Heterodimerized Ig domains

Abstract

The present invention provides a protein complex comprising heterodimerization domains HRI and HRII, each consisting of an antiparallel β-strand and an intervening domain, where HRI and HRII are interspersed fusion proteins of two human constant domains of immunoglobulin or immunoglobulin-like proteins. The present invention also provides a nucleic acid molecule comprising a sequence encoding the protein complex and a vector comprising the nucleic acid. The present invention also provides the protein complex, the nucleic acid, and the vector for use as a pharmaceutical. The present invention further provides a method for determining the amino acid sequence of HRI and / or the amino acid sequence of HRII. The present invention also provides a method for generating the amino acid chain of HRI and / or the amino acid chain of HRII. The present invention further provides the protein complex for use in the prevention, treatment, or diagnosis of a disorder or disease.

Description

[Technical Field]

[0001] The present invention provides a protein complex comprising heterodimerization domains HRI and HRII, each consisting of an antiparallel β-strand and an intervening domain, where HRI and HRII are interspersed fusion proteins of two human constant domains of immunoglobulin or immunoglobulin-like proteins. The present invention also provides a nucleic acid molecule comprising a sequence encoding the protein complex and a vector comprising the nucleic acid. The present invention also provides the protein complex, the nucleic acid, and the vector for use as a pharmaceutical. The present invention further provides a method for determining the amino acid sequence of HRI and / or the amino acid sequence of HRII. The present invention also provides a method for generating the amino acid chain of HRI and / or the amino acid chain of HRII. The present invention further provides the protein complex for use in the prevention, treatment, or diagnosis of a disorder or disease. [Background technology]

[0002] Bispecific antibodies are attracting increasing interest for diagnostic and therapeutic applications. A comprehensive review is provided in Kontermann RE and Brinkmann U (2015) Drug Discovery Today 20(7):883 and the references cited therein. While natural antibodies are monospecific, bispecific antibodies recognize two distinct epitopes on the same or different antigens. Applications of bispecific antibodies range widely, from diagnosis, imaging, and therapy. Initially, therapeutic applications focused primarily on retargeting effector cells for cancer therapy, including T cells that cannot be recruited to tumor cells by normal antibodies. However, over the past decade, many other therapeutic strategies based on bispecific antibodies have been established, including dual targeting strategies, half-life extension, and delivery across the blood-brain barrier, in addition to retargeting effector molecules, cells, and genetic vehicles. Indications include cancer, chronic inflammatory diseases, autoimmune and bleeding disorders, and infectious diseases.

[0003] Bispecific antibodies with defined specificities are artificial molecules that are not found in nature. Therefore, they must be generated by molecular or genetic means. The generation of bispecific IgG molecules faces two major challenges due to the fact that the antigen-binding site is constructed by the variable domains of the light and heavy chains (VL, VH). First, bispecific antibodies require two different heavy chains. Second, bispecific antibodies also require two different light chains. Therefore, bispecific IgG antibodies exhibit asymmetry due to the presence of at least two different variable domains (Fv). Promiscuous pairing of the heavy and light chains of two antibodies expressed in a single cell theoretically results in 10 possible combinations, of which only one combination will be bispecific, while the remaining pairings will result in nonfunctional or monospecific molecules (Schaefer et al., 2016). Directing and enforcing the correct binding site, i.e., the correct assembly of the heavy and light chains, is one of the challenges in generating bispecific antibodies.

[0004] Genetic engineering to force heavy chain heterodimerization, described in the next section, addresses one of the problems of bispecific IgG formation. Heterodimeric heavy chains can assemble with two different light chains to form four possible combinations: one bispecific molecule, one nonfunctional combination, and two monospecific molecules, reducing the possible combinations from 10 different molecules to four. Heavy chain pairing is mediated by the last domain of the constant region, i.e., CH3, of the IgG molecule, to form a high-affinity homodimeric complex. Additional interactions exist in the hinge region, which is responsible for the covalent bonding of the two heavy chains formed after heavy chain assembly.

[0005] Various strategies use either steric or electrostatic steering effects, or a combination thereof, to generate complementary interfaces that favor heterodimerization over homodimerization.

[0006] Ridgway and colleagues created a CH3 interface that favors heterodimer assembly by replacing small side chains with large ones in one CH3 interface to create a knob and large side chains with small ones in the other CH3 domain to create a hole (Ridgway et al., 1996). Testing various mutants showed preferential heterodimerization with the substitution T366Y in one chain and Y407T in the other. These initial knobs-into-holes mutations were used, for example, to produce IgG against HER2 and IGF-1R. The knobs-into-holes approach was subsequently expanded to identify additional suitable combinations by phage display. These mutations were then used to generate bispecific IgG antibodies, testing additional substitutions that allow disulfide bond formation. One mutant showed >95% heterodimer formation (S354C, T366W / Y349C, T366S, L368A, Y407V). This heterodimeric heavy chain was then applied to construct a bispecific antibody against MLP and HER3 from single-chain variable fragments (scFv) using the same VL domain, thus expressing a common light chain (Merchant et al., 1998). The heteromeric heavy chain produced a functional bispecific antibody, enabled purification by protein A chromatography, and retained Fc-mediated effector functions such as ADCC. This approach has been adopted to generate a variety of bispecific antibodies and today forms a versatile platform for generating bispecific IgG molecules and their derivatives, including trivalent Ig-like antibodies, bispecific Fc, and CH3 fusion proteins.

[0007] One example is bispecific antibodies that retarget T cells. To avoid systemic activation of T cells due to bivalent binding to CD3, molecules displaying only one binding site to CD3 were designed. These include scFv-Fc(kih), which has one scFv on each Fc chain, and tandem scFv-Fc(kih) (BiTE-KIH), in which tandem scFvs are fused to one of the Fc chains (Xu et al., 2015). In this study, the CD3-binding moiety was fused to either a knob-containing Fc chain or a hole-containing Fc chain (KIH and KIH, respectively). r ). Interestingly, BiTE-KIH r was superior to BiTE-KIH in terms of expression titer; however, no differences were observed in terms of T cell activation and tumor cell lysis. In a similar approach, Fc-KIH was used to generate a bivalent, bispecific scFv-Fc fusion protein against CD16 and HER2 to retarget NK cells to tumor cells (Xie et al., 2005).

[0008] Monovalent binding is also essential for antibodies targeting cell surface receptors such as c-MET to avoid receptor cross-linking and activation. Bispecific antibodies that bind monovalently to cell surface receptors, applicable to dual targeting and neutralization of two different receptors, were generated by fusing an Fc arm to the N-terminus of an Fc hole chain and a disulfide-stabilized scFv to the C-terminus of the same Fc chain, coexpressing it with an unfused Fc knob.

[0009] Fusing a VH domain to the C-terminus of one Fc(kih) chain and a VL domain, either expressed separately or fused to the C-terminus of the other Fc(kih) chain, resulted in a bispecific trivalent IgG-Fv (mAb-Fv) fusion protein in which the Fv fragment was stabilized by an interdomain disulfide bond (Metz et al., 2012). The flexibility of the Fv fragment in the IgG-Fv fusion could be increased by introducing a proteolytic cleavage site, e.g., for furin or MMP, into the linker connecting the VL domain to the Fc chain. After cleavage, this resulted in a bispecific molecule with a C-terminal Fv fragment connected to the IgG only via the VH domain. Similarly, an scFv-Fc-Fv fusion protein was generated that displayed two binding sites for EGFR (scFv fused to the N-terminus of the Fc chain) and one binding site for LPS (VH fused to the C-terminus of the Fc(knob) and VL fused to the C-terminus of the Fc(hole)). Further derivatives of bispecific IgG (kih) antibodies include TriMAbs. In this case, one or two disulfide-stabilized scFvs are fused to one or both Fc (kih) chains, resulting in trispecific trivalent or tetravalent antibodies, respectively. This has been demonstrated with TriMAbs targeting EGFR, IGF-1R, and either cMet or HER3. In this approach, the Fab fragments consisted of single-chain Fab fragments with disulfide-stabilized Fv domains.

[0010] Recently, the knobs-into-holes strategy has been extended to other IgG isotypes, e.g., IgG4 heterodimers (which themselves lack Fcc-mediated effector functions) were generated to generate bispecific antibodies against IL-4 and IL-13.

[0011] Using structure- and sequence-based approaches, we explored the energetics of paired mutant combinations across the CH3 dimer interface, generating the HA-TF mutant (S364H, F405A / Y349T, 394F), which was developed for simultaneous targeting of HER2 (bivalent binding) and CD3 (monovalent binding) and showed approximately 83% heterodimer formation in the context of a bispecific mAb-Fv (IgG-Fv) molecule. Further examples using this CH3 heterodimerization module include monovalent and bivalent scFv-Fc fusion proteins.

[0012] Another rational, structure-guided approach yielded a set of mutations that were reported to be highly thermostable and form pure heterodimers with no detectable homodimers. The Fc design (ZW1) contained substitutions of T350V, L351Y, F405A, and Y407V in the first Fc chain and T350V, T366L, K392L, and T394W in the second Fc chain.

[0013] While the above strategies primarily rely on hydrophobic interactions, other approaches utilize electrostatic interactions (steering) to prevent homodimerization of the CH3 domains through electrostatic repulsion and to direct heterodimerization through electrostatic attraction. In wild-type CH3 domains, two charge interactions between K409 and D399 are found at the CH3-CH3 interface. Substituting K409 with aspartic acid in one CH3 domain and D399 with lysine in the other CH3 domain was found to promote CH3 heterodimer formation. Further substitutions, such as K392D in one chain and E356K in the other, were introduced. Introducing additional charge pairs resulted in decreased productivity. This approach was used with two charge pair substitutions (K409D, K392D / D399K, E356K, CH3 charge pair) to generate bispecific scFv-Fc fusion proteins against CD3 and TARTK, and more recently bispecific IgGs against EGFR and HER2 or sclerostin and DKK-1, which involved introducing a new charge pair into the Fab arm to direct the correct light chain pairing.

[0014] The electrostatic steering effect has also been used in biclonics, a bispecific antibody that utilizes a common light chain and heterodimerized heavy chains (Geuijen et al., 2014). In this case, residues 366, 366, and 351 in one CH3 are replaced with positively charged lysine residues, and one or more residues in the second CH3 (e.g., 349, 351, 355, and 368) are replaced with negatively charged glutamic or aspartic acid residues. One bispecific antibody based on this technology (MCLA-128) against HER2 and HER3 is currently in Phase I / II clinical trials.

[0015] Preferential heavy chain heterodimerization has also been achieved by introducing charge pairs into the hinge regions of IgG1 and IgG2. For IgG1, these hinge substitutions include D221E and P228E in the first hinge and D221R and P228R in the second hinge. For IgG2, these substitutions include C223E and P228E in one hinge region and C223R, E225R, and P228R in the other hinge region. Here, only two substitutions are required in the first IgG2 hinge, as E225R can form an electrostatic interaction with the native glutamic acid at position 225. These mutations were combined with L368E and K409R, respectively, to force heterodimer assembly at the CH3 domain. Applicability was demonstrated for an anti-EGFRx anti-HER2 bispecific IgG antibody and an anti-CEx anti-CD20 antibody using separate expression of the two antibodies followed by assembly from half antibodies.

[0016] Another study identified mutations that support heterodimer assembly of the CH3 domain by first substituting charged residues around the rim of the conserved hydrophobic core (L351, T366, L368, Y407) with larger or smaller hydrophobic amino acids to replace symmetric electrostatic interactions with asymmetric hydrophobic interactions, and secondly, substituting amino acids weakly involved in interactions with amino acids with long charged side chains to form asymmetric long-range electrostatic interactions. This resulted in the final combination (EW-RVT) of K360E and K409W in one CH3 and Q347R, D399V, and F405T in the other CH3. Functionality of a bispecific scFv-Fc heterodimer targeting VEGFR-2 and Met was demonstrated. Introducing a disulfide bond into the CH3 domain (Y349C in the first domain and S354C in the second domain) improved heterodimer formation and thermodynamic stability. Furthermore, using a combinatorial Fc library displayed on the yeast surface, we selected mutants that harbored various mutations and showed high heterodimerization yields (80–90%).

[0017] Based on the observation that IgG4 antibodies can exchange Fab arms, a dynamic process involving the separation of the two heavy chains and their reassembly into a complete IgG4 molecule was proposed. This process was attributed to the IgG4 core hinge sequence linked to residues in the CH3 domain. This natural process of Fab arm exchange in IgG4 was adapted to generate bispecific IgG1 molecules by controlled Fab arm exchange (cFAE). Screening for mutations in the CH3 domain that enabled cFAE in the context of the corresponding K409R mutation in CH3 identified the F405L mutation, which enabled efficient exchange of the half-antibodies of separately expressed antibodies after mixing with R-mercaptoethanol and mild reduction. The scalability of this process was demonstrated with an anti-EGFR x anti-CD20 bispecific IgG (DuoBody), yielding bispecific molecules with >95% fidelity.

[0018] Complementarity of the CH3 interface, which allows heterodimeric assembly of Fc chains, was exploited by designing strand-exchange engineered domain (SEED) heterodimers. These SEED CH3 domains, composed of alternating segments derived from human IgA and IgG CH3 sequences (AG SEED CH3 and GA SEED CH3), were used to generate so-called SEEDbodies (Davis et al., 2010). Because molecular modeling suggested that interaction with the neonatal Fc receptor (FcRn) was impaired with AG SEED CH3, the residues at the CH2-CH3 junction were reverted to the IgG sequence. Pharmacokinetic studies confirmed the long half-life of SEEDbodies, comparable to other Fc fusion proteins and IgG1 (Muda et al., 2011). One example of SEEDbodies is the generation of a bispecific Fab-scFv-Fc fusion protein targeting two distinct epitopes on the EGFR. This biparatopic antibody exhibited enhanced activity similar to the combination of the two parent antibodies.

[0019] An additional CH3 heterodimerization interface was obtained by mimicking the natural association of the T cell receptor α and β chains. This BEAT technology (Bispecific Engagement by Antibodies based on the T cell receptor) was applied to generate Fab / scFv-Fc fusion proteins, avoiding light chain mispairing. This approach was used to generate bispecific antibodies against CD3 and HER2 for T cell retargeting.

[0020] Leaver-Fay and colleagues (Leaver-Fay et al., 2016) applied multistage design (MSD), an approach that simultaneously designs multiple protein stages, to generate sets of CH3 mutations at the Fc interface. Using two sets (7.8.60 and 20.8.34) and combining them with orthogonal Fab interface mutations, they generated bispecific IgGs derived from pertuzumab (anti-HER2), matuzumab (anti-EGFR), BHA10 (anti-LTRR), and MetMAb (anti-cMet). Bispecific antibodies were obtained in at least 93% success in all cases.

[0021] Heterodimeric assembly of heavy chains can also be achieved using a separate heterodimerization module, which is then removed from the bispecific antibody. This strategy was applied by employing a leucine zipper structure derived from the Acid.p1 (Ap1) and Base.p1 (Bp1) peptides fused to the C-terminus of the two heavy chains. Using this LUZ-Y platform, we generated not only monovalent Fab-Fc fusion proteins but also bispecific IgGs based on common light chain or scFab arms against EGFR and HER3. Introducing a proteolytic cleavage site between the C-terminus of the Fc chain and the leucine zipper sequence allows for the removal of the leucine zipper to obtain bispecific IgG antibodies with native composition.

[0022] While the use of modifications to force heterodimerization of the Fc region targets the heavy chain problem, these approaches still have issues with the light chain. Thus, by using two different light chains, four different combinations can be generated, but only one of them is bispecific. Therefore, approaches have been developed that target the correct pairing of cognate heavy and light chains in combination with Fc-modified heavy chains.

[0023] The first described approach was the use of a common light chain (Merchant et al., 1998). This was based on the observation that antibodies against diverse antigens isolated from phage display libraries often use the same VL domain, reflecting the very limited size of the phage library's light chain repertoire. In combination with knobs-into-holes heavy chain modifications, bispecific IgG molecules against, for example, HER3 and Mpl were created. Subsequently, various bispecific IgGs with a common light chain were produced using, for example, knobs-into-holes modifications as well as other Fc modifications.

[0024] Some of the Fc modifications mentioned above have circumvented the light chain issue by utilizing scFv fragments fused to the Fc chain to generate bispecific antibodies. Based on the finding that Fab fragments can be expressed as single-chain derivatives (scFabs connecting the C-terminus of the light chain to the N-terminus of the VH domain), complete IgG molecules have been generated by expressing a single polypeptide containing the light chain connected to the heavy chain. Linkers ranging in length from 30 residues (e.g., (G4S)6) to 38 residues have been utilized, including deletion of the disulfide bond connecting CH1 and CL. An improved scFab platform has been described for disulfide-linked scFab molecules using a flexible 60-residue linker. Applying the G4S6 linker in combination with knobs-into-holes mutations in the Fc region generated bispecific Fab-Fc fusion proteins, which were further modified by C-terminal fusion of scFv fragments to obtain trispecific, trivalent, and tetravalent molecules targeting, for example, EGFR, IGF-1R, and either cMet or HER3. The scFab format was also combined with the LUZ-Y Fc heterodimerization strategy. Here, a proteolytic cleavage site was introduced into the Fab linker to allow removal of the linker from correctly assembled bispecific IgG molecules. Furthermore, the scFab was combined with an unmodified Fab fragment and combined with Fc(kih) to generate bispecific Fab-scFab-Fc fusion proteins (OAscFab-IgG format), such as a bispecific IgG targeting EGFR and IGF-1R (which could be expressed in high yields).

[0025] Another approach is applied by CrossMab technology, where the light chain of one Fab arm is exchanged with the Fd fragment of the corresponding heavy chain in the context of a knobs-into-holes heavy chain (CrossMab Fab ), or the variable domain of one Fab (CrossMab VH-VL ) or constant domain (CrossMab CH1-CL) is exchanged between the light and heavy chains. This results in pairing of an unmodified light chain with the corresponding unmodified heavy chain, and pairing of a modified light chain with the corresponding modified heavy chain. Taking the bispecific CrossMab against VEGF and Ang-2 as an example, simultaneous antigen binding without alteration of affinity was demonstrated, and the CrossMab CH1-CL showed an excellent by-product profile. Subsequent studies have shown that this antibody (A2V) can reprogram tumor-associated macrophages and prolong survival in multiple extracranial tumor models. The antibody (RG7716) is currently in clinical development. Recent studies have shown that CrossMab CH1-CL We applied this format to generate bispecific antibodies against HIV Env protein and CD4 / CCR5 for virus neutralization. Here, heterogeneity was observed in the original CrossMabs due to incorrect pairing of the unmodified light chain, but this was ameliorated by introducing additional mutations into this chain. The CrossMab approach has evolved into a versatile platform technology, enabling the generation of bivalent bispecific IgG molecules as well as trivalent and tetravalent bispecific IgG fusion proteins by, for example, fusing an additional Fab fragment to the N-terminus of one of the knobs-into-holes heavy chains or fusing two CrossMab Fab arms to the C-terminus of the homodimerized heavy chain. Many other formats, including bispecific trivalent and tetravalent IgG-Fab fusion proteins, have been enabled by CrossMab technology, e.g., bispecific molecules with one binding site for CD3 and two binding sites for tumor-associated antigens have been generated. The concept was further developed to generate tetravalent, tetraspecific, four-in-one antibodies by applying knobs-into-holes Fc regions and CrossMab technology to two-in-one Fab arms (see below).

[0026] Another approach to the light chain problem is genetic engineering of the light and heavy chain interface to generate an orthogonal interface that allows the light chain to interact with its cognate heavy chain with higher affinity. In this case, the interaction between the variable domain (VH-VL pair) and the first constant domain (CH1-CL) is modified. Various modifications identified through multistep design were tested to establish a set of mutations that support orthogonal Fab fragment pairing. These modifications were applied to generate various bispecific IgG molecules, such as those directed against EGFRxcMET, EGFRxHER2, AxlxcMet, and EGFRxLTβR, or against two epitopes on HER2 by combining the binding sites of trastuzumab and pertuzumab. Here, Fc heterodimerization was achieved through electrostatic steering effects introduced into the CH3 domain (Gunasekaran et al., 2010). For example, a bispecific antibody with orthogonal Fab arms based on pertuzumab (anti-HER2) and matuzumab (anti-EGFR), both lambda light chains, was generated by combining substitutions of Q39K, R62E, H172A, F174G in the heavy chain and D1R, Q38R, L135Y, S176W in the light chain of pertuzumab (VRD1CRD2 modifications) with substitutions of Q39Y in the heavy chain and Q38R in the light chain of matuzumab (VRD2 modifications), generating 90% correct light chain assembly.

[0027] A further attempt to direct light chain pairing with the cognate heavy chain Fc fragment involved replacing the CH1 and CL domains of one Fab arm with the Cα and Cβ domains from the T cell receptor (TCR). This was applied to generate either Fab-IgG molecules in which the Fab arm was fused to the N-terminus of the heavy chain via a (G4S)5 linker, or IgG-Fab molecules in which the Fab arm was fused to the C-terminus of the heavy chain via a (G4S)4 linker, as in the case of bispecific antibodies derived from trastuzumab and pertuzumab. However, likely due to the strong VH / VL interaction in the trastuzumab binding site, only a small fraction showed correct Fab arm pairing. This was partially ameliorated by introducing an additional mutation (Y36F) in the VL domain to weaken the VH-VL interaction and introducing a charge-charge interaction (VL Q38D, VH Q39K) between the VL and VH domains in the trastuzumab Fv. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0028] However, although many approaches exist to direct heterodimerization of multispecific antibodies, these molecules are artificial. Immunogenicity, in particular, remains a significant issue. Furthermore, the degree of heterodimerization is often unsatisfactory, and the thermal stability and biophysical properties, such as half-life, are often not as expected. [Means for solving the problem]

[0029] To overcome the shortcomings of the prior art, the present inventors further explored the generation of Ig domains that assemble into heterodimers that can be used as building blocks to generate molecules with one, two, or more specificities and potencies. A key feature is that these heterodimer pairs are composed exclusively of human sequences, providing a completely natural heterodimerization interface without the need for mutations, thus minimizing the tendency to elicit an immune response. Furthermore, the heterodimerized Ig domains are connected by disulfide bonds (formed between cysteine ​​residues) contained within the naturally occurring sequences, allowing them to be presented in an evolutionarily developed and therefore optimal conformation. Furthermore, these domains are further endowed with the ability to interact with adjacent Ig domains, such as the FcRn or CH2 domains and variable domains, providing the heterodimerized domain with further advantageous properties. Surprisingly, it was found that the terminal half-life of the generated bivalent formats could be improved, resulting in increased bioavailability. Furthermore, these heterodimerized Ig building blocks were used to generate bivalent or trivalent bispecific scFv-Fc fusion proteins that retarget CD3-expressing T cells to FAP (fibroblast activation protein)- or Her3-expressing tumor cells or to tumor cells surrounded by FAP-expressing fibroblasts.

[0030] The basic principle of the present invention is the generation of novel heterodimerization regions or heterodimerization protein domains (HRI and HRII) that can be included in two or more proteins of a protein complex, where the constant immunoglobulin domains naturally heterodimerize (the first and third CRIs) and are modified to provide additional properties to the HRI and HRII. These properties are transferred to the heterodimerization constant immunoglobulin domains (the first and third CRIs) from one or more additional constant immunoglobulin domains (the second and fourth CRIs) that differ from the first and third CRIs by substituting amino acids from the heterodimerization constant immunoglobulin domains (the first and third CRIs) with amino acids from the alternative constant immunoglobulin domains (the second and fourth CRIs). These properties are, for example, interaction with the neonatal Fc receptor (FcRn) or interaction with a protein domain connected to the N- or C-terminus (e.g., an additional constant immunoglobulin domain that is not the first, second, third, or fourth CRI).

[0031] Thus, in a first aspect, the present invention provides a protein complex comprising at least two amino acid chains I and II, wherein the amino acid chains I and II are non-covalently bound to each other via a heterodimerization region I (HRI) comprised in the amino acid chain I and a heterodimerization region II (HRII) comprised in the amino acid chain II, and the HRI and HRII each comprise a heterodimerization domain of a human constant region of an immunoglobulin or immunoglobulin-like protein, wherein each heterodimerization domain has been modified so that an amino acid sequence of an additional immunoglobulin or immunoglobulin-like protein is inserted into at least two, and preferably at least three, regions of each heterodimerization domain that are outside the heterodimerization interface of each HRI and HRII, i.e., preferably so that the amino acid sequence is solvent accessible after heterodimerization of the HRI and HRII.

