Coupling module including modification EHD2 domain

Abstract

To provide a binding molecule including two polypeptide chains, where the peptide chain includes a modification EHD2 domain which enables heterodimer only, by which homodimer is prevented, a nucleic acid encoding such a binding molecule, and use thereof in treatment.SOLUTION: A binding molecule includes a first polypeptide chain including a first binding domain (BD1), and a first modification EHD2 domain and a second polypeptide chain including a second binding domain (BD2) and a second modification EHD2 domain, where amino acid sequences of EHD2-1 and EHD2-2 are different from each other, each of which is selected from an amino acid sequence which has at least 70% amino acid identity to a specific amino acid sequence, and does not have Cys at position 14 or a position 102, and BD1 and BD2 form an antigen-binding site together, wherein, EHD2-1 and EHD2-2 are covalently bonded to each other.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a binding molecule comprising two polypeptide chains, wherein the polypeptide chains comprise modified EHD2 domains that allow only heterodimerization and thereby prevent homodimers. The present invention further relates to nucleic acids encoding such binding molecules, and the use of such binding molecules or nucleic acids encoding such binding molecules in therapy.

Background Art

[0002] Antibodies with at least two different specificities (so-called bispecific antibodies) are attracting increasing interest in a wide range of applications including diagnosis, imaging, prevention and therapy. In addition to the retargeting of effector molecules, cells and gene vehicles, bispecific and pre-targeting strategies, mimicking the natural functions of proteins, extending half-life, and delivery across biological barriers such as the blood-brain barrier have been utilized (Labrijn et al., 2019, Nat. Rev. Drug. Discov. 18: 585-608). Bispecific antibodies are being evaluated as potential treatments for a variety of conditions including cancer, chronic inflammatory diseases, autoimmunity, neurodegeneration, bleeding disorders and infections.

[0003]

[0004] ​However, the production of bivalent bispecific IgG or Ig-like molecules has become complex due to the fact that the antigen-binding sites are constructed by the variable domains of the light and heavy chains (VL, VH). The random pairing of the heavy and light chains of two antibodies expressed in one cell theoretically results in 16 different combinations (10 different molecules), with only one being bispecific and the remaining pairings becoming non-functional or monospecific molecules. Directly and forcibly modifying the assembly to correct the binding sites, i.e., the heavy and light chains with the correct specificity, is one of the challenges in producing bispecific antibodies. To address such problems, various strategies have been developed and established over the past 20 years.

[0005] Fusion of two antibody-producing cell lines, for example, the production of composite hybrid-hybridomas (quadromas), enables the combination of the heavy and light chains of two different antibodies. The bispecific antibody thus obtained contains the heavy and light chains of the first antibody as well as the heavy and light chains of the second antibody. The constant regions of the heavy and light chains can be of the same isotype or different isotypes. They can even be from different species, which is the strategy utilized to produce triomabs. In this format, a mouse hybridoma is fused with a rat hybridoma, and an asymmetric hybrid IgG molecule with bispecificity is produced. The preferential pairing of the light chain with its corresponding heavy chain has been described. Importantly, the heterologous Fc portion enables fractionation and purification by protein A chromatography due to reduced binding, and elution from the column already occurs at a pH of approximately 5.8. Furthermore, cell lines producing two different heavy and light chains can be generated by genetic means. This allows for defined compositions, such as the use of specific human isotype heavy and light chains or the implementation of mutant sequences, but the random pairing of the heavy and light chains represents one of the major obstacles to these approaches.

[0006] Genetic engineering to force heavy chain heterodimerization is one of the problems of bispecific IgG formation, e.g., C HResolved the knobs-into-hole mutations and electrostatic steering mutations introduced into the 3 domains (Krah et al., 2017, N. Biotechnol. 39: 167-173). However, the heavy chain of the heterodimer can still associate with two different light chains, resulting in four possible combinations: one bispecific molecule, one non-functional combination, and two monospecific molecules. Therefore, approaches have been developed to enable the correct pairing of the homologous heavy and light chains combined with the Fc-modified heavy chain. One approach is to use a common light chain that is used for both heavy chains of the IgG molecule to form a functional binding site (Merchant et al., 1998, Nat. Biotechnol. 16: 677-681). However, this requires the identification and isolation of antigen-binding sites that utilize the same VL domain.

[0007] Alternatively, to enforce the correct pairing of the heavy and light chains, mutations or modifications have been introduced into one light chain and the Fd fragment (V H -C H 1 fragment) of the heavy chain of the binding site (Krah et al., 2017, N. Biotechnol. 39: 167-173). These strategies involve modifications of the C H 1 and C L domains of one heavy and one light chain, and in some cases, modifications of the V H and V L domains as well. Examples include the CrossMab technology that exchanges the C H 1 and C L domains between one heavy and one light chain (Klein et al., 2012, MAbs 4: 653-663), the introduction of orthogonal Fab mutations into the C H 1 and C L domains (Lewis et al., 2014, Nat. Biotechnol. 32: 191-198), or even V H and V LIntroduction of orthogonal Fab mutations into the domain (Golay et al., 2016, J. Immunol. 196: 199-211; Froning et al., 2017, Protein Sci. 26: 2021-2038; Bonisch et al., 2017, Protein Eng. Des. Sel., 2017, 30: 685-696), and the Duetmab technology (Mazor et al., 2015, MAbs 7: 461-469) that removes the native disulfide bond between CH1 and CL and replaces it with newly introduced artificial disulfide bonds.

[0008] Another approach is to replace the C H 1 and C L domains with structurally related domains from other proteins such as the C-alpha and C-beta domains from the T cell receptor (TCR) (Wu et al., 2015, MAbs 7: 470-482). Alternatively, C H 1 and C LThe domain was replaced by a mutated heavy chain domain 2 from IgE that naturally forms a homodimer. The mutations introduced into this so-called EFab module allowed the formation of a knob-into-hole-like structure that decreased homodimerization and enabled the formation of heterodimeric Fab-like molecules (Cooke et al., 2018, MAbs 10: 1248-1259). However, a common problem with these knob-into-hole mutations is the possibility of the formation of homodimers of the hole-mutation-containing domains. In the EFab format, these hole mutations are present in the light chain and thus carry the risk of the formation of light chain homodimers (Kuglstatter et al., 2017, Protein Eng. Des. Sel. 30: 649-656). Furthermore, for EFab, a decrease in thermal stability and an increase in sensitivity to protein cleavage were observed compared to wild-type Fab fragments and wild-type EHD2 Fab molecules, which was ultimately due to disordered regions at the interface between these mutated domains (Cooke et al., 2018, MAbs 10: 1248-1259).

[0009] Recently, additional mutations have been proposed for creating heterodimerizing EHD2 domains (WO 2017 / 011342 Al). Here, several mutations were described that allow heterodimerization via electrostatic or hydrophobic interactions by introducing them into either the EHD2 (EH2) domain or, alternatively, the structurally and functionally related MHD2 (MH2) domain (Seifert et al., 2014, Mol. Cancer Ther. 13: 101-111). However, in all examples carried out, only MH2 derivatives were used. Nevertheless, in these derivatives, a fairly large number of residues had to be modified from the wild-type sequence.

[0010] To a satisfactory extent, eliminate the homodimerization of the polypeptide in the binding molecule, and thus provide a solution to the pairing problem of the light and heavy chains, that is, multiple specificities, preferably the mispairing of the light and heavy chains in a bispecific antibody. The need for dimerization of the domain remains in the art. Summary of the Invention

[0011] The inventors have found that by co-expressing a first polypeptide chain containing a first modified EHD2 domain with a second polypeptide chain containing a second modified EHD2 domain, the formation of homodimers can be substantially prevented while enabling the formation of heterodimers to a satisfactory extent, and have rendered modified domains particularly suitable for the large-scale production of binding molecules.

[0012] Accordingly, in a first aspect, the present invention provides a binding molecule comprising a first polypeptide chain comprising a first binding domain (BD1) and a first modified EHD2 domain (EHD2-1), and a second polypeptide chain comprising a second binding domain (BD2) and a second modified EHD2 domain (EHD2-2), wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other, and each is an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 14, or an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 102, wherein BD1 and BD2 together form an antigen-binding site, and EHD2-1 and EHD2-2 are covalently bonded to each other.

[0013] According to one embodiment, one or both of the modified EHD2 domains further comprise a single amino acid substitution at position N39.

[0014] According to a further embodiment, BD1 and BD2 are different from each other, and each is a variable heavy chain (V H ) and a variable light chain (V L) is selected from, or is selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain.

[0015] According to yet another embodiment, the binding molecule of the present invention further comprises a first Fc chain.

[0016] According to a further embodiment, the binding molecule of the present invention further comprises a third binding domain (BD3) and a fourth binding domain (BD4), wherein BD3 and BD4 together form an antigen binding site.

[0017] According to a further embodiment, BD3 and BD4 are different from each other, and each is H and V L is selected from, or is selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain.

[0018] According to a further embodiment, the binding molecule of the present invention further comprises a third polypeptide chain. According to a preferred embodiment, the binding molecule of the present invention further comprises a third polypeptide chain and a fourth polypeptide chain.

[0019] According to yet another embodiment, the binding molecule of the present invention further comprises a second Fc chain.

[0020] According to one embodiment, the first Fc chain and the second Fc chain are different from each other and form a heterodimeric Fc.

[0021] According to a further embodiment, the binding molecule of the present invention is monospecific or bispecific.

[0022] According to yet another embodiment, the third polypeptide chain comprising BD3 is (i) C H 1 domain, (ii) C L domain, (iii) a first modified EHD2 domain (EHD2-1), and (iv) further comprises one of the second modified EHD2 domains (EHD2-2), and, wherein the fourth polypeptide chain comprising BD4, in case of (i) C L domain, in case of (ii) C H 1 domain, in case of (iii) the second modified EHD2 domain (EHD2-2), and, in case of (iv) the first modified EHD2 domain (EHD2-1), further comprises, wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other, and each has an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and no Cys at position 14, or an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and no Cys at position 102, and is selected from.

[0023] According to one embodiment, one or both of the modified EHD2 domains of the third polypeptide chain or the fourth polypeptide chain further comprises a single amino acid substitution at position N39.

[0024] According to a further embodiment, the binding molecule of the present invention further comprises a fifth binding domain (BD5) and a sixth binding domain (BD6), wherein BD5 and BD6 together form an antigen binding site.

[0025] According to yet another embodiment, the binding molecule of the present invention, binds to BD5, (i) C H 1 domain, (ii) C L domain, (iii) the first modified EHD2 domain (EHD2-1), or (iv) the second modified EHD2 domain (EHD2-2), and binds to BD6, (i) in case of C L domain, In the case of (ii), C H 1 domain, in the case of (iii), the second modified EHD2 domain (EHD2-2), and in the case of (iv), the first modified EHD2 domain (EHD2-1), wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other and each has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 14, or has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 102, and is selected from the amino acid sequences.

[0026] According to one embodiment, one or both of the modified EHD2 domains that bind to BD5 or BD6 further contain a single amino acid substitution at position N39.

[0027] According to another embodiment, BD5 and BD6 are different from each other and each is selected from V H and V L or is selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain.

[0028] According to yet another embodiment, the C H 1 domain, C L domain, EHD2-1 or EHD2-2 that binds to BD5 or BD6 binds to BD1, BD2, BD3 or BD4 via a linker.

[0029] According to one embodiment, the binding molecule of the present invention is monospecific, bispecific or trispecific.

[0030] According to a further embodiment, the binding molecule of the present invention further comprises a seventh binding domain (BD7) and an eighth binding domain (BD8), wherein BD7 and BD8 together form an antigen binding site.

[0031] According to one embodiment, the binding molecule of the present invention is Binding to BD7, (i) C H 1 domain, (ii) C L domain, (iii) The first modified EHD2 domain (EHD2-1), or (iv) The second modified EHD2 domain (EHD2-2), and, Binding to BD8, In the case of (i), C L domain, (ii) In the case of, C H 1 domain, (iii) In the case of, the second modified EHD2 domain (EHD2-2), and (iv) In the case of, the first modified EHD2 domain (EHD2-1), further comprising, wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other, and each has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 14, or has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 102, and is selected from the amino acid sequences.

[0032] According to yet another embodiment, one or both of the modified EHD2 domains that bind to BD7 or BD8 further comprise a single amino acid substitution at position N39.

[0033] According to one embodiment, BD7 and BD8 are different from each other, and each is selected from V H and V L or is selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain.

[0034] According to a further embodiment, the C H 1 domain, C L domain, EHD2-1 or EHD2-2 that binds to BD7 or BD8 binds to any one of BD1, BD2, BD3 or BD4 that does not bind to BD5 or BD6 via a linker.

[0035] According to yet another embodiment, the binding molecule of the present invention is monospecific, bispecific, trispecific or quadrispecific.

[0036] According to a general embodiment, none of the modified EHD2 domains carry one N-glycan, or one or more of the modified EHD2 domains carry one N-glycan.

[0037] According to a further general embodiment, the Cys at position 14 of SEQ ID NO:1 is substituted by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr (C14S, C14G, C14A, C14T, C14Q, C14N, C14Y), preferably Ser (C14S).

[0038] According to a further general embodiment, the Cys at position 102 is substituted by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr (C102S, C102G, C102A, C102T, C102Q, C102N, C102Y), preferably Ser (C102S).

[0039] According to yet another general embodiment, the single amino acid substitution at position N39 is N39Q.

[0040] According to a further aspect, the present invention provides a nucleic acid or a set of nucleic acids encoding the binding molecule of the present invention.

[0041] According to a further aspect, the present invention provides a vector comprising the nucleic acid or the set of nucleic acids of the present invention.

[0042] According to a further aspect, the present invention provides a host cell comprising the vector of the present invention.

[0043] According to a further aspect, the present invention provides a pharmaceutical composition comprising a binding molecule, nucleic acid or set of nucleic acids, vector, or host cell of the present invention, and a pharmaceutically acceptable carrier.

[0044] Further aspects and embodiments will become apparent from the following detailed description of the invention.

Brief Description of the Drawings

[0045] The contents of the figures constituted in this specification will be described below. In this context, reference is also made to the above and / or the following detailed description of the invention.

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Figure 19

[0065] Sequence Listing SEQ ID NO: 1 (wt human EHD2 core amino acid sequence; underlined at positions C14, N39, and C102) DFTPPTVKIL QSS C DGGGHF PPTIQLLCLV SGYTPGTI N I TWLEDGQVMD VDLSTASTTQ EGELASTQSE LTLSQKHWLS DRTYTCQVTY QGHTFEDSTK K C ADSN

[0066] SEQ ID NO: 2 (Modified EHD2 amino acid sequence with a Cys substitution at position 14; X = any amino acid other than Cys, preferably Ser, Gly, Ala, Thr, Gln, Asn, or Tyr, most preferably Ser) DFTPPTVKIL QSS X DGGGHF PPTIQLLCLV SGYTPGTINI TWLEDGQVMD VDLSTASTTQ EGELASTQSE LTLSQKHWLS DRTYTCQVTY QGHTFEDSTK KCADSN

[0067] SEQ ID NO: 3 (Modified EHD2 amino acid sequence with a Cys substitution at position 102; X = any amino acid other than Cys, preferably Ser, Gly, Ala, Thr, Gln, Asn, or Tyr, most preferably Ser) DFTPPTVKIL QSSCDGGGHF PPTIQLLCLV SGYTPGTINI TWLEDGQVMD VDLSTASTTQ EGELASTQSE LTLSQKHWLS DRTYTCQVTY QGHTFEDSTK K X ADSN

[0068] SEQ ID NO: 4 (Modified EHD2 amino acid sequence containing Cys at position 14) DFTPPTVKIL QSS S DGGGHF PPTIQLLCLV SGYTPGTINI TWLEDGQVMD VDLSTASTTQ EGELASTQSE LTLSQKHWLS DRTYTCQVTY QGHTFEDSTK KCADSN

[0069] SEQ ID NO: 5 (Modified EHD2 amino acid sequence containing Ser at position 14 and Gln at position 39) DFTPPTVKIL QSS S DGGGHF PPTIQLLCLV SGYTPGTI Q I TWLEDGQVMD VDLSTASTTQ EGELASTQSE LTLSQKHWLS DRTYTCQVTY QGHTFEDSTK KCADSN

[0070] SEQ ID NO: 6 (Modified EHD2 amino acid sequence containing Ser at position 102) DFTPPTVKIL QSSCDGGGHF PPTIQLLCLV SGYTPGTINI TWLEDGQVMD VDLSTASTTQ EGELASTQSE LTLSQKHWLS DRTYTCQVTY QGHTFEDSTK K S ADSN

[0071] SEQ ID NO: 7 (Modified EHD2 amino acid sequence containing Ser at position 102 and Gln at position 39) DFTPPTVKIL QSSCDGGGHF PPTIQLLCLV SGYTPGTI Q I TWLEDGQVMD VDLSTASTTQ EGELASTQSE LTLSQKHWLS DRTYTCQVTY QGHTFEDSTK K S ADSN

[0072] SEQ ID NO: 8 (Igκ leader sequence) METDTLLLWVLLLWVPGSTG

[0073] SEQ ID NO: 9 (V L 5D5-EHD2-1 (N39Q)) DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0074] SEQ ID NO: 10 (V H 5D5-EHD2-2-Fc hole ) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0075] Sequence number: 11 (V L 5D5-EHD2-2) DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSN

[0076] Sequence number: 12 (V H 5D5-EHD2-1(N39Q)-Fc hole ) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0077] Sequence number: 13 (V L 3-43-C L λ) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0078] Sequence number: 14 (V H 3-43-C H 1-Fc knob ) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0079] Sequence number: 15 (V L huU3-EHD2-1(N39Q)) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKLEIKRTDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0080] Sequence number: 16 (V H huU3-EHD2-2-Fc hole ) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNGKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0081] Sequence number: 17 (V H 5D5-EHD2-2-Linker-V H 3-43-C H 1) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTGGSGGGGSGGQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0082] Sequence number: 18 (V H 3-43-C H 1-Linker-V H 5D5-EHD2-2) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTGGSGGGGSGGEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSN

