid sequence of Anti-EGFR scFv-L3-EHD2

[0052] FIG. 42: Nucleic acid and amino acid sequence of Anti-EGFR scFv-L3-EHD2-scFv

[0053] FIG. 43: Nucleic acid and amino acid sequence of MHD2-scTNF.sub.R2

[0054] FIG. 44: Nucleic acid and amino acid sequence of EHD2-scTNF.sub.R2-L16aa

[0055] FIG. 45: Nucleic acid and amino acid sequence of EHD2-scTNF.sub.R2-L28aa

DETAILED DESCRIPTION

[0056] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0057] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H. G. W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0058] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS

[0059] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0060] The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bond-linked chain of amino acids, regardless of length or post-translational modification. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitops and protein domains) can be further modified by chemical modification. This means such a chemically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility.

[0061] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. The nucleic acids, can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann & Peyman (1990) Chemical Reviews 90:543-584).

[0062] As used herein, the term "variant" is to be understood as a polynucleotide or protein which differs in comparison to the polynucleotide or protein from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a protein or nucleic acid variant is derived is also known as the parent or parental polypeptide or polynucleotide. "Dimerizing variants" as referred to herein are variants of a parental dimerization domain which differ from said parental dimerization domain by one or more changes in its length or sequence as detailed above. The term "variant" or "dimerizing variants" comprises "fragments" or "derivatives" of the parent molecule. Typically, "fragments" are smaller in length or size than the parent molecule, whilst "derivatives" exhibit one or more differences in their sequence in comparison to the parent molecule. Also encompassed are modified molecules such as but not limited to post-translationally modified proteins (e.g. glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. Also mixtures of different molecules such as but not limited to RNA-DNA hybrids, are encompassed by the term "variant" or "dimerizing variants". Typically, a variant or dimerizing variants is constructed artificially, preferably by gene-technological means whilst the parent polypeptide or polynucleotide is a wild-type protein or polynucleotide. However, also naturally occurring variants are to be understood to be encompassed by the term "variant" or "dimerizing variants" as used herein. Further, the variants or dimerizing variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant or dimerizing variants exhibits at least one biological activity of the parent molecule, i.e. is functionally active.

[0063] The changes in the nucleotide or amino acid sequence may be nucleotide or amino acid exchanges, insertions, deletions, 5'- or 3' truncations, N- or C-terminal truncations, or any combination of these changes, which may occur at one or several sites. In preferred embodiments, a variant usable in the present invention exhibits a total number of up to 150 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150) changes in the nucleotide or amino acid sequence (i.e. exchanges, insertions, deletions, and/or truncations). Amino acid exchanges may be conservative and/or non-conservative. Typical substitutions are among the aliphatic amino acids, among the amino acids having aliphatic hydroxyl side chain, among the amino acids having acidic residues, among the amide derivatives, among the amino acids with basic residues, or the amino acids having aromatic residues. Typical semi-conservative and conservative substitutions are:

TABLE-US-00001 Amino Conservative acid substitution Semi-conservative A G; S; T N; V; C C A; V; L M; I; F; G D E; N; Q A; S; T; K; R; H E D; Q; N A; S; T; K; R; H F W; Y; L; M; H I; V; A G A S; N; T; D; E; N; Q H Y; F; K; R L; M; A I V; L; M; A F; Y; W; G K R; H D; E; N; Q; S; T; A L M; I; V; A F; Y; W; H; C M L; I; V; A F; Y; W; C; N Q D; E; S; T; A; G; K; R P V; I L; A; M; W; Y; S; T; C; F Q N D; E; A; S; T; L; M; K; R R K; H N; Q; S; T; D; E; A S A; T; G; N D; E; R; K T A; S; G; N; V D; E; R; K; I V A; L; I M; T; C; N W F; Y; H L; M; I; V; C Y F; W; H L; M; I; V; C

[0064] Changing from A, F, H, I, L, M, P, V, W or Y to C is semi-conservative if the new cysteine remains as a free thiol. Furthermore, the skilled person will appreciate that glycines at sterically demanding positions should not be substituted and that P should not be introduced into parts of the protein which have an alpha-helical or a beta-sheet structure.

[0065] Alternatively or additionally, a "variant" or "dimerizing variant" as used herein, can be characterized by a certain degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 70% sequence identity to its parent polypeptide. A polynucleotide variant in the context of the present invention exhibits at least 70% sequence identity to its parent polynucleotide. Preferably, the sequence identity of protein variants is over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, the sequence identity of polynucleotide variants is over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides.

[0066] The term "at least 70% sequence identity" is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide.

[0067] In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID, if not specifically indicated otherwise. For example, a peptide sequence consisting of 90 amino acids compared to the amino acid sequence of a human MHD2 domain may exhibit a maximum sequence identity percentage of 81.08% (90/111) while a sequence with a length of 78 amino acids may exhibit a maximum sequence identity percentage of 70.03% (78/111). The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680) available e.g. on http://www.ebi.ac.uk/Tools/clustalw/ or on http://www.ebi.ac.uk/Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw- .html. Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, word length=12. BLAST protein searches are performed with the BLASTP program, score=50, word length=3. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M. (2003b) Bioinformatics 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

[0068] "Hybridization" can also be used as a measure of sequence identity or homology between two nucleic acid sequences. Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. "Moderate hybridization conditions" are defined as equivalent to hybridization in 2.times. sodium chloride/sodium citrate (SSC) at 30.degree. C., followed by a wash in 1.times.SSC, 0.1% SDS at 50.degree. C. "Highly stringent conditions" are defined as equivalent to hybridization in 6.times. sodium chloride/sodium citrate (SSC) at 45.degree. C., followed by a wash in 0.2.times.SSC, 0.1% SDS at 65.degree. C.

[0069] The term "immunoglobulin (Ig)" as used herein refers to immunity conferring glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells by their transmembrane region and encompass molecules such as but not limited to B-cell receptors, T-cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (.beta.2M), CD3, CD4 and CD8. Typically, the term "antibody" as used herein refers to secreted immunoglobulins which lack the transmembrane region and can thus, be released into the bloodstream and body cavities. Human antibodies are grouped into different isotypes based on the heavy chain they possess. There are five types of human Ig heavy chains denoted by the Greek letters: .alpha., .delta., .epsilon., .gamma., and .mu.. The type of heavy chain present defines the class of antibody, i.e. these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively, each performing different roles, and directing the appropriate immune response against different types of antigens. Distinct heavy chains differ in size and composition; .alpha. and .gamma. comprise approximately 450 amino acids, while .mu. and .epsilon. have approximately 550 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas, such as the gut, respiratory tract and urogenital tract, as well as in saliva, tears, and breast milk and prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD mainly functions as an antigen receptor on B cells that have not been exposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898). IgE is involved in allergic reactions via its binding to allergens triggering the release of histamine from mast cells and basophils. IgE is also involved in protecting against parasitic worms (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides the majority of antibody-based immunity against invading pathogens and is the only antibody isotype capable of crossing the placenta to give passive immunity to fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans there are four different IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum with IgG1 being the most abundant (.about.66%), followed by IgG2 (.about.23%), IgG3 (.about.7%) and IgG (.about.4%). The biological profile of the different IgG classes is determined by the structure of the respective hinge region. IgM is expressed on the surface of B cells in a monomeric form and in a secreted pentameric form with very high avidity. IgM is involved in eliminating pathogens in the early stages of B cell mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437).

[0070] Antibodies are not only found as monomers but are also known to form dimers of two Ig units (e.g. IgA), tetramers of four Ig units (e.g. IgM of teleost fish), or pentamers of five Ig units (e.g. mammalian IgM). Antibodies are typically made of four polypeptide chains comprising two identical heavy chains and identical two light chains which are connected via disulfide bonds and resemble a "Y"-shaped macro-molecule. Each of the chains comprises a number of immunoglobulin domains out of which some are constant domains and others are variable domains. Immunoglobulin domains consist of a 2-layer sandwich of between 7 and 9 antiparallel .beta.-strands arranged in two .beta.-sheets. Typically, the "heavy chain" of an antibody comprises four Ig domains with three of them being constant (C.sub.H domains: C.sub.H1, C.sub.H2, C.sub.H3) domains and one of the being a variable domain (V.sub.H), with the exception of IgM and IgE which contain one variable (VH) and four constant regions (C.sub.H1, C.sub.H2, C.sub.H3, C.sub.H4). The additional domain (C.sub.H2: C.mu.2, C.epsilon.2) in the heavy chains of IgM and IgE molecules connects the two heavy chains instead of the hinge region contained in other Ig molecules (Perkins et al., 1991; Beavil et al., 1995; Wan et al., 2002). The "light chain" typically comprises one constant Ig domain (C.sub.L) and one variable Ig domain (V.sub.L). Exemplified, the human IgM heavy chain is composed of four Ig domains linked from N- to C-terminus in the order V.sub.H-C.sub.H1-C.sub.H2-C.sub.H3-C.sub.H4 (also referred to as V.sub.H-C.mu.1-C.mu.2-C.mu.3-C.mu.4), whereas the human IgM light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order V.sub.L-C.sub.L, being either of the kappa or lambda type (V.kappa.-C.kappa. or V.lamda.-C.lamda.).

[0071] Exemplified, the constant chain of human IgM comprises 452 amino acids. Throughout the present specification and claims, the numbering of the amino acid positions in an immunoglobulin are that of the "EU index" as in Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th ed. U.S. Department of Health and Human Service, National Institutes of Health, Bethesda, Md. The "EU index as in Kabat" refers to the residue numbering of the human IgM EU antibody. Accordingly, C.sub.H domains in the context of IgM are as follows: "C.sub.H1" refers to amino acid positions 118-215 according to the EU index as in Kabat; "C.sub.H2" refers to amino acid positions 231-340 according to the EU index as in Kabat; "C.sub.H3" refers to amino acid positions 341-446 according to the EU index as in Kabat. "C.sub.H4" refers to amino acid positions 447-558 according to the OU index as in Kabat.

[0072] Whilst in human IgA, IgG, and IgD molecules two heavy chains are connected via their hinge region, IgE and IgM antibodies do not comprise such hinge region. Instead, IgE and IgM antibodies possess an additional Ig domain, their C.sub.H2 domain, which functions as dimerization domain between two heavy chains. In contrast to rather flexible and linear hinge regions of other antibodies, the C.sub.H2 domain of IgE and IgM are composed of two beta sheets stabilized by an intradomain disulfide bond forming a c-type immunogloublin fold (Bork et al., 1994; Wan et al., 2002). Furthermore, the MHD2 and EHD2 domains contain one N-glycosylation site.

[0073] The "human IgE heavy chain domain 2" ("EHD2") consists of 106 amino acid residues. The domain contains an intradomain disulfide bond formed between Cys261 and Cys321 (EU numbering as in Kabat). Typically, two EHD2 domains are covalently linked by two interdomain disulfide bonds between Cys247 and Cys337. The EHD2 contains an N-glycosylation site at Asn275 (FIG. 2).

[0074] The amino acid sequence of human EHD2 is provided in SEQ ID NO: 2.

[0075] The "IgM heavy chain domain 2" ("MHD2") consists of 111 amino acid residues (12.2 kDa) forming a homodimer covalently held together by a disulfide bond formed between cysteine residue 337 of two domains (Davis et al., 1989; Davis & Shulman, 1989). The domain is further stabilized by an intradomain disulfide bond formed between Cys261 and Cys321. Typically, two MHD2 domains are covalently linked by an interdomain disulfide bond between Cys337. The MHD2 contains an N-glycosylation site at Asn333 (FIG. 2).

[0076] The amino acid sequence of human MHD2 is provided in SEQ ID NO: 1.

[0077] The human MHD2 and EHD2 have approximately 25% sequence identity (FIG. 2).

[0078] Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab fragments" (also referred to as "Fab portion" or "Fab region") each with a single antigen binding site, and a residual "Fc fragment" (also referred to as "Fc portion" or "Fc region") whose name reflects its ability to crystallize readily. The crystal structure of the human IgG Fc region has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In IgG, IgA and IgD isotypes, the Fc region is composed of two identical protein fragments, derived from the C.sub.H2 and C.sub.H3 domains of the antibody's two heavy chains; in IgM and IgE isotypes, the Fc regions contain three heavy chain constant domains (C.sub.H2-4) in each polypeptide chain. In addition, smaller immunoglobulin molecules exist naturally or have been constructed artificially. The term "Fab' fragment" refers to a Fab fragment additionally comprising the hinge region of an Ig molecule whilst "F(ab').sub.2 fragments" are understood to comprise two Fab' fragments being either chemically linked or connected via a disulfide bond. Whilst "single domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811) and "Nanobodies" only comprise a single V.sub.H domain, "single chain Fv (scFv)" fragments comprise the heavy chain variable domain joined via a short linker peptide to the light chain variable domain (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs (scFvA-scFvB). This can be done by producing a single peptide chain with two V.sub.H and two V.sub.L regions, yielding "tandem scFvs" (V.sub.HA-V.sub.LA-V.sub.HB-V.sub.LB). Another possibility is the creation of scFvs with linkers that are too short for the two variable regions to fold together, forcing scFvs to dimerize. Usually linkers with a length of 5 residues are used to generate these dimers. This type is known as "diabodies". Still shorter linkers (one or two amino acids) between a V.sub.H and V.sub.L domain lead to the formation of monospecific trimers, so-called "triabodies" or "tribodies". Bispecific diabodies are formed by expressing to chains with the arrangement V.sub.HA-V.sub.LB and V.sub.HB-V.sub.LA or V.sub.LA-V.sub.HB and V.sub.LB-V.sub.HA, respectively. Single-chain diabodies (scDb) comprise a V.sub.HA-V.sub.LB and a V.sub.HB-V.sub.LA fragment which are linked by a linker peptide (P) of 12-20 amino acids, preferably 14 amino acids, (V.sub.HA-V.sub.LB-P-V.sub.HB-V.sub.LA). "Bi-specific T-cell engagers (BiTEs)" are fusion proteins consisting of two scFvs of different antibodies wherein one of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244). Dual affinity retargeting molecules ("DART" molecules) are diabodies additionally stabilized through a C-terminal disulfide bridge.

[0079] The term "pharmaceutically active moiety" as used herein, is understood to refer to a part or moiety of a macromolecule or complex, i.e. a polypeptide, polynucleotide or complex thereof, which mediates a pharmaceutical effect including but not limited to prophylactic, therapeutic, and/or diagnostic effects.

[0080] As used herein, "prevent", "preventing", "prevention", or "prophylaxis" of a disease or disorder means preventing that such disease or disorder occurs in a patient. Accordingly, a moiety having a prophylactic effect prevents the onset of a disease or disorder in a patient.

[0081] As used herein, "treat", "treating", "treatment" or "therapy" of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in an individual that has previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in individuals that were previously symptomatic for the disorder(s). Accordingly, a moiety having a therapeutic effect treats the symptoms of a disease or disorder by accomplishing one or more of above named effects (a)-(e).

[0082] The terms "identify", "identifying", "identification" or "diagnosis" of a disease or disorder are used herein to refer to the determination of the nature and the cause of a disease or disorder. Accordingly, a moiety having a diagnostic effect allows for the determination of the nature and the cause of a disease or disorder.

[0083] "Symptoms" of a disease or disorder are implication of the disease or disorder noticeable by the tissue, organ or organism having such disease or disorder and include but are not limited to pain, weakness, tenderness, strain, stiffness, and spasm of the tissue, an organ or an individual as well as the presence, absence, increase, decrease, of specific indicators such as biomarkers or molecular markers. The term "disease" and "disorder" as used herein, refer to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a tissue, an organ or an individual is not able to efficiently fulfil its function anymore. Typically, but not necessarily, a disease or disorder is associated with specific symptoms or signs indicating the presence of such disease or disorder. Diseases or disorders include but are not limited to autoimmune diseases, allergic diseases, cancer type diseases, cutaneous conditions, endocrine diseases, blood diseases and disorders, eye diseases and disorders, genetic disorders, inflammatory diseases, infectious diseases, intestinal diseases, neurological disorders, and mental illness. Exemplified, autoimmune diseases include but are not limited to Diabetes mellitus type 1, rheumatoid arthritis, psoriasis, Crohns Disease, autoimmune cardiomyopathy, autoimmune hepatitis, Hashimoto's thyroiditis, and Sjogern's syndrome. Exemplified, allergic diseases include but are not limited to allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom allergies, drug allergies, and food allergies. Exemplified, cancer type diseases include but are not limited to Basal cell carcinoma, Bladder cancer, Bone cancer, Brain tumor, Breast cancer, Burkitt lymphoma, Cervical cancer, Colon Cancer, Cutaneous T-cell lymphoma, Esophageal cancer, Retinoblastoma, Gastric (Stomach) cancer, Gastrointestinal stromal tumor, Glioma, Hodgkin lymphoma, Kaposi sarcoma, Leukemias, Lymphomas, Melanoma, Oropharyngeal cancer, Ovarian cancer, Pancreatic cancer, Pleuropulmonary blastoma, Prostate cancer, Throat cancer, Thyroid cancer, and Urethral cancer. Exemplified, cutaneous conditions include but are not limited to Acne, Dermatitis, Eczema, conditions of the skin appendages, conditions of the subcutaneous fat, disturbances of pigmentation, epidermal nevi, epidermal neoplasms, epidermal cysts, erythemas, frostbites genodermatoses, mucinoses, neurocutaneous conditions (e.g. Wiskott-Aldrich syndrome), and psoriasis. Exemplified, endocrine diseases include but are not limited to Diabetes mellitus type 1 and type 2, Osteoporosis, and Cushing's disease. Exemplified, blood diseases and disorders include but are not limited to coagulopathies (hemophilia A, hemophila B, etc.), fibrinolytic disorders, complement deficiencies, immunoglobulin deficiencies, and anemia. Exemplified, genetic disorders include but are not limited to color blindness, cystic fibrosis, Down syndrome, Sickle-cell disease, and Turner syndrome. Exemplified, inflammatory diseases include but are not limited to rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, atheriosclerosis, osteoarthrisis, and asthma. Exemplified, infectious diseases include but are not limited to infections diseases caused by viruses, bacteria, worms, prions or other pathogens or parasites such as African sleeping sickness, AIDS, HIV infection, Anthrax, Borreliosis, Calicivirus infection (Norovirus and Sapovirus), Chickenpox, Chlamydia infection, Cholera, Clostridium infection, Colorado tick fever (CTF), common cold, Creutzfeldt-Jakob disease, Dengue fever (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola, Enterovirus infection, infections with Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Gonorrhea, Streptoccocal infections (group A and B), Hand, foot and mouth disease (HFMD), Helicobacter pylori infection, Hepatitis (A, B, C, and D), Herpes infection, Papillomavirus infection, Parainfluenza virus infection, Influenza, Lassa fever, Marburg fever, Measles, Meningitis, Mumps, Pasteurellosis, Pediculus infection, Plague, Pneumococcal infection, Respiratory syncytial virus infection, Rotavirus infection, Rubella virus infection, Salmonella food poisoning and infection, SARS, Scabies infections, Schistosomiasis, Smallpox, Staphylococcal food poisoning and infection, Syphilis, Tetanus, Trichophyton infection, Tuberculosis, Typhus, Venezuelan equine encephalitis, and Yellow fever. Exemplified, intestinal diseases include but are not limited to Gastroenteritis, Ileus, Ileitis, Colitis, Appendicitis, Coeliac disease, Irritable bowel syndrome, Diverticular disease, Diarrhea, Polyp, and Ulcerative colitis. Exemplified, neurological disorders include but are not limited to Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Brain damage, Creutzfeldt-Jakob disease, Cushing's syndrome, Dyslexia, Encephalitis, Epilepsy, Headache, Huntington's disease, Migraine, Multiple sclerosis, Parkinson's disease, Polio, Rabies, Schizophrenia, and Stroke. Exemplified, mental illness include but are not limited to Acute stress disorder, attention-deficit hyperactivity disorder (ADHD), Autistic disorder, Borderline personality disorder, Bulimia nervosa, Burn Out, Schizophrenia, Depression, Cognitive disorder, Communication disorder, Eating disorder, Kleptomania, Learning disorders, Male erectile disorder, Melancholia, Obsessive-compulsive disorder (OCD), Paranoia Pathological gambling, Posttraumatic stress disorder (PTSD), Psychotic disorder, Hypersomnia, Insomnia, and Tourette's syndrome.

