Novel antigen-binding chimeric proteins and methods and uses thereof

Abstract

The present invention relates to the field of structural biology. More specifically, the present invention relates to novel antigen-binding chimeric proteins and their use and methods in macromolecular three-dimensional structural analysis, such as X-ray crystallography and high-resolution cryo-EM, and their use as therapeutic, diagnostic, or imaging tools. Even more specifically, the present invention relates to fusions of a scaffold protein and an antigen-binding domain, where the scaffold protein of the fusion disrupts the immunoglobulin domain topology to form a rigid chimera.

Description

[Technical Field]

[0001] The present invention relates to the field of structural biology. More specifically, the present invention relates to novel antigen-binding chimeric proteins and their uses and methods in macromolecular three-dimensional structural analysis, such as X-ray crystallography and high-resolution cryo-EM, and their use as therapeutic, diagnostic, or imaging tools. Even more specifically, the present invention relates to fusions of a scaffold protein and an antigen-binding domain, in which the scaffold protein disrupts the immunoglobulin domain topology to form a rigid chimera that maintains its antigen-binding ability. [Background technology]

[0002] Proteins and their complexes play crucial roles in all aspects of life, yet solving the three-dimensional structures of many of these macromolecular components remains challenging. The generation of diffraction-quality crystals remains a major obstacle in macromolecular X-ray crystallography. One promising approach is the use of crystallization chaperones (Hunte & Michel, 2002). These crystallization chaperones are developed as antibody fragments or other proteins engineered to specifically bind to a given macromolecular target. The basis of this strategy is, first, to minimize conformational heterogeneity in the target by binding to a specific conformation, and, second, to increase the probability of obtaining well-ordered crystals by adding additional protein surface that can facilitate primary intermolecular contacts within the crystal lattice. Another unique attribute of the crystallization chaperone approach is that chaperones can provide initial phasing information based on models (Koide, 2009). Nanobodies® are widely used to stabilize specific conformations and thus facilitate structural analysis, especially crystallography, of their targets (Pardon et al., 2014; Rostislavleva et al., 2015). One example is the use of powerful nanobody® technology in the context of GPCR crystallization, demonstrating the potential of these tools to advance structural studies (Manglick et al., 2017; Staus et al., 2016).

[0003] However, X-ray crystallography has several inherent drawbacks, including the necessity of high-quality purified proteins, the need for relatively large amounts of protein, and the difficulty of obtaining large numbers of protein crystals of suitable quality for diffraction. Single-particle cryo-electron microscopy (cryo-EM) has recently been developed as a versatile alternative technique for structural analysis of macromolecular complexes at atomic resolution (Nogales, 2016). While data analysis instruments and methods are steadily improving, tools for resolving small, less symmetric, and more flexible particles to high resolution are lacking. In addition to the essential homogeneity of a given sample, the highest achievable resolution of three-dimensional reconstructions relies heavily on the ability to repeatedly refine the orientation parameters of individual particles with high accuracy. Preferential particle orientation due to surface properties of polymers that preferentially attach certain regions to the air-water interface or to the substrate support is a recurring issue in cryo-EM. Thus, while it is relatively easy to recognize large molecules in noisy, low-dose images of frozen-hydrated samples, and these particles possess sufficient structural features to facilitate accurate determination of their orientation parameters (Henderson, 1995), acquiring and processing images of small particles is significantly more challenging. One fundamental problem is that image signal-to-noise ratios decrease with particle size, primarily because images of small proteins or complexes embedded in vitreous ice do not contain sufficient features for accurate image alignment. Attempts to address these issues have been made by creating fusion proteins or adding chemical linkers to these proteins. For example, optimized junctions of glutamine synthetase that form multimers with target proteins as homo-oligomers to achieve symmetry have been used, enabling nanometer-level resolution of these small target proteins by cryo-EM (Coscia et al., 2016). The problem with such fusions is that they are created using flexible linkers, which lack rigidity and limit conformational uniformity. A more rigid fusion was reported by Zhang et al. (2015), in which a humanized antibody was fused with a cytokine to form a humanized agonist.These rigid fusions have been created by using a coiled-coil "stalk" motif based on the bovine Fab structural motif to ensure proper folding. Linking cytokines to such stalk motifs in fusions with antibody complementarity-determining regions (CDRs) appears to reduce the antigen-binding capacity of the antibody but maintain the cytokine's folding and biological activity. Antibody fragments, such as Fabs (~50 kDa), have been utilized to elucidate the structures of small proteins by cryo-EM (Wu et al., 2012; Lin et al., 2013). One drawback of using Fabs is that while they can also bind to linear epitopes, they are known to exhibit reduced rigidity. Therefore, proper selection of Fabs is necessary to achieve rigid binding and facilitate structural analysis. Furthermore, Fabs have internal flexibility (between the variable and constant regions), are still relatively small, and are more difficult to engineer than nanobodies. Although nanobodies are an excellent tool for reducing conformational heterogeneity for structural analysis, nanobodies (15 kDa) are still small proteins, do not add a large amount of protein surface to facilitate primary intermolecular contacts within the crystal lattice, and are so small that they are still not suitable for the size-related requirements necessary for high-resolution cryo-EM of small proteins. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hunte & Michel, 2002 [Non-patent document 2] Koide, 2009 [Non-patent document 3] Pardon et al., 2014 [Non-patent document 4] Rostislavleva et al., 2015 [Non-Patent Document 5] Manglick et al., 2017 [Non-patent document 6] Staus et al., 2016 [Non-Patent Document 7] Nogales, 2016 [Non-patent document 8] Henderson, 1995 [Non-Patent Document 9] Coscia et al., 2016 [Non-Patent Document 10] Zhang et al. (2015) [Non-Patent Document 11] Wu et al., 2012 [Non-Patent Document 12] Lin et al., 2013 Summary of the Invention [Problem to be solved by the invention]