[0032] More specifically, the protein complex of the present invention comprises at least two amino acid chains, namely, an amino acid chain I and an amino acid chain II, which are non-covalently bound to each other via a heterodimerization region I (HRI) contained in the amino acid chain I and a heterodimerization region II (HRII) contained in the amino acid chain II; (a) HRI comprises seven antiparallel β-strands AI, BI, CI, DI, EI, FI, and GI, six intervening regions bI, cI, dI, eI, fI, and gI, an N-terminal region aI, and a C-terminal region hI, located in the following order from N-terminus to C-terminus: aI-AI-bI-BI-cI-CI-dI-DI-eI-EI-fI-FI-gI-GI-hI, An HRI is a fusion protein in which a first human constant region of an immunoglobulin or immunoglobulin-like protein (first CRI, acceptor) is interspersed with amino acids from a second human constant region of an immunoglobulin or immunoglobulin-like protein (second CRI, donor), wherein the first CRI comprises seven antiparallel β-strands A1, B1, C1, D1, E1, F1, and G1, six intervening regions b1, c1, d1, e1, f1, and g1, an N-terminal region a1, and a C-terminal region h1, located in the following order from N-terminus to C-terminus: a1-A1-b1-B1-c1-C1-d1-D1-e1-E1-f1-F1-g1-G1-h1, wherein the second CRI comprises seven antiparallel β-strands A2, B2, C2, D2, E2, F2, and G2, six intervening regions b2, c2, d2, e2, f2, and g2, an N-terminal region a2, and a C-terminal region h2, located in the following order from N-terminus to C-terminus: a2-A2-b2-B2-c2-C2-d2-D2-e2-E2-f2-F2-g2-G2-h2, wherein the HRI has the amino acid sequence of a first CRI, with at least the following amino acids of the first CRI substituted with the following amino acids of a second CRI: (i) at least one amino acid in a1 is substituted with at least one amino acid in a2 (substitution 1); (ii) at least one amino acid of c1 is substituted with at least one amino acid of c2 (substitution 2); and (iii) at least one amino acid of g1 is substituted with at least one amino acid of g2 (substitution 3), and (b) HRII comprises seven antiparallel β-strands AII, BII, CII, DII, EII, FII, and GII, six intervening regions bII, cII, dII, eII, fII, and gII, an N-terminal region aII, and a C-terminal region hII, located in the following order from N- to C-terminus: aII-AII-bII-BII-cII-CII-dII-DII-eII-EII-fII-FII-gII-GII-hII, HRII is a fusion protein consisting of the third human constant region of an immunoglobulin or immunoglobulin-like protein (third CRI, acceptor) interspersed with amino acids from the fourth human constant region of an immunoglobulin or immunoglobulin-like protein (fourth CRI, donor); wherein the third CRI comprises seven antiparallel β-strands A3, B3, C3, D3, E3, F3, and G3, six intervening regions b3, c3, d3, e3, f3, and g3, an N-terminal region a3, and a C-terminal region h3, located in the following order from N-terminus to C-terminus: a3-A3-b3-B3-c3-C3-d3-D3-e3-E3-f3-F3-g3-G3-h3, wherein the fourth CRI comprises seven antiparallel β-strands A4, B4, C4, D4, E4, F4, and G4, six intervening regions b4, c4, d4, e4, f4, and g4, an N-terminal region a4, and a C-terminal region h4, located in the following order from N-terminus to C-terminus: a4-A4-b4-B4-c4 -C4-d4-D4-e4-E4-f4-F4-g4-G4-h4, wherein HRII has the amino acid sequence of a third CRI, and at least the following amino acids of the third CRI are substituted with the following amino acids of a fourth CRI: (i) at least one amino acid in a3 is substituted with at least one amino acid in a4 (substitution 4); (ii) at least one amino acid in c3 is substituted with at least one amino acid in c4 (substitution 5); and (iii) at least one amino acid in g3 is substituted with at least one amino acid in g4 (substitution 6); wherein the first CRI and the third CRI are different from each other and specifically bind to each other under physiological conditions.

[0033] In a second aspect, the present invention provides a nucleic acid encoding amino acid chain I and / or amino acid chain II of the first aspect.

[0034] In a third aspect, the present invention provides a vector comprising the nucleic acid of the second aspect.

[0035] In a fourth aspect, the present invention provides a method for determining (defining) the amino acid sequence of an HRI of amino acid chain I and / or an HRII of amino acid chain II, the method comprising the steps of: (i) Select a first CRI, a second CRI, a third CRI, and a fourth CRI; (ii) determine (define) the seven β-strands A, B, C, D, E, F, and G of the first CRI, the second CRI, the third CRI, and the fourth CRI, the intervening sequences b, c, d, e, f, and g of the first CRI, the second CRI, the third CRI, and the fourth CRI, and the N-terminal and C-terminal sequences a and h of the first CRI, the second CRI, the third CRI, and the fourth CRI; (iii) substitution of at least one amino acid in a of the first CRI with at least one amino acid in a of the second CRI (substitution 1); substitution of at least one amino acid in c of the first CRI with at least one amino acid in c of the second CRI (substitution 2); substitution of at least one amino acid in g of the first CRI with at least one amino acid in g of the second CRI (substitution 3); substitution of at least one amino acid in a of the third CRI with at least one amino acid in a of the fourth CRI (substitution 4); substitution of at least one amino acid in c of the third CRI with at least one amino acid in c of the fourth CRI (substitution 5); and substitution of at least one amino acid in c of the third CRI with at least one amino acid in c of the fourth CRI (substitution 6), wherein the first CRI and the third CRI are different from each other and specifically bind to each other under physiological conditions.

[0036] In a fifth aspect, the present invention provides a method for producing an amino acid chain I comprising an HRI sequence determined (defined) according to the fourth aspect and / or an amino acid chain II comprising an HRII sequence determined (defined) according to the fourth aspect.

[0037] In a sixth aspect, the present invention provides a protein complex of the first aspect for use as a medicament. [Brief explanation of the drawings]

[0038] [Figure 1] Schematic representation of constant Ig domain sequences. A cartoon representation of the sequence of an Ig domain, with arrows indicating the positions of β-strands and bars indicating loop regions.

[0039] [Figure 2]Schematic diagram of heterodimerization Ig domains. An exemplary set of human constant regions (CRIs) and heterodimerization regions I and II (HRIs and HRIIs) of immunoglobulin or immunoglobulin-like proteins. Possible first and third CRI sequences are derived from two different heterodimerization Ig domains (white areas, first and third CRIs) that retain the potential to form interchain disulfide bonds (black bars). Possible second and fourth CRI sequences (second and fourth CRIs) of Ig domains that interact with, for example, FcRn or other Ig domains are shown in black.

[0040] [Figure 3] Graphical study of a series of Ig domains. A cartoon of the superposition of the amino acid backbones of a series of Ig domains is shown (see Figure 4). β-strands are shown in black and loop regions in grey.

[0041] [Figure 4] Sequence alignment of several Ig domains. Residues predicted to form β-strands are highlighted in black font with a dark gray background. Residues predicted to form β-strands but excluded from β-strand AG by structural or sequence alignment are highlighted in black font with a light gray background. Residues not predicted to form β-strands but included in β-strand AG by structural or sequence alignment are highlighted in white font with a dark gray background.

[0042] [Figure 5] Schematic diagram of the second / fourth CRI and first / third CRI sequence definitions. The sequences of heterodimerized Ig domains are shown, with arrows indicating the positions of β-strands and bars indicating loop regions. White indicates the first / third CRI sequence, and black indicates the sequence stretch starting from the second / fourth CRI sequence. The lengths of the different second / fourth CRI sequences indicate specific applications (A: CH31, B: CH3k, C: other domains).

[0043] [Figure 6] Imaging studies of additional substitutions of the first / third CRI residues with the second / fourth CRI amino acids. a) Interaction of Val37 (gray stick) with Ile46 and Val48 (gray spheres) from IgG1 CH3 grafted from IgG1 CH3 (gray) to the first / third CRI domain (black). b) Interaction of Glu49, Ala47, and Met107 (gray sticks) from IgG CH3 with residues (gray spheres) from the IgG CH2 domain fused to the N-terminus of the heterodimerized Ig domain (black). c) Interaction of Tyr81 (gray stick) from IgG CH1 or any light chain constant domain with the variable domain (gray sphere) fused to the N-terminus of the heterodimerized Ig domain (black).

[0044] [Figure 7] Extension of Heterodimerized Ig Domains. The N- and C-termini of a multifunctional protein complex (e.g., a covalent heterodimer with FcRn-binding ability) may be fused to additional protein domains, including different Ig domains, TNFSF members, other cytokines, toxins, etc.

[0045] [Figure 8]Sequence alignment of the CH31 (HRI) and CH3k (HRII) Ig domains. Shown are the alignments of IgG1-CH1 (first CRI) with IgG1-CH3 (second CRI, top panel) and Igκ-CL (third CRI) with IgG1-CH3 (fourth CRI, bottom panel). The portions of the sequences used to generate the heterodimerized Ig domains with FcRn binding capacity are underlined. Residues predicted to form β-strands are highlighted in black font with a dark gray background. Residues predicted to form β-strands but excluded from β-strand AG by structural or sequence alignment are highlighted in black font with a light gray background. Residues not predicted to form β-strands but included in β-strand AG by structural or sequence alignment are highlighted in white font with a dark gray background.

[0046] [Figure 9] Heterodimerized Fc portion. Example 1 demonstrates the application of the proposed system to generate the Fc portion of a heterodimerized antibody. Thus, IgG1 CH1 and Igκ CL were used as the first and third CRI, respectively, with the second and fourth CRI sequences of a grafted IgG1-CH3 (fused to the C-terminus of the IgG1 hinge and CH2 sequences) (designated Fc1k).

[0047] [Figure 10] Genotypes of the control constructs scFv13.7-Fc1k and scFv13.7-Fc. a) Genetic arrangement of the heterodimerized IgG domains of the scFv13.7-Fc1k heavy and light chains, including the restriction sites used for cloning. b) Genetic arrangement of the homodimerized IgG domains of the scFv13.7-Fc heavy and light chains, including the restriction sites used for cloning.

[0048] [Figure 11]Expression of the control constructs scFv13.7-Fc1k and scFv13.7-Fc. a) Schematic of scFv13.7-Fc1k. b) SDS-PAGE of purified scFv13.7-Fc1k (5% stacking gel, 12% separating gel). c) Size-exclusion chromatography of scFv13.7-Fc1k. d) Schematic of scFv13.7-Fc. e) SDS-PAGE of purified scFv13.7-Fc (5% stacking gel, 12% separating gel). f) Size-exclusion chromatography of scFv13.7-Fc.

[0049] [Figure 12] Schematic diagram of Example 2; Fv13.7-Fc1k. Top panel: Generation of multifunctional Ig domains. Thus, IgG1-CH1 and Igκ CL were used as the first and third CRIs, respectively, with the second and fourth CRI sequences of grafted IgG1-CH3 (fused to the C-terminus of IgG1-CH2) (designated Fc1k). Fusion of either VH13.7 or VL13.7 to each CH2 domain connected via a linker sequence generated the monovalent Fv13.7-Fc1k. Bottom panel: Gene sequences of the Fv13.7-Fc1k heavy and light chain IgG domains, including restriction sites used for cloning.

[0050] [Figure 13] Expression of Fv13.7-Fc1k. a) Schematic diagram of Fv13.7-Fc1k. b) SDS-PAGE of purified Fv13.7-Fc1k (5% stacking gel, 12% separating gel). c) Size exclusion chromatography of Fv13.7-Fc1k.

[0051] [Figure 14] Equilibrium binding of Fv13.7-Fc1k to human TNFR1-Fc. Increasing concentrations were tested for binding to huTNFR1-Fc in ELISA (n=3, mean ± SD). Fab13.7 served as a control.

[0052] [Figure 15]Biological activity of Fv13.7-Fc1k. a) IL-8 release from HT1080 cells induced by Fv13.7-Fc1k. Unstimulated cells, TNF (33 nM), ATROSAB, and Fab13.7 served as controls. Mean values ​​and standard deviations of two separate experiments are shown. b) Inhibition of IL-8 induced by 0.1 nM TNF is shown. ATROSAB, Fab13.7, 0.1 nM TNF, and unstimulated cells served as controls. Mean values ​​and standard deviations of two separate experiments are shown.

[0053] [Figure 16] Pharmacokinetic study of Fv13.7-Fc1k. Early and terminal plasma half-lives and bioavailability (area under the curve) after a single dose (25 μg) were determined using C57BL / 6J mice (n=3 / n=4 for ATROSAB) homozygously carrying the extracellular domain of human TNFR1 at the mouse locus. Residual active antibody in serum samples was detected by ELISA. ATROSAB and Fab13.7 served as controls.

[0054] [Figure 17] Schematic diagram of Example 3—FAPN-CH3N-hFc and FAPN-CH3N-Fc. Two scFv fragments targeting either FAP or CD3 were N-terminally fused to the hinge region of the heterodimerized Fc moiety Fc1k (Example 3a). To avoid covalent cross-linking through the hinge, the same construct was made using a cysteine-free hinge sequence (Example 3b).

[0055] [Figure 18] Schematic diagram of Example 4—FAPN-CH3C-hFc and FAPN-CH3C-Fc. Two scFv fragments targeting either FAP or CD3 were fused at the N-terminus (FAP) or C-terminus (CD3) to the heterodimerized Fc moiety Fc1k (Example 4a). To avoid covalent cross-linking via the hinge, the same construct was made using a cysteine-free hinge sequence (Example 4b).

[0056] [Figure 19] Schematic diagram of Example 5—FAPNN-CH3C-hFc and FAPNN-CH3C-Fc. Two scFv fragments targeting FAP were fused N-terminally to the heterodimerized Fc moiety Fc1k, and another scFv fragment targeting CD3 was fused C-terminally to Fc1k (Example 5a). To avoid covalent cross-linking via the hinge, the same construct was made using a cysteine-free hinge sequence (Example 5b).

[0057] [Figure 20] Schematic diagram of Example 6—FAPNC-CH3C-hFc and FAPNC-CH3C-Fc. Two scFv fragments targeting FAP were fused to the heterodimerized Fc moiety Fc1k at the N- or C-terminus, and another scFv fragment targeting CD3 was fused to Fc1k at the C-terminus (Example 6a). To avoid covalent cross-linking via the hinge, the same construct was made using a cysteine-free hinge sequence (Example 6b).

[0058] [Figure 21]Sequence alignment of the first CRI / third CRI and second CRI / fourth CRI sequences for generating bispecific IgG. The first and third CRI domain sequences of FcRnα3 and β2 microglobulin were aligned with the second and fourth CRI domain sequences of IgG CH3 (a), and the first and third CRI domain sequences of TCRα2 and TCRβ2 were aligned with the second and fourth CRI sequences of IgG CH1 and Igκ constant domain, respectively (b). The portions of the sequences used to generate heterodimerized Ig domains with FcRn binding (a) or Ig domain-to-domain interaction (b) are underlined. Residues predicted to form β-strands are highlighted in black font with a dark gray background. Residues predicted to form β-strands but excluded from β-strand AG due to structural or sequence alignment are highlighted in black font with a light gray background. Residues that were not predicted to form a β-strand but are included in β-strand AG by structural or sequence alignment are highlighted in white font and a dark grey background.

[0059] [Figure 22] Schematic diagram of Example 7—Bispecific IgG. To solve the problems of Fc heterodimerization and light chains, two multifunctional Ig domains must be created. Fcb2Rn is composed of the first and third CRI domain sequences of FcRn α3 and β2 microglobulin and the second and fourth CRI sequences of the grafted IgG CH3 (7a). FabTCR is composed of the first and third CRI domain sequences of TCR α2 and TCR β2 and the second and fourth CRI sequences of the grafted IgG CH1 and Igκ constant domain (7b).

[0060] [Figure 23]Schematic diagram of Example 8—Fv13.7X-Fc1k. Top panel: Generation of multifunctional Ig domains. Thus, IgG1-CH1 and Igκ CL were used as the first and third CRIs, respectively, with the second and fourth CRI sequences of grafted IgG1-CH3 (fused to the C-terminus of IgG1-CH2) (designated Fc1k). Fusion of either VH13.7 or VL13.7 to each CH2 domain connected via a linker sequence generated the monovalent Fv13.7x-Fc1k. Bottom panel: Gene sequences of the Fv13.7x-Fc1k heavy and light chain IgG domains, including restriction sites used for cloning.

[0061] [Figure 24] Production and biological activity of Fv13.7X-Fc. a) Genotyping of Fv13.7X Fc1k. b) Purified proteins were analyzed by SDS-PAGE (10%, Coomassie staining) followed by SEC (c, Yarra SEC-3000 column, flow rate 0.5 ml / min). d) Fv13.7X Fc1k was tested for binding to human TNFR1-Fc by ELISA (n=1, mean ± SD of duplicates, ATROSAB and Fab13.7 served as controls). e) IL-8 release from HT1080 cells triggered by Fv13.7X Fc1k (n=1, mean ± SD of duplicates; ATROSAB, Fab13.7, unstimulated cells, and recombinant human TNF served as controls) and TNF-induced IL-8 release using 0.1 nM recombinant human TNF (f, n=1, mean ± SD of duplicates; ATROSAB, Fab13.7, unstimulated cells, and recombinant human TNF served as controls) were analyzed.

[0062] [Figure 25]Schematic diagram of Example 9—FvCD3-Fc1k-scFvHer32. Top panel: Generation of multifunctional Ig domains. Thus, IgG1-CH1 and Igκ CL were used as the first and third CRIs, respectively, with the second and fourth CRI sequences of grafted IgG1-CH3 (fused to the C-terminus of IgG1-CH2) (designated Fc1k). By fusing either VHCD3 or VLCD3 to each CH2 domain connected via a linker sequence and fusing one Her3-targeting scFv fragment to each C-terminus of Fc1k, the bispecific and trivalent FvCD3-Fc1k-scFvHer32 was generated. Bottom panel: Gene sequences of the IgG domains of the FvCD3-Fc1k-scFvHer32 chains, including restriction sites used for cloning.

[0063] [Figure 26] Schematic diagram of Example 10 - 13.7NCD3N-hFc1k. Top panel: Generation of a multifunctional Ig domain. Thus, IgG1-CH1 and Igκ CL were used as the first and third CRIs, respectively, with the second and fourth CRI sequences of grafted IgG1-CH3 (fused to the C-terminus of IgG1-CH2) (designated Fc1k). Two scFv fragments targeting human TNFR1 (scFv13.7) or CD3 were fused N-terminally to the hinge region of the heterodimerized Fc moiety Fc1k. Bottom panel: Gene sequence of the IgG domain of the 13.7NCD3N-hFc1k chain, including restriction sites used for cloning.

[0064] [Figure 27]Production and biological activity of 13.7NCD3N-hFc1k. a) Genotype of 13.7NCD3N-hFc1k. b) Purified protein was analyzed by SDS-PAGE (10%, Coomassie staining) followed by SEC (c, Yarra SEC-3000 column, flow rate 0.5 ml / min). d) 13.7NCD3N-hFc1k was tested for binding to human TNFR1-Fc by ELISA (n = 1, mean ± SD of duplicates). e) Binding of 13.7NCD3N-hFc1k to TNFR1-expressing HT1080 cells (n = 1, mean ± SD of duplicates) and CD3-expressing Jurkat cells was analyzed by flow cytometry (f, n = 1, mean ± SD of duplicates). g) Recruitment and activation of human peripheral blood mononuclear cells to TNFR1-expressing HT1080 cells was examined in vitro by crystal violet staining of surviving target cells 5 days after stimulation (n=1, mean ± SD of duplicates).

[0065] [Figure 28] Schematic diagram of Example 10 - Her3NCD3N-hFc1k. Top panel: Generation of multifunctional Ig domains. Thus, IgG1-CH1 and Igκ CL were used as the first and third CRIs, respectively, with the second and fourth CRI sequences of grafted IgG1-CH3 (fused to the C-terminus of IgG1-CH2) (designated Fc1k). Two scFv fragments targeting either Her3 or CD3 were fused N-terminally to the hinge region of the heterodimerized Fc moiety Fc1k. Bottom panel: Gene sequence of the IgG domains of the Her3NCD3N-hFc1k chain, including restriction sites used for cloning.

[0066] [Figure 29]Production and biological activity of Her3NCD3N-hFc1k. a) Genotype of Her3NCD3N-hFc1k. b) Purified protein was analyzed by SDS-PAGE (10%, Coomassie staining) followed by SEC (c, Yarra SEC-3000 column, flow rate 0.5 ml / min). d) Her3NCD3N-hFc1k was tested for binding to Her3-Fc by ELISA (n=1, mean ± SD of duplicates).

[0067] [Figure 30] Schematic diagram of Example 10 - MSCPNCD3N-hFc1k. Top panel: Generation of a multifunctional Ig domain. Thus, IgG1-CH1 and Igκ CL were used as the first and third CRIs, respectively, with the second and fourth CRI sequences of grafted IgG1-CH3 (fused to the C-terminus of IgG1-CH2) (designated Fc1k). Two scFv fragments targeting either MCSP or CD3 were fused N-terminally to the hinge region of the heterodimerized Fc moiety Fc1k. Bottom panel: Gene sequence of the IgG domain of the MSCPNCD3N-hFc1k chain, including restriction sites used for cloning.

[0068] [Figure 31] Production and biological activity of MSCPNCD3N-hFc1k. a) Genotype of MSCPNCD3N-hFc1k. b) Purified protein was analyzed by SDS-PAGE (10%, Coomassie staining) followed by SEC (c, Yarra SEC-3000 column, flow rate 0.5 ml / min). d) Recruitment and activation of human peripheral blood mononuclear cells to WM35 cells expressing MCSP was examined in vitro by crystal violet staining of surviving target cells 5 days after stimulation (n = 1, mean ± SD of duplicates).

[0069] [Figure 32]Schematic diagram of Example 10 - Her3NCD3C-hFc1k. Top panel: Generation of multifunctional Ig domains. Thus, IgG1-CH1 and Igκ CL were used as the first and third CRIs, respectively, with the second and fourth CRI sequences of grafted IgG1-CH3 (fused to the C-terminus of IgG1-CH2) (designated Fc1k). Two scFv fragments targeting either Her3 or CD3 were fused at the N- or C-terminus, respectively, to the hinge region of the heterodimerized Fc moiety Fc1k. Bottom panel: Gene sequence of the IgG domains of the Her3NCD3C-hFc1k chain, including restriction sites used for cloning.

[0070] [Figure 33] Production and biological activity of Her3NCD3C-hFc1k. a) Genotype of Her3NCD3C-hFc1k. b) Purified protein was analyzed by SDS-PAGE (10%, Coomassie staining) followed by SEC (c, Yarra SEC-3000 column, flow rate 0.5 ml / min). d) Her3NCD3C-hFc1k was tested for binding to Her3-Fc by ELISA (n=1, mean ± SD of duplicates).

[0071] [Figure 34] array.