[0084] A "pharmaceutically active moiety" typically comprises a biological and/or chemical pharmaceutical, e.g. ligands, effector molecules, half-life extension modules and imaging molecules. The term "ligand" refers to a chemical or biological substance that forms a complex with another molecule to fulfil a specific biological function. Ligands include but are not limited to substrates, inhibitors, and activators, such as antigen-binding molecules, scaffold proteins, natural ligands, ligand-binding receptor fragments, and apatamers. The term "effector molecule" typically refers to small molecules, peptides or polypeptides that bind to a protein and thereby alter the activity of that protein. They include but are not limited to cytokines, chemokines, immuno(co)-stimulatory molecules, immunosuppressive molecules, death ligands, apoptosis-inducing proteins, kinases, prodrug-converting enzymes, RNases, agonistic antibody or antibody fragment, antagonistic antibody or antibody fragment, toxins, growth factors, hormone, coagulation factor, fibrinolytic protein, peptides mimicking these, and fragments, fusion proteins or derivatives thereof. "Half-life extension modules" prolong the half-life, e.g. the "plasma half-life" or the "serum half-life", of a chemical or biological substance. Imaging molecules are those binding to specific target molecules thereby, allowing the visualization of the location of that molecule.

[0085] "Chemical pharmaceuticals" are typically understood to refer to chemical compounds synthesized artificially which are effective in the prevention, treatment or diagnosis of disorders or diseases.

[0086] "Biologicals" are typically understood to refer to medical drugs produced using biotechnological means and are used for prophylactic, therapeutic, and/or in vivo diagnostic purposes. Biologicals include but are not limited to peptides, polypeptides, proteins and nucleic acids (e.g. DNA, RNA, or hybrids thereof). Approved therapeutic biologicals include but are not limited to hormones (e.g. insulin, hGH, FSH, Glucagon-like peptide 1, parathyroid hormone, calcitonin, lutropin, glucagon), growth factors (e.g. erythropoietin, G-CSF/GM-CSF, IGF-1), interferons (e.g. IFN-.alpha., IFN-.beta., IFN-.gamma.), interleukins (e.g. IL-2, IL-11, IL-1Ra), coagulation factors (e.g. factor VIII, factor IX, factor VIIa, thrombin), thrombolytics and anti-coagulants (e.g. t-PA, hirudin, activated protein C), enzymes (e.g. .alpha.-glucosidase, glucocerebrosidase, iduronate-2-sulfatase, galactosidase, urate oxidase, DNase), antigen-binding molecule such as antibodies and antibody fragments (e.g. IgG, Fab), and fusion proteins thereof (e.g. TNFR2-Fc, TMP-Fc, CTLA-4-Fc, IL-1R-Fc, LFA-3-Fc, IL-2-DT).

[0087] A "peptide linker" in the context of the present invention refers to an amino acid sequence which sterically separates two parts or moieties of a complex, e.g. two peptides or proteins. Typically such linker consists of between 1 and 100 amino acids having a minimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, and a maximum length of at least 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less. The indicated preferred minimum and maximum lengths of the peptide linker according to the present invention may be combined, if such a combination makes mathematically sense, e.g. such linker may consist of 1-15, or 12-40, or 25-75, or 1-100 amino acids. Peptide linkers may also provide flexibility among the two moieties that are linked together. Such flexibility is generally increased if the amino acids are small. Accordingly, flexible peptide linkers comprise an increased content of small amino acids, in particular of glycins and/or alanines, and/or hydrophilic amino acids such as serines, threonines, asparagines and glutamines. Preferably, more than 20%, 30%, 40%, 50%, 60% or more of the amino acids of the peptide linker are small amino acids.

[0088] The term "cleavage site" as used herein refers to an amino acid sequence or nucleotide sequence wherein this sequence directs the division of a complex or a macromolecule (e.g. a nucleic acid or a protein), e.g. because it is recognized by a cleaving enzyme, and/or can be divided. Typically, a polypeptide chain is cleaved by hydrolysis of one or more peptide bonds that link the amino acids and a polynucleotide chain is cleaved by hydrolysis of one or more of the phosphodiester bond between the nucleotides. Cleavage of peptide- or phosphodiester-bonds may originate from chemical or enzymatic cleavage. Enzymatic cleavage refers to such cleavage being attained by proteolytic enzymes including but not limited to restriction endonuclease (e.g. type I, type II, type II, type IV or artificial restriction enzymes) and endo- or exo-peptidases or -proteases (e.g. serine-proteases, cysteine-proteases, metallo-proteases, threonine proteases, aspartate proteases, glutamic acid proteases). Typically, enzymatic cleavage occurs due to self-cleavage or is affected by an independent proteolytic enzyme. Enzymatic cleavage of a protein or polypeptide can happen either co- or post-translational. Accordingly, the term "endopeptidase cleavage site" used herein, refers to a cleavage cite within the amino acid or nucleotide sequence where this sequence is cleaved or is cleavable by an endopeptidase (e.g. trypsin, pepsin, elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8, cathepsins).

[0089] The term "self-cleavage site" as used herein refers to a cleavage site within the amino acid sequence where this sequence is cleaved or is cleavable without such cleavage involving any additional molecule. It is understood that cleavage sites typically comprise several amino acids. Thus, the cleavage site may also serve the purpose of a peptide linker, i.e. sterically separating two peptides or proteins.

[0090] As used herein, the term "vector" refers to a protein or a polynucleotide or a mixture thereof which is capable of being introduced or of introducing the proteins and/or nucleic acid comprised therein into a cell. In the context of the present invention it is preferred that the genes of interest encoded by the introduced polynucleotide are expressed within the cell upon introduction of the vector or vectors. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[0091] The term "complex" as used herein, refers to a whole that comprehends a number of individual components, parts or moieties which are in close proximity to each other and fulfil a common or interrelated function. The individual moieties of a complex may be of the same or of differing nature, i.e. they may be composed of the same, a similar or of differing chemical entities such as but not limited to nucleotides, amino acids, nucleic acids, peptides, polypeptides, proteins, carbohydrates, and/or lipids. Exemplified, a complex may comprise a number of associated proteins, or a mixture of one or more proteins and one or more nucleic acids or a mixture of one or more proteins and one or more lipids and/or carbohydrates. It is understood that any other combination of identical, similar or differing chemical entities is also encompassed. The individual moieties of a complex may or may not be interconnected. Typically, the individual parts of a complex are connected via covalent or non-covalent bonds.

[0092] The term "multimerization" as used herein refers to the formation of a macromolecular complex of two or more molecules, e.g. proteins or nucleic acids, (i.e. two, three, four, five or more molecules), whilst the term "dimerization" as used herein refers specifically to the formation of a macromolecular complex of two molecules, e.g. proteins or nucleic acids. A homodimer is formed by two identical molecules (so-called "homodimerization"), whilst a hetero-dimer is formed by two different macromolecules (so-called "heterodimerization"). Typically, in a "dimer", the two macromolecules are bound via non-covalent or covalent bonds, e.g. disulfid-bonds (R--S--S--R). In the context of the present invention, the MHD2 or the EHD2 may serve as a "dimerization tool" in that homodimers are formed between two MHD2 domains or between two EHD2 domains via disulfid-bonds between the two Cys337 of the two MHD2 domains or between the two Cys247 and Cys337 of the two EHD2 domains (Cys247 of one domain pairs with Cys337 of the other domain), respectively. Because of the central location of the MHD2 or EHD2 within the heavy chain of the IgM or IgE molecule, further modules can be fused to either the N- and/or the C-Terminus of these HD2 domains. These modules may comprise one or more pharmaceutically active moieties as defined above and/or below. Accordingly, the MHD2 and EHD2 are used as covalently linked "homodimerization module" allowing for the generation of multivalent and bifunctional molecules held together by the covalently linked dimerization domains.

[0093] The term "modular system" as used herein refers to a system subdivided into smaller parts ("modules") that can be independently created and then used in different systems to drive multiple functionalities. Typically, the individual modules are isolated, self-contained functional elements which are functionally partitioning into discrete scalable, reusable modules. In the context of the present invention, the term "modules" refers in particular to self-sufficient parts or separable component of a macromolecule, e.g. polynucleotide or polypeptide, or a complex. Typically, a module can evolve, function, and/or exist independently of the rest of the macromolecule or complex and consists of one or several domains with each of them being a three-dimensional structure being stably and independently folded. Such module typically forms an independent functional unit within the macromolecule or complex.

[0094] The terms "pharmaceutical", "medicament" and "drug" are used interchangeably herein, referring to a substance and/or a combination of substances being used for the identification, prevention or treatment of a disease or disorder.

[0095] The terms "preparation" and "composition" are intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with the active compound.

[0096] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0097] The term "active ingredient" refers to the substance in a pharmaceutical composition or formulation that is biologically active, i.e. that provides pharmaceutical value. A pharmaceutical composition may comprise one or more active ingredients which may act in conjunction with or independently of each other. The active ingredient can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as but not limited to those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as but not limited to those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0098] The term "carrier", as used herein, refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, surfactants, stabilizers, physiological buffer solutions or vehicles with which the therapeutically active ingredient is administered. Such pharmaceutical carriers can be liquid or solid. Liquid carrier include but are not limited to sterile liquids, such as saline solutions in water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0099] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0100] "Surfactants" include anionic, cationic, and non-ionic surfactants such as but not limited to sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, and polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0101] "Stabilizers" include but are not limited to mannitol, sucrose, trehalose, albumin, as well as protease and/or nuclease antagonists.

[0102] "Physiological buffer solution" include but are not limited to sodium chloride solution, demineralized water, as well as suitable organic or inorganic buffer solutions such as but not limited to phosphate buffer, citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4 (2 hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (3 morpholino-1 propanesulphonic acid). The choice of the respective buffer in general depends on the desired buffer molarity. Phosphate buffer are suitable, for example, for injection and infusion solutions.

[0103] The term "adjuvant" refers to agents that augment, stimulate, activate, potentiate, or modulate the immune response to the active ingredient of the composition at either the cellular or humoral level, e.g. immunologic adjuvants stimulate the response of the immune system to the actual antigen, but have no immunological effect themselves. Examples of such adjuvants include but are not limited to inorganic adjuvants (e.g. inorganic metal salts such as aluminium phosphate or aluminium hydroxide), organic adjuvants (e.g. saponins or squalene), oil-based adjuvants (e.g. Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g. IL-1.beta., IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-.gamma.) particulate adjuvants (e.g. immuno-stimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g. monophosphoryl lipid A, or muramyl peptides), synthetic adjuvants (e.g. non-ionic block copolymers, muramyl peptide analogues, or synthetic lipid A), or synthetic polynucleotides adjuvants (e.g. polyarginine or polylysine).

[0104] An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.

EMBODIMENTS

[0105] In a first aspect the present invention provides a polypeptide comprising a heavy chain domain 2 (HD2) from IgM or IgE and at least one pharmaceutically active moiety. The pharmaceutically active moiety is envisaged not to be a Fab or Fc fragment from IgM or IgE or a part thereof, i.e. the invention does not comprise naturally occurring IgM or IgE.

[0106] In preferred embodiments such polypeptide does not comprise one or more of the heavy chain constant domains C.sub.H1, C.sub.H3 and/or C.sub.H4 of the IgM or IgE isotypes, i.e. the polypeptide does not comprise C.sub.H1, C.sub.H3, C.sub.H4, C.sub.H1 and C.sub.H3, C.sub.H1 and C.sub.H4, C.sub.H3 and C.sub.H4, or C.sub.H1, C.sub.H3 and C.sub.H4.

[0107] It is particularly preferred that said polypeptide comprises only the heavy chain domain 2 C.sub.H2 (HD2) of IgE or IgM. Preferably, no additional part(s) of IgE or IgM are comprised. Accordingly, it is envisaged that said polypeptide comprises the HD2 domain of IgE or IgM as sole domain of IgE or IgM.

[0108] Preferably such polypeptide resembles a peptide-bond-linked chain of amino acids which may optionally be chemically modified, e.g. may comprise glycosylated amino acids and/or phosphorylated amino acids and/or may be PEGylated, HESylated, and/or polysialiated. In preferred embodiments the HD2 from IgM or IgE comprises one or more cysteine residues. It is particularly preferred that the HD2 of IgM (MHD2) comprises at least three cysteine residues, preferably two cysteine residues forming an intradomain disulfide bond and the third one allowing for the formation of interdomain disulfide bonds. In preferred embodiments these disulfide bonds improve the intradomain and/or interdomain stability of the MHD2. Preferably, these cysteine residues are located at positions 261, 321 and 337 of the MHD2 (EU numbering as in Kabat). In further embodiments, it is preferred that the HD2 of IgE (EHD2) comprises at least four cysteine residues, preferably two cysteine residues forming an intradomain disulfid bond and two cysteine residues allowing for the formation of interdomain disulfid bonds. In preferred embodiments these disulfide bonds improve the intradomain and/or interdomain stability of the EHD2. Preferably, these cysteine residues are located at positions 261 and 321, and at position 247 and 337, respectively, of the EHD2 (EU numbering as in Kabat).

[0109] In preferred embodiments, the HD2 domain comprises an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 or dimerizing variants thereof.

[0110] Preferably, the MHD2 comprises the amino acid sequence:

[0111] AELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTT DQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPD as given in SEQ ID NO: 1 or variants thereof.

[0112] Preferably, the EHD2 comprises the amino acid sequence: DFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQE GELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN as given in SEQ ID NO: 2 or dimerizing variants thereof.

[0113] In preferred embodiments a dimerizing variant has at least 70% sequence identity to the MHD2 or EHD2 of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. It is particularly preferred that a variant has at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the MHD2 or EHD2 of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. In particularly preferred embodiments a variant has at least 80%, at least 90% or at least 95% sequence identity to the MHD2 or EHD2 of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. It is preferred that said dimerizing variants exhibits at least one biological activity of the parent MHD2 or EHD2, i.e. is functionally active. It is particularly preferred that said dimerizing variant remains functionally active by being able to dimerize with a second MHD2 or EHD2 or dimerizing variant thereof.

[0114] In preferred embodiments, said dimerizing variants may exhibit amino acid exchanges, preferably conservative and/or non-conservative amino acid exchanges, amino acid deletions and/or amino acid additions. Preferably, these amino acid exchanges, deletions and/or additions are in regions of the sequence which are less or not conserved in orthologous or paralogous sequences. Preferably, conserved and/or highly conserved regions in the sequence are not altered. It is particularly preferred that the cysteine residues in MHD2 and EHD2, respectively, are maintained.

[0115] In an IgM or IgE molecule the HD2 occupies a central position within the heavy chain with further heavy chain sequences being connected at the C- and N-terminal ends of the HD2 domain. These connected heavy chain sequences do not influence, alter, or inhibit the ability of the HD2 to fulfil its function to dimerize. Accordingly, it is envisaged that further modules may be fused to the N- and/or C-Terminus of either HD2 domain without influencing, altering or inhibiting the ability of the HD2 to dimerize. Thus, both domains are suitable as anchor points in a modular system comprising further individual or a plurality of functionally distinct modules, such as but not limited to chemical and biological molecules (e.g. small chemical entities, peptides, proteins, protein domains, protein fragments, nucleic acids, or hybrids thereof). Preferably, said modules exhibit a size, surface charge, and function such as not to influence, alter or inhibit the function of the HD2 in dimerization. Modules which would interfere with the function of the HD2 may be fused via linker peptide to minimize and/or abolish the interfering effect. In preferred embodiments modules fused to the C- and/or N-Terminus of the HD2 of IgM or IgE comprise at least one pharmaceutically active moiety. Thus, in preferred embodiments at least one pharmaceutically active moiety is connected to the N- and/or C-Terminus of the HD2.

[0116] The number of fused modules, preferably comprising at least one pharmaceutically active moiety, may be 1 or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, at the N- and/or C-Terminus of the HD2. It is understood by the skilled person that the number of fused modules is primarily limited by the desired function of the resulting polypeptide as well as by functional or sterical limitations due to size or surface charge of the resulting polypeptide. Typically such resulting polypeptide is up to 1000 kDa in size, i.e. 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 kDa, but may even be bigger if suitable in the respective context.

[0117] In further embodiments, at least two modules, preferably comprising at least two pharmaceutically active moieties, are connected to either or both of the N- and/or C-Terminus of the HD2 of IgM or IgE. Each X and Y module, respectively, may be identical or different modules, preferably identical or different pharmaceutically active moieties. Thus, it is envisaged that X.sub.m modules are connected to the N-Terminus of the MHD2 or EHD2 domain and Y.sub.n modules are connected to the C-Terminus of the MHD2 or EHD2 domain, wherein m is preferably between 0 and 10, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n is preferably between 0 and 10, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, with the proviso that at least one of m and n is 1 or more. M and n may be identical or different.