[0083] Sequence number: 19 (V H 3-43-C H 1-Linker-V H 5D5-EHD2-2-Fc hole ) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTGGSGGGGSGGEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] Sequence number: 20 (V H 3-43-C H 1-Linker-V H 3-43-C H 1-Fc knob ) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTGGSGGGGSGGQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0085] Sequence number: 21 (V H 5D5-EHD2-2-Linker-V H 3-43-C H 1-Fc hole ) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTGGSGGGGSGGQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0086] Sequence number: 22 (V H 3-43-EHD2-2-Fc) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0087] Sequence number: 23 (V L 3-43-EHD2-1 (N39Q)) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLGTDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0088] Sequence number: 24 (V H hu225-C H 1-Linker-V H 3-43-EHD2-2-Fc) EVQLVESGGGLVQPGGSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSGGGGSGGQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0089] Sequence number: 25 (V L hu225-C L k) DIQLTQSPSFLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQNNNWPTTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0090] Sequence number: 26 (V H 3 - 43 - EHD2 - 2 - linker - V H hu225 - C H 1 - Fc) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTGGSGGGGSGGEVQLVESGGGLVQPGGSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0091] Sequence number: 27 (V H 3 - 43 - EHD2 - 2 (C102A) - His) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKK A ADSNAAAHHHHHH

[0092] Sequence number: 28 (V H 3-43-EHD2-2(C102W)-His) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKK W ADSNAAAHHHHHH

[0093] Sequence number: 29 (V H 3-43-EHD2-2(C102N)-His) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKK N ADSNAAAHHHHHH

[0094] Accession number: 30 (V H 3-43-EHD2-2(C102T)-His) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKK T ADSNAAAHHHHHH

[0095] Accession number: 31 (V H 3-43-EHD2-2(C102S)-His) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKK S ADSNAAAHHHHHH

[0096] Accession number: 32 (V L 3-43-EHD2-1(C14A, N39Q)) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLDFTPPTVKILQSS A DGGGHFPPTIQLLCLVSGYTPGTI QITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0097] Accession number: 33 (V L 3-43-EHD2-1 (C14T, N39Q)) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLDFTPPTVKILQSS T DGGGHFPPTIQLLCLVSGYTPGTI Q ITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0098] Accession number: 34 (V L 3-43-EHD2-1 (C14N, N39Q)) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLDFTPPTVKILQSS N DGGGHFPPTIQLLCLVSGYTPGTI Q ITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0099] Accession number: 35 (V L 3-43-EHD2-1 (C14W, N39Q)) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLDFTPPTVKILQSSW DGGGHFPPTIQLLCLVSGYTPGTI Q ITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0100] Sequence number: 36 (V L 3-43-EHD2-1(C14S, N39Q)) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLDFTPPTVKILQSS S DGGGHFPPTIQLLCLVSGYTPGTI Q ITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0101] Sequence number: 37 (V L 3-43-EHD2-1(C14S)) QAGLTQPPAVSVAPGQTASITCGRDNIGSRSVHWYQQKPGQAPVLVVYDDSDRPAGIPERFSGSNYENTATLTISRVEAGDEADYYCQVWGITSDHVVFGGGTKLTVLDFTPPTVKILQSS S DGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0102] Sequence number: 38 (V H 3-43-EHD2-2(N39Q, C102S)-His) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTI Q ITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKK S ADSNAAAHHHHHH

[0103] Sequence number: 39 (V H huU3-EHD2-2-Linker-V H 3-43-C H 1-Fc knob ) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNGKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGGSGGGGSGGQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0104] Sequence number: 40 (V H 3-43-C H 1-Fc hole ) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0105] Sequence number: 41 (V H 3-43-C H 1-Linker-V H huU3-EHD2-2-Fc hole ) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSGGGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYTMNWVRQAPGQGLEWMGLINPYKGVSTYNGKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0106] Accession number: 42 (V H hu36-EHD2-2-Fc hole ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEKFKDRVTMTADTSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGTLVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0107] Sequence number: 43 (VLhu36-EHD2-1(N39Q)) DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIKRDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0108] Sequence number: 44 (V H hu225-C H 1 - linker - V H (3 - 43 - EHD2 - 2 - His) EVQLVESGGGLVQPGGSLRLSCAASGFSLTNYGVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSGGGGSGGQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAQSLKSRITINPDTPKNQFSLQLNSVTPEDTAVYYCARDGQLGLDALDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNAAAHHHHHH

[0109] Sequence number: 45 (V L hu36-C L κ) DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIKRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0110] Sequence number: 46 (V H hu36-C H 1-Fc hole ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEKFKDRVTMTADTSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGTDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0111] Sequence number: 47 (V H 2C11-EHD2-1(N39Q)) EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0112] Sequence number: 48 (V L 2C11-EHD2-2-Fc knob ) DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0113] Sequence number: 49 (V L 2C11-EHD2-1(N39Q)) DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0114] Sequence number: 50 (V H 2C11-EHD2-2-Fc knob ) EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNGTDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0115] Sequence number: 51 (V L S309-C L κ) EIVLTQSPGTLSLSPGERATLSCRASQTVSSTSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDTSLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0116] Sequence number: 52 (V L (P2B-2F6-EHD2-1(N39Q)) QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSNNLVCGGGTKLTVLDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTIQITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSN

[0117] Sequence number: 53 (V H S309-C H 1-V H (P2B-2F6-EHD2-2-Fc) QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSGGGGSGGQVQLQESGPGLVKPSETLSLTCTVSGYSISSGYYWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKTRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAVVGIVVVPAAGRRAFDIWGQGTMVTVSSDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKSADSNDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0118] (Detailed Description of the Invention) Before describing the present invention in detail below, it is understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein. Also, 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 is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0119] Preferably, the terms used herein are described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, Band Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0120] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted to mean the inclusion of a stated integer or step or group of integers or steps and not the exclusion of any other integer or step or group of integers or steps. In the following passages, various aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless the contrary is clearly indicated. In particular, any feature indicated as optional, preferred, or advantageous may be combined with any other feature or features indicated as optional, preferred, or advantageous.

[0121] Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing in this specification should be construed as an admission that the invention has no right to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being "incorporated by reference". In the event of any conflict between the definition or teaching of such incorporated documents and the definition or teaching set forth herein, the text of this specification shall control.

[0122] The following describes the elements of the present invention. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This specification is to be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Further, any permutation and combination of all the elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0123] Definition

[0124] The following provides some definitions of terms frequently used in this specification. In each example of their use, these terms have the meanings and preferred meanings defined elsewhere in the remainder of this specification.

[0125] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

[0126] As used herein, the term "binding molecule" means any molecule or part of a molecule that can specifically bind to a target molecule or target epitope. The binding properties of the binding molecules of the present invention can be derived from (a) an antibody or an antigen-binding fragment thereof; (b) an oligonucleotide; (c) an antibody-like protein; (d) a T cell receptor or (e) a peptidomimetic.

[0127] As used herein, the term "binding" preferably relates to specific binding. "Specific binding" means that a binding protein (e.g., an antibody) binds more strongly to a target, such as an epitope, to which it is specific, compared to its binding to another target. A binding protein binds more strongly to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) that is lower than the dissociation constant for the second target. Preferably, the dissociation constant (Kd) for the target to which the binding protein specifically binds is more than 10-fold, preferably more than 20-fold, more preferably more than 50-fold, still more preferably 100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociation constant (Kd) for a target to which the binding protein does not specifically bind.

[0128] As used herein, the term "K d " (measured in "mol / L" and sometimes abbreviated as "M") is intended to mean the dissociation equilibrium constant of a particular interaction between a binding protein (e.g., an antibody or a fragment thereof) and a target molecule (e.g., an antigen or an epitope thereof). Methods for determining the binding affinity of a compound, i.e., for determining the dissociation constant K D , are known to those skilled in the art and can be selected, for example, from the following methods known in the art: surface plasmon resonance (SPR)-based techniques, biolayer interferometry (BLI), quartz crystal microbalance (QCM), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). In the context of the present application, the "K d " value is determined by surface plasmon resonance spectroscopy (Biacor TM ) or quartz crystal microbalance (QCM) at room temperature (25 °C).

[0129] As used herein, the term "binding domain" means an amino acid sequence having the ability to specifically bind to an antigen, and can be derived, for example, from an antibody or an antigen-binding fragment thereof, a T cell receptor and an antigen-binding fragment thereof. Examples included within the term "binding domain" include Fab fragments, monovalent fragments consisting of V L and V H domains; Fv fragments consisting of the V L and V H domains of a single arm of an antibody, dAb fragments consisting of the V H domain or V L domain, VHH, nanobody, or variable domain of IgNAR (Ward et al, (1989) Nature 341: 544-546); isolated complementarity-determining regions (CDRs), and combinations of any two or more isolated CDRs linked by an optional synthetic peptide linker. The term "binding domain" also means the variable domains or regions of TCR α- and TCR β-chains, or TCR γ- and TCR δ-chains.

[0130] As used herein, the term "TCR" or "T cell receptor" refers to a molecule found on the surface of a T cell. TCRs consist of two separate peptide chains and, in humans, are most often produced from the independent T cell receptor α and β (TCRα and TCRβ) genes. These chains are referred to as the α-chain and β-chain. The TCR α-chain and TCR β-chain each have a variable domain and a constant domain. Each variable domain bears three CDRs for binding to an antigen. TCR γ- and TCR δ-chains are less common in humans, but bear variable and constant domains, and each variable domain bears three CDRs for binding to an antigen.

[0131] As used herein, the term "antigen" relates to a substance that includes an epitope recognized by an antigen-binding domain. The term "antigen" includes, in particular, proteins and peptides. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include, or be derived from, allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or the antigen can also be a tumor antigen. According to the present invention, the antigen can correspond to a naturally occurring product, such as a viral protein, or a part thereof, or a tumor protein.

[0132] As used herein, the term "dimerization domain" refers to a domain capable of forming a dimer of two polypeptide or protein chains, wherein at least one dimerization domain is present on the first chain and at least a second dimerization domain is present on the second chain. The dimerization domain is selected from the group consisting of the Fc regions of IgE and IgM, heterodimerizing Fc regions, C H 1, C L , the second heavy chain constant domain (C H 2) (EHD2, MHD2), the modified EHD2 according to the present invention, the last heavy chain constant domain (C H 3 or C H 4) of IgG, IgD, IgA, IgM or IgE and its heterodimerizing derivatives, and the constant domains C-α and C-β of the T cell receptor. Depending on each dimerization domain, the C-terminus and N-terminus of the dimerization domain can vary. When the dimerization domain is derived from a naturally occurring protein, such as an immunoglobulin, the dimerization domain can preferably bind directly to the variable domain in the sense of the present invention, i.e., it can bind without a peptide linker if there are no amino acids not naturally present at its C-terminus or N-terminus.

[0133] V used in the context of the present invention H and V LThe domain is preferably derived from an antibody or immunoglobulin. Such "antibody" or "immunoglobulin" has a "Y" - shaped form and consists of four polypeptide chains; it may be a natural or conventional antibody in which two identical heavy chains and two identical light chains are joined by disulfide bonds. Each chain consists of structural domains. The two heavy chains are joined to each other by disulfide bonds, and each heavy chain is joined to a light chain by a disulfide bond. There are two types of light chains, lambda (λ) and kappa (κ), and there are five main heavy - chain classes (or isotypes): IgM, IgD, IgG, IgA, IgE that determine the functional activity of the antibody molecule. Each chain contains different sequence domains. The light chain contains two domains or regions, a variable domain (V L ) and a constant domain (C L ). The heavy chain contains four domains, a variable domain (V H ) and three constant domains (CH1, CH2, CH3, collectively referred to as CH), and in the case of IgE and IgM, it contains five domains, a variable domain (V H ) and four constant domains (CH1, CH2, CH3, CH4). The light chain (V L ) and the heavy chain (V H) The binding recognition and specificity for the antigen are determined by the variable regions of both. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties such as the binding, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR) of the antibody chains. The "arms" of the Y-shaped antibody contain sites that can bind to specific molecules and can recognize specific antigens. This region of the antibody is called the Fab (fragment, antigen-binding) region. It consists of one constant domain and one variable domain from each of the heavy and light chains of the antibody. The base of the Y plays a role in regulating the activity of immune cells. This region is called the Fc (fragment, crystalline) region and consists of two heavy chains that contribute two or three constant domains depending on the class of the antibody. The Fv fragment is the N-terminal part of the Fab region of the immunoglobulin and consists of the variable parts of one light chain and one heavy chain. The specificity of the antibody lies in the structural complementarity between the antibody-binding site and the antigen determinant. The antibody-binding site mainly consists of residues from the hypervariable regions or complementarity-determining regions (CDR). Occasionally, residues from non-hypervariable regions or framework regions (FR) affect the structure of the entire domain and thus affect the binding site. The complementarity-determining region or CDR refers to the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of the immunoglobulin each have three CDRs called CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, the conventional antibody-antigen binding site contains six CDRs including the CDR sets from each of the V regions of the heavy and light chains.

[0134] The antibodies and antigen-binding fragments thereof that can be used in the present invention can be derived from any animal, including birds and mammals. Preferably, the antibody or fragment is derived from a human, chimpanzee, rodent (e.g., mouse, rat, guinea pig, or rabbit), chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog. It is particularly preferred that the antibody is of human or mouse origin. The antibodies of the present invention also include chimeric molecules in which the constant region of an antibody, preferably derived from a human, is combined with an antigen-binding site derived from another species, such as a mouse. Furthermore, the antibodies of the present invention include humanized molecules in which the antigen-binding site of an antibody derived from a non-human species (e.g., mouse) is combined with a human-derived constant region and framework region.

[0135] As exemplified herein, antibodies can be obtained directly from hybridomas expressing the antibody or can be cloned and recombinantly expressed in host cells (e.g., CHO cells, or lymphocyte cells). Further examples of host cells are microorganisms such as Escherichia coli and fungi such as yeast. Alternatively, they are recombinantly produced in transgenic non-human animals or plants.

[0136] As used herein, the terms "EHD2" and "EHD2 domain" mean the second constant domain of the heavy chain of IgE (Seifert et al., 2012, Protein Eng Des Sel.; 25:603-12; and Seifert et al., 2014, Mol Cancer Ther.;13:101-11). Similarly, the term "modified EHD2 domain" means a portion of the EHD2 domain whose sequence is modified as compared to the original EHD2 sequence from which it is derived. Modifications include amino acid deletions, substitutions, and insertions. The modification preferably includes one or more amino acid substitutions at the positions shown with respect to SEQ ID NO:1. The substitutions according to the invention are made with respect to Cys at position 14 or Cys at position 102. Preferred substitutions include C14S and C102S, respectively, with respect to SEQ ID NO:1. As used herein, the term "hetEHD2" refers to a heterodimer of two modified EHD2 domains, i.e., a dimer comprising EHD2-1 and EHD2-2 as defined herein. Although reference is made in some examples to specific amino acid sequences, it is emphasized that the terms "EHD2-1" and "EHD2-2" as used herein do not mean specific amino acid sequences, but are mainly used to emphasize the differences between the amino acid sequences of the first modified EHD2 domain and the second EHD2 domain. Using the amino acid numbering scheme defined by Lefranc et al., 2005 (Developmental and Comparative Immunology 29, 185-203) for the constant domains of immunoglobulins, C14 of SEQ ID NO:1 corresponds to C11, C28 of SEQ ID NO:1 corresponds to C23, C86 of SEQ ID NO:1 corresponds to C104, C102 of SEQ ID NO:1 corresponds to C124, and N39 of SEQ ID NO:1 corresponds to N38.

[0137] As used herein, the term "monoclonal antibody" means a preparation of antibody molecules of single molecular composition. Monoclonal antibodies exhibit single binding specificity and affinity for a particular epitope. In one embodiment, monoclonal antibodies are produced by hybridomas including those produced by fusing B cells obtained from a non-human animal, such as a mouse, with immortalized cells.

[0138] As used herein, the term "recombinant antibody" means any antibody prepared, expressed, made or isolated by recombinant means, e.g., (a) an antibody isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to an immunoglobulin gene or a hybridoma prepared therefrom, (b) an antibody isolated from a host cell transformed to express an antibody, e.g., a transfectoma, (c) an antibody isolated from a recombinant, combinatorial antibody library, and (d) an antibody prepared, expressed, made or isolated by other means involving splicing of immunoglobulin gene sequences with other DNA sequences.

[0139] Accordingly, "antibodies and antigen-binding fragments thereof" suitable for use in the present invention include polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, chimeric, heterohybrid, humanized (particularly CDR-grafted), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, Fd, Fv, disulfide-bonded Fv (dsFv), single-chain antibodies (e.g., scFv), bispecific antibodies or tetrabodies (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90(14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotype (anti-Id) antibodies (e.g., including anti-Id antibodies to the antibodies of the present invention), and epitope-binding fragments of any of the above, but are not limited thereto.

[0140] As used herein, the term "bispecific" refers to a binding molecule, such as an antibody, that can bind to two different antigens or two different epitopes within one antigen. Similarly, the terms "trispecific" and "tetraspecific" refer to binding molecules that can bind to three and four different antigens, respectively, or epitopes within an antigen.

[0141] As used herein, the term "naturally occurring" as applied to an object means the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including a virus) isolated from a natural source and has not been intentionally modified by a person in the laboratory is naturally occurring.

[0142] As used herein, the term "nucleic acid aptamer" means a nucleic acid molecule engineered to bind to a target molecule through iterative rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) (see Brody E.N. and Gold L. (2000), Aptamers as therapeutic and diagnostic agents. J. Biotechnol. 74(1):5-13 for a review). The nucleic acid aptamer may be a DNA molecule or an RNA molecule. The aptamer may contain modifications, such as modified nucleotides, for example, 2'-fluorine-substituted pyrimidines.

[0143] As used herein, the term "antibody-like protein" means a protein engineered (e.g., by loop mutagenesis) to specifically bind to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide with both ends attached to a protein scaffold. This dual structural constraint significantly improves the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The length of the variable peptide loop is usually 10-20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, and designed ankyrin repeat proteins (see Binz H.K. et al. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23(10):1257-1268 for a review). Antibody-like proteins can be derived from large libraries of variants, e.g., selected by panning from a large phage display library and isolated in a manner similar to that of conventional antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues of globular proteins. Antibody-like proteins are sometimes referred to as "peptide aptamers."

[0144] The "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequences in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions). This percentage is determined by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences, determining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0145] As used herein, the term "identical" is used to mean two or more sequences or subsequences that are identical, i.e., contain the same sequence of nucleotides or amino acids, in the context of two or more nucleic acid or polypeptide sequences. Sequences are considered to be "identical" to each other when they have the same specific percentage of nucleotides or amino acid residues. According to the present invention, at least 70% identity is at least 75%, 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% identity over a particular sequence when measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, comparing and aligning for maximum correspondence over a comparison window or designated region. These definitions also mean the complement of the test sequence. Thus, throughout this specification, the term "at least 70% sequence identity" is used with respect to the sequence comparison of polypeptides and polynucleotides. This expression preferably means 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 each respective reference polypeptide or each respective reference polynucleotide.