[0072] Sequence Listing - Free Text Information SEQ ID NO: 1 Amino acid sequence of CH1 of IgG1 SEQ ID NO: 2 Amino acid sequence of CH1 of IgG2 SEQ ID NO: 3 Amino acid sequence of CH1 of IgG3 SEQ ID NO: 4 Amino acid sequence of CH1 of IgG4 SEQ ID NO: 5 Amino acid sequence of CH1 of IgA1 SEQ ID NO: 6 Amino acid sequence of CH1 of IgA2 SEQ ID NO: 7 Amino acid sequence of CH1 of IgD SEQ ID NO: 8 Amino acid sequence of CH1 of IgE SEQ ID NO: 9 Amino acid sequence of CH1 of IgM SEQ ID NO: 10 Amino acid sequence of TCRα SEQ ID NO: 11 Amino acid sequence of TCRβ SEQ ID NO: 12 Amino acid sequence of FcRnα3 SEQ ID NO: 13 Amino acid sequence of β2 microglobulin SEQ ID NO: 14 Amino acid sequence of HLA-A SEQ ID NO: 15 Amino acid sequence of HLA-Bα3 SEQ ID NO: 16 Amino acid sequence of HLA-Dα2 SEQ ID NO: 17 Amino acid sequence of HLA-Dβ2 SEQ ID NO: 18 Amino acid sequence of Igκ constant region SEQ ID NO: 19 Amino acid sequence of Igλ constant region SEQ ID NO: 20: Amino acid sequence of CH31, first CRI:CH1 (IgG1), second CRI:CH3 (IgG1) SEQ ID NO: 21 Amino acid sequence of CH3κ, third CRI:CLκ, second CRI:CH3 (IgG1) SEQ ID NO: 22 CH1H3, amino acid sequence of the first CRI:CH1 (IgG1), and the second CRI:CH3 (IgG1) SEQ ID NO: 23: Amino acid sequence of CLkH3, third CRI:CLκ, fourth CRI:CH3 (IgG1) SEQ ID NO: 24 b2mH3, 1st CRI: β2 microglobulin, 2nd CRI: amino acid sequence of CH3 (IgG1) SEQ ID NO: 25 Amino acid sequence of FcRnH3, third CRI:FcRnα3 domain, fourth CRI:CH3 (IgG1) SEQ ID NO: 26 TCRaH3, 1st CRI: TCR α chain constant domain, 2nd CRI: amino acid sequence of CH3 (IgG1) SEQ ID NO: 27: Amino acid sequence of TCRbH3, 3rd CRI: TCR β chain constant domain, 4th CRI: CH3 (IgG1) SEQ ID NO: 28 TCRaH1, 1st CRI: TCR α chain constant domain, 2nd CRI: amino acid sequence of CH1 (IgG1) SEQ ID NO: 29 TCRaLk, 3rd CRI: TCR α chain constant domain, 4th CRI: CLκ amino acid sequence SEQ ID NO: 30 TCRaLL, 3rd CRI: TCR α chain constant domain, 4th CRI: CL λ amino acid sequence SEQ ID NO: 31 TCRbH1, 1st CRI: TCR β chain constant domain, 2nd CRI: amino acid sequence of CH1 (IgG1) SEQ ID NO: 32 TCRb1Lk, 3rd CRI: TCR β chain constant domain, 4th CRI: CLk amino acid sequence SEQ ID NO: 33 TCRbLL, 3rd CRI: TCR β chain constant domain, 4th CRI: CL λ amino acid sequence SEQ ID NO: 34 Amino acid sequence of VH13.7-CH2-CH31 SEQ ID NO: 35 Amino acid sequence of VL13.7-CH2-CH3κ SEQ ID NO: 36 Amino acid sequence of scFv13.7-hinge-CH2-CH31 SEQ ID NO: 37 Amino acid sequence of hinge-CH2-CH3κ SEQ ID NO: 38 Amino acid sequence of scFvhuU3-hinge-CH2-CH3κ SEQ ID NO: 39 Amino acid sequence of scFv3-43-hinge-CH2-CH31 SEQ ID NO: 40 Amino acid sequence of scFhuMCSP-hinge-CH2-CH31 SEQ ID NO: 41 Amino acid sequence of VH13.7-CH2-CH3k SEQ ID NO: 42 Amino acid sequence of VL13.7-CH2-CH31 SEQ ID NO: 43 Amino acid sequence of VHCD3-CH2-CH3k-scFvHer3 SEQ ID NO: 44 Amino acid sequence of VLCD3-CH2-CH31-scFvHer3 SEQ ID NO: 45 Amino acid sequence of IgG1 CH3 SEQ ID NO: 46 Amino acid sequence of IgG2 CH3 SEQ ID NO: 47 Amino acid sequence of IgG3 CH3 SEQ ID NO: 48 Amino acid sequence of IgG4 CH3 SEQ ID NO: 49 Amino acid sequence of IgM CH4 SEQ ID NO: 50 Amino acid sequence of IgA1 CH3 SEQ ID NO: 51 Amino acid sequence of IgA2 CH3 SEQ ID NO: 52 Amino acid sequence of IgD CH3 SEQ ID NO: 53 Amino acid sequence of IgE CH4 SEQ ID NO: 54 Amino acid sequence of Igλ constant region SEQ ID NO: 55 Amino acid sequence of Igλ constant region SEQ ID NO: 56 Amino acid sequence of Igλ constant region SEQ ID NO: 57 Amino acid sequence of TCRβ1 SEQ ID NO: 58 Amino acid sequence of HLA-Dβ2

[0073] [Figure 35] Biochemical characterization of Fv13.7X-Fc1k. Fv13.7X-Fc1k was produced from a CHO cell pool after stable lentiviral transduction and purified by protein A chromatography followed by preparative SEC. Characterization was performed by analytical SEC (a, TSKgel SuperSW mAb HR, flow rate 0.5 ml / min, mobile phase NaHPO / NaHPO) and SDS-PAGE (b, NuPAGE™ 4-12% Bis-TRIS Midi Gel) under reducing (R) and nonreducing (NR) conditions. M: marker. c) Melting temperatures were determined by dynamic light scattering and visual interpretation of the results. Plasma stability analysis was performed after incubation with human plasma at the indicated time points, followed by analysis of the EC50 value of the residual binding protein by ELISA (d).

[0074] [Figure 36]Antigen and Fc receptor binding of Fv13.7X-Fc1k. Binding of Fv13.7X-Fc1k to human TNFR1-Fc was analyzed by ELISA (a, n = 3, mean ± SD) and QCM (b). Five concentrations between 128 nM and 4 nM were used to generate the kinetic data in b), and a one-to-one binding algorithm was used for fitting. C) Binding of human Fcγ receptors I, IIb, and III and complement protein C1q to immobilized Fv13.7X-Fc1k was analyzed by ELISA. Rituximab (wild-type Fc moiety) and ATROSAB (silenced Fc) served as controls (n = 2, mean ± SD).

[0075] [Figure 37] Lack of agonist and antagonist biological activity of Fv13.7X-Fc1k. The inherent lack of agonist activity of Fv13.7X-Fc1k with respect to TNFR1 activation was demonstrated in three separate assays: (a) IL-6 release from HeLa cells, (b) IL-8 release from HT1080 cells, and (c) a cell death induction assay using Kym-1 cells. The inhibitory ability of Fv13.7X-Fc1k was also demonstrated in (d) an IL-6 release assay using HeLa cells, (e) an IL-8 release assay using HT1080 cells, and (f) a cell death induction assay using Kym-1 cells, all of which were performed in the presence of a fixed concentration of 0.1 nM TNF (d and e) or 0.01 nM TNF (f). Fab13.7, ATROSAB, and TNF alone served as control molecules for TNFR1 activation (TNF and ATROSAB in a, b, and c) and inhibition of TNF-induced TNFR1 activation (Fab 13.7 and ATROSAB in d, e, and f). All graphs represent the average of three separate experiments, and error bars indicate SD.

[0076] [Figure 38]Lack of agonistic biological activity of Fv13.7X-Fc1k in the presence of anti-human IgG antibodies. Activation of TNFR1 on the surface of HT1080 cells by Fv13.7X-Fc1k in the presence of three different anti-human IgG serum preparations (a, b, c) at a fixed concentration (approximately 15.8 nM) was determined by detecting IL-8 release into the culture supernatant. Cells alone and 33 nM TNF served as controls. The agonistic effects of potentially cross-linking antibodies were compared with those of Fab13.7 and ATROSAB. All experiments show the mean ± standard deviation of three individual experiments.

[0077] [Figure 39] Pharmacokinetic analysis of Fv13.7X-Fc1k. 400 μg of Fv13.7X-Fc1k was injected into C56BL / 6J knock-in mice carrying the gene for the human TNFR1 extracellular domain connected to the mouse transmembrane and intracellular domains in place of the wild-type mouse gene. Intact protein remaining in serum was determined by ELISA upon binding to TNFR1 at the indicated time points. Mean values ​​± SD for five mice are shown. DETAILED DESCRIPTION OF THE INVENTION

[0078] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, which may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which 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.

[0079] Preferably, the terms used herein are as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Koelbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0080] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number sequence submissions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0081] I. Definition The word "comprise" and variations such as "comprises" and "comprising" are meant to include a stated integer or step or group of integers or steps, but not to exclude other integers or steps or groups of integers or steps.

[0082] 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.

[0083] Concentrations, amounts, and other numerical data may be expressed or presented in a "range" format herein. It should be understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range boundaries, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. For example, a numerical range of "150 mg to 600 mg" is interpreted not only to include the explicitly recited value of 150 mg to 600 mg, but also to include each individual numerical value and subrange within the stated range. Thus, this numerical range includes individual numerical values ​​such as 150, 160, 170, 180, 190, ... 580, 590, 600 mg, and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges reciting only a single numerical value. Moreover, such interpretation should be applied regardless of the breadth of scope or characteristics being described.

[0084] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0085] The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein and are understood to refer to single- or double-stranded oligomers or polymers of deoxyribonucleotides or ribonucleotide bases, or both. A nucleotide monomer is composed of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, 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, as well as 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- or double-stranded molecules and are composed of naturally occurring nucleotides. A description of a single strand of a nucleic acid also defines (at least in part) the sequence of the complementary strand. Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequence. For example, a single-stranded nucleic acid molecule may have a 3' or 5' overhang, and therefore is not necessarily or intended to be completely double-stranded throughout its entire length. Nucleic acids can be obtained by any method known in the art, including, but not limited to, biological, biochemical, or chemical synthesis, or amplification, and reverse transcription of RNA. The term nucleic acid includes chromosomes or chromosome 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). Nucleic acids may be, for example, single-stranded, double-stranded, or triple-stranded and are not limited to a particular length. Unless otherwise specified, a particular nucleic acid sequence may include or encode complementary sequences in addition to the sequence explicitly indicated.

[0086] Nucleic acids may be degraded by endonucleases or exonucleases, particularly DNases and RNases found in cells. Therefore, it is advantageous to modify the nucleic acids of the present invention to stabilize them against degradation, thereby ensuring that high concentrations of nucleic acids are maintained in cells over long periods of time. Typically, such stabilization can be achieved by introducing one or more internucleotide phosphate groups or by introducing one or more non-phosphorus internucleotides. Thus, nucleic acids can be composed 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 nucleic acids include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate, phosphorodithioate, and / or phosphate ester, and 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 5' or 3' nucleotides to allow for ligation or prevention of exonuclease degradation / polymerase extension, respectively; amino, thiol, alkyne, or biotinyl modifications for covalent and near-covalent attachment; fluorophores and quenchers; and deoxyinosine (dI), 5-bromodeoxyuridine (5-bromo-dU), deoxyuridine, 2-aminopurine, 2,6-diaminopurine, inverted dT, inverted dideoxy-T, dideoxycytidine (ddC), 5-methyldeoxycytidine (5-methyl-dC), locked nucleic acid (LNA), 5-nitroindole, iso-dC and isodG bases, 2'-O-methyl RNA bases, hydroxymethyl-dC, 5-hydroxybutynyl-2'-deoxyuridine, 8-aza-7-deazaguanosine, and fluorine-modified bases.Thus, the nucleic acid may also be an artificial nucleic acid, including but not limited to polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), and glycol nucleic acid (GNA) and threose nucleic acid (TNA).

[0087] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0088] The term "polynucleotide", as used in the context of the present invention, refers to a nucleic acid of more than about 50 nucleotides in length, for example, 51 or more nucleotides in length.

[0089] Polypeptides of the invention are prepared by any suitable method, including, but not limited to, isolation of existing or naturally occurring sequences, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99, 1979); the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucci et al. (J. Am. Chem. Soc. 103:3185-3191, 1981); automated synthesis methods; or the solid support method of U.S. Pat. No. 4,458,066, or other methods known to those skilled in the art.

[0090] As used herein, the term "vector" refers to a protein or polynucleotide, or mixture thereof, that is introduced or capable of introducing the proteins and / or nucleic acids contained therein into a cell. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, viruses, or artificial chromosomes. In particular, vectors are used to deliver a gene product of interest, e.g., foreign or heterologous DNA, into a suitable host cell. A vector may contain a "replicon" polynucleotide sequence that facilitates autonomous replication of the vector in the host cell. Foreign DNA is defined as DNA not naturally found in the host cell, e.g., heterologous DNA that replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. Once inside a host cell, a vector can replicate independently of or simultaneously with the host chromosomal DNA, generating several copies of the vector and its inserted DNA. Additionally, a vector may contain necessary elements that enable transcription of the inserted DNA into an mRNA molecule or cause replication of the inserted DNA into multiple RNA copies. A vector may further contain an "expression control sequence" that regulates expression of the gene of interest. Typically, an expression control sequence is a polypeptide or polynucleotide, such as, but not limited to, a promoter, enhancer, silencer, insulator, or repressor. In a vector containing multiple polynucleotides encoding one or more gene products of interest, expression can be controlled jointly or separately by one or more expression control sequences. More specifically, each polynucleotide contained in a vector can be controlled by a separate expression control sequence, or all polynucleotides contained in a vector can be controlled by a single expression control sequence. Polynucleotides contained in a single vector controlled by a single expression control sequence form an open reading frame. Some expression vectors further contain sequence elements adjacent to the inserted DNA that extend the half-life of the expressed mRNA and / or enable translation of the mRNA into a protein molecule.Thus, many molecules of mRNA and polypeptides encoded by the inserted DNA can be rapidly synthesized.

[0091] The term "amino acid" generally refers to a monomeric unit comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids containing photoactivatable crosslinkers, metal-binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that interact covalently or noncovalently with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids containing polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioacid-containing amino acids, and amino acids containing one or more toxic moieties. As used herein, the term "amino acid" includes the following 20 naturally occurring or genetically encoded alpha amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), and ribozyme (RI).), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). If the "X" residue is not defined, it should be defined as "any amino acid." The structures of these 20 naturally occurring amino acids are shown, for example, in Stryer et al., Biochemistry, 5th Edition, Freeman and Company (2002). Additional amino acids, such as selenocysteine ​​and pyrrolysine, can also be genetically encoded (Stadtman (1996) "Selenocysteine", Annu Rev Biochem. 65: 83-100 and Ibba et al. (2002) "Genetic code: introducing pyrrolysine," Curr Biol. 12(13):R464-R466). The term "amino acid" also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and / or backbones), and amino acid analogs. For example, Zhang et al., (2004) “Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad. Sci. USA 101(24):8882-8887; Anderson et al., (2004) “An expanded genetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci. USA 101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novel histidine analogue and its efficient incorporation into a protein invivo,” Protein Eng. Des. Sel. 16(9):699-706、Chinら、(2003)“An Expanded Eukaryotic Genetic Code,” Science 301(5635):964-967、Jamesら、(2001)“Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues,” Protein Eng. Des. Sel. 14(12):983-991、Kohrerら、(2001)“Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins,” Proc. Natl. Acad. Sci. U.S.A. 98(25):14310-14315、Bacherら、(2001)“Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol. 183(18):5414-5425、Hamano-Takakuら、(2000)“A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than Tyrosine,” J. Biol. Chem. 275(51):40324-40328、およびBudisaら、(2001)“Proteins with {beta}-(thienopyrrolyl) alanines as alternative chromophores and pharmaceutically active amino acids,” ProteinSci. 10(7):1281-1292. The amino acids can be fused to peptides, polypeptides, or proteins.

[0092] In the context of the present invention, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. Peptides have the same chemical (peptide) bonds as proteins, but are generally shorter in length. The shortest peptides are dipeptides, in which two amino acids are joined by a single peptide bond. Tripeptides, tetrapeptides, pentapeptides, etc. are also possible. Typically, peptides are up to 8, 10, 12, 15, 18, or 20 amino acids in length. Peptides, unless they are cyclic, have an amino terminus and a carboxyl terminus.

[0093] In the context of the present invention, the term "polypeptide chain" or "amino acid chain" refers to a single linear chain of amino acids joined together by peptide bonds, typically comprising at least about 21 amino acids.

[0094] The term "protein complex" as used herein refers to a group of two or more related polypeptides or amino acid chains. Different polypeptide chains may have different functions. A protein complex is a form of quaternary structure. Proteins within a protein complex are linked by non-covalent protein-protein interactions and optionally by covalent bonds formed between two adjacent Cys residues of two different polypeptide chains, for example. Depending on the stability of the non-covalent and optionally covalent bonds, different protein complexes have different degrees of stability over time.

[0095] The term "human constant region of an immunoglobulin or immunoglobulin-like protein (CRI)" is used in the context of the present invention to refer to an amino acid sequence of 60 to 150 amino acids in length, comprising seven antiparallel beta strands that form a sandwich-like globular structure of two cysteine-linked beta sheets. An exemplary sheet structure is shown in Figure 3. The most preferred CRI amino acid sequence for use in connection with the present invention is shown in Figure 4. The term also includes variants of the specifically indicated sequences, so long as they are able to fold into a beta sheet-based Ig domain-like sandwich conformation.

[0096] As used herein, the term "heterodimerization region" (HRI, HRII) refers to an amino acid sequence stretch within the larger amino acid chain I and amino acid chain II, respectively, that specifically binds to each other, preferably under physiological conditions, and thus participates in the heterodimerization of amino acid chain I and amino acid chain II. The term heterodimerization region also refers to the amino acid sequences of amino acid chains I and II being different. With respect to the protein complexes defined above, the binding between HRI and HRII is primarily mediated by noncovalent interactions. However, if HRI and HRII contain Cys residues at adjacent positions within the binding interface between HRI and HRII, respectively, a covalent bond between these Cys residues can stabilize the binding between HRI and HRII. HRI and HRII each form a β-sheet structure as shown in Figure 3.

[0097] The term "Fc function" refers to the ability of an immunoglobulin to stimulate phagocytes or cytotoxic cells to destroy microorganisms or infected cells through antibody-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, or complement-mediated cytolysis. This function is based on the binding of an antibody to Fc receptors present on the surface of certain immune cells, particularly B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells. "Fc function" also refers to the ability to bind to the neonatal Fc receptor (FcRn), which mediates half-life extension. Those skilled in the art are familiar with methods for measuring the Fc function of an antibody or antibody fragment. The Fc function of an immunoglobulin resides primarily in the CH2 and / or CH3 domains, particularly the CH2 and / or CH3 domains of IgG.

[0098] In the context of this invention, the "primary structure" of a protein or polypeptide is the sequence of amino acids in the polypeptide chain. The "secondary structure" of a protein is the general three-dimensional format of a local segment of the protein. However, "secondary structure" does not describe specific atomic positions in three-dimensional space, which is considered a tertiary structure. In proteins, the secondary structure is defined by the pattern of hydrogen bonds between the amide and carboxyl groups of the main chain. The "tertiary structure" of a protein is the three-dimensional structure of the protein determined by atomic coordinates. The "quaternary structure" is the arrangement of multiple folded or coiled protein or polypeptide molecules in a multi-subunit complex.

[0099] As used herein, the term "folding" or "protein folding" refers to the process by which a protein assumes its three-dimensional shape or conformation, i.e., the process by which a protein forms a particular three-dimensional shape through non-covalent and / or covalent interactions, such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, electrostatic effects, intramolecular Cys bonds, etc. Thus, the term "folded protein" refers to the three-dimensional shape, such as the secondary, tertiary, or quaternary structure, of a protein.

[0100] The term "beta strand" as used in the context of the present invention refers to a 5-10 amino acid long section within a polypeptide chain in which the N-Cα-C-N torsion angle in the main chain is approximately 120 degrees. The beta strands within a given protein sequence can be predicted after (multiple) sequence alignments (e.g., using Clustal Omega) by retrieving annotations from the PDB file. If not all seven beta strands can be assigned (e.g., as indicated by stars in Figure 4), additional beta strand predictions can be performed using publicly available software tools such as JPred (the alignment in Figure 4 used the longest cumulative prediction with default settings). The start and end positions of the seven beta strands can be confirmed by additional structural alignment of the PDB file using pyMol. This allows for refinement of the multiple sequence alignment according to structurally conserved residues by (i) including unassigned residues in a strand, (ii) excluding previously assigned residues from a strand, (iii) deleting gaps inside the β-strands introduced into the sequence by multiple sequence alignment, or (iv) inserting new gaps outside the β-strands (as performed for the four exceptions in Figure 4: d_CH3: position 111; m_CH1: position #72; HLAA / HLAB: position #60). The inserted / extended or deleted / shortened gap positions must be compensated for by deletion or insertion, respectively, of the gap positions of the following existing gaps introduced into the sequence during multiple sequence alignment to maintain alignment of the sequences located closer to the C-terminus. Thus, the seven β-strands can be defined by these measures as follows: A) The six residues following the conserved proline residue at the N-terminus of the predicted first β-strand (positions 13–18 in Figure 4 ). B) The four residues adjacent to the N-terminus and five residues adjacent to the C-terminus of the conserved cysteine ​​residue in the second predicted β-strand (positions 13–18 in Figure 4 31–40). C) Four residues adjacent to the N-terminus and two adjacent to the C-terminus of the tryptophan residue contained in the third predicted β-strand (positions 46–52 in Figure 4). D) Positions 63–70 in Figure 4. However, connecting to residues conserved throughout the alignment was not feasible. E) Eight residues (positions 81–89 in Figure 4 ) starting with a conserved tyrosine / phenylalanine residue located at the beginning or N-terminus of the fourth predicted β-strand. F) Defined as the two residues adjacent to the N-terminus and four residues adjacent to the C-terminus of the conserved cysteine ​​residue in the sixth predicted β-strand (positions 102–108 in Figure 4 ). G) Positions 128–133 in Figure 4. However, connecting to residues conserved throughout the alignment was not feasible.

[0101] Thus, for a given region of an immunoglobulin or immunoglobulin-like protein (CRI), one skilled in the art can easily determine the seven β-strands. Alternatively, a given CRI not included in Figure 4 can be added to the alignment in Figure 4. β-strand A spans positions 13-18 of the IgLCRC, β-strand B spans positions 31-40 of the IgLCRC, β-strand C spans positions 46-52 of the IgLCRC, β-strand D spans positions 63-70 of the IgLCRC, β-strand E spans positions 81-89 of the IgLCRC, β-strand F spans positions 102-108 of the IgLCRC, and β-strand G spans positions 128-133 of the IgLCRC.

[0102] The term "β-sheet" as used in the context of this invention refers to the antiparallel-oriented β-strands that form a β-sheet. β-sheets are a common motif in the normal secondary structure of proteins. Because peptide chains are oriented by their N- and C-termini, β-strands are also said to be oriented. They are typically represented in protein topology diagrams by arrows pointing to the C-termini. Adjacent β-strands can form hydrogen bonds in antiparallel, parallel, or mixed configurations. In an antiparallel arrangement, successive β-strands alternate direction, with the N-terminus of one strand adjacent to the C-terminus of the next. This configuration provides the strongest interstrand stability because it allows interstrand hydrogen bonds between carbonyls and amines to be planar. β-structure is characterized by elongated polypeptide chains. The amino acid composition of β-strands tends to favor hydrophobic (water-fearing) amino acid residues. The side chains of these residues tend to be less soluble in water than those of more hydrophilic (water-loving) residues. β-structures tend to be found within the core structure of proteins, where interstrand hydrogen bonds are protected from competition with water molecules. As is evident from Figure 3, the seven antiparallel β-strands contained in each CRI, and therefore in the HRI and HRII derived from the two CRIs, form a β-sheet structure.

[0103] The term "intervening region" as used in the context of human immunoglobulins or human immunoglobulin-like proteins refers to the stretch of amino acids between two beta strands. The intervening region may be unstructured, e.g., forming a loop or containing a short alpha helix. For a given CRI, one skilled in the art can determine the intervening region by structural and / or sequence alignment. CRIs not included in Figure 4 can be added to the alignment. Once aligned, intervening regions "b," "c," "d," "e," "f," and "g" span positions 19-30 of the IgLCRC, 41-45 of the IgLCRC, 53-62 of the IgLCRC, 71-80 of the IgLCRC, 90-101 of the IgLCRC, and 109-127 of the IgLCRC, respectively. Each intervening region may contain one or more amino acid deletions, as long as the seven antiparallel beta strands are able to form a beta sheet. Thus, an intervening region may have fewer or more amino acids than indicated by the IgLCRC position of the intervening region. The N- and C-terminal amino acids of the intervening region, and therefore its length, are determined by the N- and C-terminal amino acids of the beta sheets of the human immunoglobulin or human immunoglobulin-like protein of the present invention. Thus, the length of intervening region "b" is 5 to 12, particularly 6 to 11, particularly 7 to 10, and more particularly 8 to 9 amino acids; the length of intervening region "c" is 1 to 5, particularly 2 to 4, and more particularly 3 amino acids; the length of intervening region "d" is 1 to 10, particularly 3 to 8, and more particularly 4 to 6 amino acids; the length of intervening region "e" is 1 to 10, particularly 2 to 8, and more particularly 4 to 6 amino acids; the length of intervening region "f" is 1 to 12, particularly 3 to 10, and more particularly 5 to 8 amino acids; and the length of intervening region "g" is 4 to 19, particularly 5 to 15, and more particularly 7 to 11 amino acids. As noted above, the intervening region of some human immunoglobulins or human immunoglobulin-like proteins may be longer than the typical lengths listed above, where the intervening region contains more amino acids than the IgL CRC positions of that intervening region, e.g., intervening region "b" of a particular human immunoglobulin or human immunoglobulin-like protein has a length of 15 amino acids, and intervening region "b" only spans positions 19-30 of the IgL CRC.In this case, the amino acids in the intervening region are aligned, and any extra amino acids that do not align well are given the additional designation "a," "b," etc., IgLCRC position. Thus, the 15 amino acid intervening region "b" spans the following IgLCRC designations: 19, 19a, 19b, 19c, and 20-30.