[0118] Accordingly, such polypeptide may have one of the following structures: X.sub.m-MHD2-Y.sub.n, more preferably X.sub.1-MHD2, MHD2-Y.sub.1, X.sub.1-X.sub.2-MHD2, X.sub.1-[X].sub.m-1-MHD2, MHD2-Y.sub.1-Y.sub.2, MHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-MHD2-Y.sub.1, X.sub.1-X.sub.2-MHD2-Y.sub.1-Y.sub.2, X.sub.1-[X].sub.m-1-MHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-MHD2-Y.sub.1-Y.sub.2, X.sub.1-MHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-X.sub.2-MHD2-Y.sub.1, or X.sub.1-[X].sub.m-1-MHD2-Y.sub.1; X.sub.m-EHD2-Y.sub.n, more preferably X.sub.1-EHD2, EHD2-Y.sub.1, X.sub.1-X.sub.2-EHD2, X.sub.1-[X].sub.m-1-EHD2, EHD2-Y.sub.1-Y.sub.2, EHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-EHD2-Y.sub.1, X.sub.1-X.sub.2-EHD2-Y.sub.1-Y.sub.2, X.sub.1-[X].sub.m-1-EHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-EHD2-Y.sub.1-Y.sub.2, X.sub.1-EHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-X.sub.2-EHD2-Y.sub.1, or X.sub.1-[X].sub.m-1-EHD2-Y.sub.1 wherein m is preferably between 0 and 10, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n is preferably between 0 and 10, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, with the proviso that at least one of m and n is 1 or more. M and n may be identical or different. Particularly preferred combinations of m and n are the following: m=1 and n=0; m=0 and n=1, m=1 and n=1; m=1 and n=2; m=1 and n=3; m=1 and n=4; m=1 and n=5; m=1 and n=6; m=1 and n=7; m=1 and n=8; m=1 and n=9; m=1 and n=10; m=2 and n=1; m=2 and n=2; m=2 and n=3; m=2 and n=4; m=2 and n=5; m=2 and n=6; m=2 and n=7; m=2 and n=8; m=2 and n=9; m=2 and n=10; m=3 and n=1; m=3 and n=2; m=3 and n=3; m=3 and n=4; m=3 and n=5; m=3 and n=6; m=3 and n=7; m=3 and n=8; m=3 and n=9; m=3 and n=10; m=4 and n=1; m=4 and n=2; m=4 and n=3; m=4 and n=4; m=4 and n=5; m=4 and n=6; m=4 and n=7; m=4 and n=8; m=4 and n 9; m=4 and n=10; m=5 and n=1; m=5 and n=2; m=5 and n=3; m=5 and n=4; m=5 and n=5; m=5 and n=6; m=5 and n=7; m=5 and n=8; m=5 and n=9; m=5 and n=10; m=6 and n=1; m=6 and n=2; m=6 and n=3; m=6 and n=4; m=6 and n=5; m=6 and n=6; m=6 and n=7; m=6 and n=8; m=6 and n=9; m=6 and n=10; m=7 and n=1; m=7 and n=2; m=7 and n=3; m=7 and n=4; m=7 and n=5; m=7 and n=6; m=7 and n=7; m=7 and n=8; m=7 and n=9; m=7 and n=10; m=8 and n=1; m=8 and n=2; m=8 and n=3; m=8 and n=4; m=8 and n=5; m=8 and n=6; m=8 and n=7; m=8 and n=8; m=8 and n=9; m=8 and n=10; m=9 and n=1; m=9 and n=2; m=9 and n=3; m=9 and n=4; m=9 and n=5; m=9 and n=6; m=9 and n=7; m=9 and n=8; m=9 and n=9; m=9 and n=10; m=10 and n=1; m=10 and n=2; m=10 and n=3; m=10 and n=4; m=10 and n=5; m=10 and n=6; m=10 and n=7; m=10 and n=8; m=10 and n=9; m=10 and n=10.

[0119] The module, preferably the pharmaceutically active moiety, may be connected directly to the MHD2 or EHD2 or may be connected indirectly via one or more linkers (L). In embodiments wherein more than one module is connected to the HD2 domain, the individual modules may be connected directly to each other or may be connected indirectly via one or more linkers (L). Modules that interfere with the dimerization of MHD2 and EHD2, respectively, or with the function of a further connected module, are preferably connected indirectly via a linker. Similarly, it is preferred to use an indirect connection through a linker, if the dimerization of MHD2 and EHD2 interferes with the pharmaceutical activity of the pharmaceutically active moiety. Thus, in preferred embodiments at least one pharmaceutically active moiety is connected to the HD2 directly or indirectly via one or more linkers.

[0120] Preferably, the one or more linkers comprise peptide linkers which sterically separate the connected module, preferably the pharmaceutically active moiety, from the HD2 domain. In embodiments wherein more than one module, preferably pharmaceutically active moieties, is connected to the HD2 domain, linkers comprise peptide linkers which sterically separate the connected modules from the HD2 domain, and peptide linkers which sterically separate different connected module from one another.

[0121] Preferably, peptide linkers have a length between 5 and 40 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids), more preferably between 5 and 20 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids), most preferably 8 to 15 amino acids (i.e. 8, 9, 10, 11, 12, 13, 14, 15 amino acids). Linkers of suitable length allowing for the sterical seperation of fused modules from the HD2 domain or from further connected modules can be selected by the skilled person using routine methodology well-known in the art (Arai et al., 2001; George & Heringa et al., 2003; Wriggers et al., 2005; Tanaka et al., 2005).

[0122] Particularly preferred are flexible peptide linkers. Flexible linkers are composed of amino acids without bulky side chains that impede rotation or bending of the amino acid chain. Flexible linkers preferably comprise G, S, T, and A residues. Preferably at least 50% of the amino acids of the flexible linker peptide consists of amino acids selected from the group consisting of G, S, T, and A. More preferably at least 60%, 70%, 80%, 90%, 95% or 100% of the amino acids of the linker consists of amino acids selected from the group consisting of G, S, T, and A.

[0123] A large number of peptide linkers, suitable to sterically separate the HD2 domain from the module, preferably the pharmaceutically active moiety, are described in the art (Robinson & Sauer, 1998; Volkel et al., 2001; Kavoosi et al., 2007; Watanabe et al., 2011). Preferred peptide linkers include but are not limited to linker peptide 1: GGGGS (SEQ ID NO: 18), linker peptide 2: GGGGSGGGGS (SEQ ID NO: 19), linker peptide 3: GGGGSGGGGSGGGGS (SEQ ID NO: 20), linker peptide 4: GSLGGSGG (SEQ ID NO: 21), linker peptide 5: GGGSGGGT (SEQ ID NO: 22), linker peptide 6: GGGSGGGTGS (SEQ ID NO: 23), linker peptide 7: GGGSGGGTGSGG (SEQ ID NO: 24), linker peptide 8: GGGSGGGS (SEQ ID NO: 25), linker peptide 9: EFTRG (SEQ ID NO: 26), and linker peptide 10: AAA (SEQ ID NO: 27), or multimers, derivatives and fragments thereof.

[0124] In further preferred embodiments the one or more linkers comprise one or more cleavage sites, i.e. one or more sequence areas wherein the linker sequence may be chemically or enzymatically cleaved by division of one or more peptide-bonds. It is preferred that the cleavage site allows for the release of the pharmaceutically active moiety once the intended destination is reached. Enzymatic cleavage may be attained by proteolytic enzymes including but not limited to restriction endonuclease (e.g. type I, type II, type II, type IV or artificial restriction enzymes) and endo- or exo-peptidases or -proteases (e.g. serine-proteases, cysteine-proteases, metallo-proteases, threonine proteases, aspartate proteases, glutamic acid proteases). In particularly preferred embodiments the one or more cleavage sites comprise one or more endopeptidase cleavage sites, i.e. wherein the sequence is cleaved or is cleavable by an endopeptidase such as but not limited to trypsin, pepsin, elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8, and/or cathepsins.

[0125] It is envisaged that depending on how many modules are attached to the HD2 peptide linkers may be positioned between the C- and/or N-Terminus of the HD2 domain as well as between the different modules. In embodiments wherein more than one linker is present between different modules and/or the HD2 domain, these linkers may be identical or differ from each other. Accordingly, such polypeptide may have one of the following structures:

[0126] X.sub.1-L-MHD2, X.sub.1-X.sub.2-L-MHD2, X.sub.1-L-X.sub.2-L-MHD2, X.sub.1-L-X.sub.2-MHD2, X.sub.1-[X].sub.m-1-L-MHD2, X.sub.1-L-[X].sub.m-1-L-MHD2, X.sub.1-L-[X].sub.m-1-MHD2, Y.sub.1-L-MHD2, Y.sub.1-Y.sub.2-L-MHD2, Y.sub.1-L-Y.sub.2-L-MHD2, Y.sub.1-L-Y.sub.2-MHD2, Y.sub.1-[Y].sub.n-1-L-MHD2, Y.sub.1-L-[Y].sub.n-1-L-MHD2, Y.sub.1-L-[Y].sub.n-1-MHD2, X.sub.1-L-MHD2-L-Y.sub.1, X.sub.1-MHD2-L-Y.sub.1, X.sub.1-L-MHD2-Y.sub.1, X.sub.1-X.sub.2-L-MHD2-L-Y.sub.1-Y.sub.2, X.sub.1-X.sub.2-MHD2-L-Y.sub.1-Y.sub.2, X.sub.1-X.sub.2-L-MHD2-Y.sub.1-Y.sub.2, X.sub.1-L-X.sub.2-L-MHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-X.sub.2-L-MHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-MHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-MHD2-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-MHD2-L-Y.sub.1-Y.sub.2, X.sub.1-L-X.sub.2-MHD2-Y.sub.1-L-Y.sub.2, X.sub.1-X.sub.2-MHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-MHD2-Y.sub.1-Y.sub.2, X.sub.1-[X].sub.n-1-L-MHD2-L-Y.sub.1-[Y].sub.n-1, X.sub.1-[X].sub.m-1-MHD2-L-Y.sub.1-[Y].sub.n-1, X.sub.1-[X].sub.m-1-L-MHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-L-MHD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-[X].sub.m-1-L-MHD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-MHD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-X.sub.2-L-MHD2-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-MHD2-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-MHD2-Y.sub.1-L-[Y].sub.n-1, X.sub.1-[X].sub.m-1-MHD2-L-Y.sub.1-L-[Y].sub.n-1, or X.sub.1-L-[X].sub.m-1-L-MHD2-Y.sub.1-[Y].sub.m-1;

[0127] X.sub.1-L-EHD2, X.sub.1-X.sub.2-L-EHD2, X.sub.1-L-X.sub.2-L-EHD2, X.sub.1-L-X.sub.2-EHD2, X.sub.1-[X].sub.m-1-L-EHD2, X.sub.1-L-[X].sub.m-1-L-EHD2, X.sub.1-L-[X].sub.m-1-EHD2, Y.sub.1-L-EHD2, Y.sub.1-Y.sub.2-L-EHD2, Y.sub.1-L-Y.sub.2-L-EHD2, Y.sub.1-L-Y.sub.2-EHD2, Y.sub.1-[Y].sub.n-1-L-EHD2, Y.sub.1-L-[Y].sub.n-1-L-EHD2, Y.sub.1-L-[Y].sub.n-1-EHD2, X.sub.1-L-EHD2-L-Y.sub.1, X.sub.1-EHD2-L-Y.sub.1, X.sub.1-L-EHD2-Y.sub.1, X.sub.1-X.sub.2-L-EHD2-L-Y.sub.1-Y.sub.2, X.sub.1-X.sub.2-EHD2-L-Y.sub.1-Y.sub.2, X.sub.1-X.sub.2-L-EHD2-Y.sub.1-Y.sub.2, X.sub.1-L-X.sub.2-L-EHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-X.sub.2-L-EHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-EHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-EHD2-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-EHD2-L-Y.sub.1-Y.sub.2, X.sub.1-L-X.sub.2-EHD2-Y.sub.1-L-Y.sub.2, X.sub.1-X.sub.2-EHD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-EHD2-Y.sub.1-Y.sub.2, X.sub.1-[X].sub.m-1-EHD2-L-Y.sub.1-[Y].sub.n-1, X.sub.1-[X].sub.m-1-L-EHD2-Y.sub.1-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-L-EHD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-[X].sub.m-1-L-EHD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-EHD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-X.sub.2-L-EHD2-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-L-EHD2-L-Y.sub.1-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-EHD2-Y.sub.1-L-[Y].sub.n-1, X.sub.1-[X].sub.m-1-EHD2-L-Y.sub.1-L-[Y].sub.n-1, or X.sub.1-L-[X].sub.m-1-L-EHD2-Y.sub.1-[Y].sub.n-1. m and n have in each case the above indicated preferred and particularly preferred meanings.

[0128] In preferred embodiments the at least one pharmaceutically active moiety is a chemical pharmaceutical or a biological. In embodiments wherein the at least one pharmaceutically active moiety is a biological it is preferred that such biological is a peptide, polypeptide, protein and/or nucleic acid (e.g. DNA, RNA, or hybrids thereof). In particularly preferred embodiments such biological is selected from the group consisting of hormones (e.g. insulin, hGH, FSH, Glucagon-like peptide 1, parathyroid hormone, calcitonin, lutropin, glucagon); growth factors (e.g. erythropoietin, thrombopoetin, G-CSF/GM-CSF, IGF-1); cytokines (e.g. TNF, TRAIL, TGF-.beta.) such as interferons (e.g. IFN-.alpha., IFN-.beta., IFN-.gamma.) and interleukins (e.g. IL-2, IL-11, IL-1Ra); coagulation factors (e.g. factor VIII, factor IX, factor VIIa, thrombin); thrombolytics and anti-coagulants (e.g. t-PA, hirudin, activated protein C); enzymes (e.g. .alpha.-glucosidase, glucocerebrosidase, iduronate-2-sulfatase, galactosidase, urate oxidase, DNase); antigen-binding molecule such as antibodies and antibody fragments (e.g. IgG, Fab, Fc); and fusion proteins thereof (e.g. TNFR2-Fc, TMP-Fc, CTLA-4-Fc, IL-1R-Fc, LFA-3-Fc, IL-2-DT).

[0129] In further preferred embodiments, the at least one pharmaceutically active moiety is selected from the group consisting of ligands, effector molecules, half-life extension modules, and imaging molecules. Preferably, ligands are any chemical or biological substance that forms a complex with another molecule to fulfil a specific biological function such as substrates, inhibitors, and activators. More preferably, ligands include but are not limited to antigen-binding molecules, scaffold proteins, natural ligands (e.g. EGF, VEGF, PDGF, FGF, EPO, TPO, TGF-.beta., TNF, TRAIL), ligand-binding receptor fragments (e.g. TNFR1, TNFR2, VEGFR, CTLA-4, LFA-3, BR3, CD95R, IL-1R, FGFR1), and apatamers (e.g. anti-Thrombin, anti-FIXa, anti-C3b, anti-VEGF, anti-CD40L).

[0130] Preferably, scaffold proteins are regulators of key signalling pathways including but not limited to KSR, MEKK1, BCL-10, MAPK, AHNAK-1, HOMER, Pellino, NLRP, DLG1, Spinophilin, Plant FLU regulatory protein.

[0131] Preferably, the antigen-binding molecule is selected from the group consisting of an antibody fragment, a Fab fragment (excluding those from IgM or IgE), a Fab' fragment (excluding those from IgM or IgE), a heavy chain antibody, a single-domain antibody (sdAb), variable domain of a heavy chain antibody, VHH, Nanobodies, a single-chain variable fragment (scFv), a tandem scFv, a bispecific T-cell engager (BITEs), a diabody, a single-chain diabody, a DART molecule, a triple body, a nanoantibody, an alternative scaffold protein (e.g. DARPins, Anticalins, Affibody molecules, Microbodies, Monobodies, Fynomers, Adnetins, Tetranectins, Kunitz domains, Affilins, Avimers), and a fusion protein thereof. It is preferred that the antigen-binding molecule binds to an antigen that is pharmaceutically relevant, i.e. which is suitable to prevent, diagnose and/or treat a disease or the symptoms of a disease or disorder. In preferred embodiment the disease is a cancer type disease. Preferably, the antigen-binding molecule recognises a tumor-associated antigen such as but not limited to EGFR, HER2, HER3, HER4, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), CA-125, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), and abnormal products of ras and p53, estrogen receptors, 5-alpha-reductase, prostaglandin-endoperoxide synthase 2, VEGFRs, integrin receptor family, fibroblast activation protein, galectin, EpCAM, CEA, CD44, CD44v, CD2, CD5, CD7, CD19, CD20, CD21, CD22, CD24, CD25, CD30, CD33, CD38, CD40, CD52, CD56, CD71, CD72, CD73, CD105, CD117, CD123, claudins, c-Met, PDGFR, IGF1-R, HMW-MAA, TAG-72, GD2, GD3, GM2, folate receptor, L&', MUC-1, MUC-2, PSMA, PSCA and uPAR. In preferred embodiments the antigen-binding molecule is envisaged not to be a Fab or Fc fragment from IgM or IgE.

[0132] In particularly preferred embodiments the antigen-binding molecule is a scFv, preferably an anti-HER2 scFv or an anti-EGFR scFv, more preferably according to SEQ ID NO: 3 or 4, or variants thereof.

[0133] In preferred embodiments, effector molecules, i.e. small molecules, peptides or polypeptides that bind to a protein and thereby alter the activity of that protein, include but are not limited to cytokines, chemokines, immuno(co)-stimulatory molecules, immunosuppressive molecules, death ligands, apoptosis-inducing proteins, enzymes (e.g. kinases) prodrug-converting enzymes, RNases, agonistic antibody or antibody fragment, antagonistic antibody or antibody fragment, toxins, growth factors, hormone, coagulation factor, fibrinolytic protein, peptides mimicking these, and fragments, fusion proteins or derivatives thereof.

[0134] In preferred embodiments, cytokines are interleukins and/or interferons. Interleukins (IL) include but are not limited to Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9, Interleukin-10, Interleukin-11, Interleukin 12, Interleukin-13, Interleukin-14, Interleukin-15, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, Interleukin-24, Interleukin-25, Interleukin-26 Interleukin-27, Interleukin-28, Interleukin-29, Interleukin-30, Interleukin-31, Interleukin-32, Interleukin-33, Interleukin-34 and Interleukin-35. Interferons (IFN) include but are not limited to interferon type I (e.g. IFN-.alpha., IFN-.beta. and IFN-.omega.), interferon type II (e.g. IFN-.gamma.), and interferon type III. In particular included are interferon A1, interferon A2, interferon A4, interferon A5, interferon A6, interferon A7, interferon A8, interferon A10, interferon A13, interferon A14, interferon A16, interferon A17, interferon A21, interferon B1, TNF, TRAIL, and FasL.

[0135] In preferred embodiments chemokines include but are not limited to CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines. In particular chemokine include but are not limited to CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1.

[0136] In preferred embodiments, immuno-(co)stimulatory proteins include but are not limited to B7.1, B7.2, 4-1BBL, LIGHT, ICOSL, GITRL, CD40L, OX40L, and CD70.

[0137] Immuno-suppressive proteins preferably include but are not limited to IL1-Ra, IL-10, CTLA-4, PD-L1, and PD-L2, and toxins preferably include but are not limited to Pseudomonas exotoxin A, Diphtheria toxin and ricin.