[0146] As used herein, "array comparison" means a process of comparing a certain array as a reference array with a test array. When using an array comparison algorithm, the test array and the reference array are input into a computer, the coordinates of the partial array are specified as necessary, and the program parameters of the array algorithm are specified. Although default program parameters are common, alternative parameters can also be specified. Thereafter, the array comparison algorithm calculates the percentage of array identity of the test array with respect to the reference array based on the program parameters. When two arrays are compared and the reference array is not specified in the comparison where the percentage of array identity is calculated, the array identity is calculated based on the longer of the two arrays being compared, unless otherwise indicated. When the reference array is indicated, the array identity is determined based on the full length of the reference array indicated by the array number, unless otherwise indicated.

[0147] In sequence alignment, the term "comparison window" refers to those stretches of adjacent positions of an array that are compared with an adjacent stretch of positions of the reference array having the same number of positions. Typically, the number of adjacent positions ranges from about 20 to 100 adjacent positions, about 25 to 90 adjacent positions, about 30 to 80 adjacent positions, about 40 to about 70 adjacent positions, about 50 to about 60 adjacent positions. According to the present invention, when comparing for percent identity with an array of the present invention such as SEQ ID NO: 1, if the reference array has the same length or a longer length than the SEQ ID NO of the present invention, preferably, the full length of the SEQ ID NO, for example, 106 amino acids of SEQ ID NO: 1 are compared with the reference array. When the reference array is shorter than the SEQ ID NO of the present invention, the full length of the reference array must be compared with the full length of the SEQ ID NO of the present invention.

[0148] Methods for aligning arrays for comparison are well known in the art. Optimal alignment of arrays for comparison can be carried out, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). In this algorithm, first, short words of length W in the query sequence are identified that match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database sequence, thereby identifying high-scoring sequence pairs (HSPs). T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them.Word hits are extended in both directions along each sequence as long as the cumulative alignment score increases. The cumulative score is calculated using the parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction defaults when: the cumulative alignment score drops by an amount X from the maximum value achieved; the cumulative score becomes zero or less due to the accumulation of one or more negative-score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program defaults to a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989), alignment (B) 50, expectation value (E) 10, M = 5, N = -4, and comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurred by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in a comparison of the test nucleic acid and the reference nucleic acid is less than about 0.2, generally less than about 0.01, and more generally less than about 0.001.

[0149] The terms "nucleic acid" and "nucleic acid molecule" are used synonymously herein and are understood to be polymers of single-stranded or double-stranded oligo- or deoxyribonucleotide or ribonucleotide bases, or both. Nucleotide monomers consist of a nucleic acid base, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, nucleic acids are formed via phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, and also includes synthetic forms of nucleic acids containing other linkages (e.g., peptide nucleic acids as described by Nielsen et al. (Science 254:1497-1500, 1991)). Typically, nucleic acids are single-stranded or double-stranded molecules consisting of naturally occurring nucleotides. The description of a single strand of a nucleic acid also (at least partially) defines the sequence of the complementary strand. Nucleic acids can be single-stranded or double-stranded, and can contain portions of both double-stranded and single-stranded sequences. The exemplified double-stranded nucleic acid molecules can have 3' or 5' overhangs, and as such, it is not required or assumed that they be completely double-stranded over their entire length. Nucleic acids can be obtained by any of the methods known in the art, including, but not limited to, biological, biochemical or chemical synthesis methods, or amplification methods, and reverse transcription methods for RNA. The term nucleic acid includes, for example, chromosomes or chromosomal segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA) or small interfering RNA (siRNA), etc. Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, and are not limited to any particular length. Unless otherwise specified, a particular nucleic acid sequence includes or encodes a complementary sequence in addition to any of the explicitly shown sequences.

[0150] As used in the context of the present invention, the term "C-terminus" (also known as the carboxyl terminus, carboxy terminus, C-terminal tail, C-terminus, or COOH-terminus) refers to the end of an amino acid chain (protein or polypeptide) that is terminated by a free carboxyl group (-COOH). When a protein is translated from messenger RNA, it is created from the N-terminus to the C-terminus. The term "N-terminus" (also known as the amino terminus, NH 2 -terminus, N-terminus, or amine terminus) refers to the start of a protein or polypeptide that ends with an amino acid having a free amine group (-NH 2 ). The convention for writing a peptide sequence is to place the N-terminus on the left and write the sequence from the N-terminus to the C-terminus.

[0151] The term "linker" or "peptide linker" in the context of the present invention refers to an amino acid sequence that spatially separates two parts within the engineered polypeptide of the present invention, i.e., a polypeptide. Typically, such a peptide linker consists of 1 to 100, preferably 3 to 50, more preferably 5 to 20 amino acids. Thus, such a peptide linker has 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. A peptide linker can provide flexibility between the two parts that are joined together. Such flexibility generally increases when the amino acids are small. Thus, a flexible peptide linker contains an increased content of small amino acids, particularly glycine and / or alanine, and / or hydrophilic amino acids such as serine, threonine, asparagine, and glutamine. Preferably, 20%, 30%, 40%, 50%, 60% or more of the amino acids of the peptide linker are small amino acids. Preferred peptide linkers have the sequences GGGGS, [G 4 S] 2 、[G 4 S] 3 、[G 2 SG 2 、 [G 2 SG 2 2 、[G 2 SG 2 3 . One of ordinary skill in the art can readily determine the appropriate length of a linker that avoids or hinders intramolecular interactions within a single polypeptide chain.

[0152] ​​​As used herein, the term "substantially the same" means a deviation of up to 10% and including 10% of the disclosed value or numerical value, or a deviation of up to 15% and including 15% of the disclosed value or numerical value. In particular, this term encompasses a deviation of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% of the disclosed value or numerical value.

[0153] As used herein, the term "Fc chain" refers to an Fc portion that can form homodimers or heterodimers and preferably binds to respective effector molecules with either increased or decreased affinity, thus altering effector functions such as ADCC, CMC, or FcRn-mediated recycling. The literature (Presta et al., 2008, Curr Opin Immunol. 20: 460-470), e.g., IgG1-DE (S239D, I332E), describes various IgG variants with altered interactions with human FcγRIIIa (CD16), such as IgG1-DE (S239D, I332E) that results in a 10-fold increase in ADCC, or IgG1-DE (S239D, I332E, A330L) that results in a 100-fold increase in ADCC. The literature also describes Fc portions that decrease effector functions in addition to increasing effector functions. For the IgG1-P329G LALA variant (L234A, L235A, P329G), it has been reported that the interaction with the entire Fcγ receptor family is almost completely lost, resulting in an effector-silent molecule (Schlothauer et al., 2016, Protein Eng Des Sel. 29; 457-466). Furthermore, the reduced binding to FcγRI described for the IgG-Δab variant (E233P, L234V, L235A, Δ236G, A327G, A330S, P331S) also led to a decrease in effector function (Armour et al., 1999; Eur J Immunol. 29: 2612-2624) (also described in Strohl et al., 2009; Curr Opin Biotechnol; 20: 685-691). In addition to altering the binding to receptors on immune cells (e.g., human FcγRIIIa), the binding to FcRn can also be altered by introducing substitutions into the Fc portion.An increase (or decrease) in the binding to the FcRn molecule affects the half-life of the Fc-containing molecule. For example, in IgG1-YTE (M252Y, S254T, T256E), the terminal half-life of the protein increases 3- to 4-fold, and in IgG1-QL (T250Q, M428L), the terminal-phase half-life increases 2.5-fold (Presto et al, 2008; Strohl et al, 2009).

[0154] The terms "heterodimeric Fc" or "Fc of heterodimer" relate to variants of the Fc portion that can form heterodimers. In addition to the knob-into-hole technique, there are other variants of the Fc portion described in the literature for the generation of heterodimeric Fc portions (Krah et al., 2017, N. Biotechnol. 39: 167-173; Ha et al., 2016, Front Immuno. 7: 394; Mimoto et al., 2016, Curr Pharm Biotechnol. 17: 1298-1314; Brinkmann & Kontermann, 2017, MAbs 9: 182-212). The technique, also referred to as "Knob-into-Hole" or "Knobs-into-Holes", means both the mutations Y349C, T366S, L368A and Y407V (Hole) and S354C and T366W (Knob) at the CH3-CH3 interface to promote heteromultimer formation, and is described in particular in patents US5,731,168 and US8,216,805 (both of which are incorporated herein by reference).

[0155] The dimerization domain C used in the context of the present invention H 1 and C L is based on immunoglobulins and can be derived from any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0156] As used herein, the term "trigger molecule on immune effector cells" refers to a molecule that binds to a receptor molecule of the immune system, such as a pattern recognition receptor (PRR), toll-like receptor (TLR), killer activation and killer inhibitory receptors (KAR and KIR), complement receptor, Fc receptor, B cell receptor, and T cell receptor. Binding to these receptors causes a reaction in the immune system via the trigger molecule. Non-limiting examples of trigger molecules on immune effector cells are selected from the group consisting of, but not limited to, CD2, CD3, CD16, CD44, CD64, CD69, CD89, Mel14, Ly-6.2C, TCR complex, Vy9V52 TCR, and NKG2D.

[0157] As used herein, the term "pharmaceutical composition" refers to a substance and / or combination of substances used for the identification, prevention, or treatment of a disease or tissue condition. A pharmaceutical composition is formulated for administration to a patient for the prevention and / or treatment of a disease. Further, a pharmaceutical composition means a combination of an active agent and an inert or active carrier that renders the composition suitable for therapeutic use. Depending on its chemical and physical properties, a pharmaceutical composition can be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous, or vaginal routes of administration. Pharmaceutical compositions include solids, semi-solids, liquids, and transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powders, granules, pellets, capsules, effervescent tablets, or transdermal therapeutic systems. Also included are liquid compositions selected from the group consisting of solutions, syrups, drip solutions, extracts, solutions for intravenous application, drip solutions, or solutions of the carrier systems of the present invention. Semi-solid compositions that can be used in the context of the present invention include emulsions, suspensions, creams, lotions, gels, microspheres, buccal tablets, and suppositories.

[0158] The term "active agent" means a biologically active, i.e., pharmaceutically valuable, substance in a pharmaceutical composition or formulation. According to the present invention, the pharmaceutical composition comprises at least the binding molecule according to the present invention as an active agent. However, the pharmaceutical composition may comprise more than one active agent that can act in concert or independently of one another. The active agent may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed from free amino groups, for example, those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and those formed from free carboxyl groups, for example, but not limited to, those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0159] The terms used in this document are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0160] The practice of the present invention, unless otherwise specified, uses conventional methods of biochemistry, cell biology, immunology, and recombinant DNA technology as described in the literature of this field (e.g., see Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0161] All of the methods described in this specification can be performed in any suitable order, unless otherwise specified herein or clearly inconsistent with the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not otherwise limit the scope of the claimed invention. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0162] Description of Embodiments In the following, various aspects of the invention are defined in more detail. Each aspect so defined can be combined with any other aspect(s), unless the contrary is clearly indicated. In particular, any feature(s) shown as being preferred or advantageous can be combined with any other feature(s) shown as being preferred or advantageous.

[0163] In a first aspect, the present invention provides a binding molecule comprising a first polypeptide chain comprising a first binding domain (BD1) and a first modified EHD2 domain (EHD2-1), and a second polypeptide chain comprising a second binding domain (BD2) and a second modified EHD2 domain (EHD2-2). The amino acid sequences of EHD2-1 and EHD2-2 are different from each other. One has an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and no Cys at position 14, and the other has an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and no Cys at position 102. The Cys is preferably replaced by one selected from the group consisting of various amino acids, preferably Ser, Gly, Ala, Thr, Gln, Asn, and Tyr, most preferably Ser. The substitution of Cys at position 14 or 102 of SEQ ID NO: 1 prevents the formation of a disulfide bridge between the amino acid at position 14 on the amino acid sequence of the first modified EHD2 domain and the amino acid at position 102 of the second modified EHD2 domain. Nevertheless, the modified EHD2 domains can form a covalent bond, preferably one disulfide bridge, between each other, thereby functioning as a dimerization domain for the binding domains. In the binding molecule of the present invention, the binding domains BD1 and BD2 together form an antigen-binding site, giving rise to the binding properties and characteristics of the binding molecule.

[0164] Accordingly, according to the present invention, EHD2-1 and EHD2-2 are covalently bonded to each other, preferably by means of a disulfide bond. Both modified EHD2 domains (EHD2-1 and EHD2-2) preferably have the same or at least substantially the same length, i.e., the same or substantially the same number of amino acids.

[0165] While not wishing to be bound by any theory, substitution of Cys at position 14 in one modified EHD2 domain and substitution of Cys at position 102 in another modified EHD2 domain prevent the formation of a disulfide bridge between these two positions. In the native EHD2 domain, the disulfide bond confers symmetry and enables the formation of homodimers. Deleting one disulfide bond between modified EHD2 domains results in asymmetry and allows only the formation of heterodimers. Thus, substituting Cys at position 14 of the first modified EHD2 domain with, for example, Ser and substituting Cys at position 102 of the other modified EHD2 domain with, for example, Ser, surprisingly, by applying this heterodimer domain, fusing a first binding domain to the first modified EHD2 domain (carrying the C14 substitution) of the first polypeptide chain and a second binding domain to the second modified EHD2 domain (having the C102 substitution) of the second polypeptide chain, it has been found that a heterodimeric binding molecule can be produced. Furthermore, by fusing one of the modified EHD2 domains to the Fc region, it is possible to produce divalent, trivalent, and tetravalent molecules with various specificities. Substituting amino acids one by one in the two EHD2 domains minimizes the number of mutations and, while maintaining the native interface between the two domains, heterodimerization can be forced by directed or induced disulfide bond formation.

[0166] Compared to the state of the art, the binding molecules of the invention using alternative EHD2 modifications (hetEHD2) have one or more of the following advantages:

[0167] The thermal stability of the heterodimer does not decrease, or at least does not decrease as much, as determined, for example, by dynamic light scattering or differential scanning calorimetry.

[0168] The sensitivity of the heterodimer to proteolysis does not decrease, or at least does not decrease as much.

[0169] The immunogenicity of the heterodimer does not increase, or at least does not increase as much.

[0170] Using the modified EHD2 domain results in an increase in the fraction of heterodimers compared to homodimers.

[0171] The modified EHD2 domain does not interfere with the cysteines in the hinge region.

[0172] It should be noted that the intradomain disulfide bond between positions C28 and C86 of SEQ ID NO: 1 preferably results in a disulfide bond within one EHD2 domain and is maintained in the modified EHD2 domains (EHD2-1 and EHD2-2).

[0173] The basic structure of the binding molecule according to the present invention is schematically shown in FIG. 2, where the binding domain BD1 is bound to EHD2-1 and the binding domain BD2 is bound to EHD2-2. Both modified EHD2 domains together form a heterodimeric EHD2 shown as hetEHD2.

[0174] According to a preferred embodiment, the Cys at position 14 is preferably substituted by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr (C14S, C14G, C14A, C14T, C14Q, C14N, and C14Y). In the most preferred embodiment of the present invention, the Cys at position 14 of SEQ ID NO: 1 is substituted by Ser (C14S).

[0175] According to a further preferred embodiment, the Cys at position 102 is preferably replaced by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr (C102S, C102G, C102A, C102T, C102Q, C102N, C102Y). In the most preferred embodiment of the present invention, the Cys at position 102 of SEQ ID NO: 1 is replaced by Ser (C102S). The substitutions mentioned above can be combined in any way, but the most preferred set of mutations is C14S in one modified EHD2 domain and C102S in the other modified EHD2 domain. Thus, according to a particularly preferred embodiment, one of the modified EHD2 domains (EHD2-1 and EHD2-2) contains the substitution C14S and the other of the modified EHD2 domains contains the substitution C102S.

[0176] Each of the modified EHD2 domains (EHD2-1 and EHD2-2) can have at least 70% amino acid identity to SEQ ID NO: 1, but it is noted that it is preferred that the amino acid sequences of both modified EHD2 domains are substantially the same with respect to the amino acid sequences ignoring the specific substitutions at positions 14 and 102 respectively.

[0177] Furthermore, it is noted that in all cases sequences that are at least 70% identical to SEQ ID NO: 1 contain specific substitutions such as the substitutions at positions C14 and C102. It is further preferred that the cysteine residues at positions 28 and 86 are maintained and do not undergo any mutations such as single amino acid substitutions.

[0178] According to a preferred embodiment of the present invention, one or both of the modified EHD2 domains further contain a single amino acid substitution at position N39. The preferred substitution of Asn at this position is Gln (N39Q).

[0179] According to a further preferred embodiment, the binding modules BD1 and BD2 are different from each other and each, V H and V LIt is selected from. Alternatively, BD1 and BD2 are different from each other and are each selected from the TCR α-chain and the TCR β-chain.

[0180] The binding molecule of the present invention may further comprise a first Fc chain. The effector function of the Fc chain can be increased or decreased. This first Fc chain preferably binds to one of the first polypeptide or the second polypeptide.

[0181] According to a preferred embodiment of the present invention, the binding molecule of the present invention further comprises a third binding domain (BD3) and a fourth binding domain (BD4). Both the binding domains BD3 and BD4 together form an antigen binding site. This antigen binding site may be the same as or different from the binding site formed by BD1 and BD2.

[0182] According to one embodiment, BD3 and BD4 are different from each other and each, V H and V L It is selected from. Alternatively, BD3 and BD4 are different from each other and are each selected from the TCR α-chain and the TCR β-chain.

[0183] The third binding domain (BD3) can be located on the same polypeptide as the first binding domain or the second binding domain (BD1, BD2). Similarly, the fourth binding domain (BD4) can be located on the same polypeptide as the first binding domain or the second binding domain (BD1, BD2), preferably on a polypeptide different from the third binding domain (BD3).

[0184] According to a further preferred embodiment, the binding molecule of the present invention further comprises a second Fc chain. This second Fc chain preferably binds to one of a third polypeptide or a fourth polypeptide. The Fc chains in the binding molecule of the present invention enable dimerization of BD1 and BD2 with BD3 and BD4, resulting in a bivalent binding molecule. The Fc chain may be a homodimerizing Fc chain or a heterodimerizing Fc chain. According to a preferred embodiment, the first Fc chain and the second Fc chain are different from each other and form a heterodimeric Fc.