[0104] The terms "N-terminal region a" and "C-terminal region h" refer to the unstructured portions at the ends of CRI and its derivatives, HRI and HRII, respectively. N-terminal region a may span positions 1 to 12 of the IgLCRC (see Figure 4). In particular, it may be 4 to 12 amino acids long, particularly 5 to 10 amino acids long, and more particularly 6 to 8 amino acids long. C-terminal region h may span positions 134 to 144 of the IgLCRC (see Figure 4). In particular, it may be 4 to 11 amino acids long, particularly 5 to 10 amino acids long, and more particularly 6 to 8 amino acids long.

[0105] The term "fragment" as used herein refers to naturally occurring fragments (e.g., splice variants) and artificially constructed fragments, particularly fragments obtained by genetic engineering. Typically, a fragment has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acids at its N-terminus and / or C-terminus and / or internally, preferably at its N-terminus, its N- and C-terminus, or its C-terminus, compared to the parent polypeptide.

[0106] An "epitope," also known as an antigenic determinant, is a segment of a macromolecule that is recognized by the immune system, particularly antibodies, B cells, or T cells. Such an epitope is a portion or segment of a macromolecule that can bind to an antibody or its antigen-binding fragment. In this context, the term "binding" preferably refers to specific binding. In the context of the present invention, the term "epitope" preferably refers to a segment of a protein or polyprotein that is recognized by the immune system. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not.

[0107] As used herein, a "conformational epitope" refers to an epitope of a linear polymer (e.g., a polypeptide) that is formed by the three-dimensional structure of said polymer. In the context of the present application, a "conformational epitope" is a "discontinuous epitope," i.e., a conformational epitope on a polymer (e.g., a polypeptide) that is formed from at least two distinct regions of the primary sequence of the polymer (e.g., the amino acid sequence of the polypeptide). In other words, if an epitope consists of at least two distinct regions to which a binding moiety of the invention (e.g., an antibody or antigen-binding fragment thereof) simultaneously binds, and these at least two distinct regions are interrupted by another region in the primary sequence to which a binding moiety of the invention does not bind, then the epitope is considered to be a "conformational epitope" in the context of the present invention. In particular, such a "conformational epitope" is present on a polypeptide, wherein two distinct regions in the primary sequence are two distinct amino acid sequences to which a binding moiety of the invention (e.g., an antibody or antigen-binding fragment thereof) binds, and these at least two distinct amino acid sequences are interrupted by another amino acid sequence in the primary sequence to which the binding moiety of the invention does not bind. In particular, the interrupted amino acid sequence is a contiguous amino acid sequence comprising two or more amino acids to which the binding moiety of the invention does not bind. The at least two distinct amino acid sequences to which the binding moiety of the invention binds are not particularly limited in terms of their length. Such distinct amino acid sequences can consist of only one amino acid, as long as the total number of amino acids in the at least two distinct amino acid sequences is large enough to provide specific binding between the binding moiety and the conformational epitope.

[0108] A "paratope" is the portion of an antibody that recognizes an epitope. In the context of the present invention, a "paratope" is the portion of a binding moiety described herein (e.g., an antibody or antigen-binding fragment thereof) that recognizes an epitope.

[0109] A "peptide linker" in the context of the present invention refers to an amino acid sequence that sterically separates two parts or moieties of a conjugate, for example, two peptides or proteins. Typically, such linkers consist of 1 to 100 amino acids, with a minimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, and a maximum length of at least 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less. The indicated preferred minimum and maximum lengths of peptide linkers according to the present invention may be combined if such combinations are mathematically reasonable; for example, such linkers may consist of 1 to 15, or 12 to 40, or 25 to 75, or 1 to 100 amino acids. Peptide linkers also provide flexibility between the two moieties being linked together. Such flexibility is generally enhanced when the amino acids are small. Thus, flexible peptide linkers contain 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 in the peptide linker are small amino acids.

[0110] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from the polypeptide or polynucleotide from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a polypeptide or polynucleotide variant is derived is also known as the parent polypeptide or polynucleotide. The term "variant" includes "fragments" or "derivatives" of the parent molecule. Typically, "fragments" are smaller in length or size than the parent molecule, while "derivatives" exhibit one or more differences in their sequence compared to the parent molecule. Also included are modified molecules, including, but not limited to, post-translationally modified proteins (e.g., glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids, such as methylated DNA. The term "variant" also encompasses mixtures of different molecules, including, but not limited to, RNA-DNA hybrids. Typically, variants are artificially constructed, preferably by genetic engineering, while the parent protein or polynucleotide is a wild-type protein or polynucleotide or a consensus sequence thereof. However, naturally occurring variants should also be understood to be encompassed by the term "variant" as used herein. Furthermore, variants usable in the present invention may be derived from homologs, orthologs, or paralogs of the parent molecule or artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent molecule, i.e., is functionally active. In particular, the terms "peptide variant," "polypeptide variant," and "protein variant" are understood to refer to a peptide, polypeptide, or protein that differs by one or more changes in amino acid sequence compared to the peptide, polypeptide, or protein from which it is derived. The peptide, polypeptide, or protein from which a peptide, polypeptide, or protein variant is derived is also known as the parent peptide, polypeptide, or protein.Furthermore, variants that can be used in the present invention may be derived from homologs, orthologs, or paralogs of the parent peptide, polypeptide, or protein, or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent peptide, polypeptide, or protein. The changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal truncations, C-terminal truncations, or a combination of these changes, occurring at one or several sites.

[0111] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions with identical nucleic acid bases or amino acid residues in both sequences to obtain 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.

[0112] The term "identical" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same, i.e., contain the same sequence of nucleotides or amino acids. Sequences are "substantially identical" to one another if a specified percentage of nucleotides or amino acid residues are the same (e.g., at least 70%, 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 specified region) when compared over a comparison window, i.e., a designated region determined using one of the sequence comparison algorithms below or by manual alignment and visual inspection, and aligned for maximum correspondence. These definitions also refer to the complement of a test sequence. Thus, the term "at least 80% sequence identity" is used throughout this specification in reference to comparisons of polypeptide and polynucleotide sequences. This expression preferably refers to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the respective reference polypeptide or respective reference polynucleotide.

[0113] The term "sequence comparison" refers to a process in which one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are often used, although alternative parameters can also be designated. The sequence comparison algorithm then calculates the percent sequence identity or similarity of the test sequence relative to the reference sequence based on the program parameters. When two sequences are compared and no reference sequence is designated for comparison, sequence identity is calculated with reference to the longer of the two sequences being compared, unless otherwise specified. When a reference sequence is provided, sequence identity is determined based on the entire length of the reference sequence, as indicated by the SEQ ID NO:, unless otherwise specified.

[0114] In sequence alignment, the term "comparison window" refers to a stretch of contiguous positions of a sequence that is compared to a reference stretch of contiguous positions of a sequence having the same number of positions. The number of contiguous positions selected can range from 4 to 1000 and can include 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 contiguous positions. Typically, the number of contiguous positions ranges from about 20 to 800 contiguous positions, about 20 to 600 contiguous positions, about 50 to 400 contiguous positions, about 50 to about 200 contiguous positions, or about 100 to about 150 contiguous positions.

[0115] Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by manual alignment and visual inspection (e.g., Ausubel et al., Current Protocols in Molecular Biology, vol. 1, pp. 111-114, 1997). This can be done by the method described in the American Journal of Biology (1995 Supplement). Suitable algorithms 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 from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in a query sequence that, when aligned with words of the same length in a database sequence, match or meet a positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them.Word hits are extended in both directions along each sequence for as far as possible to increase the cumulative alignment score. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is halted when: the cumulative alignment score decreases by a quantity X from the maximum achieved value; the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall to zero or below; 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) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program defaults to a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989): alignment (B) 50, expectation (E) 10, M=5, N=-4, 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 smallest sum probability (P(N)), which indicates the probability that two nucleotide or amino acid sequences would match by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0116] Semi-conservative, and especially conservative, amino acid substitutions, in which an amino acid is replaced with a chemically related amino acid, are preferred. Typical substitutions are between aliphatic amino acids, between amino acids with aliphatic hydroxyl side chains, between amino acids with acidic residues, between amide derivatives, between amino acids with basic residues, or between amino acids with aromatic residues. Typical semi-conservative and conservative substitutions are as follows: TIFF0007822587000001.tif169160

[0117] Provided the new cysteine ​​remains a free thiol, changing A, F, H, I, L, M, P, V, W, or Y to C is semi-conservative. Furthermore, one skilled in the art will understand that one should not substitute a glycine in a sterically demanding position, nor should one introduce P into a portion of a protein that has alpha-helical or beta-sheet structure.

[0118] A tag (or marker or label) is any type of substance that can indicate the presence of another substance or a complex of substances. A marker can be a substance that is bound to or introduced into the substance to be detected. Detectable markers are used in molecular biology and biotechnology to detect, for example, proteins, products of enzymatic reactions, second messengers, DNA, molecular interactions, etc. Examples of suitable tags or labels include fluorophores, chromophores, radioactive labels, metal colloids, enzymes, or chemiluminescent or bioluminescent molecules. In the context of the present invention, a suitable tag is preferably a protein tag whose peptide sequence is genetically grafted into or onto a recombinant protein. Protein tags include, for example, affinity tags, solubilization tags, chromatography tags, epitope tags, or fluorescent tags.

[0119] "Affinity tags" are added to proteins so that they can be purified from crude biological sources using affinity techniques. These include chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST). The poly(His) tag is a versatile protein tag that binds to metal matrices.

[0120] "Solubilization tags" are used, particularly for recombinant proteins expressed in chaperone-deficient species, to aid in proper folding of the protein and prevent precipitation. These include thioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role as both a solubilizer and a GST, such as MBP.

[0121] "Chromatography tags" are used to modify the chromatographic properties of proteins and provide different resolution in certain separation techniques. They are often composed of polyanionic amino acids, such as the FLAG tag.

[0122] The term "epitope tag" as used in the context of this invention refers to a short peptide sequence selected to reliably generate high-affinity antibodies in many different species. These are usually derived from viral genes, which account for their high immunoreactivity. Epitope tags include V5 tags, Myc tags, and HA tags. These tags are particularly useful for Western blot, immunofluorescence, and immunoprecipitation experiments, but can also be used for antibody purification.

[0123] "Fluorescent tags" are used to visually read proteins. GFP and its variants are the most commonly used fluorescent tags. More advanced applications of GFP include its use as a folding reporter (fluorescent when folded, colorless when not). Further examples of fluorophores include fluorescein, rhodamine, and the sulfoindocyanine dye Cy5.

[0124] The term "binding" according to the present invention preferably relates to specific binding. The term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., a target or antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y is generally determined by the dissociation constant (K D ) "Specific binding" means that a binding moiety (e.g., an antibody) binds more strongly to a target, such as an epitope, for which it is specific compared to binding to another target. A binding moiety has a dissociation constant (K) that is lower than the dissociation constant of the second target. D ) binds to the first target, it binds more strongly to the first target than to the second target. The dissociation constant (K D ) is the dissociation constant (K D ) is at least 10 times, preferably at least 20 times, more preferably at least 50 times, even more preferably at least 100 times, at least 200 times, at least 500 times, or at least 1000 times lower.

[0125] Hence the term "K D " (measured in "mol / L" (sometimes abbreviated as "M")) refers to the dissociation equilibrium constant of a particular interaction between a binding moiety (e.g., an antibody or fragment thereof) and a target molecule (e.g., an antigen or epitope thereof). Affinity can be measured by common methods known in the art, including, but not limited to, surface plasmon resonance-based assays (such as BIAcore assays); quartz crystal microbalance assays (such as Attana assays); enzyme-linked immunosorbent assays (ELISAs); and competitive assays (such as RIAs). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens quickly and tend to bind longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0126] Typically, antibodies or antibody mimetics contained in amino acid chain I and / or amino acid chain II have sufficient binding affinity to their target, e.g., a K of 500 nM-1 pM, i.e., 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 1 pM. D Join by value.

[0127] As used herein, the term "immunoglobulin (Ig)" refers to immunity-conferring glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" are attached by a transmembrane region to the plasma membrane of, for example, an effector cell or endothelial cell, and include molecules such as, but not limited to, neonatal Fc receptors, B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (β2M), CD3, CD4, and CD8.

[0128] Typically, the term "antibody" as used herein refers to a secretory immunoglobulin that lacks a transmembrane region and can therefore be released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, designated by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, each of which fulfills a different role and induces the appropriate immune response to different types of antigens. Different heavy chains vary in size and composition and contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the intestine, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and protects against pathogen colonization (Underdown & Schiff (1986) Annu. Rev. Immunol. 4: 389-417). IgD primarily functions as an antigen receptor for unexposed B cells and is involved in the activation of basophils and mast cells to produce antibacterial factors (Geisberger et al. (2006) Immunology 118: 429-437; Chen et al. (2009) Nat. Immunol. 10: 889-898). IgE is involved in allergic responses through binding to allergens, causing the release of histamine from mast cells and basophils. IgE is also involved in protection against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides the majority of antibody-based immunity against invading pathogens and is the only antibody isotype that can cross the placenta and confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). There are four distinct IgG subclasses in humans (IgG1, 2, 3, and 4), named in order of abundance in serum, with IgG1 being the most abundant (~66%), followed by IgG2 (~23%), IgG3 (~7%), and IgG4 (~4%). The biological profiles of the different IgG classes are determined by the structure of their respective hinge regions.IgM is expressed on the surface of B cells in both a monomeric form and a secreted pentameric form with extremely high avidity. IgM is involved in the early stages of B cell-mediated (humoral) immunity, eliminating pathogens before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118: 429-437). Antibodies are known to form not only as monomers but also as dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM from bony fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies typically consist of four polypeptide chains, including two identical heavy chains and two identical light chains, connected via disulfide bonds and resembling a "Y"-shaped polymer. Each chain contains several immunoglobulin domains, some of which are constant domains and others variable domains. The immunoglobulin domain consists of a two-layer sandwich of seven to nine antiparallel β-strands arranged in two β-sheets. Typically, antibody heavy chains contain four Ig domains, three of which are constant (CH domains: CHI, CH2, and CH3) and one of which is a variable domain (VH). Light chains usually contain one constant Ig domain (CL) and one variable Ig domain (VL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or factors, including the first component of the classical complement system (C1q).

[0129] The term "Ig-like constant region consensus (IgLCRC)" refers to a numbering system derived from an alignment of human immunoglobulins or human immunoglobulin-like proteins, as shown in Figure 4. Those skilled in the art know how to add additional human immunoglobulins or human immunoglobulin-like proteins to this alignment using sequence identity and / or structural information. Thus, those skilled in the art can determine the respective IgLCRC number for each amino acid of a given human immunoglobulin or human immunoglobulin-like protein. For example, the six intervening regions "b," "c," "d," "e," "f," and "g" of the CH3 of IgG1 span IgLCRC19-30 (excluding amino acids 26 and 28 of the IgLCRC), IgLCRC41-45 (excluding amino acids 43 of the IgLCRC), IgLCRC53-62 (excluding amino acids 59-61 of the IgLCRC), e spans IgLCRC71-80 (excluding amino acids 72-76 of the IgLCRC), IgLCRC90-101 (excluding amino acids 98 and 99 of the IgLCRC), and IgLCRC109-127 (excluding amino acids 112-124 of the IgLCRC). The CH3 of IgG1 lacks amino acids at some positions of the IgLCRC. This is due to the high variability of these less structured regions of human immunoglobulins or human immunoglobulin-like proteins, which makes them suitable for amino acid deletion and insertion without altering the overall structure of human immunoglobulins or human immunoglobulin-like proteins. Similarly, the seven beta strands "A," "B," "C," "D," "E," "F," and "G" of the CH3 of IgG1 span positions 13-18, 31-40, 46-52, 63-70, 81-89, 102-108, and 128-133 of the IgLCRC. Furthermore, the N-terminal region "a" of the CH3 of IgG1 spans positions 7-12 of the IgLCRC, and the C-terminal region "h" spans positions 134-139 of the IgLCRC.In a similar manner, one skilled in the art can determine the N- and C-termini of each element within a given human immunoglobulin or human immunoglobulin-like protein for each given human immunoglobulin or human immunoglobulin-like protein. The number of gaps in the amino acid sequence of a given aligned human immunoglobulin or human immunoglobulin-like protein may vary. However, beta strand A spans positions 13-18 of the IgLCRC of CH3, beta strand B spans positions 31-40 of the IgLCRC, beta strand C spans positions 46-52 of the IgLCRC, beta strand D spans positions 63-70 of the IgLCRC, beta strand E spans positions 81-89 of the IgLCRC, beta strand F spans positions 102-108 of the IgLCRC, and beta strand B spans positions 103-109 of the IgLCRC. Region G spans positions 128-133 of the IgLCRC, region b spans positions 19-30 of the IgLCRC, region c spans positions 41-45 of the IgLCRC, region d spans positions 53-62 of the IgLCRC, region e spans positions 71-80 of the IgLCRC, region f spans positions 90-101 of the IgLCRC, and region g spans positions 109-127 of the IgLCRC. N-terminal region a spans positions 1-12 of the IgLCRC of the respective human immunoglobulin or human immunoglobulin-like protein, and C-terminal region h spans from position 134 to the C-terminus of the IgLCRC.

[0130] For amino acid sequences not included in Figure 4 that contain one or more intervening regions longer than intervening regions b (22 residues), c (5 residues), d (10 residues), e (12 residues), f (19 residues) as defined herein, or that contain an N-terminal region longer than N-terminal region a (12 residues) as defined herein, additional residues may be added by specifying additional residues 6a, 6b, 6c, etc. after position 6, or additional residues 25a, 25b, 25c, etc. after position 25, or additional residues 26a, 26b, 26c, etc. after position 42. Additional residues can be introduced into the IgLCRC numbering scheme by designating additional residues 42a, 42b, 42c, etc. after position 58, or by designating additional residues 58a, 58b, 58c, etc. after position 58, or by designating additional residues 71a, 71b, 71c, etc. after position 71, or by designating additional residues 91a, 91b, 91c, etc. after position 91, or by designating additional residues 111a, 111b, 111c, etc. after position 111. Furthermore, the positions of the IgLCRCs do not directly reflect the amino acid positions within the sequences contained in the sequence listing, because the respective amino acid sequences of a given human immunoglobulin or human immunoglobulin-like protein do not necessarily start at position 1 of the IgLCRC and may have gaps when aligned as shown in Figure 4. For example, a preferred CH3 of IgG1 has the amino acid sequence according to SEQ ID NO:45. The N-terminal region spans positions 7-12 of the IgL CRC, which corresponds to amino acids 1-6 of SEQ ID NO: 45, and IgG1 CH3 β-strands "A," "B," "C," "D," "E," "F," and "G" span positions 13-18, 31-40, 46-52, 63-70, 81-89, 102-108, and 128-133 of the IgL CRC, which correspond to amino acids 7-12, 23-32, 37-43, 51-58, 64-72, 83-88, and 95-100, respectively, of SEQ ID NO: 45. The C-terminal region spans amino acids 101-107.The five intervening regions "b," "c," "d," "e," "f," and "g" of the CH3 of IgG1 span positions 19-30, 41-45, 53-62, 71-80, 90-101, and 109-127 of an IgLCRC, which correspond to amino acids 13-22, 33-36, 44-50, 53-63, 59-63, 73-82, and 89-94 of SEQ ID NO: 45, respectively. Taking these considerations into account, one skilled in the art can determine the first, second, third, and fourth CRI elements without undue burden. Once the elements have been determined and the sequences of the first and second CRIs and the third and fourth CRIs have been aligned, one skilled in the art can easily replace the amino acids of the first and third CRIs with those of the third and fourth CRIs, respectively, according to the following instructions.

[0131] As used herein, the term "antigen-binding protein" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. It also includes immunoglobulin-like proteins that are selected to specifically bind to a target molecule or target epitope by techniques including, for example, phage display. In assessing the binding and / or specificity of an antigen-binding protein, e.g., an antibody or immunologically functional fragment thereof, an antibody or fragment can substantially inhibit binding of a ligand to its binding partner if the excess antibody reduces the amount of binding partner bound to the ligand by at least about 1-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (e.g., as measured in an in vitro competitive binding assay). Neutralizing capacity is measured using the IC 50 or EC 50 It can be written as a value.

[0132] "I C 50 The IC value refers to half the maximal inhibitory concentration of a substance and is therefore a measure of the effectiveness of the substance in inhibiting a particular biological or biochemical function. The value is usually expressed as a molar concentration. The IC of a drug50 IC can be determined in a functional antagonism assay by constructing a dose-response curve and examining the inhibitory effect of a test substance at various concentrations. Alternatively, a competitive binding assay can be performed to determine the IC 50 Typically, inhibitory antibodies of the present invention have an IC value of 50 nM to 1 pM, i.e., 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 1 pM. 50 Indicates the value.

[0133] "EC 50 The EC value refers to half the maximum effective concentration of a substance and is therefore a measure of the concentration of that substance that elicits a response halfway between the baseline and maximum after a specific exposure time. It is commonly used as a measure of drug potency. Therefore, the EC value of a graded dose-response curve is 50 represents the concentration of a substance at which 50% of the maximum effect is observed. EC 50 represents the concentration of a compound at which 50% of a population shows a response after a specified exposure period. Typically, inhibitory antibodies of the invention have an EC of 50 nM to 1 pM, i.e., 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or 1 pM. 50 Indicates the value.

[0134] The term "antigen-binding fragment" of an antibody (or simply "binding portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0135] As used herein, "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Human antibodies of the invention include antibodies isolated from human immunoglobulin libraries, or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described, for example, in U.S. Patent No. 5,939,598 by Kucherlapati & Jakobovits.

[0136] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal (e.g., a mouse) fused to an immortalized cell.

[0137] The term "recombinant antibody" as used herein includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody, (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) antibodies prepared, expressed, created or isolated by other means, including splicing immunoglobulin gene sequences into other DNA sequences.

[0138] The term "chimeric antibody" refers to an antibody in which portions of the amino acid sequences of the heavy and light chains are homologous to corresponding sequences in antibodies from a particular species or class, while the remaining segments of the chains are homologous to corresponding sequences in another species or class. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from one mammalian species, while the constant regions are homologous to sequences in antibodies from another species. One distinct advantage of such chimeric antibody types is that the variable regions can be conveniently derived from known sources, for example, using readily available B cells or hybridomas from non-human host organisms in combination with constant regions derived from human cell preparations. While variable regions have the advantage of being easy to prepare and source-independent in their specificity, human constant regions are less likely to elicit an immune response from a human subject when injected with the antibody than constant regions from non-human sources. However, the definition is not limited to this specific example.

[0139] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, with the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise complete variable domains fused to constant domains, or only the complementarity-determining regions (CDRs) grafted into appropriate framework regions within the variable domains. The antigen-binding site may be wild-type or may be modified by one or more amino acid substitutions, e.g., to resemble a human immunoglobulin more closely. Some forms of humanized antibodies retain all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs of a mouse antibody). Other forms have one or more CDRs that are altered with respect to the original antibody.

[0140] As reviewed by Almagro & Fransson, 2008 (the contents of which are incorporated herein by reference in their entirety), different methods for antibody humanization are known to those skilled in the art. The review article by Almagro & Fransson is briefly summarized below. Almagro & Fransson distinguish between rational and empirical approaches. Rational approaches are characterized by generating several variants of an engineered antibody and evaluating their binding and other relevant properties. If the designed variants do not produce the expected results, a new cycle of design and binding evaluation is initiated. Rational approaches include CDR grafting, resurfacing, superhumanization, and human string content optimization. In contrast, empirical approaches are based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening. Therefore, empirical approaches rely on reliable selection and / or screening systems that can search a vast space of antibody variants. In vitro display techniques such as phage display and ribosome display meet these requirements and are well known to those skilled in the art. Empirical approaches include FR libraries, guided selection, framework-shuffling, and humaneering.