[0138] In preferred embodiments apoptosis-inducing proteins include but are not limited to Bid, Bik, Puma, and Bim, and proapoptotic cytokines (death ligands) such as but not limited to TNF, scTNF, TRAIL, scTRAIL, and FasL.

[0139] In preferred embodiments enzymes include but are not limited to oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. Kinases include but are not limited to AGC kinases such as PKA, PKC and PKG, CaM kinases such as calcium/calmodulin-dependent protein kinases and serine/threonine protein kinases (e.g. DAPK2), CK1 such as the casein kinase 1 group, CMGC such as CDK, MAPK, GSK3 and CLK kinases, STE such as homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases, tyrosine kinases (TK), the tyrosine-kinase like group of kinases (TKL), receptor-associated tyrosine kinases, MAP kinases, and histidine kinases.

[0140] Pro-drug-converting enzymes include but are not limited to esterases such as but not limited to acetylesterase, thiolester hydrolases, phosphoric monoester hydrolases, phosphoric diester hydrolases, triphosphoric monoester hydrolases, sulfuric ester hydrolases (sulfatases), diphosphoric monoester hydrolases, and phosphoric triester hydrolases; phosphatases such as but not limited to tyrosine-specific phosphatases, serine/threonine specific phosphatases, dual specificity phosphatases, histidine phosphatase, and lipid phosphatase; and reductases such as but not limited to 5-alpha reductase, dihydrofolate reductase, HMG-CoA reductase, methemoglobin reductase, ribonucleotide reductase, thioredoxin reductase, E. coli nitroreductase, methylenetetrahydrofolate reductase, and carboxypeptidase G2, cytosine deaminase, nitroreductase, thymidine kinase.

[0141] RNAses include endoribonucleases such as but are not limited to RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V1, and RNase V, and exoribonucleases such as but not limited to Polynucleotide Phosphorylase (PNPase), RNase PH, RNase II, RNase R, RNase D, RNase T, Oligoribonuclease Exoribonuclease I, and Exoribonuclease II.

[0142] Agonistic antibodies or antibody fragments include those that cause an action in a tissue, organ or individual such as but not limited to receptor-signalling, gene expression, protein synthesis, and protein degradation, e.g. directed against TRAIL receptors, anti-glucocorticoid-induced tumor necrosis factor family receptor (GITR), and CD40. Typically, Agonistic antibody or antibody fragment act by binding to the active site or to allosteric sites of a receptor molecule thereby, triggering a specific reaction.

[0143] Antagonistic antibodies or antibody fragments include those blocking the action of an agonist. Typically, antagonistic antibodies or antibody fragments act by binding to the active site or to allosteric sites of a receptor molecule, or interact with unique binding sites not normally involved in the regulation of the activity of the receptor, e.g. anti-CTLA-4, anti-TNFR1, anti-VEGFR, anti-PDGFR, anti-EGFR, anti-Her2. Typically, an antagonistic antibody or antibody fragment competes with the agonist at structurally-defined binding sites or alters the binding site of the agonist in a manner that the agonist is not able to cause the action it would normally cause due to its binding.

[0144] In preferred embodiments growth factors include but are not limited to Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha (TGF-.alpha.), Transforming growth factor beta (TGF-.beta.), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, and placental growth factor (PlGF).

[0145] In preferred embodiments, coagulation factors include but are not limited to Thrombin, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII and Factor XIII, and active fragments thereof.

[0146] In preferred embodiments fibrinolytic proteins include but are not limited to plasmin, urokinase, plasminogen, .alpha.2-antiplasmin, tissue-plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1).

[0147] In particularly preferred embodiments, the cytokine is the tumor-necrosis factor (TNF), more preferably according to SEQ ID NO: 5 or variants thereof. In further preferred embodiments the cytokine is the TNF-relative apoptosis-inducing factor (TRAIL), more preferably according to SEQ ID NO: 6 or variants thereof.

[0148] Mimicking peptides and proteins include peptides and proteins which mimic activities of other peptides or proteins, in particular of peptides or proteins named herein above or below, such as but not limited to thrombopoietin-mimetic peptides, erythropoietin-mimetic peptides.

[0149] In further embodiments, half-life extension modules are chemical or biological substances that alter the half-life, e.g. the "plasma half-life" or the "serum half-life", of the polypeptide of the present invention. Preferably, the half-life extension module is selected from the group consisting of immunoglobulin binding domains (IgBD), albumin, albumin-binding domains (ABD), peptides, small molecules, fatty acids, antibody fragments, single-domain antibodies, VHH, scaffold proteins, and natural ligands exhibiting affinity for a long-circulating plasma protein, either of which are optionally PEGylated, HESylated, Polysialylated, N-glycosylated, O-glycosylated, or PEG-mimicking polypeptides. Preferably, an IgBD may bind to any of the domains of an Ig molecule, i.e. to the variable domains VH or VL and/or to the constant domains CH1, CH2, CH3 CH4 and/or CL of an Ig molecule. IGBDs include but are not limited to domains derived from protein A (SpA) of Staphylococcus aureus, streptococcal protein G (SpG), protein L (PpL) from peptostreptococcus magnus, protein Eib from Escherichia coli, protein Sbi from Staphylococcus, and streptococcal proteins MAG, MIG, H, M and ZAG.

[0150] In further embodiments, imaging molecules are those binding to specific target molecules thereby, allowing the visualization of the location of that molecule. Preferably, the imaging molecule is selected from the group consisting of bioluminescent reagents, chemiluminescent reagents, fluorescent imaging reagents, photosensitizers, chelating reagents, and radioactive moieties.

[0151] Imaging molecule include bioluminescent, chemiluminescent and fluorescent imaging reagent such as but not limited to luciferase from Renilla reniformis and/or Metridia Longa, peroxalate, polymethines (e.g. cyanine dyes such as Cy3, Cy5, Cy5.5, Cy7) squaraine derivatives, phthalocyanine, porphhyrin derivatives, and BODIPY analogous (BODIPY FL, BODIPY R6G, BODIPY TR, BODIPY TMR, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), as well as fluorescent proteins such as but not limited to GFP, EGPF, CFP, BFP, YFP, DsRED (Chudakov et al. (2010) Physiol. Rev. 90:1103-1163).

[0152] Chelating reagents are capable of binding at least one metal ion, such as but not limited to calcium, magnesium, iron, aluminium, zinc, copper, arsenic, lead, thallium, and mercury ions, by chelation. Such chelating reagents may comprise ethylenediamine tetraacetic acid (EDTA), ethylenediamine tetraacetic acid (calcium disodium versante) (CaNa2-EDTA), dimercaprol (BAL), dimercaptosuccinic acid (DMSA), dimercapto-propane sulfonate (DMPS), ferritin, deferoxamine and deferasirox, deferiprone (1,2-dimethyl-3-hydroxyl-4-pyridinone), DOTA, DTPA, DADT, DADS, DO3A, N2S2MAMA, Triamidethiol, phosphonates, organic gadolinium complexes, penicillamine, and antibiotic drugs of the tetracycline family.

[0153] In preferred embodiments the radioactive moiety comprises a radionuclide. The radioactive moiety may be an isotope of F, Br, Mn, Co, Ga, As, Zr, P, C, S, H, I, In, Lu, Cu, Rh, Bi, At, Y, Re, Ac, Tc, or Hg atom. The radioactive moiety labels polypeptide of the present invention radioactively allowing for its detection, e.g in the human body, rendering it not only useful for diagnostic approaches (radioimmunodetection: RAID) but also suitable in therapeutic applications (radioimmunotherapy: RAIT).

[0154] Photosensitizers are chemical compounds capable of light emission or formation of free radicals and singlet oxigen after being excited by light of a specific wavelength. Photosensitizers are used e.g. for photodynamic therapy. In preferred embodiments photosensitizers include but are not limited to compounds of the porphyrin family, texaphyrin family, the chlorin family and the phthalocyanine family, in particular including HpD, ALA, M-ALA, Vertiporfin, Lutexaphyrin, Temoporfin, Talaporfin, HPPH, Phthalocyanine, and Napthalocyanine.

[0155] In particularly preferred embodiments the polypeptide comprises an MHD2 domain to which C-Terminus a scFv.sub.EGFR is fused and/or to which N-Terminus a scFV.sub.HER2 is fused. In further preferred embodiments a scTRAIL or a scTNF is fused to the C- and/or N-Terminus of the MHD2 domain. In particularly preferred embodiments, scFv.sub.EGFR is fused to the N-terminus of MHD2 and scFv.sub.HER2 is fused to the C-Terminus of MHD2. In further preferred embodiments scFv.sub.EGFR is fused to the N-terminus of MHD2 and scTNF is fused to the C-Terminus of MHD2. In further preferred embodiments scFv.sub.EGFR is fused to the N-terminus of MHD2 and scTRAIL is fused to the C-Terminus of MHD2. In further preferred embodiments scDb.sub.EpCAMxEGFR is fused to the N-terminus of MHD2 and scTRAIL is fused to the C-Terminus of MHD2. Highly preferred are polypeptides according to SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14.

[0156] In particularly preferred embodiments the polypeptide comprises an EHD2 domain to which C-Terminus a scFv.sub.EGFR is fused and/or to which N-Terminus a scFV.sub.HER2 is fused. In further preferred embodiments a scTRAIL or a scTNF is fused to the C- and/or N-Terminus of the EHD2 domain. In particularly preferred embodiments, scFv.sub.EGFR is fused to the N-terminus of EHD2 and scFv.sub.HER2 is fused to the C-Terminus of EHD2. In further preferred embodiments scFv.sub.EGFR is fused to the N-terminus of EHD2 and scTNF is fused to the C-Terminus of EHD2. In further preferred embodiments scFv.sub.EGFR is fused to the N-terminus of EHD2 and scTRAIL is fused to the C-Terminus of EHD2. In further preferred embodiments scDb.sub.EpCAMxEGFR is fused to the N-terminus of EHD2 and scTRAIL is fused to the C-Terminus of EHD2. Highly preferred are polypeptides according to SEQ ID NO: 15, 16, or 17.

[0157] In a second aspect, the present invention provides a nucleic acid molecule comprising a sequence encoding the polypeptide of the first aspect of the present invention. Preferably, such nucleic acid molecule comprises a DNA and/or RNA molecule.

[0158] In a third aspect the present invention provides a vector comprising the polynucleotide of the second aspect of the present invention. It is understood that suitable vectors include but are not limited to plasmids, cosmids, phages, viruses and/or artificial chromosomes.

[0159] In a fourth aspect the present invention provides a complex comprising at least two polypeptides of the first aspect of the present invention. In preferred embodiments the at least two polypeptides are connected via their HD2 domains, preferably via covalent or non-covalent bonds. It is particularly preferred that the covalent bond is a disulfide bond which is preferably formed between two cysteine residues, each residing within the HD2 domain of the respective polypeptide. The at least two polypeptides forming the complex may comprise the same or different HD2 domains. It is particularly preferred that the at least two polypeptides forming the complex comprise the same HD2, i.e. the at least two polypeptides forming a complex comprise an MHD2 or the at least two polypeptides forming a complex comprise an EHD2.

[0160] The at least two polypeptides may be identical or different with regard to the modules fused to the N- and/or C-Terminus of the HD2 domain or with regard to the linker peptides connecting the HD2 domains and the fused modules, i.e. they may comprise identical or differing modules, preferably pharmaceutically active moieties, attached to their HD2 domain and may comprise different peptide linkers connecting different modules, as described in detail above. Thus, in preferred embodiments the complex comprises at least two polypeptides of the following structure, wherein the at least two polypeptides are identical or different:

[0161] X.sub.1-HD2, HD2-Y.sub.1, X.sub.1-X.sub.2-HD2, X.sub.1-[X].sub.m-1-HD2, HD2-Y.sub.1-Y.sub.2, HD2-Y.sub.1-[Y].sub.n-1, X.sub.1-HD2-Y.sub.1, X.sub.1-X.sub.2-HD2-Y.sub.1-Y.sub.2, X.sub.1-[X].sub.m-1-HD2-Y.sub.1-[Y].sub.n-1, X.sub.1-HD2-Y.sub.1-Y.sub.2, X.sub.1-HD2-Y.sub.1-Y.sub.n, X.sub.1-X.sub.2-HD2-Y.sub.1, X.sub.1-[X].sub.m-1-HD2-Y.sub.1, X.sub.1-L-HD2, X.sub.1-X.sub.2-L-HD2, X.sub.1-L-X.sub.2-L-HD2, X.sub.1-L-X.sub.2-HD2, X.sub.1-[X].sub.m-1-L-HD2, X.sub.1-L-[X].sub.m-1-L-HD2, X.sub.1-L-[X].sub.m-1-HD2, Y.sub.1-L-HD2, Y.sub.1-Y.sub.2-L-HD2, Y.sub.1-L-Y.sub.2-L-HD2, Y.sub.1-L-Y.sub.2-HD2, Y.sub.1-[Y].sub.n-1-L-HD2, Y.sub.1-L-[Y].sub.n-1-L-HD2, Y.sub.1-L-[Y].sub.n-1-HD2, X.sub.1-L-HD2-L-Y.sub.1, X.sub.1-HD2-L-Y.sub.1, X.sub.1-L-HD2-Y.sub.1, X.sub.1-X.sub.2-L-HD2-L-Y.sub.1-Y.sub.2, X.sub.1-X.sub.2-HD2-L-Y.sub.1-Y.sub.2, X.sub.1-X.sub.2-L-HD2-Y.sub.1-Y.sub.2, X.sub.1-L-X.sub.2-L-HD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-X.sub.2-L-HD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-HD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-HD2-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-HD2-L-Y.sub.1-Y.sub.2, X.sub.1-L-X.sub.2-HD2-Y.sub.1-L-Y.sub.2, X.sub.1-X.sub.2-HD2-L-Y.sub.1-L-Y.sub.2, X.sub.1-L-X.sub.2-L-HD2-Y.sub.1-Y.sub.2, X.sub.1-[X].sub.m-1-L-HD2-L-Y.sub.1-[Y].sub.n-1, X.sub.1-[X].sub.m-1-HD2-L-Y.sub.1-[Y].sub.n-1, X.sub.1-[X].sub.m-1-L-HD2-Y.sub.1-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-L-HD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-[X].sub.m-1-L-HD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-HD2-L-Y.sub.1-L-[Y].sub.n-1, X.sub.1-L-X.sub.2-L-HD2-Y.sub.1-L-[Y].sub.m-1, X.sub.1-L-[X].sub.m-1-L-HD2-L-Y.sub.1-[Y].sub.n-1, X.sub.1-L-[X].sub.m-1-HD2-Y.sub.1-L-[Y].sub.n-1, X.sub.1-[X].sub.m-1-HD2-L-Y.sub.1-L-[Y].sub.n-1, or X.sub.1-L-[X].sub.m-1-L-HD2-Y.sub.1-[Y].sub.n-1. m and n have in each case the above indicated preferred and particularly preferred meanings.

[0162] In particularly preferred embodiments, complexes are formed between two polypeptides each of which comprises an MHD2 domain to which C-Terminus a scFv.sub.EGFR is fused and/or to which N-Terminus a scFV.sub.HER2 is fused; an MHD2 domain to which C- and/or N-Terminus a scTRAIL or a scTNF is fused; an MHD2 to which N-Terminus a scFv.sub.EGFR is fused and to which C-Terminus a cFv.sub.HER2 is fused, an MHD2 domain to which N-terminus scFv.sub.EGFR is fused and to which C-Terminus scTNF is fused; an MHD2 to which N-terminus scFv.sub.EGFR is fused and to which C-Terminus scTRAIL; and an MHD2 to which N-terminus scDb.sub.EpCAMxEGFR is fused and to which C-Terminus scTRAIL. Highly preferred are complexes formed between two polypeptides according to SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14.

[0163] In particularly preferred embodiments, complexes are formed between two polypeptides each of which comprises an EHD2 domain to which C-Terminus a scFv.sub.EGFR is fused and/or to which N-Terminus a scFV.sub.HER2 is fused; an EHD2 domain to which C- and/or N-Terminus a scTRAIL or a scTNF is fused; an EHD2 to which N-Terminus a scFv.sub.EGFR is fused and to which C-Terminus a scFv.sub.HER2 is fused, an EHD2 domain to which N-terminus scFv.sub.EGFR is fused and to which C-Terminus scTNF is fused; an EHD2 to which N-terminus scFv.sub.EGFR is fused and to which C-Terminus scTRAIL; and an EHD2 to which N-terminus scDb.sub.EpCAMxEGFR is fused and to which C-Terminus scTRAIL. Highly preferred are complexes formed between two polypeptides according to SEQ ID NO: 15, 16, or 17.

[0164] In a fifth aspect the present invention provides a cell comprising the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, or the complex of the fourth aspect. It is understood that such cell includes but is not limited to prokaryotic (e.g. a bacterial cell) or eukaryotic cells (e.g. a fungal, plant or animal cell).

[0165] In a sixth aspect the present invention provides a composition comprising the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the complex of the fourth aspect, or the cell of the fifth aspect and a pharmaceutical acceptable carrier and/or excipient. Preferably, such composition is a pharmaceutical composition.

[0166] In preferred embodiments the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient and optionally one or more additional active substances. Preferably, the composition of the fifth aspect contains a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier and/or excipient so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0167] The pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0168] For preparing pharmaceutical compositions of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form compositions include powders, tablets, pills, capsules, lozenges, cachets, suppositories, and dispersible granules. A solid excipient can be one or more substances, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the excipient is preferably a finely divided solid, which is in a mixture with the finely divided inhibitor of the present invention. In tablets, the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. Suitable excipients are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0169] Liquid form compositions include solutions, suspensions, and emulsions, for example, water, saline solutions, aqueous dextrose, glycerol solutions or water/propylene glycol solutions. For parenteral injections (e.g. intravenous, intraarterial, intraosseous infusion, intramuscular, subcutaneous, intraperitoneal, intradermal, and intrathecal injections), liquid preparations can be formulated in solution in, e.g. aqueous polyethylene glycol solution. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0170] Preferably, the pharmaceutical composition is in unit dosage form. In such form the composition may be subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged composition, the package containing discrete quantities of the composition, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, an injection vial, a tablet, a cachet, or a lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0171] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0172] Furthermore, such pharmaceutical composition may also comprise other pharmacologically active substance such as but not limited to adjuvants and/or additional active ingredients. Adjuvants in the context of the present invention include but are not limited to inorganic adjuvants, organic adjuvants, oil-based adjuvants, cytokines, particulate adjuvants, virosomes, bacterial adjuvants, synthetic adjuvants, or synthetic polynucleotides adjuvants.