[0185] According to a preferred embodiment, the third polypeptide chain of the binding molecule of the present invention comprising BD3 further comprises a dimerization domain selected from the group consisting of a C H 1 domain, a C L domain, a first modified EHD2 domain (EHD2-1), and a second modified EHD2 domain (EHD2-2). Accordingly, the fourth polypeptide chain of the binding molecule of the present invention comprises a C L domain, a C H 1 domain, a second modified EHD2 domain (EHD2-2), and a respective counter binding domain selected from the group consisting of a first modified EHD2 domain (EHD2-1). In other words, if BD3 comprises a C H 1 domain, BD4 comprises a C L domain. If BD3 comprises a C L domain, BD4 comprises a C HIt contains 1 domain. When BD3 contains the first modified EHD2 domain (EHD2-1), BD4 contains the second modified EHD2 domain (EHD2-2). When BD3 contains the second modified EHD2 domain (EHD2-2), BD4 contains the first modified EHD2 domain (EHD2-1). The modified EHD2 domains, as defined above, i.e., the amino acid sequences of EHD2-1 and EHD2-2 are different from each other. One amino acid sequence of the modified EHD2 domains (EHD2-1 and EHD2-2) has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 14, and the other one amino acid sequence of the modified EHD2 domains (EHD2-1 and EHD2-2) has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 102. As defined above, the Cys at the indicated position is preferably replaced by a different amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr, most preferably Ser.

[0186] Thus, the present invention encompasses bivalent binding molecules that are either monospecific or bispecific.

[0187] These binding molecules include a first polypeptide and a second polypeptide chain comprising BD1 and BD2 and the modified EHD2 domains EHD2-1 and EHD2-2. Further, these binding molecules (i) BD3-C H 1 and BD4-C L ; (ii) BD3-C L and BD4-C H 1; (iii) BD3-EHD2-1 and BD4-EHD2-2; or (iv) BD3-EHD2-2 and BD4-EHD2-1, may include a third polypeptide chain and a fourth polypeptide chain.

[0188] Non-limiting examples for embodiments (i) and (ii) are shown in FIGS. 3B and 3C. Non-limiting examples for embodiments (iii) and (iv) are shown in FIG. 3G, which is exemplified as a bivalent monospecific binding molecule. According to an exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 22 + 23.

[0189] In the case of generating a bispecific binding molecule, the Fc region is preferably a heterodimer, which means that one of the first polypeptide chain and the second polypeptide chain contains a first Fc chain, and one of the third polypeptide chain and the fourth polypeptide chain contains a second different Fc chain, forming a heterodimer Fc region in the binding molecule. Non-limiting examples of such bivalent bispecific binding molecules containing a heterodimer Fc region are shown in the top row of FIGS. 3B and 3C.

[0190] According to one embodiment, when the third polypeptide chain and the fourth polypeptide chain contain a modified EHD2 domain, one or both of the modified EHD2 domains further contain a single amino acid substitution at position N39. The preferred substitution of asparagine at this position is glutamine (N39Q).

[0191] According to the present invention, when four different polypeptide chains are expressed in one cell, one chain has a C H domain, and the other chain uses a heterodimerizing polypeptide chain containing a first modified EHD2 domain (EHD2-1) to co-express a polypeptide chain containing a C L domain, and a polypeptide chain containing a second modified EHD2 domain (EHD2-2), correct assembly of the heavy and light chains is observed, and a bivalent bispecific antibody that completely retains the antigen-binding specificity for each antigen is obtained.

[0192] Alternatively, the binding molecule of the present invention may comprise four binding domains on three polypeptide chains. According to each embodiment, two binding domains are each located on a first polypeptide chain, and the other two binding domains are located on the second and third polypeptide chains.

[0193] An exemplary embodiment of such a bivalent binding module having three polypeptide chains is shown in Figure 3A and may have the following polypeptide chains: (i) BD1-(optional linker)-EHD2-2-linker-BD3-(optional linker)-C H 1 (ii) BD2-(optional linker)-EHD2-1 (iii) BD4-(optional linker)-C L

[0194] As shown on the left of Figure 3A, when all of the above three polypeptide chains are co-expressed intracellularly, in the resulting binding molecule according to the present invention, polypeptide chain (ii) pairs with the EHD2-1 domain of (i), and polypeptide chain (iii) pairs with the C H 1 domain of (i). According to an exemplary embodiment, the binding molecule has a sequence represented by SEQ ID NO: 9 + 13 + 17, as schematically shown in Figure 3A.

[0195] In an alternative embodiment, the binding molecule according to the present invention comprising three polypeptide chains may have the following polypeptide chains: (i) BD3-(optional linker)-C H 1-linker-BD1-(optional linker)-EHD2-2 (ii) BD2-(optional linker)-EHD2-1 (iii) BD4-(optional linker)-C L

[0196] The linker is preferably a peptide linker as defined above.

[0197] As shown to the right of FIG. 3A, when all three of the above polypeptide chains are co-expressed intracellularly, in the binding molecule obtained according to the present invention, polypeptide chain (ii) pairs with the EHD2-1 domain of (i), and polypeptide chain (iii) pairs with the C H domain 1 of (i). According to an exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 9 + 13 + 18.

[0198] It is understood that the order or arrangement and selection of the dimerization domain and the binding domain within the tetravalent binding molecule can be varied and adapted according to individual needs as long as there is at least one pair of modified EHD2 domains as defined herein that results in a heterodimeric EHD2 domain.

[0199] According to a further embodiment of the present invention, the binding molecule is bivalent and comprises four polypeptide chains. For purposes of illustration only, exemplary embodiments of such trivalent binding molecules comprising four polypeptide chains are shown in FIGS. 3B and 3C. This binding molecule can have the following polypeptide chains: (i) BD1-(optional linker)-EHD2-1-Fc (ii) BD2-(optional linker)-EHD2-2 (iii) BD3-(optional linker)-C H 1-Fc (iv) BD4-(optional linker)-C L .

[0200] As shown in FIGS. 3B and 3C, when all four polypeptide chains are co-expressed intracellularly, the binding molecule obtained according to the present invention comprises polypeptide chains (i) and (iii) as "heavy chains", and corresponding polypeptide chains (ii) and (iv) as "light chains", where (ii) pairs with the EHD2-1 domain of (i), and (iv) pairs with the CH1 domain of (iii). According to an exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 9 + 10 + 13 + 14, as schematically shown in FIG. 3B.

[0201] According to a further embodiment of the present invention, the binding molecule is trivalent and thus further comprises a fifth binding domain (BD5) and a sixth binding domain (BD6). Both the binding domains BD5 and BD6 together form an antigen-binding site.

[0202] According to a preferred embodiment of the present invention, the binding molecule further comprises a C H 1 domain, a C L domain, a first modified EHD2 domain (EHD2-1), or a second modified EHD2 domain (EHD2-2). In this embodiment of the present invention, BD6 binds to a respective counter part selected from a C L domain, a C H 1 domain, the second modified EHD2 domain (EHD2-2), and the first modified EHD2 domain (EHD2-1). In other words, if BD5 binds to a C H 1 domain, BD6 binds to a C L domain. If BD5 binds to a C L domain, BD6 binds to a C H1 binds to a domain. When BD5 binds to the first modified EHD2 domain (EHD2-1), BD6 binds to the second modified EHD2 domain (EHD2-2). When BD5 binds to the second modified EHD2 domain (EHD2-2), BD6 binds to the first modified EHD2 domain (EHD2-1). The modified EHD2 domains are as defined above, i.e., the amino acid sequences of EHD2-1 and EHD2-2 are different from each other. One of the amino acid sequences of the modified EHD2 domains (EHD2-1 and EHD2-2) has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 14, and the other amino acid sequence of the modified EHD2 domains (EHD2-1 and EHD2-2) has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 102. As defined above, the Cys at the indicated position is preferably replaced by a different amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr, most preferably Ser.

[0203] As used herein, the term "binds to" means a bond between two polypeptides using a direct peptide bond or via a peptide linker as defined above. Thus, according to one embodiment, C that binds to BD5 or BD6 H 1 domain, C L domain, EHD2-1 or EHD2-2 binds to one of BD1, BD2, BD3 or BD4 via a linker.

[0204] According to one embodiment, one or both of the modified EHD2 domains that bind to BD5 or BD6 further contain a single amino acid substitution at position N39. The substitution of asparagine at this position is glutamine (N39Q).

[0205] According to a preferred embodiment, BD5 and BD6 are different from each other and each, V H and V Lselected from. Alternatively, BD5 and BD6 are different from each other and are each selected from the TCR α-chain and the TCR β-chain.

[0206] The binding molecules according to the invention, which contain three binding sites, i.e., are trivalent, can bind to the same or different targets, resulting in trivalent monospecific, bispecific or trispecific binding molecules. Preferably, in these trivalent binding molecules according to the invention, the C that binds to BD5 or BD6 H 1 domain, C L domain, EHD2-1 or EHD2-2 preferably binds to one of BD1, BD2, BD3 or BD4 via a linker. It is emphasized that in these trivalent molecules, one or two sets of modified EHD2 domains can be used to provide the spatial arrangement of the binding domains for forming the antigen-binding sites.

[0207] Thus, the trivalent binding molecules according to the invention can essentially contain three units, each forming an antigen-binding site: (i) A first and a second polypeptide chain comprising BD1 and BD2, and the modified EHD2 domains EHD2-1 and EHD2-2; (ii) A third and a fourth polypeptide chain comprising BD3 and BD4, and either the modified EHD2 domains EHD2-1 and EHD2-2 or the domain C H 1 and C L either one; and (iii) BD5 and BD6 that bind to either the modified EHD2 domains EHD2-1 and EHD2-2 or the domain C H 1 and C L either one.

[0208] According to a preferred embodiment of the trivalent binding molecule, the dimerization domains EHD2-1, EHD2-2, C H 1 and C LOne of BD5 and BD6, which is combined with one of them, forms part of a further fifth polypeptide chain. Correspondingly, the other one of BD5 and BD6, together with the corresponding dimerization domain (EHD2-1, EHD2-2, C H 1, and C L the other one of) binds to any one of BD1, BD2, BD3 or BD4.

[0209] By way of example only, an exemplary embodiment of such a trivalent binding molecule comprising five polypeptide chains is shown in FIG. 3D. This binding molecule has the following polypeptide chains: (i) BD5-(optional linker)-C H 1-linker-BD1-(optional linker)-EHD2-1-(optional linker)-Fc (ii) BD2-(optional linker)-EHD2-2 (iii) BD3-(optional linker)-C H 1-Fc (iv) BD4-(optional linker)-C L (v) BD6-(optional linker)-C L .

[0210] As shown in FIG. 3D, when all five polypeptide chains are co-expressed intracellularly, the resulting binding molecule according to the invention comprises polypeptide chains (i) and (iii) as "heavy chains" and corresponding polypeptide chains (ii), (iv) and (v) as "light chains", where (ii) pairs with the EHD2-1 domain of (i), (iii) pairs with (iv), and (v) pairs with the C H 1 domain of (i). According to one exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 9 + 13 + 14 + 19, as schematically shown in FIG. 3D.

[0211] Similarly, other constructs can be made according to individual requirements. Further non-limiting embodiments are shown in FIG. 3E. Such a binding molecule according to one embodiment of the invention also comprises five polypeptide chains: (i) BD1-(Any linker)-EHD2-1-(Any linker)-Fc (ii) BD2-(Any linker)-EHD2-2 (iii) BD5-(Any linker)-C H 1-Linker-BD3-(Any linker)-C H 1-(Any linker)-Fc (iv) BD4-(Any linker)-C L (v) BD6-(Any linker)-C L 。

[0212] As shown in Figure 3E, when all five of the above polypeptide chains are co-expressed intracellularly, the binding molecule obtained according to the present invention comprises polypeptide chains (i) and (iii) as "heavy chains" and corresponding polypeptide chains (ii), (iv), and (v) as "light chains", where (ii) pairs with the EHD2-1 domain of (i), (iv) pairs with the C H 1 domain of (iii), and (v) pairs with the C H 1 domain of (iii). According to an exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 9 + 10 + 13 + 20, as schematically depicted in Figure 3E.

[0213] Further non-limiting examples are shown in Figure 3F. Such binding molecules according to one embodiment of the present invention also comprise five polypeptide chains: (i) BD1-(Any linker)-EHD2-1-Linker-BD3-(Any linker)-C H 1-(Any linker)-Fc (ii) BD2-(Any linker)-EHD2-2 (iii) BD5-(Any linker)-C H 1-(Any linker)-Fc (iv) BD4-(Any linker)-C L (v) BD6-(Any linker)-C L 。

[0214] As illustrated in Fig. 3F, when all five of the above polypeptide chains are co-expressed intracellularly, the binding molecule obtained according to the present invention comprises polypeptide chains (i) and (iii) as "heavy chains" and polypeptide chains (ii), (iv) and (v) as corresponding "light chains", where (ii) pairs with the EHD2-1 domain of (i), (iv) pairs with the C H 1 domain of (i), and (v) pairs with the C H 1 domain of (iii). According to an exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 9+13+14+21, as schematically shown in Fig. 3F.

[0215] It is emphasized that the order or arrangement and selection of the dimerization domain and the binding domain within the construct can be varied and adapted to individual requirements as long as at least one pair of modified EHD2 domains, namely EHD2-1 and EHD2-2 as defined herein, are present. Thus, the binding molecule according to the present invention can have two pairs of modified EHD2 domains. Examples of such binding molecules according to the present invention are not limited to trivalent binding molecules but also include tetravalent binding molecules.

[0216] According to a further embodiment of the present invention, the binding molecule is trivalent and thus further comprises a seventh binding domain (BD7) and an eighth binding domain (BD8). Both the binding domains BD7 and BD8 together form an antigen-binding site.

[0217] According to a preferred embodiment of the present invention, the binding molecule further comprises a C H 1 domain, a C L domain, a first modified EHD2 domain (EHD2-1), or a second modified EHD2 domain (EHD2-2). In this embodiment of the present invention, BD8 binds to a counter part selected from a C L domain, a C H 1 domain, a second modified EHD2 domain (EHD2-2), and a first modified EHD2 domain (EHD2-1). In other words, if BD7 is C HWhen binding to 1 domain, BD8 binds to the C L domain. When BD7 binds to the C L domain, BD8 binds to the C H 1 domain. When BD7 binds to the first modified EHD2 domain (EHD2-1), BD8 binds to the second modified EHD2 domain (EHD2-2). When BD7 binds to the second modified EHD2 domain (EHD2-2), BD8 binds to the first modified EHD2 domain (EHD2-1). The modified EHD2 domains are as defined above, that is, the amino acid sequences of EHD2-1 and EHD2-2 are different from each other. One amino acid sequence of the modified EHD2 domains (EHD2-1 and EHD2-2) has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 14, and the other one amino acid sequence of the modified EHD2 domains (EHD2-1 and EHD2-2) has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 102. As defined above, the Cys at the indicated position is preferably substituted by a different amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr, most preferably by Ser.

[0218] As used herein, the term "binds to" means a bond between two polypeptides using a direct peptide bond or via a peptide linker as defined above. Thus, according to one embodiment, the C H 1 domain, C L domain, EHD2-1 or EHD2-2 binds to one of BD1, BD2, BD3, BD4, BD5 or BD6 via a linker.

[0219] According to one embodiment, one or both of the modified EHD2 domains that bind to BD7 or BD8 further contain a single amino acid substitution at position N39. The preferred substitution target for asparagine at this position is glutamine (N39Q).

[0220] According to a preferred embodiment, BD7 and BD8 are different from each other and each is selected from V H and V L Alternatively, BD7 and BD8 are different from each other and each is selected from TCR α-chain and TCR β-chain.

[0221] The binding molecules according to the invention, which contain four binding sites, i.e., are tetravalent, can bind to the same or different targets, resulting in tetravalent monospecific, bispecific, trispecific or tetravalent specific binding molecules. Preferably, in these tetravalent binding molecules according to the invention, the C H 1 domain, C L domain, EHD2-1 or EHD2-2 preferably binds to one of BD1, BD2, BD3, BD4, BD5 or BD6 via a linker. It is emphasized that in these tetravalent molecules, one or two sets of modified EHD2 domains can be used to provide the spatial arrangement of the binding domains for forming the antigen binding sites.

[0222] Thus, the tetravalent binding molecules according to the invention essentially contain four units, each forming an antigen binding site: (i) a first and a second polypeptide chain comprising BD1 and BD2, and the modified EHD2 domains EHD2-1 and EHD2-2; (ii) a third and a fourth polypeptide chain comprising BD3 and BD4, and either the modified EHD2 domains EHD2-1 and EHD2-2 or one of the domains C H 1 and C L ; (iii) BD5 and BD6 that bind to either the modified EHD2 domains EHD2-1 and EHD2-2 or one of the domains C H 1 and C L ; and (iv) the modified EHD2 domains EHD2-1 and EHD2-2, or one of the domains C H 1 and C LBD7 and BD8 that bind to either one of them.

[0223] According to a preferred embodiment of the tetravalent binding molecule, one of BD7 and BD8 combined with one of the dimerization domains EHD2-1, EHD2-2, C H 1 and C L forms part of a further sixth polypeptide chain. Correspondingly, the other one of BD7 and BD8, together with the corresponding dimerization domain (EHD2-1, EHD2-2, C H 1, and the other one of C L ) that binds to it, binds to any one of BD1, BD2, BD3, BD4, BD5 or BD6.

[0224] Non-limiting examples of the tetravalent binding molecules of the present invention are shown in FIGS. 3H and 3I. A non-limiting embodiment of the tetravalent binding molecule according to the present invention may include six polypeptide chains. A non-limiting embodiment of such a binding molecule may have the following polypeptide chains: (i) BD5-(optional linker)-C H 1-linker-BD1-(optional linker)-EHD2-1-(optional linker)-Fc (ii) BD2-(optional linker)-EHD2-2 (iii) BD7-(optional linker)-C H 1-linker-BD3-(optional linker)-EHD2-1-(optional linker)-Fc (iv) BD4-(optional linker)-EHD2-2 (v) BD6-(optional linker)-C L (vi) BD8-(optional linker)-C L .

[0225] As shown in FIG. 3H, when all six of the above polypeptide chains are co-expressed intracellularly, the binding molecule obtained according to the present invention comprises polypeptide chains (i) and (iii) as "heavy chains" and corresponding polypeptide chains (ii), (iv), (v) and (vi) as "light chains", where (ii) pairs with the EHD2-1 domain of (i), (iv) pairs with the EHD2-1 domain of (iii), (v) pairs with the C H 1 domain of (i), and (vi) pairs with the C H 1 domain of (iii). According to an exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 23+24+25, as schematically shown in FIG. 3H.