[0141] A "bivalent antibody" contains two antigen-binding sites. Bivalent antibodies can be monospecific or bispecific. When a bivalent antibody is monospecific, the two binding sites of the antibody have the same antigen specificity. A "bispecific" or "bifunctional" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody that has two different antigen-binding sites. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes present on the same or different antigens. Bispecific antigen-binding proteins and antibodies are a type of multispecific antigen-binding protein antibody and can be produced by a variety of methods, including, but not limited to, hybridoma fusion, chemical conjugation of IgG or IgG fragments such as Fab', or genetic means. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553; Kontermann, 2014, MAbs 4:182-197.

[0142] A "trifunctional antibody" is a type of bispecific antibody that contains two binding sites targeting different antigens and an intact Fc portion that can bind to Fc receptors on accessory cells (e.g., monocytes / macrophages, natural killer cells, dendritic cells, etc.). For example, a trifunctional antibody contains a binding site that targets an epitope on the surface of a cancer cell, a second binding site that targets an epitope on the surface of a T cell (e.g., CD3), and the Fc portion binds to an Fc receptor on the surface of a macrophage. Thus, such a trifunctional antibody can bind T cells and macrophages to tumor cells, resulting in their destruction.

[0143] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab fragments" (also called "Fab portions" or "Fab regions"), each containing a single antigen-binding site, and an "Fc fragment" (also called "Fc portion" or "Fc region"; the name reflects its ability to crystallize easily). The crystal structure of the human IgG Fc region has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In IgG, IgA, and IgD isotypes, the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the antibody's two heavy chains; in IgM and IgE isotypes, the Fc region contains three heavy-chain constant domains (CH2-CH4) on each polypeptide chain. Additionally, smaller immunoglobulin molecules exist naturally or have been artificially constructed. The term "Fab' fragment" refers to a Fab fragment that further contains the hinge region of an Ig molecule, and a "F(ab')2 fragment" is understood to include two Fab' fragments that are chemically linked or connected via disulfide bonds. While "single-domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811) and "nanobodies" contain only a single VH domain, "single-chain Fv (scFv)" fragments contain a heavy-chain variable domain connected to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Bivalent single-chain variable fragments (di-scFvs) can be designed by combining two scFvs (scFvA-scFvB). This can be done by generating a single peptide chain with two VH and two VL regions, creating a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create scFvs with a linker that is too short to allow the two variable regions to fold together, forcing the scFv to dimerize. Typically, a five-residue long linker is used to generate these dimers. This type is known as a "diabody."Shorter linkers (one or two amino acids) between the VH and VL domains form monospecific trimers known as "triabodies" or "tribodies." Bispecific diabodies are formed by expressing VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. Single-chain diabodies (scDbs) contain VHA-VLB and VHB-VLA fragments linked by a linker peptide (P) of 12–20 amino acids, preferably 14 amino acids (VHA-VLB-P-VHB-VLA). Bispecific T-cell engagers (BiTEs) are fusion proteins consisting of two scFvs from different antibodies; one scFv binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244). Dual affinity retargeting molecules ("DART" molecules) are diabodies that are further stabilized via a C-terminal disulfide bridge.

[0144] As used herein, the term "antibody-like protein" or "immunoglobulin-like protein" refers to a protein engineered to specifically bind to a target molecule (e.g., by loop mutagenesis). Typically, such antibody-like proteins contain at least one variable peptide loop attached at both ends to a protein scaffold. This dual structural constraint significantly increases the binding affinity of antibody-like proteins to a level comparable to that of antibodies. The variable peptide loop is typically 10 to 20 amino acids in length. 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 engineered ankyrin repeat proteins (for a review, see Binz HK et al. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23(10):1257-1268). Antibody-like proteins can be derived from large libraries of mutants, for example, by panning from large phage display libraries and isolating them in the same way as conventional antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues of globular proteins. Antibody-like proteins are sometimes called "peptide aptamers."

[0145] As used herein, a "peptidomimetic" is a small protein-like chain designed to mimic a peptide. Peptidomimetics typically result from modifying existing peptides to change the properties of the molecule. For example, peptidomimetics can result from modifications to alter the stability or biological activity of a molecule. This plays a role in the development of drug-like compounds from existing peptides. These modifications include changes to peptides that do not occur in nature, such as altering the backbone or incorporating unnatural amino acids.

[0146] The term "target" refers to a molecule or portion of a molecule to which an antigen-binding protein can bind. In certain embodiments, a target may have one or more epitopes. In certain embodiments, a target is an antigen. The use of "antigen" in the phrase "antigen-binding protein" simply indicates that the protein sequence comprising the antigen can be bound by an antibody. In this context, it is not necessary that the protein be foreign or capable of inducing an immune response.

[0147] The term "recombinant" refers to an amino acid sequence or a nucleotide sequence that has been intentionally modified by recombinant methods. As used herein, the term "recombinant nucleic acid" refers to a nucleic acid formed in vitro and, optionally, further manipulated by endonucleases to form a nucleic acid molecule not normally found in nature. By way of example, recombinant nucleic acids include linear cDNA and vectors formed in vitro by joining DNA molecules that are not normally linked. It is understood that once a recombinant nucleic acid is created and introduced into a host cell, it replicates non-recombinantly, i.e., using the host cell's in vivo cellular machinery rather than through in vitro manipulation. Thus, a recombinantly produced nucleic acid can subsequently be replicated non-recombinantly. A "recombinant protein" is a protein created using recombinant techniques, for example, by expression of a recombinant nucleic acid as described above. As used herein, the term "recombinant vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). The vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors, and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of an operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific desired DNA fragments and lack the functional sequences necessary for the expression of the desired DNA fragment.

[0148] The term "host cell" refers to a cell harboring a vector (e.g., a plasmid or virus). Such host cells can be either prokaryotic (e.g., bacterial) or eukaryotic (e.g., fungal, plant, or animal) cells. Host cells include both unicellular prokaryotes and eukaryotes (e.g., bacteria, yeast, and actinomycetes), as well as unicellular cells from higher plants or animals when grown in cell culture. As used herein, a "recombinant host cell" refers to a host cell containing a polynucleotide encoding a polypeptide fragment of interest, i.e., a fragment of a viral PA subunit or variant thereof, according to the present invention. This polynucleotide can be found within the host cell (i) as is, freely divergent, (ii) incorporated into a recombinant vector, or (iii) integrated into the genomic or mitochondrial DNA of the host cell. Recombinant cells can be used to express a polynucleotide of interest or to amplify a polynucleotide or recombinant vector of the invention. The term "recombinant host cell" includes the progeny of an original cell transformed, transfected, or infected with a polynucleotide or recombinant vector of the invention. Recombinant host cells can be bacterial cells such as Escherichia coli cells, yeast cells such as Saccharomyces cerevisiae or Pichia pastoris cells, plant cells, insect cells such as SF9 or High Five cells, or mammalian cells. Preferred examples of mammalian cells are Chinese hamster ovary (CHO) cells, African green monkey kidney (COS) cells, human embryonic kidney (HEK293) cells, HELA cells, etc.

[0149] The terms "individual," "subject," or "patient" are used interchangeably herein and refer to a mammal, reptile, or bird that may benefit from the present invention. In particular, the individual is selected from the group consisting of a laboratory animal (e.g., a mouse, rat, or rabbit), a farm animal (including, for example, a guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, snipe, camel, cat, dog, turtle, tortoise, snake, or lizard), or a primate, including a chimpanzee, bonobo, gorilla, and human. In particular, an "individual" is a human.

[0150] The terms "disease" and "disorder" are used interchangeably herein and refer to an abnormal condition in which a tissue, organ, or individual can no longer perform its function efficiently, particularly an abnormal medical condition such as a disease or injury. A disease is usually, but not always, associated with specific symptoms or signs that indicate the presence of such a condition. The presence of such symptoms or signs is therefore indicative of a tissue, organ, or individual suffering from a disease. Changes in these symptoms or signs are indicative of the progression of such a disease. Disease progression is typically characterized by an increase or decrease in such symptoms or signs, which may indicate a "worsening" or "improvement" of the disease. "Worsening" of a disease is characterized by a decrease in the ability of a tissue, organ, or organism to effectively perform its function, whereas "improvement" of a disease is typically characterized by an increase in the ability of a tissue, organ, or individual to effectively perform its function. A tissue, organ, or individual at "risk for developing" a disease is one that is healthy but exhibits the potential for the onset of a disease. Typically, the risk of developing a disease is associated with early or mild signs or symptoms of such a disease. In such cases, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, infectious diseases, traumatic diseases, inflammatory diseases, skin diseases, endocrine diseases, intestinal diseases, neurological diseases, joint diseases, genetic diseases, autoimmune diseases, and various types of cancer.

[0151] "Tumor" means an abnormal group of cells or tissue that grows by rapid, uncontrolled cell proliferation and continues to grow after the stimulus that initiated the new proliferation has ceased. A tumor exhibits partial or complete lack of structural organization and functional coordination with normal tissue, usually forming a distinct mass of tissue that can be benign or malignant.

[0152] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process, dependent on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane into body cavities and blood vessels, and subsequent transport via the blood, followed by invasion of the target organ. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis," which refers to metastasis away from the primary tumor and regional lymph node system.

[0153] "Symptoms" of a disease or disorder refer to symptoms of the disease or disorder that are noticeable in a tissue, organ, or organism having such a disease or disorder, including, but not limited to, pain, weakness, tenderness, tension, stiffness, and spasms in a tissue, organ, or individual, as well as the presence, absence, increase, or decrease of specific indicators such as biomarkers and molecular markers. As used herein, the terms "disease" and "disorder" refer to an abnormal condition in which a tissue, organ, or individual can no longer effectively perform its function, particularly an abnormal medical condition such as a disease or injury. A disease or disorder is usually, but not necessarily, associated with specific symptoms or signs that indicate the presence of such a disease or disorder. Diseases or disorders include, but are not limited to, autoimmune diseases, allergic diseases, cancer-type diseases, skin diseases, endocrine diseases, blood diseases and disorders, eye diseases and disorders, genetic disorders, inflammatory diseases, infectious diseases, intestinal diseases, neurological disorders, and psychiatric disorders. Exemplary cancer-type diseases include, but are not limited to, basal cell carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, Burkitt's lymphoma, cervical cancer, colon cancer, cutaneous T-cell lymphoma, esophageal cancer, retinoblastoma, gastric cancer, gastrointestinal stromal tumor, glioma, Hodgkin's lymphoma, Kaposi's sarcoma, leukemia, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pleuropulmonary blastoma, prostate cancer, pharyngeal cancer, thyroid cancer, and urethral cancer.

[0154] As used herein, "treat," "treating," "treatment," or "therapy" of a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting or preventing the recurrence of the disorder in individuals who previously suffered from the disorder; and (e) limiting or preventing the recurrence of symptoms in individuals who previously exhibited symptoms of the disorder. Thus, a moiety having a therapeutic effect treats the symptoms of a disease or disorder by achieving one or more of the above effects (a)-(e).

[0155] As used herein, "prevent," "preventing," "prevention," or "prophylaxis" of a disease or disorder means to prevent such disease or disorder from occurring in a patient.

[0156] As used herein, the term "IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM Igκ, Igλ, TCR" refers to antibodies or molecules comprising a domain of the Ig superfamily, and may be of human, murine, rat, other rodent, bovine or other origin, particularly human origin.

[0157] As used herein, the term "HLA" refers to human leukocyte antigens, also known as MHC (major histocompatibility complex), and includes MHC class I and II molecules containing domains of the Ig superfamily, and may be of human, murine, rat, other rodent, bovine, or other origin.

[0158] As used herein, the term "ASL" refers to an antigen-specific ligand. Thus, ASLAn1 refers to an antigen-specific ligand specific for antigen 1, ASLAn2 is specific for antigen 2, ASLAn3 is specific for antigen 3, and so on.

[0159] The terms "pharmaceutical," "medicament," and "drug" are used interchangeably herein and refer to a substance and / or combination of substances used in the identification, prevention, or treatment of a disease or disorder.

[0160] Atrosab is a humanized monoclonal antibody that specifically blocks the pro-inflammatory TNF receptor 1 (TNFR1) without interacting with TNF receptor 2 (TNFR2). Atrosab is currently in development for further clinical studies.

[0161] In a first aspect, the present invention provides a protein complex comprising at least two amino acid chains I and II, wherein the amino acid chains I and II are non-covalently bound to each other via a heterodimerization region I (HRI) contained in the amino acid chain I and a heterodimerization region II (HRII) contained in the amino acid chain II, (a) HRI comprises seven antiparallel β-strands AI, BI, CI, DI, EI, FI, and GI, six intervening regions bI, cI, dI, eI, fI, and gI, an N-terminal region aI, and a C-terminal region hI, located in the following order from N-terminus to C-terminus: aI-AI-bI-BI-cI-CI-dI-DI-eI-EI-fI-FI-gI-GI-hI, An HRI is a fusion protein in which a first human constant region of an immunoglobulin or immunoglobulin-like protein (first CRI, acceptor) is interspersed with amino acids from a second human constant region of an immunoglobulin or immunoglobulin-like protein (second CRI, donor), wherein the first CRI comprises seven antiparallel β-strands A1, B1, C1, D1, E1, F1, and G1, six intervening regions b1, c1, d1, e1, f1, and g1, an N-terminal region a1, and a C-terminal region h1, located in the following order from N-terminus to C-terminus: a1-A1-b1-B1-c1-C1-d1-D1-e1-E1-f1-F1-g1-G1-h1, wherein the second CRI comprises seven antiparallel β-strands A2, B2, C2, D2, E2, F2, and G2, six intervening regions b2, c2, d2, e2, f2, and g2, an N-terminal region a2, and a C-terminal region h2, located in the following order from N-terminus to C-terminus: a2-A2-b2-B2-c2-C2-d2-D2-e2-E2-f2-F2-g2-G2-h2, wherein the HRI has the amino acid sequence of a first CRI, with at least the following amino acids of the first CRI substituted with the following amino acids of a second CRI: (i) at least one amino acid of a1 is substituted with at least one amino acid of a2 (substitution 1), in a preferred embodiment, at least 4 to 12 amino acids of a1, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8 amino acids, are substituted with at least 4 to 12 amino acids of a2, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8 amino acids; (ii) at least one amino acid of c1 is substituted with at least one amino acid of c2 (Substitution 2); in a preferred embodiment, a contiguous amino acid stretch comprising or consisting of 1 to 5 amino acids of c1, i.e. 1, 2, 3, 4, or 5, and 1 to 6 amino acids of C1, i.e. 1, 2, 3, 4, 5, or 6, more preferably 2 to 5 amino acids of c1 and 4 to 6 amino acids of C1 is substituted with a contiguous amino acid stretch comprising 1 to 5 amino acids of c2, i.e. 1, 2, 3, 4, or 5, and 1 to 6 amino acids of C2, i.e. 1, 2, 3, 4, 5, or 6, more preferably 2 to 5 amino acids of c2 and 4 to 6 amino acids of C2; More preferably, 1 to 5 amino acids of c1 are substituted with 1 to 5 amino acids of c2, Preferably, the residues substituted in substitution 2 include positions 47 and 49 of IgLCRC; Preferably, the total length of the substituted stretch of consecutive amino acids is 5 to 11, more preferably 5 to 9, even more preferably 5 to 7 amino acids; and (iii) at least one amino acid of g1 is substituted with at least one amino acid of g2 (substitution 3), in a preferred embodiment, a contiguous amino acid stretch comprising or consisting of 1 to 10 amino acids of g1, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1 to 6 amino acids of F1, i.e. 1, 2, 3, 4, 5, or 6, 1 to 10 amino acids of g1, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and 0 to 3 amino acids of G1, i.e. 0, 1, 2, or 3. , is substituted with a contiguous amino acid stretch comprising or consisting of 1 to 10 amino acids of g2, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1 to 6 amino acids of F2, i.e. 1, 2, 3, 4, 5 or 6, 1 to 10 amino acids of g1, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and 0 to 3 amino acids of G2, i.e. 0, 1, 2 or 3, wherein the total length of the substituted contiguous amino acid stretch is 3 to 20, more preferably 3 to 18, even more preferably 3 to 15; and (b) HRII comprises seven antiparallel β-strands AII, BII, CII, DII, EII, FII, and GII, six intervening regions bII, cII, dII, eII, fII, and gII, an N-terminal region aII, and a C-terminal region hII, located in the following order from N- to C-terminus: aII-AII-bII-BII-cII-CII-dII-DII-eII-EII-fII-FII-gII-GII-hII, HRII is a fusion protein consisting of the third human constant region of an immunoglobulin or immunoglobulin-like protein (third CRI, acceptor) interspersed with amino acids from the fourth human constant region of an immunoglobulin or immunoglobulin-like protein (fourth CRI, donor); wherein the third CRI comprises seven antiparallel β-strands A3, B3, C3, D3, E3, F3, and G3, six intervening regions b3, c3, d3, e3, f3, and g3, an N-terminal region a3, and a C-terminal region h3, located in the following order from N-terminus to C-terminus: a3-A3-b3-B3-c3-C3-d3-D3-e3-E3-f3-F3-g3-G3-h3, wherein the fourth CRI comprises seven antiparallel β-strands A4, B4, C4, D4, E4, F4, and G4, six intervening regions b4, c4, d4, e4, f4, and g4, an N-terminal region a4, and a C-terminal region h4, located in the following order from N-terminus to C-terminus: a4-A4-b4-B4-c4 -C4-d4-D4-e4-E4-f4-F4-g4-G4-h4, wherein HRII has the amino acid sequence of a third CRI, and at least the following amino acids of the third CRI are substituted with the following amino acids of a fourth CRI: (i) at least one amino acid of a3 is substituted with at least one amino acid of a4 (substitution 4); in a preferred embodiment, at least 4 to 12 amino acids of a3, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 10 amino acids, are substituted with at least 4 to 12 amino acids of a4, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 10 amino acids; (ii) at least one amino acid of c3 is substituted with at least one amino acid of c4 (Substitution 5); in a preferred embodiment, a contiguous amino acid stretch of 1 to 5 amino acids of c3, i.e., 1, 2, 3, 4, or 5, and 1 to 6 amino acids of C3, i.e., 1, 2, 3, 4, 5, or 6, more preferably a contiguous amino acid stretch of 2 to 5 amino acids of c3 and 4 to 6 amino acids of C3 is substituted with a contiguous amino acid stretch of 1 to 5 amino acids of c4, i.e., 1, 2, 3, 4, or 5, and 1 to 6 amino acids of C4, i.e., 1, 2, 3, 4, 5, or 6, more preferably a contiguous amino acid stretch of 2 to 5 amino acids of c4 and 4 to 6 amino acids of C4; More preferably, 1 to 5 amino acids of c3 are substituted with 1 to 5 amino acids of c4, Preferably, the residues substituted in substitution 5 include positions 47 and 49 of IgLCRC; Preferably, the total length of the substituted stretch of consecutive amino acids is 5 to 11, more preferably 5 to 9, even more preferably 5 to 7; and (iii) at least one amino acid of g3 is substituted with at least one amino acid of g4 (Substitution 6), in a preferred embodiment a contiguous amino acid stretch comprising or consisting of 1 to 10 amino acids of g1, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1 to 6 amino acids of F3, i.e. 1, 2, 3, 4, 5 or 6, 1 to 10 amino acids of g1, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and 0 to 3 amino acids of G3 is substituted with a contiguous amino acid stretch comprising or consisting of 1 to 10 amino acids of F4, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1 to 6 amino acids of F4, i.e. 1, 2, 3, 4, 5 or 6, and 0 to 3 amino acids of G4, i.e. 0, 1, 2 or 3, Preferably, the total length of the substituted stretch of consecutive amino acids is 3 to 20 amino acids, more preferably 3 to 18 amino acids, even more preferably 3 to 15 amino acids; wherein the first CRI and the third CRI are different from each other and specifically bind to each other under physiological conditions. A protein complex is provided.

[0162] Thus, the HRI preferably has the following amino acid structure: (i) at least one amino acid of a1 spanning positions 1 to 12 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein is substituted with at least one amino acid of a2 spanning positions 1 to 12 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, in preferred embodiments at least 4 to 12 amino acids of a1 spanning positions 1 to 12 of the IgLCRC, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8 amino acids, are substituted with at least 4 to 12 amino acids of a2 spanning positions 1 to 12 of the IgLCRC, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8 amino acids; and (ii) at least one amino acid of c1 spanning positions 41 to 45 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein is substituted with at least one amino acid of c2 spanning positions 41 to 45 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, in a preferred embodiment a contiguous amino acid stretch consisting of 1 to 5 amino acids, i.e., 1, 2, 3, 4, or 5 amino acids, of c1 spanning positions 41 to 45 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, and 1 to 6 amino acids, i.e., 1, 2, 3, 4, 5, or 6 amino acids, of C1 spanning positions 41 to 45 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, more preferably a contiguous amino acid stretch consisting of 1 to 5 amino acids, i.e., 1, 2, 3, 4, 5, or 6 amino acids, of c2 spanning positions 41 to 45 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, a contiguous amino acid stretch comprising or consisting of 2 to 5 amino acids of c1 and 4 to 6 amino acids of C1 spanning positions 46 to 52 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein is replaced with a contiguous amino acid stretch consisting of 1 to 5 amino acids of c2, i.e. 1, 2, 3, 4 or 5 amino acids, spanning positions 41 to 45 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, and 1 to 6 amino acids of C2, i.e. 1, 2, 3, 4, 5 or 6 amino acids, spanning positions 46 to 52 of the IgLCRC, more preferably a contiguous amino acid stretch consisting of 2 to 5 amino acids of c2 spanning positions 41 to 45 of the IgLCRC and 4 to 6 amino acids of C2 spanning positions 46 to 52 of the IgLCRC; More preferably, 1 to 5 amino acids of c1 are substituted with 1 to 5 amino acids of c2, Preferably, the residues substituted in substitution 2 include positions 47 and 49 of IgLCRC; Preferably, the total length of the substituted stretch of consecutive amino acids is 5 to 11, more preferably 5 to 9, even more preferably 5 to 7 amino acids; and (iii) at least one amino acid of g1 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein is substituted with at least one amino acid of g2 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, and in a preferred embodiment, 1 to 10 amino acids of g1 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably 1 to 6 amino acids of F1 spanning positions 102 to 108 of the IgLCRC, i.e., 1, 2, 3, 4, 5, or 6 amino acids, 1 to 10 amino acids of g1, i.e., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and 0 to 3 amino acids of G1 spanning positions 128 to 133 of the IgLCRC, i.e., 0, 1, 2, or 3 amino acids, are consecutively separated by 1 to 10 amino acids of g2 spanning positions 109 to 127 of the IgLCRC of an immunoglobulin or immunoglobulin-like protein, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and preferably 1 to 6 amino acids of F2 spanning positions 102 to 108 of the IgLCRC of an immunoglobulin or immunoglobulin-like protein, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. and 6 amino acids of g1 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and 0 to 3 amino acids of G2 spanning positions 128 to 133 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, i.e., 0, 1, 2, or 3 amino acids, and the total length of the substituted contiguous amino acid stretch is preferably 3 to 20 amino acids, more preferably 3 to 18 amino acids, and even more preferably 3 to 15 amino acids.