[0173] In a seventh aspect, the present invention provides the polypeptide of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the complex of the fourth aspect, or the cell of the fifth aspect as described in detail above for use as a medicament. In preferred embodiments the complex is for use in medicine, i.e. for use in the prophylaxis, treatment or diagnosis of a disorder or disease such as but not limited to autoimmune diseases, allergic diseases, cancer type diseases, cutaneous conditions, endocrine diseases, eye diseases and disorders, genetic disorders, infectious diseases, intestinal diseases, neurological disorders, and mental illness. Exemplified, autoimmune diseases include but are not limited to Diabetes mellitus type 1, rheumatoid arthritis, psoriasis, Crohns Disease, autoimmune cardiomyopathy, autoimmune hepatitis, Hashimoto's thyroiditis, and Sjogern's syndrome. Exemplified, allergic diseases include but are not limited to allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom allergies, drug allergies, and food allergies. Exemplified, cancer type diseases include but are not limited to Basal cell carcinoma, Bladder cancer, Bone cancer, Brain tumor, Breast cancer, Burkitt lymphoma, Cervical cancer, Colon Cancer, Cutaneous T-cell lymphoma, Esophageal cancer, Retinoblastoma, Gastric (Stomach) cancer, Gastrointestinal stromal tumor, Glioma, Hodgkin lymphoma, Kaposi sarcoma, Leukemias, Lymphomas, Melanoma, Oropharyngeal cancer, Ovarian cancer, Pancreatic cancer, Pleuropulmonary blastoma, Prostate cancer, Throat cancer, Thyroid cancer, and Urethral cancer. Exemplified, cutaneous conditions include but are not limited to Acne, Dermatitis, Eczema, conditions of the skin appendages, conditions of the subcutaneous fat, disturbances of pigmentation, epidermal nevi, epidermal neoplasms, epidermal cysts, erythemas, frostbites genodermatoses, mucinoses, neurocutaneous conditions (e.g. Wiskott-Aldrich syndrome), and psoriasis. Exemplified, endocrine diseases include but are not limited to Diabetes mellitus type 1 and type 2, Osteoporosis, and Cushing's disease. Exemplified, genetic disorders include but are not limited to color blindness, cystic fibrosis, Down syndrome, Sickle-cell disease, and Turner syndrome. Exemplified, infectious diseases include but are not limited to infections diseases caused by viruses, bacteria, worms, prions or other pathogens or parasites such as African sleeping sickness, AIDS, HIV infection, Anthrax, Borreliosis, Calicivirus infection (Norovirus and Sapovirus), Chickenpox, Chlamydia infection, Cholera, Clostridium infection, Colorado tick fever (CTF), common cold, Creutzfeldt-Jakob disease, Dengue fever (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola, Enterovirus infection, infections with Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Gonorrhea, Streptoccocal infections (group A and B), Hand, foot and mouth disease (HFMD), Helicobacter pylori infection, Hepatitis (A, B, C, and D), Herpes infection, Papillomavirus infection, Parainfluenza virus infection, Influenza, Lassa fever, Marburg fever, Measles, Meningitis, Mumps, Pasteurellosis, Pediculus infection, Plague, Pneumococcal infection, Respiratory syncytial virus infection, Rotavirus infection, Rubella virus infection, Salmonella food poisoning and infection, SARS, Scabies infections, Schistosomiasis, Smallpox, Staphylococcal food poisoning and infection, Syphilis, Tetanus, Trichophyton infection, Tuberculosis, Typhus, Venezuelan equine encephalitis, and Yellow fever. Exemplified, intestinal diseases include but are not limited to Gastroenteritis, Ileus, Ileitis, Colitis, Appendicitis, Coeliac disease, Irritable bowel syndrome, Diverticular disease, Diarrhea, Polyp, and Ulcerative colitis. Exemplified, neurological disorders include but are not limited to Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Brain damage, Creutzfeldt-Jakob disease, Cushing's syndrome, Dyslexia, Encephalitis, Epilepsy, Headache, Huntington's disease, Migraine, Multiple sclerosis, Parkinson's disease, Polio, Rabies, Schizophrenia, and Stroke. Exemplified, mental illness include but are not limited to Acute stress disorder, attention-deficit hyperactivity disorder (ADHD), Autistic disorder, Borderline personality disorder, Bulimia nervosa, Burn Out, Schizophrenia, Depression, Cognitive disorder, Communication disorder, Eating disorder, Kleptomania, Learning disorders, Male erectile disorder, Melancholia, Obsessive-compulsive disorder (OCD), Paranoia Pathological gambling, Posttraumatic stress disorder (PTSD), Psychotic disorder, Hypersomnia, Insomnia, and Tourette's syndrome.

[0174] The following examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

EXAMPLES

Example 1

Production of scFv-MHD2 Fusion Proteins

[0175] A humanized anti-EGFR scFv (hu225) was generated from the antibody C225 (Goldstein et al., 1995) by CDR grafting. The anti-HER2 scFv 4D5 was reproduced from published sequences (Carter et al., 1992). Both scFvs as well as the sequence of the human IgM heavy chain domain 2 (MHD2) were condon-optimized for expression in human cells and synthesized by Geneart, now a Life Technologies subsidiary (Darmstadt, Germany), adding appropriate cloning sites. Two bivalent antibody-MHD2 fusion proteins were generated fusing either a humanized anti-EGFR scFv to the N-terminus of the MHD2 (scFvEGFR-MHD2; scFvA-MHD2) or an anti-HER2 scFv to the C-terminus of the MHD2 (MHD2-scFvHER2; MHD2-scFvB), respectively. In addition, a tetravalent, bispecific fusion protein was produced fusing scFvA to the N-terminus and scFvB to the C-terminus of the MHD2 (scFvEGFR-MHD2-scFvHER2; scFvA-MHD2-scFvB) (FIG. 3a, b). All constructs were cloned into the eukaryotic expression vector pSecTagA and produced in stably transfected HEK293 cells with yields in the range of 1.5 to 2.5 mg/L supernatant. SDS-PAGE analysis revealed under reducing conditions the expected molecular masses of the monomeric polypeptide chains (approximately 50 kDa for monospecific MHD2 fusion proteins and 75 kDa for bispecific MHD2 fusion protein) taking into account the presence of one potential N-glycosylation site in the MHD2 (see FIG. 3c). Under nonreducing conditions all constructs showed a band corresponding to dimeric molecules, although a second band with the size of monomers were observed. For the monospecific MHD2 fusion proteins, approximately 80-90% of the molecules were present as disulfide-linked dimers, while for the bispecific MHD2 fusion protein approximately 50% were disulfide-linked.

Example 2

Bioactivity of scFv-MHD2 Fusion Proteins

[0176] Selectivity of the antibody-MHD2 fusion proteins was analyzed by ELISA using Fc fusion proteins of the extracellular region of EGFR, HER2, and HER3, respectively (FIG. 4a). scFv.sub.EGFR-MHD2 showed a specific binding to EGFR and MHD2-scFv.sub.HER2 to HER2, while the scFv.sub.EGFR-MHD2-scFv.sub.HER2 fusion protein showed binding to both receptors. No binding was observed for the HER3-Fc fusion protein, included as negative control. Furthermore, the antibody-MHD2 fusion proteins were analyzed by flow cytometry for binding to different tumor cell lines expressing various amounts of EGFR and HER2 (FIG. 4b). The EGFR-expressing cell line A431 showed strong binding of scFv.sub.EGFR-MHD2 and scFv.sub.EGFR-MHD2-scFv.sub.HER2, while no or only marginal binding was detected for MHD2-scFv.sub.HER2 (FIG. 4b). In contrast, the HER2-positive cell line SKBR3 showed strong binding of MHD2-scFv.sub.HER2 and scFv.sub.EGFR-MHD2-scFv.sub.HER2 and only weak binding of scFv.sub.EGFR-MHD2 (FIG. 4b). The lung carcinoma cell line NCI-H460, expressing low amounts of EGFR and HER2, showed weak binding of scFv.sub.EGFR-MHD2 and MHD2-scFv.sub.HER2 but an increased binding of scFv.sub.EGFR-MHD2-scFv.sub.HER2 (FIG. 4b). Similar results were observed for the colon carcinoma cell line Colo205 (FIG. 4b). Except for SKBR3, an increased binding was observed for the bispecific MHD2 fusion protein compared with the two monospecific MHD2 fusion proteins.

Example 3

Production MHD2-scTNF Fusion Proteins

[0177] An antibody-TNF MHD2 fusion protein was generated fusing the anti-EGFR scFv to the N-terminus of the MHD2 and a single-chain TNF derivative (scTNF; Krippner-Heidenreich et al., 2008) to the C-terminus of the MHD2 (scFv-MHD2-scTNF). Furthermore, a bivalent cytokine-MHD2 molecule was generated lacking the scFv (MHD2-scTNF) (FIG. 5a, b). The two constructs were produced in HEK293 cells with yields of 5 to 12 mg/L supernatant. SDS-PAGE showed under reducing conditions single bands corresponding to the molecular mass of the monomeric polypeptides (70 kDa for MHD2-scTNF and 100 kDa for scFv-MHD2-scTNF). Dimeric assembly was seen under non-reducing conditions with approximately 40% of the MHD2-scTNF and approximately 95% of the scFv-MHD2-scTNF present as dimers (FIG. 5c). Dimeric assembly was confirmed by size exclusion chromatography (SEC) (FIG. 5d-f). ScTNF included for comparison eluted at a major peak with an apparent molecular mass of approximately 50 kDa. The MHD2-scTNF molecule revealed a major peak of approximately 200 kDa and the scFv-MHD2-scTNF showed a major peak corresponding to approximately 300 kDa. Melting points were also determined for scFv-MHD2-scTNF indicated a major melting point, at approximately 77.degree. C. (FIG. 5g).

Example 4

Bioactivity of the MHD2-scTNF Fusion Proteins

[0178] In ELISA, the scFv-MHD2-scTNF fusion protein showed specific binding to an EGFR-Fc fusion protein, while no binding was observed for MHD2-scTNF (FIG. 6a). Binding to EGFR was further confirmed by flow cytometry with EGFR-expressing cells lines A431 and HT1080 (FIG. 6b). Binding of scFv-MHD2-scTNF to both cell lines could be blocked by pre-incubation with cetuximab. No or only marginal effects were observed with trastuzumab (anti-HER2).

[0179] Next, the fusion proteins were tested for triggering cell death on mouse embryonic fibroblasts (MEF) stably transfected to express either the extracellular region of human TNFR1 (MEF-TNFR1) or TNFR2 (MEF-TNFR2) fused to the transmembrane and cytoplasmic region of Fas (Krippner-Heidenreich et al., 2002). These cell lines allow to discriminate between the action of soluble TNF and membrane-bound TNF (mTNF), with mTNF or multimeric TNF required to active MEF-TNFR2. A titration of scTNF, MHD2-TNF and scFv-MHD2 showed a strong cytotoxic activity of scTNF and MHD2-scTNF on MEF-TNFR1, while on MEF-TNFR2 cell killing was only induced by the dimeric MHD2-scTNF construct (FIG. 6c, d). ScFv-MHD2 was inactive on both cell lines.

[0180] Bioactivity of the scTNF fusion proteins was further analyzed by measuring the TNF-mediated secretion of IL-8 from HT1080 cells. ScTNF as well as the MHD2-scTNF fusion protein induced secretion of IL-8 in a concentration-dependent manner with EC50 values of around 1-10 nM (FIG. 6e). A strongly increased stimulatory activity was observed with scFv-MHD2-scTNF, with an optimum at around 10 pM. At higher concentrations, the IL-8 release declined to approximately 25% of the highest values. The scFv-MHD2 fusion protein lacking the scTNF moiety, included as negative control, showed no stimulatory activity. IL-8 secretion induced by scFv-MHD2-scTNF was almost complete blocked to the level induced by the untargeted MHD2-scTNF fusion protein and scTNF with excess amounts of Cetuximab (660 nM) directed against the same epitope, while Trastuzumab had no effect. Both antibodies did not affect IL-8 secretion induced by MHD2-scTNF and scTNF, respectively (FIG. 60.

Example 5

Production of MHD2-scTRAIL Fusion Proteins

[0181] A bivalent MHD2-scTRAIL molecule was generated fusing a single-chain derivative of TRAIL (scTRAIL; Schneider et al., 2010) to the C-terminus of the MHD2 domain (FIG. 7a, b). Furthermore, an antibody-MHD2-scTRAIL fusion protein was generated fusing the anti-EGFR scFv to the N-terminus of the MHD2 and a single-chain TRAIL derivative to the C-terminus of the MHD2 (scFv-MHD2-scTRAIL). The constructs were produced in HEK293 cells with yields of 0.3 to 0.5 mg/L supernatant. SDS-PAGE showed under reducing conditions single bands corresponding to the molecular mass of the monomeric polypeptides (100 kDa for MHD2-scTRAIL and 115 kDa for scFv-MHD2-scTRAIL). Dimeric assembly was seen under non-reducing conditions for MHD2-scTRAIL and scFv-MHD2-scTRAIL (FIG. 7c).

Example 6

Bioactivity of MHD2-scTRAIL Fusion Proteins

[0182] Bioactivity of the scTRAIL fusion proteins was further analyzed in cytotoxicity assays using the EGFR-expressing cell lines NCI-H460 (a) and Colo205 (b). To sensitize these cells for TRAIL-induced apoptosis, bortezomib, which is a clinically approved proteasome inhibitor, was added at a concentration of 250 ng/ml. ScTRAIL as well as the MHD2-scTRAIL fusion protein induced killing of these cell lines in a concentration-dependent manner (FIG. 8a, b). The EC.sub.50 values for scTRAIL were 450 pM on NCI-H460 cells and 9.8 nM on Colo205 cells. The MHD2-scTRAIL fusion protein showed a markedly increased cytotoxic potential with EC.sub.50 values of 47 pM on NCI-H460 cells and 180 pM on Colo205 cells, corresponding to a 9.6- and 54-fold increased bioactivity, depending on the cell line used.

Example 7

Production of EHD2-scTRAIL Fusion Proteins

[0183] A bivalent scFv-EHD2 was generated fusing an scFv directed against EGFR to the N-terminus of the EHD2 domain. A bivalent EHD2-scTRAIL molecule was generated fusing a single-chain derivative of TRAIL (scTRAIL) to the C-terminus of the EHD2 domain. Furthermore, an scFv-EHD2-scTRAIL fusion protein was generated fusing the anti-EGFR scFv to the N-terminus of the EHD2 and a single-chain TRAIL derivative to the C-terminus of the EHD2 (scFv-EHD2-scTNF) (FIG. 9a, b). DNA encoding the fusion proteins were cloned into mammalian expression vector pSecTag and produced in transiently transfected HEK293 cells. Secretion of soluble scFv-EHD2 (FIG. 9c), EHD2-scTRAIL (FIG. 9d) and scFv-EHD2-scTRAIL (FIG. 9e) into the cell culture supernatant was confirmed by immunoblotting (FIG. 9c-e). Under reducing conditions, single bands corresponding to the monomeric polypeptides were detected (lanes 2), while under non-reducing conditions (lane 1) dimeric molecules (upper band) were identified corresponding to disulfide-linked dimers.

Example 8

Comparison of MHD2 and EHD2 with IgG1-CH3

[0184] The individual MHD2, EHD2 as well as the CH3 domain from human IgG1 heavy chain were produced from stably transfected HEK293 and purified by IMAC. In SDS-PAGE, the CH3 (GHD3) domain showed under reducing (FIG. 10a, lane 1) as well as non-reducing conditions (FIG. 10a, lane 2) a single band corresponding to the monomeric polypeptide, confirming that the domains are not covalently linked. Size-exclusion chromatography confirmed dimeric assembly of the domains. By dynamic light scattering, a first melting point at around 50.degree. C. (probably due to domain dissociation) and a second major melting point at 75.degree. C. (probably due to complete denaturation) was observed. In SDS-PAGE, the CH2 domain of IgM (MHD2) showed under reducing (FIG. 10b, lane 1) bands corresponding to the monomeric polypeptide, while under non-reducing conditions (FIG. 10b, lane 2) bands corresponding to dimeric molecules were detected, confirming that the domains are covalently linked. This analysis also demonstrated partial N-glycosylation apparent by two bands under reducing conditions, which was confirmed by deglycosylation with PNGaseF (not shown). Size exclusion chromatography further confirmed dimeric assembly of the domains. By dynamic light scattering, no clear transition was observed but a continuous increase of aggregate formation starting at approximately 56.degree. C. was found, presumably caused by the heterogeneity of the preparation, also indicating that the domain is rather stable even at high temperatures. In SDS-PAGE, the CH2 domain of IgE (EHD2) showed under reducing conditions (FIG. 10c, lane 1) bands corresponding to the monomeric polypeptide, while under non-reducing conditions (FIG. 10c, lane 2) bands corresponding to dimeric molecules were detected, confirming that the domains are also covalently linked. Similar to MHD2, this analysis also demonstrated partial N-glycosylation apparent by two bands under reducing conditions. Size exclusion chromatography further confirmed dimeric assembly of the domains. By dynamic light scattering, a single thermal melting point of approximately 80-82.degree. C. was determined.

Example 9

scFv-EHD2 Fusion Proteins

[0185] Various scFv-EHD2 fusion proteins were generated by fusing scFvs directed against CEA, HER2, or HER3 to the N-terminus of the EHD2 (FIG. 26). All constructs were cloned into mammalian expression vector pSecTagA and produced in transciently or stably transfected HEK293 cells. The anti-HER3 scFv-EHD2 fusion protein could be detected by immunoblotting with an anti-His-tag antibody in the supernatant of transiently transfected cells (FIG. 26c). Anti-CEA scFv-EHD2 was purified from the cell culture supernatant by immobilized metal affinity chromatography. SDS-PAGE analysis of purified anti-CEA scFv-EHD2 confirmed dimeric assembly of the fusion protein (see FIG. 27c). ELISA with immobilized CEA further confirmed correct assembly of the antigen binding sites (FIG. 27d).

Example 10

A Bispecific scDb-EHD2 Fusion Proteins for T Cell Retargeting

[0186] A bispecific single-chain diabody (scDb)-EHD2 fusion protein was generated by fusing a scDb directed against CEA and human CD3 to the N-terminus of the EHD2 (FIG. 27a, b). The construct was cloned into mammalian expression vector pSecTagA and produced in stably transfected HEK293 cells. Protein was purified from the cell culture supernatant by immobilized metal affinity chromatography. For comparison we included an scFv-EHD2 fusion protein directed against CEA (see example 9). The proteins migrated in SDS-PAGE under reducing conditions with a molecular mass of 80 kDa (scDb-EHD2) and 45 kDa (scFv-EHD2), respectively, corresponding in size to the single polypeptide. Under non-reducing conditions, the proteins migrated with an apparent molecular mass corresponding to dimeric molecules, demonstrating disulfide bond formation. Both proteins showed similar binding to immobilized CEA in ELISA (FIG. 27d), confirming functionality of the antigen-binding sites.