[0226] A further non-limiting example of a tetravalent binding molecule according to the present invention is shown in FIG. 3I. A non-limiting embodiment of such a binding molecule can have the following six polypeptide chains: (i) BD1-(optional linker)-EHD2-1-linker-BD5-(optional linker)-C H 1-(optional linker)-Fc (ii) BD2-(optional linker)-EHD2-2 (iii) BD3-(optional linker)-EHD2-1-linker-BD7-(optional linker)-C H 1-(optional linker)-Fc (iv) BD4-(optional linker)-EHD2-2 (v) BD6-(optional linker)-C L (vi) BD8-(optional linker)-C L .

[0227] As shown in FIG. 3I, when all six of the above polypeptide chains are co-expressed intracellularly, the binding molecule obtained according to the present invention comprises polypeptide chains (i) and (iii) as "heavy chains" and corresponding polypeptide chains (ii), (iv), (v) and (vi) as "light chains", where (ii) pairs with the EHD2-1 domain of (i), (iv) pairs with the EHD2-1 domain of (iii), (v) pairs with the C H1 associates with the domain, and (vi) is C of (iii). H 1 associates with the domain. According to an exemplary embodiment, the binding molecule has the sequence shown by SEQ ID NO: 23 + 25 + 26, as schematically shown in FIG. 3I.

[0228] As described above for the trivalent binding molecule according to the present invention, the order or arrangement and selection of the dimerization domain and the binding domain within the tetravalent binding molecule can be changed and adapted according to individual needs, as defined herein, as long as there are at least two sets of modified EHD2 domains.

[0229] According to one embodiment of the present invention, in the binding molecule of the present invention, one or more modified EHD2 domains carry one N-glycan. This can be achieved, for example, preferably by further substituting an amino acid at position N39. A preferred amino acid substitution is to substitute asparagine with glutamine at this position (N39Q). FIG. 4 schematically and exemplarily shows modified EHD2 domains (EHD2-1, EHD2-2) in which both domains (EHD2-1, EHD2-2) carry an N-glycan on only one of the domains (EHD2-1 or EHD2-2), or the N-glycan is completely absent.

[0230] It is again emphasized that the Cys substitution required in the modified EHD2 domain is either at position 14 or 102 of SEQ ID NO: 1. Each of Cys at positions 14 and 102 can be substituted by any amino acid. The substituted amino acids may be the same or different for both positions 14 and 102. According to a preferred embodiment, the first modified EHD2 domain has a substitution selected from the group consisting of C14S, C14G, C14A, C14T, C14Q, C14N, C14Y, preferably including C14S. According to this embodiment, the second modified EHD2 domain has a substitution selected from the group consisting of C102S, C102G, C102A, C102T, C102Q, C102N, C102Y, preferably including C102S.

[0231] According to the present invention, one or more antigens to which the binding molecule of the present invention can bind can be selected from the group consisting of, but not limited to: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; ALK; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AXL; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAFF-R; BAG1; BAI1; BCL2; BCL6; BCMA; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B BMPR2; BPAG1 (plectin); BRCA1; B7-H3; C19orf10 (IL27w); C1s; C3; C4A; C5; C5R1; CA-125; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 CMJP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 / eotaxin-2); CCL25 (TECT); CCL26 (eotaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1 / HM145); CCR2 (mcp-1RB / RA); CCR3 (CKR3 CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 STRL22 / DRY6); CCR7 (CKR7 EB11); CCR8 (CMKBR81 / TER1 / CKR-L1);CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD5; CD7; CD15; CD19; CD1G; CD11a; CD20; CD200; CD22; CD23; CD24; CD25; CD27; CD28; CD30; CD33; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD41; CD4SRB; CD51; CD52; CD56; CD6; CD62L; CD70; CD72; CD73; CD74; CD79A; CD79B; CDB; CD80; CD81; CD83; CD86; CD105; CD117; CD123; CD125; CD137L; CD137; CD147; CD152; CD154; CD221; CD276; CD279; CD319; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19, CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1 / Cip1); CDKNIB (p27Kipl); CDKN1C; CDKN2A (p16INK4a); CDKN2B; CDKN2C; CDKN3; CEA; CEACAM5; CEBPB; CER1; CFD; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (Claudin-7); CLDN18.2; CLN3; CLU (clusterin); cMET; CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSFR1; CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (β-catenin); CTSB (Cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GRO3); CXCL5 (ENA-78 / LIX); CXCL6 (GCP-2); CXCL9 (MIG);CXCR3 (GPR9 / CKR-L2); CXCR4; CXCR6 (TYMSTR / STRL33 / Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1; DLL3; DPP4; DR3; DR4; DR5; DR6; E2F1; ECGF1; EDA1; EDA2; EDAR; EDA2R; EDG1; EpCAMEFNA1; EFNA3; EFNB2; EGF; EGFL7; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHA3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); F9 or F9a; F10 or F10a; FADD; FAP; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR1; FGFR2; FGFR3; FGFR4; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; folate receptor 1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-GST; GATA3; gelatinase B; GD2; GD3; GDF5; GDF8; GFI1; GGT1; GITR; GITRL; GM-CSF; GNAS1; GNRH1; GPNMB; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HER2; HER3; HER4; HGF; H1F1A; HIP1; histamine and histamine receptor; HLA-A; HLA-DRA; HM74; HMOX1; HMW-MAA Hsp-90; HVEM; TNF-RHUMCYT2A; ICAM-1; ICEBERG; ICOSL; ID2;IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1; IGF1R; IGP1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IGHE; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; TL1F9; IL1HY1; IL1R1; IL1R2; ILLRAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2; IL24; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (Glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; Integrin αvβ3; Integrin β7; IRAK1; IRAK2; TGA1; ITGA2; ITGA3; ITGA6 (a6 Integrin); ITGAV; JTGB3; ITGB4 (b4 Integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KIR2D; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (Hair-Specific Type II Keratin); LAMA5; LEP (Leptin); LEY; LIGHT; Lingo-p75; Lingo-Troy; LIV-1; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS;MAG or Omgp; MAP2K7 (c-Jun); MCAM; MCSP; MDK; MET; MER; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MSLN; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MUC2; MYC; MYD88; myostatin; NCA-2; NCK2; nectin-4; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgRLingo; NOGO-A; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOTCH-1; NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT3; NT4; NT5E; NTN4; ODZ1; OPRD1; OX40; OX40L; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCDC1; PCNA; PCSK9; PD1; PD-L1; PDGFA; PDGFB; PDGR; igfPECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); uPAR; PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PSMA; PTAFR; PTEN; PTGS2 (COX-2); PTN; PTK7; VEGFR1; VEGFR2; VEGFR3; RAC2 (p21Rac2); RANK; RANKL; RARB; RELT; RET; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; RON; ROR1; ROR2; RYK; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial monocyte activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1;SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPAK; SPP1; SPRR1B (Spr1); SOST; ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TACl; TAG-72; tau protein; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGPB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIE-1; TIE-2; TIMP3; Tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNF-b; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSP21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNPSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TNF-R1; TNF-R2; TOLLIP; Toll-like receptor; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TRAIL-R1; TRAIL-R2; TRAIL-R3; TRAIL-R4; TREM1; TREM2; TRPC6; TROY; TSLP; TWEAK; TYRO3; TYRP1; VAP-1; VEGF; VEGFB; VEGFC; versican; VHL C5; vimentin; VLA-4; VWF; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5 / CCXCR1); YY1; and ZFPM2.;

[0232] According to a preferred embodiment, the binding molecule of the present invention binds to CD3 on T cells as part of a trigger molecule on immune effector cells, such as the T cell receptor (TCR) (Brinkmann & Kontermann, 2017, mAbs 9:182-212). According to the present invention, the trigger molecule of the immune effector cell is selected from the group consisting of, but not limited to, CD2, CD3, CD16, CD44, CD64, CD69, CD89, Mel14, Ly-6.2C, TCR complex, Vy9V52 TCR, or NKG2D.

[0233] According to a further embodiment, the binding molecule of the present invention binds to MET (5D5) (Jin et al., 2008, Cancer Res. 68, 4360-4368).

[0234] The most preferred embodiments of the bivalent, trivalent or tetravalent binding molecules according to the present invention bind to HER3 at one, two or three binding sites and to CD3 at the remaining one, two or three binding sites, respectively. Example 2 provides an example of a preferred bispecific bivalent binding molecule that binds to HER3 and CD3.

[0235] A further most preferred embodiment of the bivalent, trivalent or tetravalent binding molecules according to the present invention binds to HER3 at one, two or three binding sites and to MET at the remaining one, two or three binding sites. Even more preferably, the binding molecule of the present invention is a bispecific bivalent one that binds to MET and HER3, as exemplified in Example 1 below of the present specification.

[0236] The binding molecule of the present invention may further comprise a peptide leader sequence; one or more, preferably a hexahistidyl-tag or a FLAG-tag, which helps in purification; and / or one or more costimulatory molecules.

[0237] In embodiments where the binding molecules of the present invention are used for cell-cell recruitment, for example, where immune effector cells such as T cells or macrophages are recruited to tumor cells, it is preferred that two binding valences of the binding molecule bind to the tumor cells and the remaining one or two binding valences bind to the immune effector cells. This approach provides a high avidity for binding to tumor cells on the one hand and prevents activation of immune effector cells that may result from bivalent binding of the target to the immune effector cells on the other hand.

[0238] According to the present invention, the modified EHD2 domain has an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1. Preferably, the deviation from SEQ ID NO: 1 is due to conservative substitutions. More preferably, these substitutions do not include substitution of one or more amino acid residues to cysteine. The introduced mutations preferably do not result in the formation of intermolecular disulfide bonds, i.e., additional disulfide bonds between the first modified EHD2 domain and the second modified EHD2 domain. "Conservative substitutions" may be made, for example, based on the similarity of polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the amino acid residues involved. Amino acids can be classified into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0239] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed within the same group of the six standard amino acid groups shown above. For example, replacing Asp with Glu retains one negative charge in the polypeptide so modified. Further, glycine and proline can be substituted for one another based on their ability to disrupt α-helices. Some preferred conservative substitutions within the six groups above are exchanges within the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (ii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Given the known genetic code, and recombinant and synthetic DNA technologies, a skilled scientist can readily assemble DNA encoding conservative amino acid variants.

[0240] As used herein, "non-conservative substitution" or "non-conservative amino acid exchange" is defined as the replacement of an amino acid with another amino acid listed in a different one of the six standard amino acid groups (1)-(6) shown above.

[0241] Particularly preferred molecules according to the invention are (1) a light chain V L 3-43-C L λ (SEQ ID NO: 13) and a heavy chain V H 3-43-C H 1-Fc knob (SEQ ID NO: 14) having a constant part, and (i) HC1-LC2 resulting in eIgG1 (heavy chain: V H 5D5-EHD2-1(N39Q)-Fc hole (SEQ ID NO: 12), light chain: V L 5D5-EHD2-2 (SEQ ID NO: 11)), or (ii) HC2-LC1 resulting in eIgG2 (heavy chain: V H 5D5-EHD2-2-Fc hole (SEQ ID NO: 10), light chain: V L 5D5-EHD2-1(N39Q) (SEQ ID NO: 9)) in combination with either one; (2) the V of the light chain L3-43-C L λ (Accession No.: 13) and the V of the heavy chain H 3-43-C H 1-Fc knob (Accession No.: 14) and eIgG (heavy chain: V H huU3-EHD2-2-Fc hole (Accession No.: 16), light chain: V L huU3-EHD2-1(N39Q) (Accession No.: 15)); (3) The light chain specified by Accession No.: 13 or 15, and the heavy chain specified by Accession No.: 14, 16, 20, 39, 40 or 41; (4) The V of the light chain L 3-43-C L λ (Accession No.: 13) and the V of the heavy chain H 3-43-C H 1-Fc knob (Accession No.: 14) pairing, and the V of the light chain L hu36-EHD2-1(N39Q) (Accession No.: 43) and the V of the heavy chain H hu36-EHD2-2-Fc hole (Accession No.: 42) pairing; (5) The V of the light chain L 3-43-EHD2-1(N39Q) (Accession No.: 23) and the V of the heavy chain H 3-43-EHD2-2-Fc (Accession No.: 22); (6) The V of the light chain L 3-43-EHD2-1(N39Q) (Accession No.: 23) and V L hu225-C L κ (Accession No.: 25), and the V of the heavy chain H hu225-C H 1-V H 3-43-EHD2-2-Fc (Accession No.: 24); (7) The V of the light chain L 3-43-EHD2-1(N39Q) (Accession No.: 23) and V L hu225-C L κ (Accession No.: 25), the V of the heavy chain H hu225-C H 1-V H 3-43-EHD2-2 (Accession No.: 44); (8) The V of the light chainL hu36-CLκ (SEQ ID NO: 45) and the V of the heavy chain H hu36-C H A first molecule comprising 1-Fc(hole) (SEQ ID NO: 46), wherein the V of the light chain H 2C11-EHD2-1(N39Q) (SEQ ID NO: 47) and the V of the heavy chain L A molecule having a CD3-targeting arm with 2C11-EHD2-2-Fc(knob) (SEQ ID NO: 48), and the V of the light chain L 2C11-EHD2-1(N39Q) (SEQ ID NO: 49) and the V of the heavy chain H A second molecule comprising 2C11-EHD2-2-Fc(knob) (SEQ ID NO: 50); (9) The V of the light chain L S309-CLκ (SEQ ID NO: 51) and V L P2B-2F6-EHD2-1(N39Q) (SEQ ID NO: 52), and the V of the heavy chain H S309-C H 1-V H P2B-2F6-EHD2-2-Fc (SEQ ID NO: 53).

[0242] The most preferred molecules according to the present invention are (i) SEQ ID NOs: 13, 14, 15 and 16; (ii) SEQ ID NOs: 13, 15, 16 and 20; (iii) SEQ ID NOs: 13, 15, 39 and 40; (iv) SEQ ID NOs: 13, 14, 15 and 41; (v) SEQ ID NOs: 23, 24 and 25.

[0243] Even more preferred molecules according to the present invention have at least 90% sequence identity to the sequences specified under (1)-(9) and (i)-(v) above, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences specified under (1)-(9) and (i)-(v) above, and include molecules specified as (1)-(9) and (i)-(v) above. It will be understood that this sequence identity can mean one, two, three or four of the individual sequences specified in each of the above items (1)-(9) and (i)-(v). Preferably, these molecules having at least 90% or more sequence identity to the sequences specified under (1)-(9) and (i)-(v) above are essentially the same as or maintain the same biological function as each molecule derived from those containing the sequences specified under (1)-(9) and (i)-(v) above. Preferably, the term "biological function" as used herein means binding specificity and / or affinity. Maintaining essentially the same biological function means at least 50% of the binding specificity and / or affinity of each molecule containing the sequences specified under (1)-(9) and (i)-(v) above from which it is derived, preferably at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the binding specificity and / or affinity. The determination of binding and / or affinity is well known to those skilled in the art and can be carried out, for example, by surface plasmon resonance measurement and / or in vitro release assay.

[0244] Those skilled in the art will understand that the molecules according to the present invention may or may not have a histidine-tag (His-tag, 6xHis tag, etc.) or a similar addition or tag exemplified herein to one or more polypeptides to facilitate their purification. Those skilled in the art will readily understand that such additional structures do not have a specific impact on the functional properties of the molecules described herein.

[0245] According to a further aspect, the invention provides a nucleic acid or a set of nucleic acids encoding a binding molecule according to the invention. The nucleic acid can be degraded by endonucleases or exonucleases, particularly DNase and RNase that can be found intracellularly. Thus, it can be advantageous to modify the nucleic acids of the invention to be stabilized against degradation, thereby ensuring that high concentrations of nucleic acids are maintained intracellularly for extended periods. Typically, such stabilization can be obtained by introducing one or more internucleotide linking groups, or by introducing one or more non-phosphorus internucleotides. Thus, the nucleic acid can consist of non-naturally occurring nucleotides and / or modifications to naturally occurring nucleotides, and / or changes to the backbone of the molecule. Modified internucleotide phosphate radicals and / or non-phosphorus bridges in the nucleic acid include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate, phosphorodithioate and / or phosphate ester, while non-phosphorus internucleotide analogs include, but are not limited to, siloxane bridges, carbonate bridges, carboxymethyl ester, acetamidate bridges and / or thioether bridges. Further examples of nucleotide modifications include, but are not limited to: phosphorylation of the 5' or 3' nucleotide to enable ligation or prevent exonuclease degradation / polymerase extension, respectively; amino, thiol, alkyne or biotinyl modifications for covalent and near-covalent linkages; fluorophores and quenchers; and modified bases such as deoxyinosine (dI), 5-bromo-deoxyuridine (5-bromo-dU), deoxyuridine, 2-aminopurine, 2,6-diaminopurine, inverted dT, inverted dideoxy-T, dideoxycytidine (ddC), 5-methyl-deoxycytidine (5-methyl dC), locked nucleic acids (LNA’s), 5-nitroindole, iso-dC and -dG bases, 2'-O-methyl RNA bases, hydroxymethyl dC, 5-hydroxybutynyl-2'-deoxyuridine, 8-aza-7-deazaguanosine and fluorine-modified bases.Thus, nucleic acids can also include artificial nucleic acids such as polyamides or peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA), but are not limited thereto.

[0246] In any of the above schematic basic arrangements and embodiments of the binding molecule according to the invention, it should be understood that there are free N-termini and / or C-termini of the polypeptide chain available for the attachment of additional functional groups. Alternatively, or in addition, functional groups, particularly pharmaceutically active moieties and / or imaging molecules, can be attached to the side chains of amino acids within the polypeptide chain such as Lys, Arg, Glu or Asp.

[0247] Common expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells). Specific examples include Escherichia coli, Kluyveromyces yeast or Saccharomyces yeast, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (such as those produced from lymphocytes, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), HEK293 cells, etc. Preferred host cells are HEK293-6E cells (Durocher et al., 2002, Nucl. Acids Res. 30(2)e9) or CHO cells.

[0248] Accordingly, in a further aspect, the invention provides a vector comprising the nucleic acid or set of nucleic acids of the invention. Suitable vectors for expressing nucleic acids in host cells are well known in the art.

[0249] The invention also provides a method of producing a recombinant host cell expressing a binding molecule according to the invention, the method comprising: (i) introducing the nucleic acid or set of nucleic acids or vector as described above into a competent host cell in vitro or ex vivo; (ii) culturing the resulting recombinant host cell in vitro or ex vivo; and (iii) optionally, selecting cells that express and / or secrete the binding molecule.