[0163] Similarly, HR2 preferably has the following amino acid structure: (i) at least one amino acid of a3 spanning positions 1-12 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein is substituted with at least one amino acid of a4 spanning positions 1-12 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, and in preferred embodiments, at least 4-12 amino acids of a4 spanning positions 1-12 of the IgLCRC, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6-8 amino acids, are substituted with at least 4-12 amino acids of a4 spanning positions 1-12 of the IgLCRC, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6-8 amino acids; and (ii) at least one amino acid in c3 spanning positions 41 to 45 of the IgL CRC of the immunoglobulin or immunoglobulin-like protein is substituted with at least one amino acid in c4 spanning positions 41 to 45 of the IgL CRC of the immunoglobulin or immunoglobulin-like protein, in a preferred embodiment a contiguous amino acid stretch consisting of 1 to 5 amino acids, i.e., 1, 2, 3, 4, or 5 amino acids, in c3 spanning positions 41 to 45 of the IgL CRC of the immunoglobulin or immunoglobulin-like protein, and 1 to 6 amino acids, i.e., 1, 2, 3, 4, 5, or 6 amino acids, in C3 spanning positions 46 to 52 of the IgL CRC of the immunoglobulin or immunoglobulin-like protein, more preferably an IgL CRC of the immunoglobulin or immunoglobulin-like protein. a contiguous amino acid stretch of 2 to 5 amino acids of c3 spanning positions 41 to 45 of C and 4 to 6 amino acids of C3 spanning positions 46 to 52 of the IgLCRC is replaced with a contiguous amino acid stretch of 1 to 5 amino acids of c4, i.e. 1, 2, 3, 4, or 5 amino acids, spanning positions 41 to 45 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, and 1 to 6 amino acids of C4, i.e. 1, 2, 3, 4, 5, or 6 amino acids, spanning positions 46 to 52 of the IgLCRC, more preferably with a contiguous amino acid stretch of 2 to 5 amino acids of c4 spanning positions 41 to 45 of the IgLCRC and 4 to 6 amino acids of C4 spanning positions 46 to 52 of the IgLCRC; More preferably, 1 to 5 amino acids of c3 are substituted with 1 to 5 amino acids of c4, Preferably, the residues substituted in substitution 2 include positions 47 and 49 of IgLCRC; Preferably, the total length of the substituted stretch of consecutive amino acids is 5 to 11, more preferably 5 to 9, even more preferably 5 to 7 amino acids; and (iii) at least one amino acid of g3 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein is substituted with at least one amino acid of g4 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, in a preferred embodiment, 1 to 10 amino acids, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, of g3 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, preferably 1 to 6 amino acids, i.e., 1, 2, 3, 4, 5, or 6 amino acids, of F3 spanning positions 102 to 108 of the IgLCRC, and 1 to 10 amino acids, i.e., 1, 2, 3, 4, 5, or 6 amino acids, of g1 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein. , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and 0 to 3 amino acids of G3 spanning positions 128 to 133 of the IgLCRC, i.e., 0, 1, 2, or 3 amino acids, followed by 1 to 10 amino acids of g4 spanning positions 109 to 127 of the IgLCRC of an immunoglobulin or immunoglobulin-like protein, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and preferably 1 to 6 amino acids of F4 spanning positions 102 to 108 of the IgLCRC of an immunoglobulin or immunoglobulin-like protein, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. and 6 amino acids in g3 spanning positions 109 to 127 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and 0 to 3 amino acids in G4 spanning positions 128 to 133 of the IgLCRC of the immunoglobulin or immunoglobulin-like protein, i.e., 0, 1, 2, or 3 amino acids, and the total length of the substituted consecutive amino acid stretch is preferably 3 to 20 amino acids, more preferably 3 to 18 amino acids, and even more preferably 3 to 15 amino acids.

[0164] A further preferred embodiment of the HRI has the following amino acid structure: (i) at least 4 to 12 amino acids, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8, of a3 spanning positions 1 to 12 of the IgL CRC are substituted with at least 4 to 12 amino acids, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8, of a4 spanning positions 1 to 12 of the IgL CRC; and (ii) a contiguous amino acid stretch comprising or consisting of 2 to 5 amino acids of c3 spanning positions 41 to 45 of the IgLCRC and 4 to 6 amino acids of C3 spanning positions 46 to 52 of the IgLCRC is replaced with a contiguous amino acid stretch comprising or consisting of 2 to 5 amino acids of c4 spanning positions 41 to 45 of the IgLCRC and 4 to 6 amino acids of C4 spanning positions 46 to 52 of the IgLCRC, or 1 to 5 amino acids of c3 are replaced with 1 to 5 amino acids of c4; More preferably, the residues substituted in substitution 2 include positions 47 and 49 of IgLCRC; Preferably, the total length of the substituted stretch of consecutive amino acids is 5 to 11, more preferably 5 to 9, even more preferably 5 to 7 amino acids; and (iii) a contiguous amino acid stretch comprising or consisting of 1 to 6 amino acids, i.e., 1, 2, 3, 4, 5, or 6 amino acids, of F1 spanning positions 102 to 108 of the IgLCRC, 1 to 10 amino acids, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, of g1, and 0 to 3 amino acids, i.e., 0, 1, 2, or 3 amino acids, of G1 spanning positions 128 to 133 of the IgLCRC, and 1 to 6 amino acids, i.e., 1, 2, 3, 4, 5, or 6 amino acids, of F2 spanning positions 102 to 108 of the IgLCRC, and spanning positions 109 to 127 of the IgLCRC of an immunoglobulin or immunoglobulin-like protein; and a contiguous amino acid stretch comprising or consisting of 1 to 10 amino acids of g2, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and 0 to 3 amino acids of G2 spanning positions 128 to 133 of IgLCRC, i.e., 0, 1, 2, or 3 amino acids, the total length of the substituted contiguous amino acid stretch being preferably 3 to 20 amino acids, more preferably 3 to 18 amino acids, and even more preferably 3 to 15 amino acids.

[0165] Similarly, a more preferred HR2 has the amino acid structure: (i) at least 4 to 12 amino acids, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8, of a3 spanning positions 1 to 12 of the IgL CRC are substituted with at least 4 to 12 amino acids, i.e., 4, 5, 6, 7, 8, 9, 10, 11, or 12, more preferably 6 to 8, of a4 spanning positions 1 to 12 of the IgL CRC; and (ii) a contiguous amino acid stretch comprising or consisting of 2 to 5 amino acids of c3 spanning positions 41 to 45 of the IgLCRC and 4 to 6 amino acids of C3 spanning positions 46 to 52 of the IgLCRC is replaced with a contiguous amino acid stretch comprising or consisting of 2 to 5 amino acids of c4 spanning positions 41 to 45 of the IgLCRC and 4 to 6 amino acids of C4 spanning positions 46 to 52 of the IgLCRC, or 1 to 5 amino acids of c3 are replaced with 1 to 5 amino acids of c4; More preferably, the residues substituted in substitution 2 include positions 47 and 49 of IgLCRC; Preferably, the total length of the substituted stretch of consecutive amino acids is 5 to 11, more preferably 5 to 9, even more preferably 5 to 7 amino acids; and (iii) a contiguous amino acid stretch comprising or consisting of 1 to 6 amino acids of F3, i.e., 1, 2, 3, 4, 5, or 6 amino acids, 1 to 10 amino acids of g3, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, spanning positions 102 to 108 of the IgLCRC, and 0 to 3 amino acids of G3, i.e., 0, 1, 2, or 3 amino acids, spanning positions 128 to 133 of the IgLCRC, and 1 to 6 amino acids of F4, i.e., 1, 2, 3, 4, 5, or 6 amino acids, spanning positions 102 to 108 of the IgLCRC, and spanning positions 109 to 127 of the IgLCRC of an immunoglobulin or immunoglobulin-like protein; The IgLCRC is substituted with a consecutive amino acid stretch comprising or consisting of 1 to 10 amino acids of g4, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and 0 to 3 amino acids of G4 spanning positions 128 to 133 of the IgLCRC, i.e., 0, 1, 2, or 3 amino acids, and the total length of the substituted consecutive amino acid stretch is preferably 3 to 20 amino acids, more preferably 3 to 18 amino acids, and even more preferably 3 to 15 amino acids.

[0166] Preferably, the HRI comprises an a2 of 6 to 8 amino acids; a c2 and a C2 of 5 to 11, more preferably 5 to 9, and even more preferably 5 to 7 amino acids; in substitution 3, a contiguous amino acid stretch of 3 to 20, more preferably 3 to 18, and even more preferably 3 to 15 amino acids from the first CRI is replaced with a contiguous amino acid stretch of 3 to 20, more preferably 3 to 18, and even more preferably 3 to 15 amino acids from the second CRI; preferably, the HRI comprises an a4 of 6 to 8 amino acids; a c4 and a C4 of 5 to 11, more preferably 5 to 9, and even more preferably 5 to 7 amino acids; and in substitution 6, a contiguous amino acid stretch of 3 to 20, more preferably 3 to 18, and even more preferably 3 to 15 amino acids from the third CRI is replaced with a contiguous amino acid stretch of 3 to 20, more preferably 3 to 18, and even more preferably 3 to 15 amino acids from the fourth CRI.

[0167] It is also preferred that the substitution is not a null substitution, i.e., if the amino acids of the first CRI and the second CRI are identical at the substituted IgLCRC position, this is not considered a substitution within the meaning of the present invention. Substitution of an amino acid of the first CRI with an amino acid of the second CRI, and substitution of an amino acid of the third CRI with an amino acid of the fourth CRI, changes the sequence of the regions of the first CRI and the third CRI, respectively.

[0168] In a preferred embodiment, the binding affinity of the HRI to the HRII is at least 50% of the affinity of the first CRI to the third CRI, and preferably the binding affinity is at least 60%, 70%, 80%, 90% or more. It is particularly preferred that the binding affinity of the HRI to the HRII is unchanged compared to the binding affinity of the first CRI and the third CRI. Those skilled in the art are familiar with methods for determining the binding affinity between two proteins. A preferred method for determining binding affinity in the context of the present invention is the use of BiaCore or quartz crystal microbalance (QCM) measurements.

[0169] To maintain the overall structure of the acceptor protein, i.e., the first and third CRIs, the acceptor amino acids of the first and third CRIs, which are part of the antiparallel β-strands, are preferably replaced with the same number of donor amino acids. This applies to substitution 2, which involves replacing a portion of C1 with a portion of C2; substitution 3, which involves replacing a portion of F1 and / or G1 with a portion of F2 and / or G2; substitution 5, which involves replacing a portion of C3 with a portion of C4; and substitution 6, which involves replacing a portion of F3 and / or G3 with a portion of F4 and / or G4. Because the length of the intervening region is more variable between two different human constant regions of immunoglobulin or immunoglobulin-like proteins, the lengths of the replaced intervening regions in substitutions 1 to 6 are not necessarily identical. Typically, the number of substituted amino acids is the same as the number of inserted amino acids. That is, the overall length is unchanged, or the number of amino acids substituted is 1, 2, or 3 more or fewer than the number of amino acids inserted from the corresponding intervening region, e.g., 10 amino acids of a1 are replaced with 7-13 amino acids of a2. These principles of substitutions 1-6 are illustrated in Figure 8.

[0170] Preferably, the first CRI amino acid at a given IgLCRC position is substituted with the second CRI amino acid at the same IgLCRC position. Similarly, preferably, the third CRI amino acid at a given IgLCRC position is substituted with the fourth CRI amino acid at the same IgLCRC position. In a most preferred embodiment, the first and third CRI amino acids substituted with the second and fourth CRI amino acids, respectively, are at the same IgLCRC positions for both HRI and HRII.

[0171] For example, HRI can include amino acids a2 at positions 7-13 of the IgLCRC, substituting amino acids a1 at positions 7-13 of the IgLCRC, and / or HRII can include amino acids a4 at positions 7-13 of the IgLCRC, substituting amino acids a3 at positions 7-13 of the IgLCRC.

[0172] In certain embodiments, the first CRI and / or the third CRI are not included in amino acid chain I. In another specific embodiment, the first CRI and / or the third CRI are not included in amino acid chain II. The first CRI and / or the third CRI are not included in amino acid chains I and II. Omission of the respective acceptor sequences prevents undesired heterodimerization. However, as long as only one of the first CRI or the third CRI is included in the HRI and HRII, heterodimerization does not occur. Therefore, inclusion of the first CRI or the third CRI enables heterodimerization of the protein complex of the present invention with additional protein chains or complexes. It should be noted that due to amino acid substitutions within the HRI and HRII, the HRI is no longer considered the first CRI, and the HRII is no longer considered the third CRI, in the context of the present invention. Therefore, it is preferred that the first CRI and / or the third CRI are not included in amino acid chain I outside the HRI and amino acid chain II outside the HRII.

[0173] In a preferred embodiment, the second CRI and the fourth CRI are identical and, more preferably, specifically bind to each other under physiological conditions, ie, form homodimers.

[0174] In certain embodiments in which the second and fourth CRIs have Fc function, it is preferred that the Fc function be maintained when the amino acid sequences of the second and fourth CRIs are inserted into the first CRI of the HRI and the third CRI of the HRII, respectively. In the context of the present invention, the Fc function is maintained when the HRI has at least 30% of the Fc function of the second CRI and the HRII has at least 30% of the Fc function of the fourth CRI. When the second and fourth CRIs specifically bind to each other and have Fc function, it is particularly preferred that the heterodimerized HRI and HRII have at least 30% of the Fc function of the dimerized, preferably homodimerized, second and fourth CRIs.

[0175] In a preferred embodiment, substitution 1, substitution 2, substitution 3, substitution 4, substitution 5, and / or substitution 6, preferably substitutions 1-6, do not introduce new B cell epitopes into HRI and / or HRII. Preferably, they do not introduce new human B cell epitopes. In a preferred embodiment, substitution 1, substitution 2, substitution 3, substitution 4, substitution 5, and / or substitution 6, preferably substitutions 1-6, do not introduce new T cell epitopes into HRI and / or HRII. Preferably, they do not introduce new human T cell epitopes.

[0176] In certain embodiments, the immunoglobulin or immunoglobulin-like protein is selected from IgG1, Ig kappa, T cell receptor (TCR) alpha, TCR beta, neonatal Fc receptor (FcRn), beta 2 microglobulin, Ig lambda, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM, leukocyte antigen (HLA) A or B, and HLA-D. In particular, it is selected from human IgG1, Ig kappa, T cell receptor (TCR) alpha, TCR beta, neonatal Fc receptor (FcRn), beta 2 microglobulin, Ig lambda, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM, human leukocyte antigen (HLA) A or B, and HLA-D.

[0177] In a specific embodiment, the first CRI and the third CRI are a constant region of heavy chain 1 (CH1) of IgG1, preferably having the amino acid sequence of SEQ ID NO: 1; an Igκ constant region, preferably having the amino acid sequence of SEQ ID NO: 18; a constant region of TCRα, preferably having the amino acid sequence of SEQ ID NO: 10; a constant region of TCRβ, preferably having the amino acid sequence of SEQ ID NO: 11; an FcRn alpha3, preferably having the amino acid sequence of SEQ ID NO: 12; a β2 microglobulin, preferably having the amino acid sequence of SEQ ID NO: 13; an Igλ constant region, preferably having the amino acid sequence of SEQ ID NO: 19, 54, 55, 56, 57, or 58; an IgG2, preferably having the amino acid sequence of SEQ ID NO: 2; an Igκ constant region, preferably having the amino acid sequence of SEQ ID NO: 3 IgA1 preferably having the amino acid sequence of SEQ ID NO:5; IgA2 preferably having the amino acid sequence of SEQ ID NO:6; IgD preferably having the amino acid sequence of SEQ ID NO:7; IgE preferably having the amino acid sequence of SEQ ID NO:8; IgM preferably having the amino acid sequence of SEQ ID NO:9; human leukocyte antigen (HLA) A preferably having the amino acid sequence of SEQ ID NO:14, or HLA-Bα3 preferably having the amino acid sequence of SEQ ID NO:15; HLA-Dα2 preferably having the amino acid sequence of SEQ ID NO:16; and HLA-Dβ2 preferably having the amino acid sequence of SEQ ID NO:17.

[0178] In certain embodiments, the combination of the first CRI and the third CRI is selected from the following: (i) The first CRI: CH1 of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM and the third CRI: Igκ constant region and Igλ constant region, i.e., the first CRI CH1 and the third CRI IIgκ of IgG1, the first CRI CH1 and the third CRI IIgκ of IgG2, the first CRI CH1 and the third CRI IIgκ of IgG3, the first CRI CH1 and the third CRI IIgκ of IgG4, the first CRI CH1 and the third CRI IIgκ of IgA1, the first CRI CH1 and the third CRI IIgκ of IgA2, the first CRI CH1 and the third CRI IIgκ of IgD, the first CRI CH1 and the third CRI IIgκ of IgE, the first CRI CH1 and the third CRI IIgκ of IgM, and the first CRI CH1 and third CRIIgλ, first CRI CH1 and third CRIIgλ of IgG2, first CRI CH1 and third CRIIgλ of IgG3, first CRI CH1 and third CRIIgλ of IgG4, first CRI CH1 and third CRIIgλ of IgA1, first CRI CH1 and third CRIIgλ of IgA2, first CRI CH1 and third CRIIgλ of IgD, first CRI CH1 and third CRIIgλ of IgE, or first CRI CH1 and third CRIIgλ of IgM; (ii) the first CRI:constant region of TCRα and the third CRI:constant region of TCRβ; (iii) the first CRI: FcRn alpha 3; HLA-Aα3; or HLA-Bα3 and the third CRI: β2 microglobulin; and (iv) 1st CRI:HLA-Dα2 and 3rd CRI:HLA-Dβ2.

[0179] In certain embodiments, (i) the second CRI and the fourth CRI are identical and are selected from the group consisting of CH3 of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2; CH1 of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM; or IgD; CH4 of IgE or IgM; and Igκ or Igλ constant region, or (ii) the second CRI and the fourth CRI are independently selected from the group consisting of CH1 of IgG1, Igκ or Igλ constant region, CH1 of IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM; CH3 of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD and CH4 of IgE or IgM.

[0180] In certain embodiments, the second CRI and the fourth CRI are independently selected from the group consisting of constant region 3 (CH3) of the heavy chain of IgG1, preferably having the amino acid sequence of SEQ ID NO: 45, CH3 of IgG2, preferably having the amino acid sequence of SEQ ID NO: 46, CH3 of IgG3, preferably having the amino acid sequence of SEQ ID NO: 47, CH3 of IgG4, preferably having the amino acid sequence of SEQ ID NO: 48, constant region 4 (CH4) of the heavy chain of IgM, preferably having the amino acid sequence of SEQ ID NO: 49, CH3 of IgA1, preferably having the amino acid sequence of SEQ ID NO: 50, CH3 of IgA2, preferably having the amino acid sequence of SEQ ID NO: 51, CH3 of IgD, preferably having the amino acid sequence of SEQ ID NO: 52, and CH4 of IgE, preferably having the amino acid sequence of SEQ ID NO: 53.

[0181] Thus, in certain embodiments, the present invention provides (i) in substitution 1 and / or substitution 4, preferably in substitution 1 and substitution 4, all amino acids from the N-terminus to beta sheet A (positions 1 to 12 of the Ig-like constant region consensus (IgLCRC)) of the first CRI and / or the third CRI, preferably the first CRI and the third CRI, are replaced with all amino acids from the N-terminus to beta sheet A (positions 1 to 12 of the IgLCRC) of the second CRI and / or the fourth CRI, preferably the second CRI and the fourth CRI, respectively; (ii) 41-45, 41-46, 41-47, 41-48, 41-49, 41-50, 41-51, 42-45, 42-46, 42-47, 42-48, 42-49, 41-50, 41-51, 42-45, 42-46, 42-52, 42-53, 42-54, 42-55, 42-56, 42-57, 42-58, 42-59, 43-60, 43-61, 43-62, 43-63, 43-64, 43-65, 43-66, 43-67, 43-68, 43-69, 44-70, 44-71, 44-72, 44-73, 44-74, 44-75, 44-76, 44-77, 44-78, 44-79, 44-80, 44-81, 44-82, 44-83, 44-84, 44-85, 44-86, 44-87, 44-88, 44-89, 45-90, 45-91, 45-92, 45-93, 45-94, 45-95, 45-96, 45-97, 45-98, 45-99, 46-100, 46-101, 46-102, 46-103, 46-104, 46-105, 46-106, 46- -47, 42-48, 42-49, 42-50, 42-51, 43-45, 43-46, 43-47, 43-48, 43-49, 43-50, 43-51, 44-45, 44-46, 44-47, 44-48, 44-49, 44-50, 44-51, 45-45, 45-46, 45-47, 45-48, 45-49, 45-50, and the amino acids at positions 45-51 are 41-45, 41-46, 41-47, 41-48, 41-49, 41-50, 41-51, 42-45, 42-46, 42-47, 42-48, 42-49, 42-50, 42-51, of the IgLCRC of the second CRI and / or the fourth CRI, preferably the second CRI and the fourth CRI; are substituted with an amino acid at positions 43-45, 43-46, 43-47, 43-48, 43-49, 43-50, 43-51, 44-45, 44-46, 44-47, 44-48, 44-49, 44-50, 44-51, 45-45, 45-46, 45-47, 45-48, 45-49, 45-50, or 45-51, respectively; and (iii) In substitution 3 and / or substitution 6, preferably in substitution 3 and substitution 6, 103-127, 103-128, 103-129, 103-130, 103-131, 103-132, 104-127, 104-128, 104-129, 104-130, 104-131, 104-132, 105-127, 105-128, 105-133, 105-134, 105-135, 105-136, 105-137, 105-138, 105-140, 105-141, 105-142, 105-143, 105-144, 105-145, 105-146, 105-147, 105-148, 105-149, 105-200, 105-201, 105-202, 105-203, 105-204, 105-205, 105-206, 105-207, 105-208, 105-209, 106-210, 106-211, 106-212, 106-213, 106-214, 106-215, 106-216, 106-217, 106-218, 106-219, 106-300, 106-310, 106-311, 106-320, 29, 105-130, 105-131, 105-132, 106-127, 106-128, 106-129, 106-130, 106-131, 106-132, 107-127, 107-128, 107-129, 107-130, 107-131, 107-132, 108-127, 108-128, 108-129, 108-130, 108-131, 108-132, 109-127, 109-128, 109-129, 109-130, 109-131 also the amino acids at positions 109-132 of the second CRI and / or fourth CRI, preferably 103-127, 103-128, 103-129, 103-130, 103-131, 103-132, 104-127, 104-128, 104-129, 104-130, 104-131, 104-132, 105-127, 105-128, 105-129, 105-130, 105-131, 105-132 of the IgLCRC of the second CRI and / or fourth CRI, , 106-127, 106-128, 106-129, 106-130, 106-131, 106-132, 107-127, 107-128, 107-129, 107-130, 107-131, 107-132, 108-127, 108-128, 108-129, 108-130, 108-131, 108-132, 109-127, 109-128, 109-129, 109-130, 109-131, or 109-132, respectively. A protein complex is provided.

[0182] The HRI mainly comprises or consists of the amino acid sequence of the first CRI, and the HRII mainly comprises or consists of the amino acid sequence of the third CRI, i.e., the acceptor. The total number of amino acids substituted in the first CRI and the third CRI, i.e., the number of amino acids inserted from the second CRI into the first CRI and from the fourth CRI into the third CRI, is 14 to 30 amino acids, i.e., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, particularly 15 to 29, and particularly 16 to 19.

[0183] In a specific embodiment, in addition to substitutions 1 to 6, the present invention provides that (i) the amino acids at positions 37, 47, 49, 81, and / or 107 of the IgLCRC of the first CRI or third CRI are substituted with the amino acids at positions 37, 47, 49, 81, and / or 107 of the IgLCRC of the second CRI or fourth CRI. These substitutions further improve the properties of HRI and HRII, such as dimerization stability or strength. In a specific embodiment, the amino acids at positions 37 and 47 of the IgLCRC of the first or third CRI, and optionally, the amino acids at positions 49 and / or 81 and / or 107 of the IgLCRC, are substituted with the amino acids at positions 37 and 47 of the IgLCRC of the second or fourth CRI, and optionally, the amino acids at positions 49 and / or 81 and / or 107 of the IgLCRC. In a specific embodiment, the amino acids at positions 37 and 81 of the IgLCRC of the first or third CRI, and optionally, the amino acids at positions 47 and / or 49 and / or 107 of the IgLCRC, are substituted with the amino acids at positions 37 and 81 of the IgLCRC of the second or fourth CRI, and optionally, the amino acids at positions 47 and / or 49 and / or 107 of the IgLCRC. In certain embodiments, the amino acids at positions 37 and 107 of the IgLCRC of the first or third CRI, and optionally, the amino acids at positions 47 and / or 49 and / or 81 of the IgLCRC, are substituted with the amino acids at positions 37 and 107 of the IgLCRC of the second or fourth CRI, and optionally, the amino acids at positions 47 and / or 49 and / or 81 of the IgLCRC. In certain embodiments, the amino acids at positions 47 and 49 of the IgLCRC of the first or third CRI, and optionally, the amino acids at positions 37 and / or 81 and / or 107 of the IgLCRC of the second or fourth CRI, are substituted with the amino acids at positions 47 and 49 of the IgLCRC of the second or fourth CRI, and optionally, the amino acids at positions 37 and / or 81 and / or 107 of the IgLCRC.In a specific embodiment, the amino acids at positions 47 and 81 of the IgLCRC of the first or third CRI, and optionally the amino acids at positions 37 and / or 49 and / or 107 of the IgLCRC, are substituted with the amino acids at positions 47 and 81 of the IgLCRC of the second or fourth CRI, and optionally the amino acids at positions 37 and / or 49 and / or 107 of the IgLCRC. In a specific embodiment, the amino acids at positions 47 and 107 of the IgLCRC of the first or third CRI, and optionally the amino acids at positions 37 and / or 49 and / or 81 of the IgLCRC, are substituted with the amino acids at positions 47 and 107 of the IgLCRC of the second or fourth CRI, and optionally the amino acids at positions 37 and / or 49 and / or 81 of the IgLCRC. In a specific embodiment, the amino acids at positions 49 and 81 of the IgLCRC of the first or third CRI, and optionally the amino acids at positions 37 and / or 47 and / or 107 of the IgLCRC, are substituted with the amino acids at positions 49 and 81 of the IgLCRC of the second or fourth CRI, and optionally the amino acids at positions 37 and / or 47 and / or 107 of the IgLCRC. In a specific embodiment, the amino acids at positions 49 and 107 of the IgLCRC of the first or third CRI, and optionally the amino acids at positions 37 and / or 47 and / or 81 of the IgLCRC, are substituted with the amino acids at positions 49 and 107 of the IgLCRC of the second or fourth CRI, and optionally the amino acids at positions 37 and / or 47 and / or 81 of the IgLCRC.