Example 11

Bivalent scFv-Cys-EHD2 and Tetravalent scFv-Cys-EHD2-scFv Fusion Proteins for Chemical Coupling

[0187] Antibodies can be used as carriers of molecules for diagnosis and therapy, e.g. drugs, toxins, and imaging reagents. In order to facilitate conjugation of these molecules, thiol groups can be introduced into the antibody molecule, e.g. by introducing one or more cysteine residues into the protein sequence, ideally at positions which do not interfere with antigen binding. We have recently described modified scFv molecules (Messerschmidt et al., 2008, Bioconjug. Chem. 19, 362-369) containing an additional cysteine residue either at the C-terminus of an scFv molecule or at the linker sequence connecting the VH and VL domains. Using an anti-FAP scFv (scFv-L3) containing a cysteine residue at position 3 of the 14 residue long linker (GGCGSGGGGSGGSA), a bivalent scFv-Cys-EHD2 was generated by fusing the scFv-L3 to the N-terminus of the EHD2 (FIG. 28). In addition, a tetravalent scFv-L3-EHD2-scFv fusion protein was generated by fusing an unmodified anti-FAP scFv to the C-terminus of the EHD2 domain (FIG. 28). The encoding DNA sequence was cloned into mammalian expression vector pSecTagA and stably transfected into HEK293 cells. The expressed protein was detected in the supernatant using anti-His-tag antibodies, demonstrating production and secretion of the full-length polypeptides in mammalian cells (FIG. 28c).

Example 12

EHD2-scTRAIL Fusion Proteins Targeting EGFR-Expressing Tumor Cells

[0188] Various fusion proteins were generated fusing an anti-EGFR scFv to the N-terminus (scFv-EHD2), a single-chain derivative of TRAIL (scTRAIL) to the C-terminus (EHD2-scTRAIL), or the scFv to the N-terminus and scTRAIL to the C-terminus of EHD2 (scFv-EHD2-scTRAIL) (FIG. 29a). All fusion proteins were produced in stably transfected HEK293 cells and purified by affinity chromatography with yields of 7.9 mg/L supernatant for the hexahistidyl-tagged scFv-EHD2, and 2.8 and 7.9 mg/L supernatant for the FLAG-tagged EHD2-scTRAIL and scFv-EHD2-scTRAIL fusion proteins, respectively. SDS-PAGE analysis confirmed purity and integrity of the fusion proteins as well as formation of disulfide-linked dimers, although only a fraction of the EHD2-scTRAIL and the scFv-EHD2-scTRAIL molecules showed covalent linkage (FIG. 29b). Nevertheless, correct assembly into dimeric molecules was demonstrated by SEC, indicating the presence of dimeric molecules even in the absence of interchain disulfide bonds (FIG. 29c). N-glycosylation of the EHD2 was confirmed by deglycosylation of the scFv-EHD2 fusion protein with PNGase F. After deglycosylation, only a single band was detected in SDS-PAGE under non-reducing conditions, corresponding in size to the faster migrating band seen for the untreated fusion protein (not shown). Functionality of the fusion proteins was shown by ELISA (FIG. 29d, e). Here, scFv-EHD2 and scFv-EHD2-scTRAIL bound to immobilized EGFR-Fc fusion protein, while no binding was seen for EHD2-scTRAIL. None of the fusion proteins was capable of binding to HER2-Fc included as negative control. Furthermore, EHD2-scTRAIL and scFv-EHD2-scTRAIL showed also binding to recombinant TRAIL-R1 and TRAIL-R2 in ELISA (FIG. 29d). A titration of the bivalent scFv-EHD2 in comparison with monovalent anti-EGFR scFv in ELISA demonstrated increased binding of the bivalent fusion protein (FIG. 29e). Furthermore, scFv-EHD2 and scFv-EHD2-scTRAIL showed binding to cell lines (Colo205, NCI-H460) expressing EGFR, while only marginal binding was observed for HEK293 cells lacking significant expression of EGFR (FIG. 30a, b). Only weak binding was observed for EHD2-scTRAIL to these cell lines, indicating a rather low expression of TRAIL receptors. This was confirmed by flow cytometry analysis of the cell lines with monoclonal antibodies directed against TRAIL receptors 1 to 4.

Example 13

An Anti-EGFR scFv-EHD2-scTRAIL Fusion Protein Shows Potent Tumor Cell Killing In Vitro

[0189] The cytotoxic activity of the fusion proteins were determined on NCI-H460 and Colo205 cells incubated with the fusion proteins for 1 day in the absence or presence of the proteasome inhibitor bortezomib (Velcade), which is known to sensitize tumor cells for TRAIL action (FIG. 31). In the absence of bortezomib, scTRAIL did not induce cell death over the analyzed concentration range (1 pM-10 nM). In contrast, EHD2-scTRAIL caused cell death with an IC.sub.50 of 7.2 nM (NCI-H460) and 10 nM (Colo205), respectively (Table 1). Compared with EHD2-scTRAIL, cytotoxic activity was further increased approximately 20-fold for the scFv-EHD2-scTRAIL fusion protein, supporting the contribution of targeted delivery. Cytotoxicity was improved for all proteins in the presence of 250 mg/ml bortezomib. Again EHD2-scTRAIL was more potent than scTRAIL and strongest effects were observed for scFv-EHD2-scTRAIL (Table 1). The scFv-EHD2 fusion protein showed no cytotoxic activity over the analyzed concentration range (not shown). To investigate the contribution of scFv-mediated targeting to cytotoxicity, experiments were repeated in the absence or presence of excess amounts of cetuximab, recognizing the same epitope as scFv hu225. Cytotoxic activity of scFv-EHD2-scTRAIL in the presence of cetuximab was reduced to that observed for EHD2-scTRAIL, while cetuximab had no effects on the cytotoxicity of EHD2-scTRAIL (FIG. 32; Table 1). Furthermore, we compared the cytotoxicity on NCI-460 and Colo205 cells of bivalent scFv-EHD2-scTRAIL with that of a monovalent scFv-scTRAIL fusion protein (FIG. 33). A strongly increased cytotoxic activity was observed for the dimeric scFv-EHD2-scTRAIL fusion protein for both cell lines and in the absence or presence of bortezomib. In the absence of bortezomib, scFv-scTRAIL reached approximately 50- to 60% of cell killing at the highest concentration of the fusion protein. In contrast, the scFv-EHD2-scTRAIL fusion protein mediated potent killing of both cell lines. In the presence of bortezomib, the cytotoxic activity of the scFv-EHD2-scTRAIL was increased approximately 3- to 4-fold compared with scFv-scTRAIL (FIG. 33).

TABLE-US-00002 TABLE 1 In vitro cytotoxicity IC.sub.50 (nM) construct Bortezomib Cetuximab NCI-H460 Colo205 scTRAIL - - >10 >10 + - 1.16 >10 EHD2-scTRAIL - - 7.2 10 + - 0.10 1.28 + + 0.09 2.39 scFv-EHD2-scTRAIL - - 0.32 0.50 + - 0.02 0.14 + + 0.15 1.43 scFv-EHD2 + - -- --

Example 14

Pharmacokinetics of EHD2-scTRAIL Fusion Proteins

[0190] Pharmacokinetic properties of the fusion proteins were determined in CD1 mice receiving a single i.v. injection of 25 .mu.g protein (FIG. 34). All three EHD2 fusion proteins exhibited a prolonged circulation time compared with scTRAIL. Terminal half-lives were increased from 3.3 h for scTRAIL to 7.4 to 10.8 h for the EHD2 fusion proteins resulting also in a 3- to 4-fold increased AUC.sub.0-24h (Table 2). Differences of the terminal half-life and AUC between scTRAIL and the various fusion proteins were all statistically significant (p<0.05), while the AUC of the EHD2 fusion proteins were statistically not significantly different from each other (p>0.05).

TABLE-US-00003 TABLE 2 Pharmacokinetic properties of EHD2 fusion proteins AUC.sub.0-24 h construct M.sub.r (kDa) S.sub.r (nm) t.sub.1/2b (h) (% h) scTRAIL 67.5 3.3 .sup.# 3.3 .+-. 0.3 114 .+-. 42 scFv-EHD2 82.8 4.4 10.8 .+-. 0.7 356 .+-. 76 EHD2-scTRAIL 164.6 4.6 7.4 .+-. 0.4 400 .+-. 123 scFv-EHD2-scTRAIL 218.6 5.1 8.0 .+-. 1.4 483 .+-. 166

[0191] The molecular masses were calculated for the dimeric molecules

Example 15

An Anti-EGFR scFv-EHD2-scTRAIL Fusion Protein Shows Potent Antitumor Activity

[0192] The scTRAIL fusion proteins were then tested in nude mice bearing subcutaneous Colo205 tumors for their antitumor activity. Mice received four i.v. injections of scTRAIL, EHD2-scTRAIL or scFv-EHD2-scTRAIL, respectively, over 16 days. Doses of 0.7 nmol scTRAIL and 0.35 nmol EHD2-scTRAIL and scFv-EHD2-scTRAIL were used, thus mice received equimolar doses in respect to scTRAIL. All mice, including a control group, received furthermore bortezomib (i.p.) every second day over a period of 14 days (FIG. 35a). Bortezomib at this dose does not induce any antitumor effects in this xenograft tumor model. A statistically significant reduction of tumor growth was observed for scFv-EHD2-scTRAIL, while EHD2-scTRAIL showed only a minor effect on tumor growth (FIG. 35b). At the applied doses, scTRAIL had no effect compared with the bortezomib control group. In a further experiment we compared the antitumor activity of the scFv-EHD2-scTRAIL fusion protein at a dose of 1 nmol with that of a scFv-EHD2 fusion protein lacking the scTRAIL moiety (FIG. 35c, d). Mice received four i.v. injections of the fusion proteins every second day. As before, bortezomib was included (5 .mu.g/i.p. injection). A strong anti-tumor activity was seen for the scFv-EHD2-scTRAIL fusion protein, while scFv-EHD2 had no effect compared with bortezomib treatment alone (FIG. 35d). Finally, a possible liver toxicity of the scFv-EHD2-scTRAIL fusion protein was analyzed in the presence of bortezomib after a single injection. No increase in ALT was found 4 or 24 hours after treatment and values were similar to PBS-treated mice (FIG. 35e).

Example 16

TNFR2-Selective MHD2- and EHD2-scTNF.sub.R2 Fusion Proteins

[0193] Tumor necrosis factor (TNF) exerts its biological functions via two distinct receptors. Whereas the TNF receptor (TNFR) 1 mainly mediates inflammatory responses, the TNFR2 is involved in tissue protection and regeneration. In particular, it has been demonstrated that TNFR2 can protect neurons against excitotoxic insults in vitro and promotes neuronal survival as well as oligodendrocyte regeneration after ischemic and neurotoxic insults, respectively. Accordingly, TNF variants selectively activating TNFR2 could potentially be useful as therapeutic regimen in a variety of diseases. Soluble recombinant TNF is a strong mediator of inflammation, predominantly through TNFR1 activation, as soluble TNF is not sufficient to activate TNFR2. In contrast, the membrane-bound form of TNF (memTNF) fully activates both TNFRs. Therefore, TNFR2-specific therapeutics need to comply with two basic requirements: mimicry of memTNF and, in order to avoid dose limiting severe inflammatory responses, and receptor selectivity. TNFR2 selectivity was ensured by introducing known TNFR discriminating mutations in the TNF molecule (D143N/A145R). The TNFR2-selective mutant was used in the single-chain TNF format (scTNF.sub.R2), consisting of three TNF monomers connected by short peptide linkers. Multimerization was achieved by fusion of the scTNF.sub.R2 to either MHD2 (MHD2-scTNF.sub.R2) or EHD2 (EHD2-scTNF.sub.R2), respectively. All proteins were produced in stably transfected HEK293 cells and purified by IMAC from the cell culture supernatant. SDS-PAGE analysis demonstrated dimeric assembly of the fusion proteins (FIG. 36a-c). The MHD2-scTNF.sub.R2 and EHD2-scTNF.sub.R2 fusion proteins showed selective binding to TNFR2-Fc fusion proteins in ELISA while recombinant human TNF (rhTNF) included in this study showed only weak binding to TNFR2-Fc, demonstrating an increased binding activity of the bivalent MHD2- and EHD2 fusion proteins.

TABLE-US-00004 Sequence Listing-Free Text Information SEQ ID NO: 1 Amino acid sequence of human MHD2: AELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREG KQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFT CRVDHRGLTFQQNASSMCVPD SEQ ID NO: 2 Amino acid sequence of human EHD2: DFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQ VMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQ GHTFEDSTKKCADSN SEQ ID NO: 3 Amino acid sequence of anti-HER2 scFvs (4D5) SEQ ID NO: 4 Amino acid sequence of anti-EGFR scFvs (hu225) SEQ ID NO: 5 Amino acid sequence of scTNF SEQ ID NO: 6 Amino acid sequence of scTRAIL SEQ ID NO: 7 Amino acid sequence of scFv.sub.EGFR-MHD2 SEQ ID NO: 8 Amino acid sequence of MHD2-scFv.sub.HER2 SEQ ID NO: 9 Amino acid sequence of scFv.sub.EGFR-MHD2-scFv.sub.HER2 SEQ ID NO: 10 Amino acid sequence of MHD2-scTNF SEQ ID NO: 11 Amino acid sequence of scFv.sub.EGFR-MHD2-scTNF SEQ ID NO: 12 Amino acid sequence of MHD2-scTRAIL SEQ ID NO: 13 Amino acid sequence of scFv.sub.EGFR-MHD2-scTRAIL SEQ ID NO: 14 Amino acid sequence of scDb.sub.EpCAMxEGFR-MHD2-scTRAIL SEQ ID NO: 15 Amino acid sequence of scFv.sub.EGFR-EHD2 SEQ ID NO: 16 Amino acid sequence of EHD2-scTRAIL SEQ ID NO: 17 Amino acid sequence of scFv.sub.EGFR-EHD2-scTRAIL SEQ ID NO: 18 linker peptide 1: GGGGS SEQ ID NO: 19 linker peptide 2: GGGGSGGGGS SEQ ID NO: 20 linker peptide 3: GGGGSGGGGSGGGGS SEQ ID NO: 21 linker peptide 4: GSLGGSGG SEQ ID NO: 22 linker peptide 5: GGGSGGGT SEQ ID NO: 23 linker peptide 6: GGGSGGGTGS SEQ ID NO: 24 linker peptide 7: GGGSGGGTGSGG SEQ ID NO: 25 linker peptide 8: GGGSGGGS SEQ ID NO: 26 linker peptide 9: EFTRG SEQ ID NO: 27 linker peptide 10: AAA SEQ ID NO: 28 Amino acid sequence of Anti-CEA scFv-EHD2 SEQ ID NO: 29 Amino acid sequence of Anti-HER2 scFv-EHD2 SEQ ID NO: 30 Amino acid sequence of Anti-HER3 scFv-EHD2 SEQ ID NO: 31 Amino acid sequence of Anti-CEAxCD3 scDb-EHD2 SEQ ID NO: 32 Amino acid sequence of Anti-EGFR scFv-L3-EHD2 SEQ ID NO: 33 Amino acid sequence of Anti-EGFR scFv-L3-EHD2-scFv SEQ ID NO: 34 Amino acid sequence of MHD2-scTNF.sub.R2 SEQ ID NO: 35 Amino acid sequence of EHD2-scTNF.sub.R2-L16aa SEQ ID NO: 36 Amino acid sequence of EHD2-scTNF.sub.R2-L28aa

LIST OF REFERENCES

[0194] 1. Arai, R., Ueda, H., Kitayama, A., Kamiya, N, & Nagamune, T. (2001); Design of linkers which effectively seperate domains of a bifunctional fusion protein. Protein Engineering. 14, 8, 529-532. [0195] 2. Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W. L. T., Rowland, A. M., Kotts, C., Carver, M. E. & Shepard, H. M. (1992) Humanization of an anti-p185.sup.HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. USA 89, 4285-4289. [0196] 3. Cuesta, A. M., Sainz-Pastor, N., Bonet, J., Oliva, B. & Alvarez-Vallina, L. (2010) Multivalent antibodies: when design surpasses evolution. Trends Biotechnol. 28, 355-362. [0197] 4. Deckert, P. M. (2009) Current constructs and targets in clinical development for antibody-based cancer therapy. Curr. Drug Targets 10, 158-175. [0198] 5. Deyev, S. M. & Lebedenko, E. N. (2008) Multivalency: the hallmark of antibodies used for optimiziation of tumor targeting by design. BioEssays 30, 904-918. [0199] 6. Dubel, S., Breitling, F., Kontermann, R., Schmidt, T., Skerra, A. & Little, M. (1995) Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J. Immunol. Methods 178, 201-209. [0200] 7. George, R. A., & Heringa, J. (2003), An analysis of protein domain linkers: their classification and role in protein folding. Protein Engineering. 15, 11, 871-879. [0201] 8. Goldstein, N. I., Prewett, M., Zuklys, K., Rockwell, P. & Mendelsohn, J. (1995) [0202] Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xengraft model. Clin. Cancer Res. 1, 1311-1318. [0203] 9. Hu, S. Z., Shively, L., Rautibschek, A., Sherman, M., Williams, L. E., Wong, J. Y. C., Shively, J. E. & Wu, A. M. (1996) Minibody: a novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-C.sub.H3) which exhibits rapid, high-level targeting of xenografts. Cancer Res. 56, 3055-3061. [0204] 10. Jazayeri, J. A. & Carroll, G. J. (2008) Fc-based cytokines: Prospects for engineering superior therapeutics. Biodrugs 22, 11-26. [0205] 11. Kavoosi, M., Creagh, A. L., Kilburn, D. G., & Haynes, C. A. (2007) Strategy for Selecting and Characterizing Linker Peptides for CBM9-Tagged Fusion Proteins Expressed in Escherichia coli. Biotechnology and Bioengineering, 98, 3, 599-610. [0206] 12. Kontermann, R. E. (2010) Alternative antibody formats. Curr. Opin. Mol. Ther. 12, 176-183. [0207] 13. Krippner-Heidenreich, A., Tubing, F., Bryde, S., Willi, S., Zimmermann, G. & Scheurich, P. (2002) Control of receptor-induced signaling complex formation by the kinetics of ligand/receptor interaction. J. Biol. Chem. 277, 4415544163. [0208] 14. Krippner-Heidenreich, A., Grunwald, I., Zimmermann, G., Kuhnle, M., Gerspach, J., Sterns, T., Shnyder, S. D., Gill, J. H., Mannel, D. N., Pfizenmaier, K. & Scheurich, P. (2008) Single-chain TNF, a TNF derivative with enhanced stability and antitumor activity. J. Immunol. 180, 8176-8183. [0209] 15. Muller, D. & Kontermann, R. E. (2010) Bispecific antibodies for cancer immunotherapy: current perspectives. BioDrugs 24, 89-98. [0210] 16. Pack, P. & Pluickthun, A. (1992) Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry 31, 1579-1584. [0211] 17. Rheinnecker, M., Hardt, C., Ilag, L. L., Kufer, P., Gruber, R., Hoess, A., Lupas, A., Rottenberg, C., Pluckthun, A. & Pack, P. (1996) Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen. J. Immunol. 157, 2989-2997. [0212] 18. Robinson, C. R., & Sauer, R. T. (1998) Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. PNAS USA, 95, 5929-5934. [0213] 19. Schrama, D., Reisfeld, R. A. & Becker, J. C. (2006) Antibody targeted drugs as cancer therapeutics. Nat. Rev. Drug Discov. 5, 147-159. [0214] 20. Tanaka, T., Yokoyama, S., & Kuroda, Y. (2005) Improvement of Domain Linker Prediction by Incorporating Loop-Length-Dependent Characteristics. Biopolymers (Peptide Science), 84, 161-168. [0215] 21. Ventura, E., Sassi, F., Fossati, s., Parodi, A., Blalock W., Balza, E., Castellani, P., Borsi, [0216] L., Carnemolla, B. & Zardi, L. (2009) Use of uteroglobin for the engineering of polyvalent, polyspecific fusion proteins. J. Biol. Chem. 284, 26646-26654. [0217] 22. Volkel T., Korn, T., Bach, M., Muller, R., & Kontermann, R. E. (2001) Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. Protein Engineering, 14, 10, 815-823. [0218] 23. Watanabe, H., Kanazaki, K., Nakanishi, T., Shiotsuka, H., Hatakeyama, S., Kaieda, M., Imamura, T., Umetsu, M., & Kumagai, I. (2011) Biomimetic Engineering of Modular Bispecific Antibodies for Biomolecule Immobilization. ACS Langmuir, 27, 9656-9661. [0219] 24. Wriggers W., Chakravarty, S., Jennings, P. 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Sequence CWU 1