[0250] Accordingly, the invention also provides a host cell comprising a vector according to the invention.

[0251] In a further aspect, the invention provides a pharmaceutical composition comprising a binding molecule, nucleic acid or set of nucleic acids, vector, or host cell according to the invention as an active agent, and a pharmaceutically acceptable carrier and / or suitable additive. The pharmaceutical composition further comprises one or more additional active agents. The pharmaceutical composition is preferably selected from the group consisting of solids, liquids, semi-solids or transdermal therapeutic systems. The pharmaceutical composition of the invention is envisaged to comprise one or more complexes of the first aspect of the invention.

[0252] In a further aspect, the invention relates to a binding molecule, nucleic acid or set of nucleic acids, vector, host cell, or pharmaceutical composition according to the invention for use in medicine.

[0253] The binding molecule, nucleic acid or set of nucleic acids, vector, host cell, or pharmaceutical composition of the invention is particularly suitable for treating cancer.

[0254] The invention particularly relates to the following items:

[0255] Item 1. A first polypeptide chain comprising a first binding domain (BD1) and a first modified EHD2 domain (EHD2-1), and A second polypeptide chain comprising a second binding domain (BD2) and a second modified EHD2 domain (EHD2-2), the binding molecule comprising The amino acid sequences of EHD2-1 and EHD2-2 are different from each other, and each is selected from (i) an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 14, or (ii) an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 102, BD1 and BD2 together form an antigen-binding site, wherein EHD2-1 and EHD2-2 are covalently bonded to each other, the binding molecule.

[0256] Item 2. The binding molecule according to item 1, wherein one or both of the modified EHD2 domains further comprise a single amino acid substitution at position N39.

[0257] Item 3. BD1 and BD2 are different from each other, and each is selected from V H and V L or selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain, the binding molecule according to any one of items 1 to 2.

[0258] Item 4. The binding molecule according to any one of items 1 to 3, further comprising a first Fc chain.

[0259] Item 5. The binding molecule according to any one of claims 1 to 4, further comprising a third binding domain (BD3) and a fourth binding domain (BD4), wherein BD3 and BD4 together form an antigen-binding site.

[0260] Item 6. BD3 and BD4 are different from each other, and each is selected from V H and V L or is selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain, the binding molecule according to item 5.

[0261] Item 7. Further comprising a third polypeptide chain, preferably further comprising a third polypeptide chain and a fourth polypeptide chain, the binding molecule according to item 5 or 6.

[0262] Item 8. Further comprising a second Fc chain, the binding molecule according to any one of items 5 to 7.

[0263] Item 9. The first Fc chain and the second Fc chain are different from each other and form a heterodimeric Fc, the binding molecule according to item 8.

[0264] Item 10. The binding molecule according to any one of items 5 to 9, which is monospecific or bispecific.

[0265] Item 11. The third polypeptide chain containing BD3 is (i) C H 1 domain, (ii) C L domain, (iii) the first modified EHD2 domain (EHD2-1), and (iv) the second modified EHD2 domain (EHD2-2), and further comprises one of The fourth polypeptide chain containing BD4 is In the case of (i), C L domain, (ii) In the case of, C H 1 domain, (iii) In the case of, the second modified EHD2 domain (EHD2-2), and (iv) In the case of, the first modified EHD2 domain (EHD2-1), and further comprises Here, the amino acid sequences of EHD2-1 and EHD2-2 are different from each other, and each has an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 14, or an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 102, and is selected from any one of items 5 to 10 of the binding molecule according to any one of items 5 to 10.

[0266] Item 12. The binding molecule according to item 11, wherein one or both of the modified EHD2 domains of the third polypeptide chain or the fourth polypeptide chain further contain a single amino acid substitution at position N39.

[0267] Item 13. The binding molecule according to any one of items 5 to 12, further comprising a fifth binding domain (BD5) and a sixth binding domain (BD6), wherein BD5 and BD6 together form an antigen-binding site.

[0268] Item 14. Binding to BD5, (i) C H 1 domain, (ii) C L domain, (iii) The first modified EHD2 domain (EHD2-1), or (iv) The second modified EHD2 domain (EHD2-2), and Binding to BD6, (i) In the case of C L domain, (ii) In the case of C H 1 domain, (iii) In the case of the second modified EHD2 domain (EHD2-2), and (iv) In the case of the first modified EHD2 domain (EHD2-1), further comprising, wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other and each has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 14, or has at least 70% amino acid identity to SEQ ID NO: 1 and does not have Cys at position 102, the binding molecule according to item 13, which is selected from an amino acid sequence.

[0269] Item 15. The binding molecule according to item 14, wherein one or both of the modified EHD2 domains that bind to BD5 or BD6 further comprise a single amino acid substitution at position N39.

[0270] Item 16. BD5 and BD6 are different from each other and each is selected from V H and V L or is selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain, the binding molecule according to item 14 or 15.

[0271] Item 17. The C H 1 domain, C L domain, EHD2-1 or EHD2-2 is bound to one of BD1, BD2, BD3 or BD4 via a linker, the binding molecule according to any one of items 14 to 16.

[0272] Item 18. The binding molecule according to any one of items 11 to 17, which is monospecific, bispecific, or trispecific.

[0273] Item 19. further comprising a seventh binding domain (BD7) and an eighth binding domain (BD8), wherein BD7 and BD8 together form an antigen-binding site, the binding molecule according to any one of items 11 to 18.

[0274] Item 20. Binding to BD7 (i) C H 1 domain (ii) C L domain (iii) The first modified EHD2 domain (EHD2-1), or (iv) The second modified EHD2 domain (EHD2-2), and that binds to BD8, in the case of (i), C L domain (ii) in the case of, C H 1 domain (iii) in the case of, the second modified EHD2 domain (EHD2-2), and (iv) in the case of, the first modified EHD2 domain (EHD2-1), further comprising, wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other, and each has an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 14, or an amino acid sequence having at least 70% amino acid identity to SEQ ID NO: 1 and not having Cys at position 102, the binding molecule according to item 19, which is selected from a 102-amino acid sequence.

[0275] Item 21. The binding molecule according to item 20, wherein one or both of the modified EHD2 domains that bind to BD7 or BD8 further comprise a single amino acid substitution at position N39.

[0276] Item 22. BD7 and BD8 are different from each other, and each is selected from V H and V L or selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain, the binding molecule according to item 21.

[0277] Item 23. The C H 1 domain that binds to BD7 or BD8, C LThe binding molecule according to any one of items 20 to 22, wherein the domain, EHD2-1 or EHD2-2 binds to one of BD1, BD2, BD3 or BD4 that is not bound to BD5 or BD6 via a linker.

[0278] Item 24. The binding molecule according to any one of items 19 to 23, which is monospecific, bispecific, trispecific or tetravalent.

[0279] Item 25. The binding molecule according to any one of items 1 to 24, wherein none of the modified EHD2 domains carry one N-glycan, or one or more modified EHD2 domains carry one N-glycan.

[0280] Item 26. The binding molecule according to any one of items 1 to 25, wherein the Cys at position 14 of SEQ ID NO: 1 is substituted by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr, preferably Ser.

[0281] Item 27. The binding molecule according to any one of items 1 to 26, wherein the Cys at position 102 of SEQ ID NO: 1 is substituted by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn, and Tyr, preferably Ser.

[0282] Item 28. The binding molecule according to any one of items 1 to 27, wherein the single amino acid substitution at position N39 is N39Q.

[0283] Item 29. A nucleic acid or set of nucleic acids encoding the binding molecule according to any one of items 1 to 28.

[0284] Item 30. A vector containing the nucleic acid or set of nucleic acids of item 29.

[0285] Item 31. A host cell containing the vector according to Item 30.

[0286] Item 32. A pharmaceutical composition comprising a binding molecule according to any one of Items 1 to 28, a nucleic acid or set of nucleic acids according to Item 29, a vector according to Item 30, or a host cell according to Item 31, and a pharmaceutically acceptable carrier.

[0287] Item 33. A binding molecule comprising SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0288] Item 34. A binding molecule comprising SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 20.

[0289] Item 35. A binding molecule comprising SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 39 and SEQ ID NO: 40.

[0290] Item 36. A binding molecule comprising SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 41.

[0291] Item 37. A binding molecule comprising SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.

[0292] Item 38. A nucleic acid or set of nucleic acids encoding the binding molecule according to any one of Items 33 to 37.

[0293] Item 39. A vector containing the nucleic acid or set of nucleic acids according to Item 38.

[0294] Item 40. A host cell containing the vector according to Item 39.

[0295] Item 41. A pharmaceutical composition comprising a binding molecule according to any one of items 33 to 37, a nucleic acid or set of nucleic acids according to item 38, a vector according to item 39, or a host cell according to item 40, and a pharmaceutically acceptable carrier.

[0296] Item 42. A binding molecule according to any one of items 33 to 37, a nucleic acid or set of nucleic acids according to item 38, a vector according to item 39, a host cell according to item 40, or a pharmaceutical composition according to item 41 for use in medicine, preferably for use in the treatment of cancer.

[0297] Item 43. A treatment method comprising administering to a patient in need of treatment a therapeutically effective amount of a binding molecule according to any one of items 33 to 37, a nucleic acid or set of nucleic acids according to item 38, a vector according to item 39, a host cell according to item 40, or a pharmaceutical composition according to item 41.

[0298] Item 44. A method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a binding molecule according to any one of items 33 to 37, a nucleic acid or set of nucleic acids according to item 38, a vector according to item 39, a host cell according to item 40, or a pharmaceutical composition according to item 41.

[0299] Item 45. A binding molecule according to any one of items 1 to 28, a nucleic acid or set of nucleic acids according to item 29, a vector according to item 30, a host cell according to item 31, or a pharmaceutical composition according to item 32 for use in medicine, preferably for use in the treatment of cancer.

[0300] Item 46. A treatment method comprising administering to a patient in need of treatment a therapeutically effective amount of a binding molecule according to any one of items 1 to 28, a nucleic acid or set of nucleic acids according to item 29, a vector according to item 30, a host cell according to item 31, or a pharmaceutical composition according to item 32.

[0301] Item 44. A method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a binding molecule according to any one of Items 1 to 28, a nucleic acid or set of nucleic acids according to Item 30, a vector according to Item 30, a host cell according to Item 31, or a pharmaceutical composition according to Item 32.

Example

[0302] Example 1: Bispecific bivalent anti-cMET x anti-HER3 eIgGs The bispecific bivalent eIgG molecule was prepared by combining an Fv domain specific for HER3(3-43) (Schmitt et al., 2017, mAbs 9, 831-843) with the C H 1 / C L heterodimer, and an Fv domain specific for MET(5D5) (Jin et al., 2008, Cancer Res. 68, 4360-4368) with the modified C H 2 domain (hetEHD2), and by making the Fc portion of the heterodimer (knob-into-hole technology). Thus, the bispecific molecule consists of four different polypeptide chains. The constant parts of the various antibodies are the V L 3-43-C L λ (SEQ ID NO: 13) and the V of the heavy chain H 3-43-C H 1-Fc knob (SEQ ID NO: 14). The second part results in eIgG1, HC1-LC2 (heavy chain: V H 5D5-EHD2-1(N39Q)-Fc hole (SEQ ID NO: 12); light chain: V L 5D5-EHD2-2 (SEQ ID NO: 11)), or eIgG2, resulting in HC2-LC1 (heavy chain: V H 5D5-EHD2-2-Fc hole (SEQ ID NO: 10); light chain: V LIt consists of either one of the EHD2 arrangements of 5D5-EHD2-1 (SEQ ID NO: 9). The bispecific bivalent eIgG molecule exhibits one antigen-binding site for HER3 and one binding site for MET. Each polypeptide chain is illustrated and schematically shown in FIG. 5A, and the resulting binding molecule is schematically shown in FIG. 5B.

[0303] The bispecific bivalent eIgG molecule was transiently transfected and expressed in HEK293-6E cells after co-administering four plasmids encoding both heavy and both light chains using polyethyleneimine as a transfection reagent. The protein secreted into the cell culture supernatant was purified using Protein A affinity chromatography. SDS-PAGE analysis revealed four major bands under reducing conditions: two bands of approximately 55 kDa corresponding to the two heavy chains of the antibody, and two bands of approximately 25 kDa corresponding to the two light chains of the antibody. Furthermore, a faint band of approximately 30 kDa, most likely corresponding to the glycosylated version of the light chain containing EHD2-2 (V L 5D5-EHD2-2) was also observed in eIgG1, and the glycosylated version of the heavy chain containing EHD2-2 (V H 5D5-EHD2-2-Fc hole) A faint band of approximately 65 kDa with the highest likelihood of corresponding to 2 O 2 was also observed. Under non-reducing conditions, one major band of approximately 200 kDa with the highest likelihood of corresponding to an intact antibody consisting of four different polypeptide chains was observed (Figure 5C). The purity, integrity, and homogeneity of the bispecific bivalent eIgG molecules were confirmed by size exclusion chromatography (Figure 5D). The binding of both eIgG molecules to the extracellular domains (ECDs) of HER3 (aa21 - 643) and MET (aa25 - 787) was determined by ELISA. His-tagged HER3 protein and MET Fc fusion protein were coated on polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% non-fat milk (MPBS). Next, the plates were incubated with serial dilutions of the bispecific eIgG antibody. After washing, the bound antibody was detected with an HRP-conjugated anti-human Fc antibody using the HER3-His antigen, an HRP-conjugated anti-human Fab antibody using the MET-Fc antigen, and TMB, H 50 O as the substrate. Both bispecific bivalent eIgG antibodies showed concentration-dependent binding to HER3-His and MET-Fc (EC

[0304] Example 2: Bispecific Bivalent Anti-CD3xAnti-HER3 eIgG The bispecific bivalent eIgG molecule combines an Fv domain specific for HER3 (3 - 43) (Schmitt et al., 2017, mAbs 9, 831 - 843) with the C H 1 / C L heterodimer, and an Fv domain specific for CD3 (huU3) with the modified C of IgE HIt was prepared by combining with 2 domains (hetEHD2) and combining the Fc portion of the heterodimer (knob-into-hole technology). The CD3 binding site consists of a humanized version of the anti-CD3 mAb UCHT1. Thus, the bispecific molecule consists of four different polypeptide chains. The first part of the various antibodies is the V of the light chain L 3-43-C L λ (SEQ ID NO: 13) and the V of the heavy chain H 3-43-C H 1-Fc knob (SEQ ID NO: 14). The second part results in eIgG, which is HC2-LC1 (heavy chain: V H huU3-EHD2-2-Fc hole (SEQ ID NO: 16); light chain: V L huU3-EHD2-1(N39Q) (SEQ ID NO: 15)) of the EHD2 arrangement. The bispecific bivalent eIgG molecule exhibits one antigen-binding site for HER3 and one binding site for CD3. Each polypeptide chain is illustrated and schematically shown in FIG. 6A, and the resulting binding molecule is schematically shown in FIG. 6B.

[0305] The bispecific bivalent eIgG molecule was transiently transfected and expressed in HEK293-6E cells after simultaneously administering four plasmids encoding both heavy and both light chains, using polyethyleneimine as a transfection reagent. The protein secreted into the cell culture supernatant was purified using protein A affinity chromatography. SDS-PAGE analysis revealed two major bands under reducing conditions: one band of approximately 50 kDa corresponding to the heavy chain of the antibody V H 3-43-C H 1-Fc knob and one band of approximately 25 kDa corresponding to the two light chains of the antibody. Furthermore, the heavy chain (in non-glycosylated and glycosylated forms) containing EHD2-2 (V H huU3-EHD2-2-Fc hole) Two faint bands of approximately 60 kDa and 65 kDa corresponding to 2 O 2 were observed. Under non-reducing conditions, one major band of approximately 200 kDa corresponding to intact antibodies consisting of four different polypeptide chains was observed (Figure 6C). The purity, integrity, and homogeneity of the bispecific bivalent eIgG molecules were confirmed by size exclusion chromatography (Figure 6D). The binding of the eIgG molecules to the extracellular domains (ECDs) of HER3 (aa21-643) and CD3 was determined by ELISA. His-tagged HER3 protein and CD3-Fc fusion protein were coated onto polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). Next, the plates were incubated with serial dilutions of the bispecific eIgG antibody. After washing, the bound antibodies were detected with HRP-conjugated anti-human Fc antibody using HER3-His antigen, HRP-conjugated anti-human Fab antibody using MET-Fc antigen, and TMB, H 50 O as substrate. The bispecific bivalent eIgG antibody showed concentration-dependent binding (EC 3 50 values of 2.1 nM for HER3) to HER3-His and CD3-Fc (Figure 6E). These experiments confirmed the binding of the bispecific bivalent eIgGs antibody to both antigens of HER3 and MET. The binding assay of the bispecific eIgG antibody to HER3-expressing cells (LIM1215) and CD3-expressing cells (Jurkat) was analyzed via flow cytometry. The attached LIM1215 cells were washed with PBS and immediately trypsinized at 37°C. Trypsin was quenched with FCS-containing medium and removed by centrifugation (500 x g, 5 minutes). Suspended Jurkat cells were used without trypsin. 100,000 cells per well were seeded and PBA (2% (v / v) FCS, 0.02% (w / v) NaN 3The cells were incubated for 1 hour at 4°C with serial dilutions of the bispecific eIgG antibody diluted in PBS containing []. The cells were washed twice with PBA. The bound antibody was detected using a PE-labeled anti-human Fc secondary antibody and incubated for an additional 1 hour at 4°C. After washing, the median fluorescence intensity (MFI) was measured using a Milltenyi MACSQuant® Analyzer 10. The relative MFI (relative to unstained cells) was calculated using MACSQuant® software and Excel. The eIgG antibody bound to the cells in a concentration-dependent manner (EC 50 values were 0.4 nM when using LIM1215 and 11.9 nM when using Jurkat cells) (Figure 6F).

[0306] Example 3: Protein engineering for generating the hetero-dimeric hetEHD2 domain Positions C14 on chain A and C102 on chain B were substituted with various residues (A, T, N, S, W), and their effects on the formation of functional hetero-dimeric eFab molecules (hetE-Fab) were evaluated. As a model, hetE-Fab consisting of the variable domains of IgG3-43 (anti-HER3 target) fused to two hetEHD2 domains was used (Figure 1A). The molecules were generated by site-directed mutagenesis using the Q5® Site-Directed Mutagenesis Kit (NEB). After confirming the correct substitution of various residues, different combinations of the two plasmids were co-transfected into HEK293-6E cells (Figure 7A and Table 1), and since all molecules contained a His tag on the VH-hetEHD2 chain, the proteins were purified from the cell culture supernatant by affinity chromatography using Ni-NTA resin. The concentration of the molecules was determined photometrically at 280 nM using a NanoDrop ND-1000, and the yield was calculated relative to the volume of the supernatant used.