[0184] In a particular embodiment, the following combination of first and third CRIs: (i) the first CRI: CH1 of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM and the third CRI: Igκ constant region and / or Igλ constant region; (ii) the first CRI:TCRα constant region and the third CRI:TCRβ constant region; As a result, HRI and HRII each contain at least one Cys residue positioned to form a covalent bond between HRI and HRII at position 20 (CH1 of IgG2, IgG3, IgG4, or IgM), 21 (CH1 of IgD), 135 (CH1 of IgA1 or IgA2), 138 (CH1 of IgG1 or IgE, CL of Igκ, or CL of Igλ), 139 (constant domain of TCRβ), or 141 (constant domain of TCRβ) of the IgLCRC, as shown in Figure 4.

[0185] In certain embodiments, the present invention provides protein complexes, wherein HRI and HRII are comprised in the protein complex and have the amino acid sequences of SEQ ID NO:20 and SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:32, SEQ ID NO:28 and SEQ ID NO:33, SEQ ID NO:31 and SEQ ID NO:29, and SEQ ID NO:31 and SEQ ID NO:30, respectively.

[0186] In certain embodiments, amino acid chain I and / or amino acid chain II are selected from the group consisting of two or more connected variable chain domains forming an antibody CH2 or CH3 domain; preferably an Fv, a single-chain Fv (scFv), a disulfide-stabilized Fv, a disulfide-stabilized scFv, a Fab, a single-chain Fab, a single-domain antibody, a variable heavy domain (VH), a variable light domain (VL), e.g., a diabody-like binding site, a nanobody, a VHH, one or more antibody-like binding proteins (e.g., diabody, anticalin, affibody, fibronectin-like domain, etc.). one or more antigen-specific ligands (ASL) selected from the group consisting of: an antibody hinge region (HR), one or more linker sequences (L), one or more cytokines (e.g., members of the TNF superfamily, interleukins (ILs, e.g., IL-2), interferons (e.g., IFNg), growth factors, hormones, ligands, peptides, receptor fragments with ligand-binding activity, chelators, enzymes, coagulation factors, anticoagulants, and derivatives thereof.

[0187] In certain embodiments, amino acid chain I consists of the following from N-terminus to C-terminus: (i) ASL specific for antigen 1 (ASLAn1)-L-CH2-HRI; (ii) CH2-HRI; (iii) ASLAn1-L-CH2-HRI; (iv) CH2-HRI-L-ASLAn1; (v) CH2-HRI; (vi) CH2-HRI-L-ASLAn1; (vii) ASL specific for ASLAn1-L-CH2-HRI-L-antigen 2 (ASLAn2); (viii) ASLAn1-L-CH2-HRI; (ix) CH2-HRI; (x)CH2-HRI-L-ASLAn1; (xi)ASLAn1-L-CH2-HRI-L-ASLAn2; (xii)ASLAn1-L-CH2-HRI-L-ASLAn2; (xiii) CH2-HRI-L-ASLAn1; (xiv) ASLAn1-L-CH2-HRI; (xv)ASLAn1-L-CH2-HRI-L-ASLAn2; (xvi) an amino acid chain according to any one of (i) to (xv) above, which contains a hinge region at the N-terminus of CH2; (xvii) an amino acid chain according to (i) to (xvi) above, which contains a cytokine (C) or an interleukin (IL) instead of one or more ASLs; (xviii) ASLAn1a (e.g. VL)-HRIa + ASLAn1b (e.g. VH)-HRIIa-L / HR-CH2-HRIb and amino acid chain II comprises, from N-terminus to C-terminus: (i) CH2-HRII; (ii) ASLAn1-L-CH2-HRII; (iii) ASL specific for antigen 2 (ASLAn2)-L-CH2-HRII; (iv) CH2-HRII; (v) CH2-HRI-L-ASLAn1; (vi) CH2-HRI-L-ASLAn2; (vii) CH2-HRII; (viii) CH2-HRII-L-ASLAn2; (ix)ASLAn1-L-CH2-HRII-L-ASLAn2; (x)ASLAn2-L-CH2-HRII; (xi) ASL specific for CH2-HRII-L-antigen 3 (ASLAn3); (xii) ASLAn3-L-CH2-HRII; (xiii)ASLAn2-L-CH2-HRII-L-ASLAn3; (xiv)ASLAn2-L-CH2-HRII-L-ASLAn3; (xiv) ASLAn3-L-CH2-HRII-L-antigen 4-specific ASL (ASLAn4); (xvi) an amino acid chain according to any one of (i) to (xv) above, which contains a hinge region at the N-terminus of CH2; (xvii) an amino acid chain according to (i) to (xvi) above, which contains a cytokine (C) or an interleukin (IL) instead of one or more ASLs; (xviii) ASLAn2a (e.g. VL)-HRIc + ASLAn2b (e.g. VH)-HRIIc-L / HR-CH2-HRIIb Includes.

[0188] In certain embodiments, amino acid chain I comprises one or more antigen-specific ligands (ASLs) and / or one or more effector molecules, and amino acid chain II comprises one or more antigen-specific ligands (ASLs) and / or one or more effector molecules, where the ASL modules are selected from the group of molecules that specifically bind to, for example, cell surface proteins (receptors, adhesion molecules, channels, transporters, etc.), hormones, growth factors, cytokines, ligands, serum proteins, clotting factors, fibrinolytic factors, chemokines, enzymes, etc., and the effector molecules are selected from the group of molecules that specifically bind to, for example, cell surface proteins (receptors, adhesion molecules, channels, transporters, etc.), hormones, growth factors, cytokines, ligands, serum proteins, clotting factors, fibrinolytic factors, chemokines, enzymes, etc.

[0189] In a particular embodiment, the invention provides amino acid chain I or amino acid chain II, preferably a combination of these amino acid chains, wherein chain I and chain II comprise, consist essentially of, or consist of an amino acid sequence according to SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:34 and SEQ ID NO:35, SEQ ID NO:43 and SEQ ID NO:44, SEQ ID NO:36 and SEQ ID NO:37 (scFv13.7-Fc1k), SEQ ID NO:36 and SEQ ID NO:38 (scFv13.7-CD3-hinge-Fc1k), SEQ ID NO:39 and SEQ ID NO:38 (scFv3-43-CD3-hinge-Fc1k) or SEQ ID NO:40 and SEQ ID NO:38 (scFvhuMCSP-CD3-hinge-Fc1k), respectively, and variants thereof having at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 98% sequence identity to said sequences and which are capable of heterodimerization and specific binding to the same target.

[0190] In a second aspect, the present invention provides a nucleic acid encoding amino acid chain I and / or amino acid chain II.

[0191] In a third aspect, the present invention provides a vector comprising the nucleic acid of the second aspect.

[0192] In a fourth aspect, the present invention provides a method for determining (defining) the amino acid sequence of HRI of amino acid chain I and / or HRII of amino acid chain II, the method comprising the steps of: (i) Select a first CRI, a second CRI, a third CRI, and a fourth CRI; (ii) determine (define) the seven beta strands A, B, C, D, E, F, and G of the first CRI, the second CRI, the third CRI, and the fourth CRI, the intervening sequences b, c, d, e, f, and g of the first CRI, the second CRI, the third CRI, and the fourth CRI, and the N-terminal and C-terminal sequences a and h of the first CRI, the second CRI, the third CRI, and the fourth CRI, respectively; (iii) substitution of at least one amino acid in a of the first CRI with at least one amino acid in a of the second CRI (substitution 1); substitution of at least one amino acid in c of the first CRI with at least one amino acid in c of the second CRI (substitution 2); substitution of at least one amino acid in g of the first CRI with at least one amino acid in g of the second CRI (substitution 3); substitution of at least one amino acid in a of the third CRI with at least one amino acid in a of the fourth CRI (substitution 4); substitution of at least one amino acid in c of the third CRI with at least one amino acid in c of the fourth CRI (substitution 5); and substitution of at least one amino acid in g of the third CRI with at least one amino acid in g of the fourth CRI (substitution 6), wherein the first CRI and the third CRI are different from each other and specifically bind to each other under physiological conditions.

[0193] Preferably, both HRI and HRII are defined, since both are required to allow heterodimerization of the two amino acid chains.

[0194] Step (ii) involves, for each CRI, sequence alignment and assignment of the N- and C-terminal amino acids of each of the β-strands and intervening sequences.

[0195] In a fifth aspect, the present invention provides a method for producing amino acid chain I having a determined (defined) HRI sequence and / or amino acid chain II having a determined (defined) HRII sequence, the method comprising the steps of introducing a nucleic acid encoding amino acid chain I and / or amino acid chain II into a host cell and expressing amino acid chain I and / or amino acid chain II.

[0196] In a sixth aspect, the present invention provides a protein conjugate for use as a medicament.

[0197] In one embodiment, the protein complex is for use in the prevention, treatment or diagnosis of a disorder or disease such as, but not limited to, an inflammatory disease, an autoimmune disease, an allergic disease, a proliferative disease, a cancer-type disease, a skin disease, an endocrine disease, an eye disease and disorder, a genetic disorder, a metabolic disease, an infectious disease, an intestinal disease, a neurological disorder, and a psychiatric disorder.

[0198] The following examples are merely illustrative of the present invention and should not be construed in any way as limiting the scope of the invention as set forth by the appended claims. [Example]

[0199] Example 1: Heterodimerized Fc moieties The Fc portion of the heterodimerized antibody was generated using IgG1 CH1 and Ig kappa constant domains as the second and fourth CRIs, respectively, and IgG1 CH3 residues as the first and third CRI sequences. For the CH1-CH3 combination (CH31), sequence configurations 1, 2 (#43-51) and 3 (#103-132) were used, and for the CLk-CH3 combination (CH3k), sequence configurations 1, 2 (#45-51) and 3 (#103-129) were used. In addition to the sequences shown, residue 37 of CH3 was moved to the third CRI sequence of CLk for potential involvement in structural stabilization of the CH3 elements introduced for sequences 1-3. The sequences of CH31 and CH3k are shown in Figure 8. To generate a fully functional Fc portion, designated Fc1k, both domains were fused to the C-terminus of the IgG1 hinge-CH2 domain (Figure 9). To demonstrate the heterodimerization potential of Fc1k, we cloned and generated two molecules containing scFv13.7 (anti-tumor necrosis factor receptor 1, TNFR1) linked to the first Fc chain via an IgG1 hinge sequence and a second chain (containing only the hinge sequence and Fc domain) (Figure 10). One construct had the CH2-CH31 / CH2-CH3k heterodimerizing Fc portion, while the second molecule contained an unmodified CH2-CH3 wild-type Fc portion. Both proteins were purified by protein A affinity chromatography. Under reducing conditions, scFv13.7-hinge-Fc1k showed two major bands corresponding to the calculated molecular weights (Figure 11a-c, heavy chain 50 kDa, light chain 24 kDa). Furthermore, a second band of lower molecular weight appeared below the light chain, indicating that it was unglycosylated or less glycosylated. Under non-reducing conditions, one dominant band was visible, resembling the correctly formed heterodimer (74 kDa). However, additional bands of lower molecular weight indicated the presence of monomeric heavy chains or dimerized light chains (approximately 50 kDa) and smaller degradation products or unlinked light chains (approximately 25 kDa). This observation was confirmed by SEC analysis.The dominant peak at approximately 14.5 min represented the correctly assembled scFv13.7-Fc1k protein, whereas the peak at approximately 16.5 min could again be attributed to monomeric heavy chains or dimerized light chains. In contrast, scFv13.7-Fc further formed heavy chain dimers, as seen by SDS-PAGE (approximately 160 kDa, Figure 11d-e) and SEC analysis (approximately 13.8 min). Compared to scFv13.7-Fc1k, the reduction in the minor band in SDS-PAGE (approximately 50 kDa) or the minor peak in SEC analysis (approximately 16.5 min) indicated a reduced tendency of the light chain to form homodimers, supporting the assumption that the minor band in the case of scFv13.7-Fc1k represents monomeric heavy chains rather than dimerized light chains. Additional minor peaks with shorter retention times observed in SEC analysis indicated aggregated or multimerized protein species and were observed for both proteins.

[0200] Example 2: Fv13.7-Fc1k A Fab-like antibody format containing a functional, bivalent Fc portion was created by fusing the variable domain of Fab13.7 to a single Fc chain consisting of CH2 and CH31 or CH2 and CH3k via a GTG3SG linker, respectively (Figure 12). Furthermore, mutations (A327G, A330S, and P331S, EU numbering) were introduced into CH2 to avoid binding to Fcγ receptors and the complement protein C1q (Richter et al., 2013). CH2-CH31 and CH2-CH3k containing an N-terminal linker (GTG3SG) were used (GeneArt TM ) was inserted into pSecTagA-L1 containing either VH13.7 or VL13.7 after digestion with KpnI and EcoRI.

[0201] Fv13.7-Fc1k was expressed in transiently transfected HEK293-6E cells after co-administration of two plasmids encoding either VH13.7-CH2-CH31 or VL13.7-CH2-CH3k using polyethyleneimine as a transfection reagent. Protein secreted into the cell culture supernatant was purified by protein A affinity chromatography (14.6 mg / L, see Table 1), followed by a preparative size-exclusion chromatography step (SEC, final yield 4.1 mg / L). Individual fractions were tested for their ability to induce TNFR1 activation. Negative fractions (data not shown), indicative of correctly assembled protein (see cartoon, Figure 13a), were pooled. Fv13.7-Fc1k exhibited two bands of slightly different sizes under reducing conditions and one band under non-reducing conditions on SDS-PAGE, all corresponding to the calculated molecular weight (Figure 13b). Similarly, under native conditions in SEC analysis, one major peak was observed with minor peaks representing a small proportion of aggregated or multimerized protein species (Figure 13c).

[0202] Table 1: Production and purification of Fv13.7-Fc1k TIFF0007822587000002.tif45154

[0203] Fv13.7-Fc1k had an EC of 1.2 nM, representing a 1.9-fold reduced binding affinity compared to Fab13.7 in ELISA. 50 100 nM TNF-induced TNFR1-mediated IL-8 release from HT1080 cells (Table 2, Figure 14). Consistent with Fab13.7, Fv13.7-Fc1k did not induce TNFR1-mediated IL-8 release from HT1080 cells, in contrast to the receptor activation observed with ATROSAB (Figure 15a). Furthermore, Fv13.7-Fc1k inhibited TNFR1-mediated IL-8 release from HT1080 cells induced by 0.1 nM TNF with an IC of 39.7 nM. 50Compared to Fab13.7, Fv13.7-Fc1k showed a 1.5-fold decrease in biological activity, but compared to ATROSAB, the inhibitory potency increased 2.5-fold (Fig. 15b, Table 2). To further clarify the potential application of Fv13.7-Fc1k as a therapeutic agent, we used human TNFR1, which was genetically engineered to express the extracellular domain of human TNFR1 instead of the mouse protein. ecd Pharmacokinetic properties were determined in vivo using knock-in mice. A terminal half-life of 29.1 h and an area under the curve of 526.4 h*μg / ml (representing a relative measure of the bioavailability of a therapeutic drug) were determined for ATROSAB (Figure 16, Table 2). Higher values ​​would be expected for ATROSAB due to its larger molecular weight and the potential for drug recycling via FcRn. However, under the experimental conditions of low-dose injection (25 μg / animal) and the presence of the target antigen (human TNFR1) applied here, ATROSAB was eliminated by a secondary effect of target-mediated clearance (Richter et al., in preparation). Fv13.7-Fc1k demonstrated reduced terminal half-life and area under the curve values ​​by factors of 2.1 and 2.0, respectively, compared to ATROSAB. However, more importantly, compared to Fab13.7, Fv13.7-Fc1k exhibited a 10-fold improvement in terminal half-life and a 65-fold increase in area under the curve.

[0204] Table 2: Functional data of Fv13.7-Fc1k TIFF0007822587000003.tif58170

[0205] Examples 3-6: Generation of bivalent or trivalent and bispecific scFv-Fc fusion proteins Furthermore, we generated bivalent or trivalent bispecific scFv-Fc fusion proteins using the heterodimerized Fc moiety Fc1k to retarget CD3-expressing T cells to FAP-expressing tumor cells or to tumor cells surrounded by FAP-expressing fibroblasts. Therefore, the scFvhu36 (FAP targeting) and scFvhuU3 (CD3 targeting) moieties were fused to either the N- or C-terminus of Fc1k. Furthermore, to investigate the importance of covalent linkage via the hinge, all constructs were generated using a cysteine-free IgG1 hinge region. The generated constructs contained one FAP targeting moiety and one CD3 targeting moiety at the N-terminus or one FAP targeting moiety at the C-terminus. N -CH3 N -hFc [containing cysteine ​​in the hinge region], Example 3a and FAP N -CH3 N -Fc [no cysteine ​​in the hinge region], Example 3b, Figure 17) or contained one FAP targeting moiety at the N-terminus and one CD3 targeting moiety at the C-terminus (FAP N -CH3 C -hFc, Example 4a and FAP N -CH3 C -Fc, Example 4b, Figure 18). Additionally, a construct containing two FAP targeting moieties at the N-terminus and one CD3 targeting moiety at the C-terminus (FAP NN -CH3 C -hFc, Example 5a and FAP NN -CH3 C -Fc, Example 5b, Figure 19) and a construct comprising one FAP targeting moiety at the N-terminus, one FAP targeting moiety at the C-terminus, and one CD3 targeting moiety at the C-terminus (FAP NC -CH3 C -hFc, Example 6a and FAP NC -CH3 C -Fc, Example 6b, Figure 20) have been produced. The production and characterization of these fusion proteins is still in progress.

[0206] Example 7: Generation of bivalent IgG-like antibodies The toolbox for generating heterodimerized Ig domains presented herein offers the possibility of generating bivalent IgG-like antibodies. Therefore, a heterodimerized Fc portion must be generated, and the first and third CRI sequences cannot be obtained from CH1 and CL kappa / lambda. The first and third CRI sequences can be combined with the second and fourth CRI sequences from CH3, e.g., FcRn-alpha3 and beta2-microglobulin, to generate the new domains FcRnH3 and b2mH3. The desired donor sequence composition (1, 2 [41-45], 3 [109-127], and additional residues [47, 49, 107]) is shown in Figure 21a. Similar to Fc1k, a complete Fc portion (Fcb2Rn) can also be generated.

[0207] To generate an IgG-like molecule, one of the Fab arms can be left intact. However, to avoid mispairing of the heavy and light chains, another heterodimerization domain pair must be generated as the basis for the second IgG arm of the Fab-like molecule. The first and third CRI sequences can be used, for example, with the second and fourth CRI sequences from CH1 and CLk from TCR-alpha2 and TCR-beta2, respectively, to generate new domains, TCRaH1 and TCRbLk, which assemble into FabTCRs after fusion to the C-termini of VH and VL of the desired specificity. The desired second and fourth CRI sequence compositions (1, 2 [41-45], 3 [109-127], and additional residues

[81] ) are shown in Figure 21a. The entire molecular sequence is shown in Figure 22.

[0208] Example 8: Fv13.7 X -Fc1k A Fab-like antibody format containing a functional, bivalent Fc portion was created by fusing the variable domain of Fab13.7 to a single Fc chain consisting of CH2 and CH31 or CH2 and CH3k via a GTG3SG linker, respectively (Figure 23). Furthermore, mutations (A327G, A330S, and P331S, EU numbering) were introduced into CH2 to avoid binding to Fcγ receptors and the complement protein C1q (Richter et al., 2013). CH2-CH31 and CH2-CH3k containing an N-terminal linker (GTG3SG) (GeneArt TM The codon-optimized DNA sequence of Fv13.7 was inserted into pSecTagA-L1 containing either VH13.7 or VL13.7 after digestion with KpnI and EcoRI. Unlike the previously described Fv13.7-Fc1k (Example 2), Fv13.7 X In the case of -Fc1k, VH13.7 was fused N-terminally to a polypeptide chain comprising CH2 and CH3k, and VL13.7 was fused N-terminally to a polypeptide chain comprising CH2 and CH31.

[0209] Fv13.7 was expressed in transiently transfected HEK293-6E cells after co-administration of two plasmids encoding either VH13.7-CH2-CH3k or VL13.7-CH2-CH31 using polyethyleneimine as a transfection reagent. X -Fc1k was expressed. The protein secreted into the cell culture supernatant was purified by Protein A affinity chromatography followed by a preparative size exclusion chromatography step. Collected fractions representing the peak representing intact heterodimer-assembled protein (Fig. 24a) were pooled. After Protein L purification, Fv13.7 X -Fc1k showed two bands of different sizes under reducing conditions and one band under non-reducing conditions on SDS-PAGE, all corresponding to the calculated molecular weight (Figure 24b). Similarly, under native conditions on SEC analysis, one large peak was observed, with minor peaks representing a small proportion of aggregated / multimerized protein species or a minimal proportion of free single polypeptide chains (Figure 24c).

[0210] Fv13.7 X -Fc1k has an EC of 1.9 nM, representing a 2.1-fold reduced binding affinity compared to Fab13.7 in ELISA 50 Consistent with Fab13.7, Fv13.7 bound to immobilized human TNFR1 at 100 ng / mL (Table 3, Figure 24d). X Although Fv13.7-Fc1k did not induce TNFR1-mediated IL-8 release from HT1080 cells (Fig. 24e), this contrasted with the receptor activation observed with ATROSAB (Fig. 15a). Notably, ATROSAB did not consistently activate TNFR1 on the surface of HT1080 cells in the IL-8 release assay, most likely due to variations in the materials used (ELISA kit) or cell batch-to-batch variations. However, Fv13.7-Fc1k did not induce TNFR1-mediated IL-8 release from HT1080 cells (Fig. 24e). X -Fc1k inhibited TNFR1-mediated IL-8 release induced by 0.1 nM TNF with an IC of 48 nM 50 Fv13.7 inhibited the activity of Fab13.7 at a higher level than Fab13.7. X -Fc1k showed a 3.7-fold decrease in biological activity, but a 2.8-fold increase in inhibitory potency compared to ATROSAB (Figure 24f, Table 3). Production and characterization of these fusion proteins are still in progress.

[0211] Table 3:Fv13.7 X -Fc1k functional data TIFF0007822587000004.tif51154

[0212] Example 9: FvCD3-Fc1k-scFvHer32 Bispecific molecules based on the Fab-like antibody format described in Examples 2 and 8 were generated by fusing two single-chain variable fragments (scFvs) of a Her3-targeting antibody to the C-terminus of the CH31- and CH3k-containing polypeptide chains of a CD3-specific Fv-Fc1k module (Figure 25). The connection was achieved by a hinge-derived polypeptide linker (Table 5) using the KasI and EcoRI restriction sites. In general, this format holds the potential to be used as a platform technology for the development of diverse bispecific and multispecific immune cell-engaging molecules by replacing the target-binding scFv portion with alternative scFvs or any type of binding domain against one or possibly two different tumor antigens or tumor-associated antigens. Production and characterization of this fusion protein are still ongoing.

[0213] Example 10: Bispecific scFv-Fc fusion protein specific for human TNFR1 and CD3 Based on the molecule described as Example 3a, another bispecific scFv-Fc fusion protein was generated containing scFv portions against CD3 and human TNFR1 fused to the N-terminus of the hinge-containing chain of Fc1k (13.7 N -CD3 N -hFc1k) (Figure 26).