1

361111PRTHomo sapiens 1Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp Gly 1 5 10 15 Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly 20 25 30 Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys Gln 35 40 45 Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys Glu 50 55 60 Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys Glu 65 70 75 80 Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His Arg 85 90 95 Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp 100 105 110 2106PRTHomo sapiens 2Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly 1 5 10 15 Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly 20 25 30 Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val 35 40 45 Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu 50 55 60 Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser 65 70 75 80 Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu 85 90 95 Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn 100 105 3265PRTHomo sapiens 3Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr 65 70 75 80 Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 85 90 95 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala 115 120 125 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Thr Gly Asp Ile Gln 145 150 155 160 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 165 170 175 Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp 180 185 190 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 195 200 205 Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 210 215 220 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 225 230 235 240 Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly 245 250 255 Gln Gly Thr Lys Val Glu Ile Lys Arg 260 265 4264PRTHomo sapiens 4Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp 65 70 75 80 Tyr Asn Thr Pro Phe Thr Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu 145 150 155 160 Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr 165 170 175 Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr 180 185 190 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Lys Tyr Ala Ser 195 200 205 Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220 Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 225 230 235 240 Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala 245 250 255 Gly Thr Lys Leu Glu Ile Lys Arg 260 5472PRTArtificial SequencescTNF 5Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn 1 5 10 15 Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala 20 25 30 Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro 35 40 45 Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln 50 55 60 Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile 65 70 75 80 Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser 85 90 95 Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 100 105 110 Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg 115 120 125 Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser 130 135 140 Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser 145 150 155 160 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 165 170 175 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 180 185 190 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 195 200 205 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 210 215 220 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 225 230 235 240 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 245 250 255 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 260 265 270 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 275 280 285 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 290 295 300 Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser 305 310 315 320 Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln 325 330 335 Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu 340 345 350 Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu 355 360 365 Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys 370 375 380 Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val 385 390 395 400 Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys 405 410 415 Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 420 425 430 Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser 435 440 445 Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln 450 455 460 Val Tyr Phe Gly Ile Ile Ala Leu 465 470 6580PRTArtificial SequencescTRAIL 6Thr Arg Gly Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln 1 5 10 15 Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala 20 25 30 Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro 35 40 45 Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu 50 55 60 Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn 65 70 75 80 Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln 85 90 95 Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp 100 105 110 Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro 115 120 125 Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala 130 135 140 Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys 145 150 155 160 Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp 165 170 175 Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly Gly 180 185 190 Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser Thr Val Gln 195 200 205 Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln 210 215 220 Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu 225 230 235 240 Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn 245 250 255 Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His 260 265 270 Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile 275 280 285 Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr 290 295 300 Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr 305 310 315 320 Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser 325 330 335 Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe 340 345 350 Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His 355 360 365 Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val 370 375 380 Gly Gly Gly Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser 385 390 395 400 Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg 405 410 415 Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser 420 425 430 Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg 435 440 445 Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser 450 455 460 Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe 465 470 475 480 Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys 485 490 495 Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr 500 505 510 Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser 515 520 525 Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly 530 535 540 Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr 545 550 555 560 Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala 565 570 575 Phe Leu Val Gly 580 7410PRTArtificial SequencescFvEGFR-MHD2 7Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu 20 25 30 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 35 40 45 Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp 50 55 60 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 65 70 75 80 Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Phe Thr 85 90 95 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 100 105 110 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr 115 120 125 Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr 130 135 140 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser 165 170 175 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly 180 185 190 Thr Asn Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 195 200 205 Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 225 230 235 240 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp 245 250 255 Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser 260 265 270 Leu Gly Gly Ser Gly Gly Ala Glu Leu Pro Pro Lys Val Ser Val Phe 275 280 285 Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu 290 295 300 Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp 305 310 315 320 Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val 325 330 335 Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser 340 345 350 Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr 355 360 365 Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser 370 375 380 Met Cys Val Pro Asp Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Glu 385 390 395 400 Phe Ala Ala Ala His His His His His His 405 410 8413PRTArtificial SequenceMHD2-scFvHER2 8Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Ser Ala Gly Ala Gly Ser 20 25 30 Leu Gly Gly Ser Gly Gly Ala Glu Leu Pro Pro Lys Val Ser Val Phe 35 40 45 Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu 50 55 60 Ile Cys Gln Ala Thr Gly Phe Ser

Pro Arg Gln Ile Gln Val Ser Trp 65 70 75 80 Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val 85 90 95 Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser 100 105 110 Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr 115 120 125 Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser 130 135 140 Met Cys Val Pro Asp Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Gly 145 150 155 160 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 165 170 175 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp 180 185 190 Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 195 200 205 Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser 210 215 220 Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 245 250 255 Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly 260 265 270 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 275 280 285 Gly Gly Ser Gly Gly Gly Thr Gly Asp Ile Gln Met Thr Gln Ser Pro 290 295 300 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 305 310 315 320 Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro 325 330 335 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser 340 345 350 Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr 355 360 365 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 370 375 380 Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val 385 390 395 400 Glu Ile Lys Arg Ala Ala Ala His His His His His His 405 410 9653PRTArtificial SequencescFvEGFR-MHD2-scFvHER2 9Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu 20 25 30 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 35 40 45 Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp 50 55 60 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 65 70 75 80 Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Phe Thr 85 90 95 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 100 105 110 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr 115 120 125 Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr 130 135 140 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser 165 170 175 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly 180 185 190 Thr Asn Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 195 200 205 Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 225 230 235 240 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp 245 250 255 Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser 260 265 270 Leu Gly Gly Ser Gly Gly Ala Glu Leu Pro Pro Lys Val Ser Val Phe 275 280 285 Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu 290 295 300 Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp 305 310 315 320 Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val 325 330 335 Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser 340 345 350 Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr 355 360 365 Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser 370 375 380 Met Cys Val Pro Asp Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Gly 385 390 395 400 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 405 410 415 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp 420 425 430 Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 435 440 445 Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser 450 455 460 Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 465 470 475 480 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 485 490 495 Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly 500 505 510 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 515 520 525 Gly Gly Ser Gly Gly Gly Thr Gly Asp Ile Gln Met Thr Gln Ser Pro 530 535 540 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 545 550 555 560 Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro 565 570 575 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser 580 585 590 Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr 595 600 605 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 610 615 620 Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val 625 630 635 640 Glu Ile Lys Arg Ala Ala Ala His His His His His His 645 650 10648PRTArtificial SequenceMHD2-scTNF 10Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Ser Ala Gly Ala Gly Ser 20 25 30 Leu Gly Gly Ser Gly Gly Ala Glu Leu Pro Pro Lys Val Ser Val Phe 35 40 45 Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu 50 55 60 Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp 65 70 75 80 Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val 85 90 95 Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser 100 105 110 Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr 115 120 125 Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser 130 135 140 Met Cys Val Pro Asp Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Glu 145 150 155 160 Phe Met Arg Gly Ser His His His His His His Gly Ser Ala Ser Ser 165 170 175 Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn 180 185 190 Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala 195 200 205 Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro 210 215 220 Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln 225 230 235 240 Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile 245 250 255 Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser 260 265 270 Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 275 280 285 Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg 290 295 300 Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser 305 310 315 320 Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser 325 330 335 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 340 345 350 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 355 360 365 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 370 375 380 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 385 390 395 400 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 405 410 415 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 420 425 430 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 435 440 445 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 450 455 460 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 465 470 475 480 Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser 485 490 495 Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln 500 505 510 Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu 515 520 525 Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu 530 535 540 Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys 545 550 555 560 Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val 565 570 575 Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys 580 585 590 Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 595 600 605 Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser 610 615 620 Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln 625 630 635 640 Val Tyr Phe Gly Ile Ile Ala Leu 645 11888PRTArtificial SequencescFvEGFR-MHD2-scTNF 11Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu 20 25 30 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 35 40 45 Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp 50 55 60 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 65 70 75 80 Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Phe Thr 85 90 95 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 100 105 110 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr 115 120 125 Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr 130 135 140 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser 165 170 175 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly 180 185 190 Thr Asn Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 195 200 205 Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 225 230 235 240 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp 245 250 255 Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser 260 265 270 Leu Gly Gly Ser Gly Gly Ala Glu Leu Pro Pro Lys Val Ser Val Phe 275 280 285 Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu 290 295 300 Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp 305 310 315 320 Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val 325 330 335 Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser 340 345 350 Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr 355 360 365 Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser 370 375 380 Met Cys Val Pro Asp Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Glu 385 390 395 400 Phe Met Arg Gly Ser His His His His His His Gly Ser Ala Ser Ser 405 410 415 Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn 420 425 430 Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala 435 440 445 Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro 450 455 460 Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln 465 470 475 480 Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile 485 490 495 Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser 500 505 510 Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 515 520 525 Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg 530 535 540 Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser 545 550 555 560 Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser 565 570 575 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 580 585 590 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 595 600 605 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 610 615 620 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 625 630 635

640 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 645 650 655 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 660 665 670 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 675 680 685 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 690 695 700 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 705 710 715 720 Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser 725 730 735 Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln 740 745 750 Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu 755 760 765 Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu 770 775 780 Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys 785 790 795 800 Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val 805 810 815 Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys 820 825 830 Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 835 840 845 Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser 850 855 860 Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln 865 870 875 880 Val Tyr Phe Gly Ile Ile Ala Leu 885 12724PRTArtificial SequenceMHD2-scTRAIL 12Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly 1 5 10 15 Ala His Ser Leu Asp Asp Tyr Lys Asp Asp Asp Asp Lys Glu Phe Ala 20 25 30 Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe 35 40 45 Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe 50 55 60 Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val 65 70 75 80 Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser 85 90 95 Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser 100 105 110 Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His Arg Gly 115 120 125 Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp Glu Phe 130 135 140 Thr Arg Gly Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln 145 150 155 160 Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala 165 170 175 Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro 180 185 190 Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu 195 200 205 Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn 210 215 220 Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln 225 230 235 240 Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp 245 250 255 Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro 260 265 270 Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala 275 280 285 Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys 290 295 300 Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp 305 310 315 320 Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly Gly 325 330 335 Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser Thr Val Gln 340 345 350 Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln 355 360 365 Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu 370 375 380 Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn 385 390 395 400 Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His 405 410 415 Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile 420 425 430 Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr 435 440 445 Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr 450 455 460 Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser 465 470 475 480 Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe 485 490 495 Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His 500 505 510 Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val 515 520 525 Gly Gly Gly Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser 530 535 540 Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg 545 550 555 560 Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser 565 570 575 Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg 580 585 590 Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser 595 600 605 Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe 610 615 620 Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys 625 630 635 640 Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr 645 650 655 Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser 660 665 670 Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly 675 680 685 Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr 690 695 700 Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala 705 710 715 720 Phe Leu Val Gly 13978PRTArtificial SequencescFvEGFR-MHD2-scTRAIL 13Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly 20 25 30 Ser Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly 35 40 45 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 50 55 60 Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn 85 90 95 Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Phe Thr Ile Ser Arg Asp 100 105 110 Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 115 120 125 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr 130 135 140 Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 165 170 175 Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg 180 185 190 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His 195 200 205 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Lys Tyr 210 215 220 Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 225 230 235 240 Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 245 250 255 Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe 260 265 270 Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Ala Glu Leu 275 280 285 Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly 290 295 300 Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro 305 310 315 320 Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser 325 330 335 Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro 340 345 350 Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp 355 360 365 Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr 370 375 380 Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp Glu Phe Thr Arg 385 390 395 400 Gly Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn 405 410 415 Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His 420 425 430 Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser 435 440 445 Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser 450 455 460 Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu 465 470 475 480 Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr 485 490 495 Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln 500 505 510 Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu 515 520 525 Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr 530 535 540 Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn 545 550 555 560 Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp 565 570 575 His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys 595 600 605 Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val 610 615 620 Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser 625 630 635 640 Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp 645 650 655 Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg 660 665 670 Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser 675 680 685 Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn 690 695 700 Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp 705 710 715 720 Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp 725 730 735 Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu 740 745 750 Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile 755 760 765 Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly 770 775 780 Gly Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser Thr Val 785 790 795 800 Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro 805 810 815 Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr 820 825 830 Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile 835 840 845 Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu 850 855 860 His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr 865 870 875 880 Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn 885 890 895 Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser 900 905 910 Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp 915 920 925 Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile 930 935 940 Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu 945 950 955 960 His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu 965 970 975 Val Gly 141217PRTArtificial SequencescDbEpCAMxEGFR-MHD2-scTRAIL 14Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly 20 25 30 Ser Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Gln Ser Gly 35 40 45 Pro Gly Leu Val Gln Pro Gly Gly Ser Val Arg Ile Ser Cys Ala Ala 50 55 60 Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly 85 90 95 Glu Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Phe Ser Leu 100 105 110 Asp Thr Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Ser Leu Arg Ala 115 120 125 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Ala Ile Lys Gly Asp 130 135 140 Tyr Trp Gly Gln Gly Thr Leu Leu Thr Val Ser Ser Gly Gly Gly Gly 145 150 155 160 Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val 165 170 175 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr 180 185 190 Asn Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 195 200 205 Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 225 230 235 240 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro 245 250 255 Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly 260 265 270 Gly Ser Gly Gly Arg Ala Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 275 280 285 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 290 295 300 Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp 305 310 315 320 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 325 330 335 Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg

Phe Thr 340 345 350 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 355 360 365 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr 370 375 380 Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr 385 390 395 400 Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 405 410 415 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 420 425 430 Ser Thr Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp 435 440 445 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gln Met 450 455 460 Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Ser 465 470 475 480 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 485 490 495 Ala Thr Tyr Tyr Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Phe Gly 500 505 510 Gln Gly Thr Lys Val Glu Leu Lys Arg Ala Ala Ala Ala Glu Leu Pro 515 520 525 Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn 530 535 540 Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg 545 550 555 560 Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly 565 570 575 Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr 580 585 590 Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu 595 600 605 Gly Gln Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe 610 615 620 Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp Glu Phe Thr Arg Gly 625 630 635 640 Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile 645 650 655 Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile 660 665 670 Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys 675 680 685 Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg 690 695 700 Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu 705 710 715 720 Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe 725 730 735 Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met 740 745 750 Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu 755 760 765 Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly 770 775 780 Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp 785 790 795 800 Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His 805 810 815 Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly Gly Gly Ser Gly 820 825 830 Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln 835 840 845 Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala 850 855 860 Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro 865 870 875 880 Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu 885 890 895 Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn 900 905 910 Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln 915 920 925 Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp 930 935 940 Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro 945 950 955 960 Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala 965 970 975 Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys 980 985 990 Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp 995 1000 1005 Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly 1010 1015 1020 Gly Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser Thr 1025 1030 1035 Val Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg 1040 1045 1050 Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg 1055 1060 1065 Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu 1070 1075 1080 Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser 1085 1090 1095 Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His 1100 1105 1110 Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 1115 1120 1125 Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val 1130 1135 1140 Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu 1145 1150 1155 Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr 1160 1165 1170 Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu 1175 1180 1185 Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp 1190 1195 1200 Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 1205 1210 1215 15405PRTArtificial SequencescFvEGFR-EHD2 15Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu 20 25 30 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 35 40 45 Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp 50 55 60 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 65 70 75 80 Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Phe Thr 85 90 95 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 100 105 110 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr 115 120 125 Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr 130 135 140 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser 165 170 175 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly 180 185 190 Thr Asn Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 195 200 205 Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 225 230 235 240 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp 245 250 255 Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser 260 265 270 Leu Gly Gly Ser Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile Leu 275 280 285 Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu 290 295 300 Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp 305 310 315 320 Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr 325 330 335 Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser 340 345 350 Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr 355 360 365 Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn 370 375 380 Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Glu Phe Ala Ala Ala His 385 390 395 400 His His His His His 405 16731PRTArtificial SequenceEHD2-scTRAIL 16Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly 20 25 30 Ser Ala Ala Gln Pro Ala Asp Phe Thr Pro Pro Thr Val Lys Ile Leu 35 40 45 Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu 50 55 60 Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp 65 70 75 80 Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr 85 90 95 Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser 100 105 110 Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr 115 120 125 Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn 130 135 140 Gly Gly Ser Gly Gly Glu Phe Thr Arg Gly Thr Ser Glu Glu Thr Ile 145 150 155 160 Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu 165 170 175 Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg 180 185 190 Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly 195 200 205 Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu 210 215 220 Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly 225 230 235 240 Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile 245 250 255 Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys 260 265 270 Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn 275 280 285 Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln 290 295 300 Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val 305 310 315 320 Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly 325 330 335 Ala Phe Leu Val Gly Gly Gly Gly Ser Gly Gly Gly Ser Thr Ser Glu 340 345 350 Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu 355 360 365 Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr 370 375 380 Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys 385 390 395 400 Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His 405 410 415 Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His 420 425 430 Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln 435 440 445 Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr 450 455 460 Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser 465 470 475 480 Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser 485 490 495 Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe 500 505 510 Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser 515 520 525 Phe Phe Gly Ala Phe Leu Val Gly Gly Gly Gly Ser Gly Gly Gly Ser 530 535 540 Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile 545 550 555 560 Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile 565 570 575 Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys 580 585 590 Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg 595 600 605 Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu 610 615 620 Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe 625 630 635 640 Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met 645 650 655 Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu 660 665 670 Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly 675 680 685 Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp 690 695 700 Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His 705 710 715 720 Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 725 730 17983PRTArtificial SequencescFvEGFR-EHD2-scTRAIL 17Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly 20 25 30 Ser Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly 35 40 45 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 50 55 60 Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn 85 90 95 Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Phe Thr Ile Ser Arg Asp 100 105 110 Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 115 120 125 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr 130 135 140 Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 165 170 175 Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg 180 185 190 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His 195 200 205 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Lys Tyr 210 215 220 Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 225 230 235 240 Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 245 250 255 Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe 260 265