[0307] Table 1: List of various Fv3-43-hetEHD2 (eFab) molecules with different residues at position C14 of chain A and position C102 of chain B. Molecules with different combinations were produced and analyzed for yield and binding to HER3 used as immobilized antigen in ELISA (EC 50 values). The sequence numbers are shown in the table.

Table 1

[0308] The yields varied, but all eFab combinations could be created (Table 1). The proteins were further analyzed by SDS-PAGE under reducing and non-reducing conditions (Figure 7B). Under reducing conditions, the presence of two different chains with an expected size of approximately 27 kDa for the VH-hetEHD2-2 fragment (carrying N-glycans on the hetEHD2 domain) and 23 kDa for the non-glycosylated light chain was confirmed. Under non-reducing conditions, the dimer assembly and disulfide bonds of the molecules were confirmed. Finally, using various eFab molecules, the binding to the immobilized extracellular domain (ECD) of HER3 (aa21-643) fused to the Fc portion was determined by ELISA (Figure 7C). The HER3-Fc fusion protein was immobilized on polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). Next, the plates were incubated with serial dilutions of various eFab molecules. After washing, the bound eFab molecules were detected with an HRP-conjugated anti-His antibody. TMB and H 2 O 2 were used as substrates. All eFab molecules showed concentration-dependent binding (EC 50 values in the nanomolar range) to the HER3-Fc fusion protein (Table 1).

[0309] In summary, the data show that functional eFab molecules can be generated by introducing various combinations of substitutions into the hetEHD2 domain (positions Cys14 in chain A and Cys102 in chain B) using, for example, alanine, asparagine, threonine, or tryptophan, thereby removing one of the original covalent disulfide bonds between C14 and C102. The discovery that various combinations of residues can be introduced without affecting heterodimer formation or antigen binding indicates an unexpected degree of plasticity at these positions. Interestingly, some substitutions, such as asparagine (N) in one chain and alanine (A) or threonine in another, yielded eFabs that were slightly superior to others with respect to productivity and antigen binding.

[0310] In a second experiment, four new eFab molecules (C14A,N39Q:N39,C102A; C14A,N39Q:N39,C102N; C14N,N39Q:N39,C102T; C14T,N39Q:N39,C102N) and the originally used eFab (C14S,N39Q:N39,C102S) were analyzed. Again, the molecules were produced in HEK293-6E suspension cells co-transfected and purified using Ni-NTA affinity purification. The integrity of the molecules was analyzed by size exclusion chromatography. Here, a single peak of the correct size was observed for all molecules, indicating that heterodimers were correctly formed in all variants (Figure 8).

[0311] Example 4: Various hetEHD2 molecules with various glycosylations Based on a novel design for the formation of EHD2 heterodimers, the effects of changes in N-glycosylation of the hetEHD2 domain on expression and functional heterodimer formation were investigated using serine substitutions on both strands (chain A: C14S; chain B: C102S). Naturally, the EHD2 domain carries an N-glycan at position N39. Using an eFab targeting HER3, eFab with N-glycans on both EHD2 domains (eFab1) (C14S, N39 (SEQ ID NO: 37): N39, C102S (SEQ ID NO: 31)), two eFabs with only one glycosylated EHD2 site (eFab2: C14S, N39Q (SEQ ID NO: 36): N39, C102S (SEQ ID NO: 31); eFab3: C14S, N39 (SEQ ID NO: 37): N39Q, C102S (SEQ ID NO: 38)), and one molecule without any N-glycosylation on EHD2 (eFab4) (C14S, N39Q (SEQ ID NO: 36): N39Q, C102S (SEQ ID NO: 38)) were generated. The resulting molecules are schematically shown in Figure 9A. V H - The hetEHD2 chain further contained a His-tag for purification and detection. Monovalent eFab molecules were transfected using polyethyleneimine as the transfection reagent, V H - hetEHD2-2 chain and V L - hetEHD2-1 chain were expressed in HEK293-6E cells after transient cotransfection of two plasmids encoding them. Proteins secreted into the cell culture supernatant were purified using Ni-NTA affinity chromatography. Under non-reducing conditions in SDS-PAGE analysis, one major band of approximately 50 kDa corresponding to intact eFab molecules consisting of two different polypeptide chains was observed (Figure 9B).

[0312] The binding of various Fab molecules to the extracellular domain (ECD) of HER3 (aa21-643) was determined by ELISA (Figure 9C). The HER3-Fc fusion protein was coated onto polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% non-fat milk (MPBS). Next, the plates were incubated with 100 nM of various Fab molecules. After washing, the bound molecules were detected with an HRP-conjugated anti-His antibody using HER3-Fc as the immobilized antigen. TMB and H2O2 were used as substrates. All four different Fab molecules showed similar binding to HER3-Fc (Figure 9C). These experiments confirmed that functional heterodimers are formed using various glycosylation derivatives of the hetEHD2 moiety.

[0313] Example 5: Bispecific trivalent eIg-Fab molecules targeting HER3 and CD3 For the generation of bispecific bivalent eIg-Fab molecules, i.e., the fusion of Fab fragments to the eIg molecule, two Fab domains derived from antibody 3-43 (anti-HER3; Schmitt et al, 2017, mAbs, 9:831-843), namely the C H 1 / C L Fv domain specific for HER3, fused to the heterodimer, ii) the modified C HOne Fab domain specific for CD3, iii) the Fc portion of the heterodimer (knob-into-hole technology), which was fused to 2 domains (hetEHD2), were used in various combinations (Figure 4). The CD3 binding site was derived from a humanized version of the anti-CD3 mAb UCHT1. The bispecific trivalent eIg-Fab molecule consists of four different polypeptide chains with different arrangements of the HER3 binding site and the CD3 binding site (see Figure 4 for details). Thus, the bispecific trivalent eIg-Fab molecule exhibits two antigen binding sites for HER3 and one binding site for CD3. The molecular structure of the bispecific bivalent eIg molecule (eIg1) having one binding site for HER3 and one binding site for CD3 is described in Example 2. The sequences of the light and heavy chains are described in Table 2.

[0314] Table 2: Various polypeptide chains for the production of eIg molecules. Using two light chains and two heavy chains, various bispecific eIg and eIg-Fab molecules were produced. Figure 10 shows the use of various molecules.

Table 2

[0315] Bispecific, bivalent or trivalent eIg molecules were transiently transfected into HEK293-6E cells using polyethyleneimine (PEI; linear, 25 kDa, Sigma-Aldrich, 764604) as a transfection reagent. The supernatant was collected 96 hours after transfection, and the protein was purified by protein A affinity chromatography. The purity of the protein was confirmed by SDS-PAGE analysis (Figure 10B), where all bispecific trivalent eIg-Fab molecules revealed three major bands under reducing conditions: V of the heavy chain respectively H 3-43-C H 1-linker-V H huU3-EHD2-Fc hole 、V H huU3-EHD2-linker-V H3-43-C H 1-Fc knob or V H 3-43-C H 1-Linker-V H 3-43-C H 1-Fc knob One band of about 77 kDa corresponding to 1-Fc, V H 3-43-C H 1-Fc knob , V H 3-43-C H 1-Fc hole or V L A second band of about 55 kDa corresponding to huU3-EHD2-1(N39Q), and two different light chains (V L 3-43-C L λ and V L A third band of about 26 kDa corresponding to huU3-EHD2-1(N39Q)). In the bivalent bispecific eIg molecule, the V of the heavy chain H 3-43-C H 1-Fc knob and V H huU3-EHD2-Fc hole , and the V of the light chain L huU3-EHD2-1(N39Q) and V L 3-43-C L Two bands of about 55 kDa and 26 kDa corresponding to λ were observed, respectively. In all eIg-Fab molecules, a major band of more than 180 kDa corresponding to the intact antibody was observed under non-reducing conditions. The purity, integrity and homogeneity of the bispecific bivalent and trivalent eIg molecules were determined by size exclusion chromatography using Waters 2695 HPLC and a TSKgel SuperSW mAb HR column (Tosoh Bioscience) with 0.1M Na 2 HPO 4 / NaH 2 PO 4 , 0.1M Na 2 SO 4 , pH 6.7 as the mobile phase at a flow rate of 0.5 ml / min. Here, all proteins eluted as one major peak corresponding to correctly associated molecules (Figure 10C).

[0316] The binding of eIg-Fab molecules to cancer cell lines showing various HER3 levels (LIM1215: 19,877 HER3 / cell; BT474: 11,244 HER / cell) and CD3-expressing cell line (Jurkat) was determined by flow cytometry (Figure 11). The attached cells were washed with PBS and immediately trypsinized at 37°C. Trypsin was quenched with FCS-containing medium and removed by centrifugation (500 x g, 5 minutes). 1 x 10 5 target cells were incubated with serial dilutions of eIg or eIg-Fab molecules diluted in PBA (2% (v / v) FCS, 0.02% (w / v) NaN 3 containing PBS) at 4°C for 1 hour. The bound protein was detected using a PE-conjugated anti-human Fc antibody (Jackson ImmunoResearch Laboratories Inc.). Fluorescence was measured by MACSQuant® Analyzer 10 (Miltenyi Biotec), and the data were analyzed using FlowJo (Tree Star). The relative mean fluorescence intensity (MFI) was calculated as follows: Relative MFI = ((MFI サンプル -(MFI detection - MFI 細胞 )) / MFI 細胞s ). For the two HER3-expressing cancer cell lines, the bispecific trivalent eIg-Fab molecule showed superior binding compared to the bispecific bivalent eIg1. The EC 50 value of the bispecific trivalent eIg-Fab molecule was in the low nanomolar range, while the bispecific bivalent eIg1 molecule showed a 50-fold weaker binding (Figure 11A, B) (Table 3). For the CD3-expressing human cell line Jurkat, eIg1 (SEQ ID NOs: 13, 14, 15, 16) and eIg-Fab4 (SEQ ID NOs: 13, 14, 15, 41) showed similar binding with EC 50 values of 5.1 ± 2.2 nM and 5.9 ± 0.5 nM, respectively, while eIg-Fab2 (SEQ ID NOs: 13, 15, 16, 20) and eIg-Fab3 (SEQ ID NOs: 13, 15, 39, 40) showed EC 50The values showed strong binding at 0.2 ± 0.08 nM and 1.1 ± 0.5 nM, respectively (Table 3). Thus, the trivalent bispecific eIg, having a higher valence for HER3 compared to the bivalent bispecific eIg, resulted in excellent binding to HER3-expressing cancer cells. In contrast, the different binding of the trivalent bispecific eIg molecule to CD3-expressing Jurkat cells indicates the influence of the position of the CD3-binding site within the eIg-Fab molecule on CD3 binding.

[0317] Table 3: Cellular binding of eIg molecules analyzed by flow cytometry. EC 50 The values (nM) were determined using combinations of HER3-expressing cells (LIM1215 and BT474) and CD3-expressing Jurkat cells with serial dilutions of the eIg molecule. Mean ± SD, n = 3.

Table 3

[0318] The cytotoxic effects of PBMC on target cells mediated by bispecific, bivalent, and trivalent eIg molecules were determined using HER3-positive cell lines with different antigen expressions (LIM1215: 19,877 HER3 / cell; BT474: 11,244 HER / cell). Target cells (2x10 4 cells / well) were incubated with bispecific bivalent or trivalent eIg or eIg-Fab antibodies for 15 min at RT before adding PBMC (E:T ratio 10:1). After incubation at 37 °C for 3 days, the supernatant was discarded, and viable target cells were stained with crystal violet. After washing and drying, the remaining dye was solubilized in methanol (50 μl / well), and the optical density was measured at 550 nM using a Tecan spark (Tecan). The eIg molecules were able to redirect unstimulated PBMC to lyse HER3-expressing cancer cells in a concentration-dependent manner (Figure 12). The cytotoxic activity of the eIg antibodies was evaluated by the potency (EC 50 value in cell death) (Table 4).

[0319] For the two cell lines LIM1215 and BT474, the highest efficacy was observed for the bispecific trivalent eIg-Fab4 with EC 50 values of 0.1 ± 0.09 nM and 0.2 ± 0.1 nM, respectively. For the bispecific trivalent molecule Ig-Fab3, an approximately 4-fold weaker efficacy was observed. For the bispecific bivalent eIg molecule eIg1, further decreases in efficacy were observed with EC 50 values of 5.8 ± 2.9 nM (LIM1215) and 3.8 ± 0.6 nM (BT474). Very low cell killing efficacy was seen for eIg-Fab2. In summary, these experiments show that the trivalent bispecific eIg-Fab can mediate an increase in cytotoxicity via T cell retargeting compared to the bivalent bispecific eIg, i.e., due to an additional target binding site. However, the experiments also show that the efficacy is affected by the molecular composition, i.e., the position of the HER3 and CD3 binding sites within the eIg-Fab molecule.

[0320] Table 4: Cytotoxic activities of eIg and eIg-Fab molecules against various tumor cell lines. EC 50 (nM) was determined using HER3-expressing tumor cells and serial dilutions of eIg and eIg-Fab molecules. Mean ± SD, n = 3, n.d. = not determined. [Table 4]

[0321] Example 6: Bivalent bispecific eIg molecule targeting HER3 and FAP Based on different variable domains from therapeutic antibodies, i) the Fv domain specific for HER3 (antibody 3-43; Schmitt et al, 2017, mAbs 9, 831-843) was combined with the C H 1 / C L heterodimer of IgG1, and ii) the Fv domain specific for fibroblast activation protein (FAP; hu36; Fabre et al., 2020, Clin. Cancer Res. 26, 3420-3430) was combined with the modified C HCombined with 2 domains (hetEHD2), iii) By fusing to the Fc portion of the heterodimer (knob-into-hole technology), a bispecific and bivalent eIg molecule was generated. Thus, the bispecific molecule consists of four different polypeptide chains: the V of the light chain L 3-43-C L λ (SEQ ID NO: 13) pairs with the V of the heavy chain H 3-43-C H 1-Fc knob (SEQ ID NO: 14), and the V of the light chain L hu36-EHD2-1(N39Q) (SEQ ID NO: 43)) pairs with the V of the heavy chain H hu36-EHD2-2-Fc hole (SEQ ID NO: 42). The bispecific bivalent eIg molecule exhibits one antigen-binding site for HER3 and one binding site for FAP. Each polypeptide chain is illustratively shown in Figure 13A, and the resulting antibody molecule is schematically shown in Figure 13B.

[0322] The bispecific bivalent eIg molecule was transiently transfected and expressed in HEK293-6E cells using polyethyleneimine as a transfection reagent after co-administering four plasmids encoding two heavy chains and two light chains. The protein secreted into the cell culture supernatant was purified using protein A affinity chromatography. SDS-PAGE analysis revealed four major bands under reducing conditions: two bands of approximately 55 kDa corresponding to the two heavy chains of the antibody, and two bands of approximately 23 kDa corresponding to the two light chains of the antibody. Under non-reducing conditions, one major band of approximately 200 kDa corresponding to the intact antibody consisting of four different polypeptide chains was observed (Figure 13C). The purity, integrity, and homogeneity of the eIg molecule were confirmed by size exclusion chromatography (Figure 13D). The binding of the eIg molecule to the extracellular domains (ECDs) of HER3 (aa21-643) and FAP (aa32-728) was determined by ELISA. His-tagged HER3 protein and Flag-tagged FAP protein were coated onto polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). Next, the plates were incubated with serial dilutions of the bispecific eIg antibody. After washing, the bound antibody was detected with HRP-conjugated anti-human Fc antibody using HER3-His or FAP-Flag as the immobilized antigen. TMB and H 2 O 2 were used as substrates. The eIg antibody showed concentration-dependent binding (EC 50 values in the nanomolar range (3.4 nM for HER3 and 7.6 nM for FAP)) to HER3-His and FAP-Flag (Figure 13E). These experiments confirmed that the eIg antibody binds to both antigens of HER3 and FAP.

[0323] Example 7: Bivalent monospecific eIg molecule targeting HER3 The anti-HER3 bivalent eIg molecule was generated by combining the variable domain of the anti-HER3 antibody (3-43) (Schmitt et al., 2017, mAbs 9, 831-843) with the modified CH2 domain of IgE (hetEHD2) and further fusing it to a homodimeric Fc region. Thus, this molecule consists of two different polypeptide chains: V of the light chain L 3-43-EHD2-1 (SEQ ID NO: 23) and V of the heavy chain H 3-43-EHD2-2-Fc (SEQ ID NO: 22). The monospecific bivalent eIg molecule exhibits two identical antigen-binding sites for HER3. Each polypeptide chain is exemplarily shown in Figure 14A, and the resulting antibody molecule is schematically shown in Figure 14B.

[0324] After co-administering two plasmids encoding the heavy and light chains, polyethyleneimine was used as a transfection reagent to transiently transfect HEK293-6E cells to express the monospecific bivalent eIg molecule. The protein secreted into the cell culture supernatant was purified using protein A affinity chromatography. SDS-PAGE analysis revealed two major bands under reducing conditions: one band of approximately 55 kDa corresponding to the heavy chain of the antibody and one band of approximately 25 kDa corresponding to the light chain of the antibody (Figure 14C). The binding of the eIg molecule to the extracellular domain (ECD) of HER3 (aa21-643) was determined by ELISA. The His-tagged HER3 protein was coated onto a polystyrene microtiter plate at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). Next, the plate was incubated with serial dilutions of the monospecific eIg antibody. After washing, the bound antibody was detected with an HRP-conjugated anti-human Fc antibody. TMB and H2O2 were used as substrates. The monospecific bivalent eIg antibody showed concentration-dependent binding to HER3-His (EC 50The values showed a nanomolar range (1.4 nM for HER3) (Figure 14D). The parental antibody IgG 3-43 showed a similar binding to immobilized HER3. This experiment confirmed the binding of the monospecific bivalent eIgs antibody to HER3 as an immobilized antigen. In summary, we successfully generated a monospecific bivalent eIg molecule with two antigen-binding sites for the same epitope.