[0214] Two pSecTagAL1 vectors, each encoding one of the two polypeptide chains, were transiently transfected using polyethyleneimine as a transfection reagent. N -CD3 N -hFc1k was produced in HEK293-6E cells. After purification using Protein A affinity chromatography followed by preparative SEC, SDS-PAGE analysis revealed two bands under reducing conditions and one dominant band under non-reducing conditions, both representing the calculated molecular weight (Figure 27b). The minor band observed under both conditions may represent a partial difference in the glycosylation state of the expressed protein. Furthermore, 13.7 N -CD3N -hFc1k showed one single peak in analytical SEC (Figure 27c). N -CD3 N Binding of -hFc1k to human TNFR1-Fc fusion protein was analyzed by ELISA and showed an EC 50 The values ​​were revealed (Figure 27d, Table 4). N -CD3 N -hFc1k binds to TNFR1 and CD3 on the surface of HT1080 (Fig. 27e) and CD3-transfected Jurkat cells (Fig. 27f), and induces EC 50 Finally, the ATP values ​​were 3.7 nM and 1.6 nM, respectively. Finally, the ATP values ​​were 13.7 nM, as reflected by their ability to reduce target cell viability (HT1080 cells expressing TNFR1) in the presence of peripheral blood mononuclear cells (PBMCs). N -CD3 N - Determine the biological activity of hFc1k and find an IC of 0.8 nM 50 and 40% residual viability at concentrations above 10 nM (FIG. 27g). Production and characterization of this fusion protein is still in progress.

[0215] Example 11: Bispecific scFv-Fc fusion protein specific for human Her3 and CD3 Based on the molecule described as Example 3a, another bispecific scFv-Fc fusion protein was generated containing scFv portions against CD3 and human Her3 (human epidermal growth factor receptor 3, also called ErbB3) fused to the N-terminus of the hinge-containing chain of Fc1k (Her3 N -CD3 N -hFc1k) (Figure 28).

[0216] Two pSecTagAL1 vectors, each encoding one of the two polypeptide chains, were transiently transfected using polyethyleneimine as a transfection reagent, followed by transfection of Her3 cells. N -CD3 N-hFc1k was produced in HEK293-6E cells. After purification using Protein A affinity chromatography followed by preparative SEC, SDS-PAGE analysis revealed two bands under reducing conditions and one dominant band under non-reducing conditions, both representing the calculated molecular weight (Figure 29b). The minor bands observed under both conditions may represent partial differences in the glycosylation state of the expressed protein. Furthermore, Her3 N -CD3 N -hFc1k showed one single peak in analytical SEC (Figure 29c). N -CD3 N Binding of -hFc1k to human TNFR1-Fc fusion protein was analyzed by ELISA and showed an EC 50 The values ​​were revealed (Figure 29d, Table 4). The production and characterization of this fusion protein is still in progress.

[0217] Example 12: Bispecific scFv-Fc fusion protein specific for human MCSP and CD3 Based on the molecule described as Example 3a, another bispecific scFv-Fc fusion protein was generated containing an scFv portion against CD3 and human MCSP (melanoma-associated chondroitin sulfate proteoglycan) fused to the N-terminus of the hinge-containing chain of Fc1k (MCSP N -CD3 N -hFc1k) (Figure 30).

[0218] Two pSecTagAL1 vectors, each encoding one of the two polypeptide chains, were transiently transfected using polyethyleneimine as a transfection reagent, followed by MCSP N -CD3 N-hFc1k was produced in HEK293-6E cells. After purification using Protein A affinity chromatography followed by preparative SEC, SDS-PAGE analysis revealed two bands under reducing conditions and one dominant band under non-reducing conditions, both representing the calculated molecular weight (Figure 31b). The minor bands observed under both conditions may represent partial differences in the glycosylation state of the expressed protein. Furthermore, MCSP N -CD3 N -hFc1k showed one single peak in analytical SEC (Fig. 31c). Using MCSP-expressing WM35 cells, the MCSP activity was measured as reflected by its ability to reduce target cell viability in the presence of peripheral blood mononuclear cells (PBMC). N -CD3 N - Determine the biological activity of hFc1k and find an IC of 0.8 nM 50 (Figure 31d, Table 4). Production and characterization of this fusion protein is still in progress.

[0219] Example 13: Bispecific scFv-Fc fusion proteins specific for Her3 and CD3 in reverse orientation Based on the molecule described as Example 4a, another bispecific scFv-Fc fusion protein was generated containing scFv moieties against CD3 and Her3 (human epidermal growth factor receptor 3, also known as ErbB3) fused to the N-terminus of the hinge-containing CH2-CH31 domain and the C-terminus of the CH2-CH3k chain of Fc1k, respectively (Her3 N -CD3 C -hFc1k) (Figure 32).

[0220] Two pSecTagAL1 vectors, each encoding one of the two polypeptide chains, were transiently transfected using polyethyleneimine as a transfection reagent, followed by transfection of Her3 cells. N -CD3 C-hFc1k was produced in HEK293-6E cells. After purification using Protein A affinity chromatography followed by preparative SEC, SDS-PAGE analysis revealed two bands under reducing conditions and one dominant band under non-reducing conditions, both of which represent the calculated molecular weight (Figure 33b). The minor band observed under both conditions may represent a partial difference in the glycosylation state of the expressed protein. Immobilization of Her3 to immobilized Her3-Fc fusion protein N -CD3 C -hFc1k binding was analyzed by ELISA and had an EC 50 The results demonstrated significant activity (Figure 33c, Table 4). The production and characterization of this fusion protein is still in progress.

[0221] Table 4: Functional data for Examples 10-13 TIFF0007822587000005.tif69154

[0222] Table 5: Linker mutants used in Examples 10 to 13 TIFF0007822587000006.tif120170 The subscript (N / C) indicates the position of the scFv relative to the Fc1k portion (N-terminus / C-terminus).

[0223] Example 14: Further characterization of Fv13.7X-Fc1k The Fab-like monovalent molecule described in Example 8 was expressed in CHO cells from a cell pool after stable lentiviral transfection by Catalent Pharma Solutions (Somerset, Ebbing, NJ, USA). The protein was purified primarily using Protein A, and the monomer fraction was further separated by FPLC-SEC. Fv13.7 XThe final preparation of Fv13.7 revealed a single peak on analytical HPLC-SEC (Figure 35a), corresponding to the calculated molecular weight of 72 kDa. On SDS-PAGE under reducing conditions, two bands were observed that migrated similarly to the reference bands at 35 kDa and 40 kDa, corresponding well to the calculated molecular weights of the individual chains of 35 kDa and 37 kDa. Under non-reducing conditions, the single band observed migrated similarly to the 70 kDa reference band, indicating correct formation of the interchain disulfide bond. X -Fc1k revealed an aggregation temperature of 64 °C as determined by dynamic light scattering and visual interpretation of the detected average count rate (Figure 35c). X -Fc1k was stable in human plasma at 37°C for at least 7 days, as indicated by retained binding activity determined by huTNFR1-Fc binding ELISA (Fig. 35d).

[0224] Fv13.7 X -Fc1k bound to human TNFR1-Fc with an EC50 value of 0.37 nM in ELISA (Figure 36a), demonstrating reduced binding compared to the control proteins ATROSAB and Fab13.7, which showed EC50 values ​​of 0.09 nM and 0.17 nM, respectively. Real-time binding analysis using a QCM (Attana, Stockholm, Sweden) showed that Fv13.7 X -Fc1k bound to human TNFR1-Fc with a KD value of 2.66 nM (Fig. 36b), which was accompanied by a koff value of 9.83 x 10 4 s-1 and a kon value of 3.69 x 10 5 M-1 s-1. X -Fc1k contains modifications within the Fc portion that reduce its propensity to mediate ADCP, ADCC, and CDC (Armour et al. (1999) Eur J Immunol. 29(8):2613-24, Shields et al. (2001) J Biol Chem. 276(9):6591-604). Consistently, Fv13.7 XBinding of human Fcγ receptors I, IIb, and III to Fc1k and binding of the human complement protein C1q were significantly reduced compared to the control antibody retuximab, which contains the wild-type Fc portion (Figure 36c). Similarly, FcγRI, IIb, and III, and C1q showed reduced binding to the similarly mutated antibody ATROSAB, demonstrating reduced mediation of ADCC and CDC in previously published experiments (Richter et al. (2013) PLoS One 8(8):e72156).

[0225] Fv13.7, as well as the monovalent control protein Fab13.7 X Fv13.7-Fc1k showed no signs of TNFR1 activation itself in IL-6 and IL-8 release experiments and cell death induction assays using HeLa, HT1080, and Kym-1 cells, respectively (Fig. 37a-c). This was in stark contrast to the control protein ATROSAB, which induced only a slight cellular response in IL-6 and IL-8 release experiments at concentrations of 1-100 nM (Fig. 37a and b). Furthermore, Fv13.7-Fc1k showed no signs of TNFR1 activation itself in IL-6 and IL-8 release experiments and cell death induction assays using HeLa, HT1080, and Kym-1 cells, respectively (Fig. 37a-c). X Fv13.7 demonstrated potent inhibition of TNF-mediated TNFR1 activation in IL-6 and IL-8 release assays and cell death induction assays (Figures 37d-f), with IC50 values ​​of 54.5 nM, 24.2 nM, and 16.2 nM, respectively. However, these values ​​revealed slightly weaker biological activity compared to Fab13.7, which was determined with IC50 values ​​of 31.7 nM, 12.7 nM, and 9.5 nM in IL-6 and IL-8 release experiments and cell death induction assays using HeLa, HT1080, and Kym-1 cells, respectively. Notably, Fv13.7 nevertheless demonstrated a significantly weaker biological activity compared to Fab13.7, which was determined with IC50 values ​​of 31.7 nM, 12.7 nM, and 9.5 nM in IL-6 and IL-8 release experiments and cell death induction assays using HeLa, HT1080, and Kym-1 cells, respectively. X The inhibitory activity of -Fc1k was clearly improved when compared with the bivalent control protein ATROSAB, which showed IC50 values ​​of 164.7 nM, 84.1 nM, and 64.4 nM in IL-6 and IL-8 release experiments and cell death induction assays.

[0226] Table 6: Biological activity of Fv13.7X-Fc1k TIFF0007822587000007.tif26157

[0227] Fv13.7 under antibody-mediated cross-linking conditions X -Fv13.7 to further evaluate the biological activity of Fc1k X Fv13.7-Fc1k was analyzed in IL-8 release experiments in the presence of a fixed concentration of drug-specific antibody (Fig. 38a-c). In contrast to the control antibody ATROSAB, Fv13.7 X -Fc1k and the corresponding Fab13.7 revealed a complete lack of agonist activity in an IL-8 release assay using three different goat anti-human IgG serum preparations (Figure 38a: SouthernBiotech Cat.:2010-01 [IgG_A], Figure 38b: MyBioSource Cat.:MBS571163 [IgG_B], Figure 38c: MyBioSource Cat.:MBS571678 [IgG_C]). In conclusion, these results demonstrate that Fv13.7 activates TNFR1 in vivo under conditions of an anti-drug immune response. X -May indicate a decreased risk of Fc1k.

[0228] Finally, Fv13.7 X -Fc1k revealed early and terminal half-lives of 2.2 ± 1.2 h and 41.8 ± 18.1 h, respectively, and an area under the curve of 5856 ± 1370 μg / ml*h after a single injection of 400 μg (20 mg / kg) in C56BL / 6J knock-in mice carrying the gene for the extracellular domain of human TNFR1 at each locus in the mouse.

[0229] material: Horseradish peroxidase (HRP)-conjugated anti-human IgG (Fab-specific) antibody was purchased from Sigma (Taufkirchen, Germany). HT1080wt cells were grown in RPMI 1640 medium, 5% FCS, and 2 mM L-glutamine. ATROSAB and human TNFR1-Fc fusion were provided by Baliopharm AG (Basel, Switzerland). Chemicals were purchased from Roth (Karlsruhe, Germany), and enzymes (cloning and PCR) and auxiliary reagents were purchased from ThermoFisher (Munich, Germany). The different sources of consumables are clearly stated below.

[0230] method: Heterodimerization and alignment of functional Ig and Ig-like domains 1) Multiple sequence alignment was performed using the Clustal Omega online tool.

[0231] 2) The beta sheet definitions in the pdb file are highlighted in the sequence.

[0232] 3) When no secondary structure beta sheets were assigned or not all secondary structure beta sheets were assigned (indicated by an asterisk in Figure 4 ), secondary structure prediction by JPred (longest cumulative prediction using default settings) was performed.

[0233] 4) The start and end positions of beta sheets A-G were aligned according to additional structural alignments from the PDB files by including unassigned residues or excluding previously assigned residues from the sheets, deleting gaps introduced by multiple sequence alignment, or inserting new gaps outside the beta sheets with four exceptions (d_CH3: position 111 of IgLCRC; m_CH1: position 72 of IgLCRC; HLAA / HLAB: position 60 of IgLCRC, Figure 4). Inserted / extended or deleted / shortened gap positions were compensated by deleting or inserting the gap position next to the existing gap introduced during the multiple sequence alignment, respectively, to maintain sequence alignment of the C-terminal region.

[0234] 5) Beta sheet A was defined by these measures as the six residues following the conserved proline residue at the N-terminus of the first predicted beta sheet (positions 13–18 in IgLCRC).

[0235] 6) Beta sheet B was defined as the four N-terminal and five C-terminal residues adjacent to a conserved cysteine ​​residue contained in the second predicted beta sheet (positions 31–40 in IgLCRC).

[0236] 7) Beta sheet C was defined as the four N-terminal and two C-terminal residues adjacent to a conserved tryptophan residue included in the third predicted beta sheet (positions 46–52 in IgL CRC). Exceptions were the TCR alpha chain and beta2 microglobulin. In these cases, alignments were performed to predict the beta sheet and were confirmed during structural alignment using pyMol.

[0237] 8) Beta sheet D was defined from positions 63–70 of IgLCRC, but connections to residues conserved throughout the alignment were not feasible.

[0238] 9) Beta sheet E was defined as the eight residues from positions 81–89 of IgLCRC, starting from the conserved tyrosine / phenylalanine residue and located at the start or N-terminus of the fourth predicted beta sheet.

[0239] 10) Beta sheet F was defined as the two N-terminal and four C-terminal residues adjacent to a conserved cysteine ​​residue in the sixth predicted beta sheet (positions 102–108 of IgLCRC).

[0240] 11) Beta sheet G was defined from positions 128–133 of IgLCRC, but connections to residues conserved throughout the alignment were not feasible.

[0241] Expression of Fab13.7, Fv13.7-Fc1k, scFv13.7-Fc1k, scFv13.7-Fc: 4 mM GlutaMAX-I and 0.1% Kolliphor P188 (F17 ++ HEK293-6E cells were cultured in suspension in exponential growth conditions in FreeStyle F17 medium containing 1 μg / ml of plasmid DNA (final concentration) and 3 μg / ml of polyethyleneimine (PEI, final concentration) to prepare a 1.5*10^6 cell / ml transfection medium. DNA and PEI were each added in 1 ml of F17 per 20 ml of cell suspension. ++ The DNA mixture was diluted with culture medium and then mixed together. After 15–30 minutes of incubation at room temperature, the DNA mixture was added to the cells and incubated overnight with shaking at 37°C, 5% CO2. After 24 hours, 0.5 ml of tryptone N1 was added per 20 ml of cell suspension. After an additional 4 days of incubation at 37°C, 5% CO2, protein was purified from the supernatant.

[0242] Protein Purification - Antibody and Protein A Affinity Chromatography: The HEK293-6E cells were removed from the culture supernatant by centrifugation (step 1: 500 g, 15 minutes; step 2: 5000 g, 5 minutes).R The samples were incubated overnight with either AFrProtein A-650F (Protein A resin, 22805, Tosoh, Stuttgart, Germany) or HiTrap KappaSelect (kappa chain-selective antibody fragments bound to an agarose matrix, 17-5458-12, GE Healthcare, Chalfont St Giles, GB) with rotation. The resin was collected by centrifugation and transferred to Poly-Prep™ by gravity flow. R The resin was loaded onto a chromatography column, washed with PBS, and the protein was eluted from the resin with 100 mM glycine at pH 2–3. The eluted fractions were directly pooled and immediately dialyzed against PBS.

[0243] Preparative Size Exclusion Chromatography: In the case of aggregated or multimeric protein in the preparation, an additional size exclusion step was performed using an Äkta purifier. Proteins were separated on a Superdex200 10 / 300GL column at a flow rate of 0.5 ml / min using PBS as the liquid phase. 200 μl fractions were collected, and the peak containing the sample was pooled for further analysis.

[0244] Protein characterization - polyacrylamide gel electrophoresis (SDS-PAGE): SDS-PAGE was performed strictly according to Laemmli 1970 using 3 μg of protein preparation and stacking and separating gels of the indicated proportions.

[0245] Protein characterization - Size Exclusion Chromatography (SEC): To determine the hydrodynamic radius, 30 μg of purified protein samples were analyzed using a Waters 2695 HPLC coupled to a Phenomenex Yarra SEC-2000 column (300 x 7.8 mm, flow rate 0.5 ml / min). The mobile phase was 0.1 M NaHPO / NaHPO, 0.1 M NaSO, pH 6.7. The following standard proteins were used: thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), and FLAG peptide (1 kDa).

[0246] Enzyme-linked immunosorbent assay (ELISA): Microtiter plates were coated with 100 μl of TNFFR1-Fc fusion protein (1 μg / ml in PBS) and incubated overnight at 4°C. Remaining binding sites were blocked with 2% MPBS (skim milk in PBS, 200 μl per well) for 2 hours at room temperature, followed by two washes with PBS. 100 μl of sample diluted in 2% MPBS was incubated for 1 hour at room temperature before a final incubation step with 100 μl of HRP-conjugated detection antibody in 2% MPBS. Bound proteins were detected with 100 μl of TMB substrate solution, the HRP reaction was stopped by adding 50 μl of 1 M H2SO4, and absorbance at 450 nm was measured using an Infinite microtiter plate reader (TECAN, Maennedorf, Switzerland). Between each incubation step and before detection, plates were washed three times with PBST and twice with PBS.

[0247] Flow cytometry: Cells were detached and transferred to a 96-well microtiter plate in 100 μl of PBA (2% FCS, 0.2% NaN3 in sterile PBS) at a concentration of 100,000-250,000 per well. Samples were diluted 2-fold with PBA to the final desired concentration, and 100 μl was added to the cells for 1 hour of incubation. Afterwards, a MACSQuant™ assay was performed. RThey were incubated with antibodies conjugated to fluorescent dyes before detection using an Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were washed twice by centrifugation (500 g, 5 min) and resuspended in 150 μl of PBA after each incubation step.

[0248] Interleukin release assay: 2x10 per well 4 HeLa or HT1080 cells were seeded into 96-well microtiter plates and grown overnight in 100 μl of RPMI 1640 + 5% FCS. The next day, the supernatant was replaced to remove constitutively produced cytokines. Cells were incubated with serial dilutions of the samples in RPMI 1640 + 5% FCS at 37°C and 5% CO2. For competition experiments, both analyzed protein samples were prepared individually (titrated or diluted to a single concentration) and then added to the plate. Unstimulated cells served as a control. After 16–20 h, the plates were centrifuged at 500 g for 5 min, and the cell supernatants were analyzed directly by ELISA, according to the manufacturer's protocol. The supernatants were diluted in RPMI 1640 (without FCS), and the antibodies were diluted in reagent diluent (0.1% BSA, 0.05% Tween 20, 20 mM TRIS, 150 mM NaCl, pH 7.5). The coated microtiter plates were blocked with 1% BSA (bovine serum albumin) in PBS, and washing and detection and measurement were performed as described above for ELISA. Sandwich ELISA kits for detecting IL-6 and IL-8 in cell culture supernatants were purchased from ImmunoTools (Friesoythe, Germany).

[0249] Cytotoxicity / Cell Viability Assay: Cells (2 x 10 per well) 4 The cells were seeded into a 96-well microtiter plate and incubated overnight at 37°C and 5% CO2. Protein was diluted in RPMI1640 + 10% FCS and diluted 2 x 10 5The cells were combined with PBMCs / well and added to the plate. The cytotoxicity assay was incubated at 37°C and 5% CO2 for 3–5 days before the supernatant was discarded and 50 μl of crystal violet solution was added to the cells. The plate was then washed 20 times with ddH2O and allowed to dry. The remaining violet dye, resulting from live and adherent cells fixed by the methanol in the staining solution, was dissolved by adding 100 μl of methanol with shaking for 10 min at room temperature. The plate was read using an Infinite microtiterplate reader (Tecan, Maennedorf, Switzerland).

[0250] Pharmacokinetics: Transgenic C57BL / 6J mice carrying the gene for the extracellular domain of human TNFR-1 at a specific mouse locus (C57BL / 6J-huTNFRSF1Aecdtm1UEG / izi) were intravenously injected with 25 μg of the analyzed protein. Blood samples were collected at 3 min, 30 min, 1 h, 3 h, and 6 h, and at 3 and 7 days and immediately incubated on ice. Serum was separated by centrifugation (13,000 g, 4°C, 10 min) and stored at -20°C. Residual protein in the serum was detected by binding ELISA as described above. ELISA signals were interpolated from freshly prepared standard binding curves of the analyzed proteins. Determined concentrations were plotted against time, and pharmacokinetic constants were obtained using the PKsolver add-in for Microsoft Excel.

Claims

1. A protein complex comprising at least two amino acid chains I and II, wherein the amino acid chains I and II are non-covalently bound to each other via a heterodimerization region I (hereinafter referred to as "HRI") contained in the amino acid chain I and a heterodimerization region II (hereinafter referred to as "HRII") contained in the amino acid chain II, wherein the HRI and HRII consist of the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 21, or heterodimerization variants thereof having at least 97% sequence identity to the amino acid sequences shown.

2. The protein complex of claim 1, wherein amino acid chain I and / or amino acid chain II further comprises one or more amino acid elements selected from the group consisting of an antibody CH2 or CH3 domain; one or more antigen-specific ligands (ASL); an antibody hinge region (HR), one or more linker sequences (L), one or more cytokines (C), interleukins (IL), interferons, growth factors, hormones, ligands, peptides, receptor fragments with ligand-binding activity, chelators, enzymes, coagulation factors and anticoagulants, and derivatives thereof.

3. The protein complex of claim 2, wherein the one or more antigen-specific ligands (ASLs) are selected from the group consisting of Fv, single-chain Fv (scFv), disulfide-stabilized Fv, disulfide-stabilized scFv, Fab, single-chain Fab, single-domain antibody, variable heavy domain (VH), variable light domain (VL), T-cell receptor or antigen-binding fragment thereof, nanobody, VHH, and one or more antibody-like binding proteins.

4. The protein complex of claim 1, wherein amino acid chain I further comprises one or more antigen-specific ligands (ASLs) and / or one or more effector molecules, and amino acid chain II further comprises one or more antigen-specific ligands (ASLs) and / or one or more effector molecules, wherein the ASL modules are selected from the group consisting of molecules that specifically bind to cell surface proteins, hormones, growth factors, cytokines, ligands, serum proteins, coagulation factors, fibrinolytic factors, chemokines, and enzymes, and wherein the effector molecules are selected from the group consisting of cell surface proteins, hormones, growth factors, cytokines, ligands, serum proteins, coagulation factors, fibrinolytic factors, chemokines, and enzymes.

5. A protein complex described in any one of claims 1 to 4, wherein HRI and HRII consist of the amino acid sequences of SEQ ID NOs: 20 and 21.

6. Combinations of amino acid chain I and amino acid chain II, wherein amino acid chain I and amino acid chain II consist of the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 42, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 36 and SEQ ID NO: 37 (scFv13.7-Fc1k), SEQ ID NO: 36 and SEQ ID NO: 38 (scFv13.7-CD3-hinge-Fc1k), SEQ ID NO: 39 and SEQ ID NO: 38 (scFv3-43-CD3-hinge-Fc1k), or SEQ ID NO: 40 and SEQ ID NO: 38 (scFvhuMCSP-CD3-hinge-Fc1k).

7. A nucleic acid encoding the amino acid chain I and / or the amino acid chain II according to claim 6.

8. A vector comprising the nucleic acid of claim 7.

9. A method for producing an amino acid chain I comprising the HRI sequence according to any one of claims 1 to 6 and / or an amino acid chain II comprising the HRII sequence according to any one of claims 1 to 6, the method comprising the steps of introducing a nucleic acid encoding the amino acid chain I and / or the amino acid chain II into a host cell, and then expressing the amino acid chain I and / or the amino acid chain II.

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