270 Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Gly Gly Ser 275 280 285 Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys 290 295 300 Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val 305 310 315 320 Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly 325 330 335 Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly 340 345 350 Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp 355 360 365 Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr 370 375 380 Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Gly Gly Ser Gly 385 390 395 400 Gly Glu Phe Thr Arg Gly Thr Ser Glu Glu Thr Ile Ser Thr Val Gln 405 410 415 Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln 420 425 430 Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu 435 440 445 Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn 450 455 460 Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His 465 470 475 480 Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile 485 490 495 Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr 500 505 510 Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr 515 520 525 Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser 530 535 540 Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe 545 550 555 560 Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His 565 570 575 Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val 580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu Thr Ile Ser 595 600 605 Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg 610 615 620 Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser 625 630 635 640 Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg 645 650 655 Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser 660 665 670 Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe 675 680 685 Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys 690 695 700 Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr 705 710 715 720 Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser 725 730 735 Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly 740 745 750 Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr 755 760 765 Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala 770 775 780 Phe Leu Val Gly Gly Gly Gly Ser Gly Gly Gly Ser Thr Ser Glu Glu 785 790 795 800 Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro Leu Val 805 810 815 Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg 820 825 830 Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala 835 840 845 Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser 850 855 860 Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu 865 870 875 880 Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu 885 890 895 Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile 900 905 910 Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala 915 920 925 Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile 930 935 940 Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val 945 950 955 960 Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe 965 970 975 Phe Gly Ala Phe Leu Val Gly 980 185PRTArtificial Sequencepeptide linker 1 18Gly Gly Gly Gly Ser 1 5 1910PRTArtificial Sequencepeptide linker 2 19Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 2015PRTArtificial Sequencepeptide linker 3 20Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 218PRTArtificial Sequencepeptide linker 4 21Gly Ser Leu Gly Gly Ser Gly Gly 1 5 228PRTArtificial Sequencepeptide linker 5 22Gly Gly Gly Ser Gly Gly Gly Thr 1 5 2310PRTArtificial Sequencepeptide linker 6 23Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser 1 5 10 2412PRTArtificial Sequencepeptide linker 7 24Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Gly Gly 1 5 10 258PRTArtificial Sequencepeptide linker 8 25Gly Gly Gly Ser Gly Gly Gly Ser 1 5 265PRTArtificial Sequencepeptide linker 9 26Glu Phe Thr Arg Gly 1 5 273PRTArtificial Sequencepeptide linker 10 27Ala Ala Ala 1 28405PRTArtificial SequenceAnti-CEA scFv-EHD2 28Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Met Ala Gln Val Lys Leu 20 25 30 Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr Ser Val Lys Leu 35 40 45 Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser Tyr Met His Trp 50 55 60 Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile Gly Trp Ile Asp 65 70 75 80 Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln Gly Lys Ala 85 90 95 Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser 100 105 110 Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Asn Glu Gly Thr 115 120 125 Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala 165 170 175 Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val 180 185 190 Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu 195 200 205 Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met 225 230 235 240 Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr 245 250 255 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Gly Ser 260 265 270 Leu Gly Gly Ser Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile Leu 275 280 285 Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu 290 295 300 Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp 305 310 315 320 Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr 325 330 335 Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser 340 345 350 Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr 355 360 365 Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn 370 375 380 Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Glu Phe Ala Ala Ala His 385 390 395 400 His His His His His 405 29391PRTArtificial SequenceAnti-HER2 scFv-EHD2 29Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 145 150 155 160 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 165 170 175 Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln 180 185 190 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 195 200 205 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 210 215 220 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 225 230 235 240 Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly 245 250 255 Thr Lys Val Glu Ile Lys Arg Ala Ala Ala Gly Gly Ser Gly Gly Asp 260 265 270 Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly 275 280 285 Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr 290 295 300 Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met 305 310 315 320 Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala 325 330 335 Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp 340 345 350 Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp 355 360 365 Ser Thr Lys Lys Cys Ala Asp Ser Asn Gly Gly Ser Gly Gly Ala Ser 370 375 380 Ser His His His His His His 385 390 30393PRTArtificial SequenceAnti-HER3 scFv-EHD2 30Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 35 40 45 Phe Ser His Tyr Val Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ser Ser Ile Ser Ser Ser Gly Gly Trp Thr Leu Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Thr Arg Gly Leu Lys Met Ala Thr Ile Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln 145 150 155 160 Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys 165 170 175 Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn Val Val Ser Trp Tyr 180 185 190 Gln Gln His Pro Gly Lys Ala Pro Lys Leu Ile Ile Tyr Glu Val Ser 195 200 205 Gln Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly 210 215 220 Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Thr Glu Asp Glu Ala 225 230 235 240 Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser Ser Ile Phe Val Ile Phe 245 250 255 Gly Gly Gly Thr Lys Val Thr Val Leu Ala Ala Ala Gly Gly Ser Gly 260 265 270 Gly Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp 275 280 285 Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser 290 295 300 Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln 305 310 315 320 Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu 325 330 335 Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu 340 345 350 Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe 355 360 365 Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Gly Gly Ser Gly Gly 370 375 380 Ala Ser Ser His His His His His His 385 390 31645PRTArtificial SequenceAnti-CEAxCD3 scDb-EHD2 31Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Met Ala Gln Val Lys Leu 20 25 30 Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr Ser Val Lys Leu 35 40 45 Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser Tyr Met His Trp 50 55 60 Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile Gly Trp Ile Asp 65 70 75 80 Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln Gly Lys Ala 85 90 95 Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser 100 105 110 Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Asn Glu Gly Thr 115 120 125 Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 145 150 155 160 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 165 170 175 Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys 180 185 190 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu 195 200 205 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 210 215 220 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 225 230

235 240 Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys 245 250 255 Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Arg Ala Ser Gly 260 265 270 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 275 280 285 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser 290 295 300 Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 305 310 315 320 Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr 325 330 335 Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys 340 345 350 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 355 360 365 Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr 370 375 380 Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 385 390 395 400 Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala 405 410 415 Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val 420 425 430 Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu 435 440 445 Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe 450 455 460 Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met 465 470 475 480 Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr 485 490 495 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Gly Ser 500 505 510 Leu Gly Gly Ser Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile Leu 515 520 525 Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu 530 535 540 Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp 545 550 555 560 Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr 565 570 575 Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser 580 585 590 Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr 595 600 605 Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn 610 615 620 Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Glu Phe Ala Ala Ala His 625 630 635 640 His His His His His 645 32394PRTArtificial SequenceAnti-EGFR scFv-L3-EHD2 32Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val 20 25 30 Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr 35 40 45 Phe Thr Glu Asn Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly 50 55 60 Leu Glu Trp Ile Gly Trp Phe His Pro Gly Ser Gly Ser Ile Lys Tyr 65 70 75 80 Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser 85 90 95 Ser Thr Val Tyr Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Arg His Gly Gly Thr Gly Arg Gly Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Cys Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Ser Ala Gln Ile Leu Met Thr Gln 145 150 155 160 Ser Pro Ala Ser Ser Val Val Ser Leu Gly Gln Arg Ala Thr Ile Ser 165 170 175 Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Ala Tyr Ser Tyr Met His 180 185 190 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu 195 200 205 Ala Ser Asn Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly Ser Gly 210 215 220 Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp 225 230 235 240 Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg Glu Leu Pro Tyr Thr Phe 245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Gly Gly Ser 260 265 270 Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys 275 280 285 Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val 290 295 300 Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly 305 310 315 320 Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly 325 330 335 Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp 340 345 350 Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr 355 360 365 Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Gly Gly Ser Gly 370 375 380 Gly Ala Ser Ser His His His His His His 385 390 33645PRTArtificial SequenceAnti-EGFR scFv-L3-EHD2-scFv 33Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val 20 25 30 Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr 35 40 45 Phe Thr Glu Asn Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly 50 55 60 Leu Glu Trp Ile Gly Trp Phe His Pro Gly Ser Gly Ser Ile Lys Tyr 65 70 75 80 Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser 85 90 95 Ser Thr Val Tyr Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Arg His Gly Gly Thr Gly Arg Gly Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Cys Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Ser Ala Gln Ile Leu Met Thr Gln 145 150 155 160 Ser Pro Ala Ser Ser Val Val Ser Leu Gly Gln Arg Ala Thr Ile Ser 165 170 175 Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Ala Tyr Ser Tyr Met His 180 185 190 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu 195 200 205 Ala Ser Asn Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly Ser Gly 210 215 220 Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp 225 230 235 240 Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg Glu Leu Pro Tyr Thr Phe 245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Gly Gly Ser 260 265 270 Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys 275 280 285 Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val 290 295 300 Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly 305 310 315 320 Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly 325 330 335 Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp 340 345 350 Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr 355 360 365 Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Gly Gly Ser Gly 370 375 380 Gly Ala Ser Ser Glu Phe Gln Val Gln Leu Lys Gln Ser Gly Ala Glu 385 390 395 400 Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly 405 410 415 Tyr Thr Phe Thr Glu Asn Ile Ile His Trp Val Lys Gln Arg Ser Gly 420 425 430 Gln Gly Leu Glu Trp Ile Gly Trp Phe His Pro Gly Ser Gly Ser Ile 435 440 445 Lys Tyr Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys 450 455 460 Ser Ser Ser Thr Val Tyr Met Glu Leu Ser Arg Leu Thr Ser Glu Asp 465 470 475 480 Ser Ala Val Tyr Phe Cys Ala Arg His Gly Gly Thr Gly Arg Gly Ala 485 490 495 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly 500 505 510 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ala Gln Ile Leu Met 515 520 525 Thr Gln Ser Pro Ala Ser Ser Val Val Ser Leu Gly Gln Arg Ala Thr 530 535 540 Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Ala Tyr Ser Tyr 545 550 555 560 Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 565 570 575 Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly 580 585 590 Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu 595 600 605 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg Glu Leu Pro Tyr 610 615 620 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala His 625 630 635 640 His His His His His 645 34652PRTArtificial SequenceMHD2-scTNFR2 34Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Gly Gly Gly Ala Ala Ala 20 25 30 His His His His His His Gly Gly Thr Gly Gly Gly Gly Ser Gly Gly 35 40 45 Lys Leu Gly Gly Ser Gly Gly Ala Glu Leu Pro Pro Lys Val Ser Val 50 55 60 Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys 65 70 75 80 Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser 85 90 95 Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln 100 105 110 Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr 115 120 125 Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe 130 135 140 Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser 145 150 155 160 Ser Met Cys Val Pro Asp Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser 165 170 175 Glu Phe Leu Ala Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His 180 185 190 Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg 195 200 205 Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln 210 215 220 Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu 225 230 235 240 Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr 245 250 255 Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser 260 265 270 Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala 275 280 285 Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu 290 295 300 Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn 305 310 315 320 Phe Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly 325 330 335 Gly Gly Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 340 345 350 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 355 360 365 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 370 375 380 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 385 390 395 400 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 405 410 415 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 420 425 430 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 435 440 445 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 450 455 460 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe 465 470 475 480 Arg Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly 485 490 495 Gly Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val 500 505 510 Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala 515 520 525 Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val 530 535 540 Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys 545 550 555 560 Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser 565 570 575 Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile 580 585 590 Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro 595 600 605 Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly 610 615 620 Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg 625 630 635 640 Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 645 650 35647PRTArtificial SequenceEHD2-scTNFR2-L16aa 35Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Gly Gly Gly Ala Ala Ala 20 25 30 His His His His His His Gly Gly Thr Gly Gly Gly Gly Ser Gly Gly 35 40 45 Lys Leu Gly Gly Ser Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile 50 55 60 Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln 65 70 75 80 Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr 85 90 95 Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser 100 105 110 Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu 115 120 125 Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr 130 135 140 Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser 145 150 155 160 Asn Gly Gly Gly Ser Gly Gly Gly Thr Gly Ser Glu Phe Leu Ala Ser 165 170 175 Ser Arg Thr

Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 180 185 190 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 195 200 205 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 210 215 220 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 225 230 235 240 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 245 250 255 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 260 265 270 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 275 280 285 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 290 295 300 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly 305 310 315 320 Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser 325 330 335 Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln 340 345 350 Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu 355 360 365 Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu 370 375 380 Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys 385 390 395 400 Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val 405 410 415 Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys 420 425 430 Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 435 440 445 Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser 450 455 460 Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly Gln 465 470 475 480 Val Tyr Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser Arg 485 490 495 Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala 500 505 510 Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala 515 520 525 Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly 530 535 540 Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro 545 550 555 560 Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser 565 570 575 Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln 580 585 590 Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile 595 600 605 Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala 610 615 620 Glu Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly Gln Val 625 630 635 640 Tyr Phe Gly Ile Ile Ala Leu 645 36660PRTArtificial SequenceEHD2-scTNFR2-L28aa 36Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Gly Gly Gly Ala Ala Ala 20 25 30 His His His His His His Gly Gly Thr Gly Gly Gly Gly Ser Gly Gly 35 40 45 Lys Leu Gly Gly Ser Gly Gly Asp Phe Thr Pro Pro Thr Val Lys Ile 50 55 60 Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln 65 70 75 80 Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr 85 90 95 Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser 100 105 110 Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu 115 120 125 Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr 130 135 140 Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser 145 150 155 160 Asn Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Ser Glu Phe Leu Ala Ser Ser Arg Thr 180 185 190 Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu 195 200 205 Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn 210 215 220 Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu 225 230 235 240 Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser 245 250 255 Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr 260 265 270 Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg 275 280 285 Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr 290 295 300 Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu 305 310 315 320 Ile Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly Gln Val Tyr 325 330 335 Phe Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser Arg Thr Pro 340 345 350 Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly 355 360 365 Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly 370 375 380 Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr 385 390 395 400 Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr 405 410 415 His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln 420 425 430 Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu 435 440 445 Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu 450 455 460 Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile 465 470 475 480 Asn Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly Gln Val Tyr Phe 485 490 495 Gly Ile Ile Ala Leu Gly Gly Gly Gly Ser Ser Ser Arg Thr Pro Ser 500 505 510 Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly Gln 515 520 525 Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val 530 535 540 Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu 545 550 555 560 Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr His 565 570 575 Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr 580 585 590 Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr 595 600 605 Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly 610 615 620 Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn 625 630 635 640 Arg Pro Asp Tyr Leu Asn Phe Arg Glu Ser Gly Gln Val Tyr Phe Gly 645 650 655 Ile Ile Ala Leu 660

Claims

1. A polypeptide comprising a heavy chain domain 2 (HD2) from IgM or IgE and at least one pharmaceutically active moiety, under the proviso that the pharmaceutically active moiety is not a Fab or Fc fragment from IgM or IgE.

2. The polypeptide of claim 1, wherein the HD2 domain comprises an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO:2 or dimerizing variants thereof.

3. The polypeptide of claim 1, wherein at least one pharmaceutically active moiety is connected to the N- and/or C-Terminus of the HD2.

4. The polypeptide of claim 1, wherein at least one pharmaceutically active moiety is connected to the HD2 directly or indirectly via one or more linkers.

5. The polypeptide of claim 4, wherein the one or more linkers comprise peptide linkers.

6. The polypeptide of claim 5, wherein the one or more linkers comprise one or more cleavage sites.

7. The polypeptide of claim 1, wherein at least two identical or at least two different pharmaceutically active moieties are connected to the HD2.

8. The polypeptide of claim 1, wherein at least one pharmaceutically active moiety is selected from the group consisting of ligands, effector molecules, half-life extension modules, and imaging molecules.

9. The polypeptide of claim 8, wherein the at least one pharmaceutically active moiety is a ligand, wherein the ligand is selected from the group consisting of antigen-binding molecules, scaffold proteins, natural ligands, ligand-binding receptor fragments, and apatamers.

10. The polypeptide of claim 9, wherein the ligand is an antigen-binding molecule, wherein the antigen-binding molecule is selected from the group consisting of an antibody fragment, a Fab fragment, a Fab' fragment, a heavy chain antibody, a single-domain antibody (sdAb), variable domain of a heavy chain antibody, VHH, Nanobodies, a single-chain variable fragment (scFv), a tandem scFv, a bispecific T-cell engager (BITEs), a diabody, a single-chain diabody, a DART, a triple body, a nanoantibody, an alternative scaffold protein, and a fusion protein thereof.

11. The polypeptide of claim 10, wherein the antigen-binding molecule is a scFv, and wherein the scFv is an anti-HER2 or an anti-EGFR scFv.

12. The polypeptide of claim 8, wherein the at least one pharmaceutically active moiety is an effector molecule, wherein the effector molecule is selected from the group consisting of cytokines, chemokines, immuno(co)-stimulatory molecules, immunosuppressive molecules, death ligands, apoptosis-inducing proteins, kinases, prodrug-converting enzymes, RNases, agonistic antibody or antibody fragment, antagonistic antibody or antibody fragment, toxins, growth factors, hormone, coagulation factor, fibrinolytic protein, peptides mimicking these, and fragments, fusion proteins or derivatives thereof.

13. The polypeptide of claim 12, wherein the cytokine is the tumor-necrosis factor (TNF).

14. The polypeptide of claim 12, wherein the cytokine is the TNF-relative apoptosis-inducing factor (TRAIL).

15. The polypeptide of claim 8, wherein the at least one pharmaceutically active moiety is a half-life extension module, wherein the half-life extension module is selected from the group consisting of immunoglobulin binding domains (IgBD), albumin, albumin-binding domains (ABD), peptides, small molecules, fatty acids, antibody fragments, single-domain antibodies, VHH, scaffold proteins, and natural ligands exhibiting affinity for a long-circulating plasma protein, which are optionally PEGylated, HESylated, Polysialylated, N-glycosylated, O-glycosylated, or PEG-mimicking polypeptides.

16. The polypeptide of claim 8, wherein the at least one pharmaceutically active moiety is an imaging molecule, wherein the imaging molecule is selected from the group consisting of bioluminescent reagents, chemiluminescent reagents, fluorescent imaging reagents, photosensitizers, chelating reagents, and radioactive moieties.

17. A nucleic acid molecule comprising a sequence encoding the polypeptide of claim 1.

18. A vector comprising the polynucleotide of claim 17.

19. A complex comprising at least two polypeptides according to claim 1.

20. The complex of claim 19, wherein the at least two polypeptides are connected via their HD2 domains.

21. The complex of claim 19, wherein the at least two polypeptides are connected via covalent or non-covalent bonds.

22. The complex of claim 21, wherein the covalent bond is a disulfide bond.

23. The complex of claim 19, wherein the at least two polypeptides are identical or different.

24. A cell comprising the polypeptide of claim 1.

25. A pharmaceutical composition comprising the polypeptide of claim 1.

26. The pharmaceutical composition of claim 25, which further comprises a pharmaceutically acceptable carrier and/or excipient and optionally one or more additional active substances.