[0325] Example 8: Tetravalent bispecific eIg molecule targeting HER3 and EGFR The bispecific tetravalent eIg-Fab molecule was generated by combining the variable domains of an anti-EGFR antibody (hu225; Seifert et al., 2014, Mol Cancer Ther 13, 101-111) and the C H 1 / C L heterodimer, and the variable domains of an anti-HER3 (3-43; Schmitt et al., 2017, mAbs 9, 831-843) and a modified CH2 domain of IgE (hetEHD2), and further combining with a homodimerizing Fc portion. Thus, the tetravalent bispecific molecule consists of three different polypeptide chains: the light chain V L 3-43-EHD2-1 (SEQ ID NO: 23) and V L hu225-C L κ (SEQ ID NO: 25), and the heavy chain V H hu225-C H 1-V H 3-43-EHD2-2-Fc (SEQ ID NO: 24). The two Fab portions in the heavy chain were separated by a linker (GGSGG) containing 10 amino acids 2 This bispecific tetravalent eIg molecule showed two antigen-binding sites for EGFR and two binding sites for HER3. Each polypeptide chain is exemplarily shown in Figure 15A, and the resulting binding molecule is schematically shown in Figure 15B.

[0326] The tetravalent bispecific eIg-Fab molecule was transiently transfected and expressed in HEK293-6E cells using polyethyleneimine as a transfection reagent after co-administering three plasmids encoding the heavy chain and both light chains. The protein secreted into the cell culture supernatant was purified using protein A affinity chromatography. SDS-PAGE analysis revealed three major bands under reducing conditions: one band of approximately 80 kDa corresponding to the heavy chain of the antibody and two bands of approximately 26 kDa corresponding to the two light chains of the antibody (Figure 15C). Under non-reducing conditions, one major band of approximately 250 kDa corresponding to the intact antibody consisting of three different polypeptide chains was observed. The purity, integrity, and homogeneity of the bispecific tetravalent eIg-Fab molecule were confirmed by size exclusion chromatography (Figure 15D).

[0327] The binding of this tetravalent eIg-Fab molecule to the extracellular domains of EGFR (aa20-643) and HER3 (aa21-643) was determined by ELISA (Figure 15E). His-tagged EGFR or HER3 protein was immobilized on polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% non-fat milk (MPBS). Next, the plates were incubated with serial dilutions of the bispecific eIg-Fab antibody. After washing, the bound antibody was detected with an HRP-conjugated anti-human Fc antibody. TMB and H2O2 were used as substrates. The bispecific tetravalent antibody showed concentration-dependent binding (EC 50 values in the nanomolar range (0.39 ± 0.25 nM for EGFR and 3.3 ± 2.7 nM for HER3)) (Figure 15E) (Table 5). Simultaneous binding to both antigens was demonstrated with immobilized EGFR-Fc incubated with serial dilutions of the bispecific eIg-Fab molecule followed by HER3-His protein. The bound HER3-His was detected via an HRP-conjugated anti-His detection antibody. Concentration-dependent binding was observed with an EC of approximately 0.23 ± 0.01 nM 50It was observed at the value. In summary, the data demonstrate the production of bispecific tetravalent eIg molecules with 2 + 2 binding sites using eIg platform technology.

[0328] Table 5: Binding of antibodies to EGFR and HER3 analyzed by ELISA. EC 50 values (nM) were determined using EGFR-His, HER3-His, or EGFR-Fc and HER3-His as antigens, and serial dilutions of the antibodies. Mean ± SD, n = 3, n.d. = not determined.

Table 5

[0329] Example 9: Bivalent bispecific Fab-eFab molecules targeting HER3 and EGFR The variable domain of anti-EGFR (hu225; Seifert et al., 2014, Mol Cancer Ther 13, 101 - 111) and the C H 1 / C L heterodimer, the variable domain of anti-HER3 (3 - 43; Schmitt et al., 2017, mAbs 9, 831 - 843) and the modified CH2 domain of IgE (hetEHD2) were combined to generate a bispecific bivalent Fab-eFab molecule, i.e., one without an Fc region. The Fab-eFab molecule consists of the V L 3 - 43 - EHD2 - 1 (SEQ ID NO: 23) and V L hu225 - C L κ (SEQ ID NO: 25), and the V of the "heavy chain" H hu225 - C H 1 - V H 3 - 43 - EHD2 - 2 (SEQ ID NO: 44). This bispecific bivalent Fab-eFab molecule showed one antigen-binding site for EGFR and one binding site for ER3. The heavy chain contained a His tag at the C-terminus for purification and detection of the molecule. The two Fab / eFab moieties were linked by a linker (GGSGG) containing 10 amino acids 2were joined via. Each polypeptide chain is exemplarily shown in Figure 16A, and the resulting antibody molecule is schematically shown in Figure 16B. The purity, integrity and homogeneity of the Fab-eFab molecule were confirmed by size exclusion chromatography (Figure 16C).

[0330] The binding of this Fab-eFab molecule to the extracellular domains of EGFR (aa20 - 643) and HER3 (aa21 - 643) was analyzed by ELISA. His-tagged EGFR and HER3 proteins were coated onto polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% non-fat milk (MPBS). Next, the plates were incubated with serial dilutions of the bispecific Fab-eFab antibody or parental antibodies (IgG hu225, IgG3 - 43). After washing, the bound antibodies were detected with an HRP-conjugated anti-human Fab antibody using both immobilized antigens. TMB and H 2 O 2 was used as the substrate. The Fab-eFab antibody showed concentration-dependent binding (EC 50 values were in the nanomolar range (6.5 nM for EGFR; 3.9 nM for HER3)) to EGFR-His and HER3-His (Figure 16D) (Table 6). The parental antibodies showed high binding ability to the immobilized antigens due to the presence of two antigen-binding sites in each molecule. Simultaneous binding to both antigens was demonstrated by incubating the serial dilutions of the Fab-eFab molecule with immobilized HER3-His and the EGFR-moFc protein as the second soluble antigen. The bound second antigen was detected via an HRP-conjugated anti-mouse Fc detection antibody. Concentration-dependent binding (EC 50 value was approximately 1.4 nM) was observed. These experiments confirmed the binding of the bispecific bivalent Fab-eFab antibody to EGFR and HER3.

[0331] Table 6: Binding of antibodies to EGFR and HER3 analyzed by ELISA. EC 50Values (nM) were determined using serial dilutions of the antibodies in combination with EGFR-His, HER3-His, or HER3-His and EGFR-moFc as antigens. Mean ± SD, n = 1, n.d. = not determined, -= not performed.

Table 6

[0332] Example 10: Bivalent bispecific eIg molecules targeting FAP and mouse CD3 - different compositions of variable domains binding to mouse CD3 The bispecific bivalent eIg molecules were prepared by combining an anti-fibroblast activation protein (FAP) antibody (hu36; Fabre et al., 2020, Clin Cancer Res. 26, 3420 - 3430) with the C H 1 / C L heterodimer and an anti-mouse CD3 (2C11; Leo et al., 1987, Proc Natl Acad USA 84, 1374 - 1378) with the modified C H 2 domain (hetEHD2) of IgE, and further fusing them to a heterodimerizing Fc portion (knob-into-hole technology). The composition of the polypeptide chains is shown in Figure 17A. Thus, the first part of the eIg molecule consists of a V L hu36-CLκ (SEQ ID NO: 45) of the light chain and a V H hu36-C H 1-Fc(hole) (SEQ ID NO: 46) of the heavy chain, forming the FAP-targeting arm. Molecule I additionally consists of a V H 2C11-EHD2-1 (SEQ ID NO: 47) of the light chain and a V L 2C11-EHD2-2-Fc(knob) (SEQ ID NO: 48) of the heavy chain, forming the mouse CD3-targeting arm. The second molecule consists of a V L 2C11-EHD2-1 (SEQ ID NO: 49) of the light chain and a V HIt consists of 2C11-EHD2-2-Fc(knob)(Accession No.: 50). The obtained antibody molecule is schematically shown in Fig. 17B. The purity, integrity and homogeneity of both eIg molecules were confirmed by size exclusion chromatography (Fig. 17C).

[0333] Binding of both bispecific eIg antibodies to FAP-expressing HT1080-FAP cells and mouse spleen cells (expressing mouse CD3) was analyzed by flow cytometry. The attached HT1080-FAP cells were washed with PBS and immediately trypsinized at 37 °C. Trypsin was quenched with FCS-containing medium and removed by centrifugation (500 x g, 5 min). To harvest mouse immune cells from the spleen, the spleen was extracted and a homogeneous cell suspension was prepared by crushing the spleen through a cell strainer. After collecting the cells by centrifugation (250 x g, 7 min), the red blood cells were removed and the spleen cells were resuspended in the medium. 100,000 HT1080-FAP cells or 200,000 mouse spleen cells were incubated at 4 °C for 1 h with serial dilutions of the bispecific eIg antibody diluted with PBA (PBS containing 2% (v / v) FCS, 0.02% (w / v) NaN 3 The cells were washed twice with PBA. The bound antibody was detected using a PE-labeled anti-human Fc secondary antibody, and this was incubated at 4 °C for an additional 1 h. After washing, the median fluorescence intensity (MFI) was measured with a Milltenyi MACSQuant® Analyzer 10. The relative MFI (relative to unstained cells) was calculated with Flow Jo (Tree star) and Excel. The eIg molecules bound to the cells in a concentration-dependent manner. eIg molecules I and II bound to HT1080-FAP cells with EC 50 values of 2.9 nM and 2.7 nM, respectively, and to mouse spleen cells with EC 50 values of 7.3 nM and 24.2 nM, respectively (Fig. 17D) (Table 7).

[0334] Table 7: Cell binding of the antibodies analyzed by flow cytometry. EC 50The value (nM) was determined using serial dilutions of the antibody in combination with FAP-expressing HT1080-FAP or mouse CD3-expressing mouse splenocytes as target cells. Mean ± SD, n = 1.

Table 7

[0335] Example 11: Lack of binding of the eIg molecule to Fcε-receptor I In the eIg technology, the modified C H 2 domains derived from human IgE are used as a heterodimerization module. Therefore, it was investigated whether antibody molecules containing hetEHD2 lack binding to the high-affinity IgE receptor FcεRI. FcεRI was prepared as an Fc fusion protein containing the extracellular region of FcεRI. In an ELISA experiment (Figure 18), purified FcεRI was used as an immobilized antigen incubated with various eIg derivatives including eIg specific for HER3 and CD3 (Example 5), tetravalent bispecific eIg-Fab specific for EGFR and HER3 (Example 10), and bivalent bispecific Fab-eFab specific for EGFR and HER3 (Example 9). As a control, EGFR and HER3 were used as additional immobilized antigens to confirm the binding of the antibodies to these receptors. FcεRI, EGFR, and HER3 were coated on polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). Next, the plates were incubated with 50 nM eIg, or eIg-Fab, and Fab-eFab molecules to study the binding to different immobilized receptors. Furthermore, human IgE was included in this experiment to confirm the binding to the Fcε receptor. After washing, the bound antibody was detected with an anti-human Fab antibody. TMB and H 2 O 2It was used as a substrate. IgE specifically bound to FcεRI. The three eIg derivatives did not show binding to FcεRI, but strongly recognized their respective target antigens, that is, eIg HER3xCD3 bound to HER3, and eIg-Fab EGFRxHER3 and Fab-eFab EGFRxHER3 bound to EGFR and HER3. The control antibody IgG 3-43, which is derived from the HER3 binding site, bound to HER3, and IgG hu225, which is derived from the EGFR binding site, bound to EGFR. No binding to FcεRI was observed with these two control IgG antibodies (Figure 18). Thus, this experiment demonstrated that eIg molecules lack binding to the IgE receptor FcεRI expressed by mast cells and basophils. Therefore, it can be excluded that molecules containing EHD2 or hetEHD2 cause activation of these immune cells by binding to FcεRI.

[0336] Example 12: Bispecific eIg Molecules for Targeting the SARS-CoV-2 Spike Protein To dual-target different epitopes of the spike protein of the SARS-CoV-2 virus, bispecific eIg-Fab molecules were prepared. In this approach, publicly available antibodies that bind to the RBD domain of the viral spike protein were used: P2B-2F6 (Ju et al., 2020, Nature 584, 115-119) and S309 (Pinto et al., 2020, Nature 583, 290-295). The variable domain of S309 was combined with the C H 1 / C L heterodimer, the variable domain of P2B-2F6 was combined with the hetEHD2 domain, and further combined with the homodimerized Fc portion to prepare a symmetric tetravalent bispecific eIg-Fab molecule. This antibody has the V L S309-CLκ (SEQ ID NO: 51) and V L P2B-2F6-EHD2-1 (SEQ ID NO: 52), as well as the V H S309-C H 1-V HIt consists of P2B-2F6-EHD2-2-Fc (Accession No.: 53). Each polypeptide chain is exemplified in Fig. 19A, and the obtained antibody molecule is schematically shown in Fig. 19B.

[0337] The tetravalent bispecific eIg-Fab molecule was transiently transfected and expressed in HEK293-6E cells by co-administering three plasmids encoding the heavy chain and both light chains and using polyethyleneimine as a transfection reagent. The protein secreted into the cell culture supernatant was purified using protein A affinity chromatography. SDS-PAGE analysis revealed three major bands under reducing conditions: one band of approximately 85 kDa corresponding to the heavy chain of the antibody and two bands of approximately 26 kDa corresponding to the two light chains of the antibody (Fig. 19C). Under non-reducing conditions, one major band of approximately 250 kDa corresponding to the intact antibody consisting of three different polypeptide chains was observed. The purity, integrity, and homogeneity of the bispecific tetravalent eIg-Fab molecule were confirmed by size exclusion chromatography (Fig. 19D).

[0338] The binding of the spike protein of this eIg-Fab molecule to the RBD domain was determined by ELISA. His-tagged RBD was coated onto a polystyrene microtiter plate at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). Next, the plate was incubated with serial dilutions of the bispecific eIg-Fab antibody. After washing, the bound antibody was detected with an HRP-conjugated anti-human Fc antibody. TMB and H 2 O 2 were used as substrates. The bispecific tetravalent eIg-Fab antibody showed concentration-dependent binding (EC 50 values in the nanomolar range (1.0 nM)) to the RBD-His antigen (Fig. 19E). These experiments confirmed the binding of the bispecific tetravalent eIg-Fab antibody to the RBD.

Claims

1. a first polypeptide chain comprising a first binding domain (BD1) and a first modified EHD2 domain (EHD2-1); and a second polypeptide chain comprising a second binding domain (BD2) and a second modified EHD2 domain (EHD2-2), the amino acid sequences of EHD2-1 and EHD2-2 are different from each other and each is selected from: (i) an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having a Cys at position 14; or (ii) an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having a Cys at position 102; BD1 and BD2 together form an antigen-binding site, and EHD2-1 and EHD2-2 are covalently bound to each other, binding molecule.

2. BD1 and BD2 are different from each other, and each is V H and V L or selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain.

3. The binding molecule of any one of claims 1 to 2, further comprising a first Fc chain.

4. and a third binding domain (BD3) and a fourth binding domain (BD4), wherein BD3 and BD4 together form an antigen-binding site, and preferably BD3 and BD4 are different from each other and each has the structure: V H and V L or selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain.

5. The binding molecule of claim 4 or 6, further comprising a third polypeptide chain, preferably further comprising a third polypeptide chain and a fourth polypeptide chain, more preferably further comprising a second Fc chain.

6. a third polypeptide chain comprising BD3, (I C H 1 domain, (ii) C L domain, (iii) a first modified EHD2 domain (EHD2-1), and (iv) a second modified EHD2 domain (EHD2-2), a fourth polypeptide chain comprising BD4, In case (i), C L domain, In case (ii), C H 1 domain, In the case of (iii), a second modified EHD2 domain (EHD2-2), and In the case of (iv), a first modified EHD2 domain (EHD2-1), wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other and each is selected from an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having Cys at position 14, or an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having Cys at position 102. The binding molecule of claim 5.

7. and a fifth binding domain (BD5) and a sixth binding domain (BD6), wherein BD5 and BD6 together form an antigen-binding site, and preferably BD5 and BD6 are different from each other and each has the structure: V H and V L or selected from the variable region of the TCR α-chain and the variable region of the TCR β-chain, more preferably Binds to BD5, (I C H 1 domain, (ii) C L domain, (iii) a first modified EHD2 domain (EHD2-1), or (iv) a second modified EHD2 domain (EHD2-2); and Binds to BD6, In case (i), C L domain, In case (ii), C H 1 domain, In case of (iii), a second modified EHD2 domain (EHD2-2), and In case (iv), the first modified EHD2 domain (EHD2-1), Further comprising: wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other and each is selected from an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having Cys at position 14, or an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having Cys at position 102. The binding molecule according to any one of claims 4 to 6.

8. and a seventh binding domain (BD7) and an eighth binding domain (BD8), wherein BD7 and BD8 together form an antigen-binding site, preferably BD7 and BD8 are different from each other and each has the sequence H and V L or selected from the TCR α-chain variable region and the variable region of the TCR β-chain.

9. Binds to BD7, (I C H 1 domain, (ii) C L domain, (iii) a first modified EHD2 domain (EHD2-1), or (iv) a second modified EHD2 domain (EHD2-2); and Binds to BD8, In case (i), C L domain, In case (ii), C H 1 domain, In case of (iii), a second modified EHD2 domain (EHD2-2), and In case (iv), the first modified EHD2 domain (EHD2-1), Further comprising: wherein the amino acid sequences of EHD2-1 and EHD2-2 are different from each other and each is selected from an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having Cys at position 14, or an amino acid sequence having at least 70% amino acid identity to SEQ ID NO:1 and not having Cys at position 102. The binding molecule of claim 8.

10. 10. The binding molecule of any one of claims 1 to 9, wherein none of the modified EHD2 domains carries a single N-glycan or one or more of the modified EHD2 domains carries a single N-glycan.

11. and / or Cys at position 14 of SEQ ID NO:1 is replaced by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn and Tyr, preferably Ser; and / or 11. The binding molecule according to any one of claims 1 to 10, wherein Cys at position 102 of SEQ ID NO:1 is substituted by an amino acid selected from the group consisting of Ser, Gly, Ala, Thr, Gln, Asn and Tyr, preferably Ser.

12. The binding molecule according to any one of claims 1 to 11, further comprising a single amino acid substitution at position N39 in one or more modified EHD2 domains, preferably wherein the single amino acid substitution is N39Q.

13. A nucleic acid or a set of nucleic acids encoding a binding molecule according to any one of claims 1 to 12.

14. A vector comprising the nucleic acid or set of nucleic acids according to claim 13.

15. A host cell comprising the vector of claim 14.

16. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 12, a nucleic acid or a set of nucleic acids according to claim 13, a vector according to claim 14, or a host cell according to claim 15, and a pharma- ceutically acceptable carrier.

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