[0005] Accurate structural analysis by X-ray crystallography or cryo-EM requires a generalized prototyping solution for rigid chaperones. Therefore, designing novel chimeras fused via rigid methods, rather than via flexible linkers, would be advantageous for constructing large, conformationally stable protein structures. In summary, there is a clear need for next-generation chaperones that enable structural analysis of small proteins, protein complexes, or protein-protein interactions by crystallization or single-particle analysis, particularly cryo-EM. It would be advantageous to design and generate such chaperones in a generalized manner to reduce the conformational flexibility of targets. First, such chaperones would be useful as auxiliary tools for adding mass and / or specific features to targets to improve resolution for obtaining high-resolution structures. Consequently, they would be advantageous for structure-based drug design and aid in the discovery and development of novel compounds. Second, such chaperones would be useful for providing novel types of drugs for biophysical, medical, and crop protection applications, including diagnostic or therapeutic products that utilize enhanced rigidity. [Means for solving the problem]

[0006] The present invention relates to the design and generation of novel functional antigen-binding chimeric proteins and their uses, such as their role as next-generation chaperones in structural analysis and their use as therapeutic and diagnostic molecules. In particular, immunoglobulin or immunoglobulin-like domains have advantageous functional characteristics, such as antigen binding and specific binding to conformational epitopes on targets, and an advantageous structural feature, such as the ability to fused scaffold proteins to immunoglobulin domains in a versatile manner to disrupt the topology of the immunoglobulin domain without compromising its folding or functionality. By combining these features, antigen-binding chimeric proteins comprising an immunoglobulin or immunoglobulin-like domain and a scaffold protein have been designed. Such antigen-binding chimeric proteins are obtained by expressing a genetic fusion of the antigen-binding domain and the scaffold protein, and are designed so that the scaffold or a fragment thereof is inserted within the topology of the antigen-binding domain. The resulting novel antigen-binding chimeric proteins are characterized by high rigidity in their fusion regions and surprisingly maintain their typical fold and functionality, i.e., high binding affinity for their antigens or target proteins. Indeed, genetic fusions between antigen-binding domains and scaffold proteins do not result in fluctuations or changes in the complementarity-determining region (CDR) structure, which is the antigen-binding site. While designing a fusion with a scaffold protein to be rigid and inflexible is not straightforward, the present invention provides a novel and unique type of antigen-binding chimeric protein by placing perfectly selected sites in the exposed interior region of the antigen-binding domain, making this possible. As a result, antigen-binding chimeric proteins add mass and structural features, making them a novel tool for facilitating the structural analysis of small proteins by high-resolution cryo-EM and X-ray crystallography. Indeed, atomic-level characterization of primarily large- to medium-sized macromolecular assemblies from homogeneous samples by cryo-EM has not yet been possible.Thus, the antigen-binding chimeric proteins of the present invention may increase particle size and help address preferential particle orientation (depending on the scaffold), allowing for better fragment alignment, thereby lowering the size limit and improving resolution. Therefore, by designing and creating these next-generation chaperones for the structural analysis of any conceivable antigen or target, it becomes possible to apply the antigen-binding chimeric proteins as an auxiliary tool for adding mass and / or specific features to the target of interest in order to improve resolution to obtain high-resolution structures. Indeed, antigen-binding chimeric proteins are therefore advantageous as tools not only for structural analysis but also for structure-based drug design and screening, adding value to the discovery and development of novel biologics and small molecule drugs. Furthermore, the creation of such rigid fusions may lead to novel applications, such as the use of antigen-binding chimeras as active ingredients in medicine as novel rigid fusion therapeutic molecules or as protectants in crop protection, depending on the type of scaffold, e.g., a scaffold comprising the antigen-binding or Ig domain itself. When antigen-binding chimeras are generated using labeled scaffolds or labeled antigen-binding domains, the invention also encompasses novel diagnostic tools to accelerate technological improvements in in vivo imaging, e.g., by non-covalent probing of targets.

[0007] A first aspect of the present invention relates to an antigen-binding chimeric or fusion protein in which an antigen-binding domain is linked to a scaffold or fusion partner protein, where the scaffold protein is linked to the antigen-binding domain at one or more amino acid sites that are accessible or exposed on the surface of the domain, thereby disrupting the topology of the antigen-binding domain. The antigen-binding chimeric protein is further characterized in that it maintains its antigen-binding functionality compared to an antigen-binding domain that is not fused to the scaffold protein. Another embodiment discloses an antigen-binding chimeric protein of the invention in which the fusion of the antigen-binding domain with the scaffold protein disrupts the primary topology of the antigen-binding domain, thereby maintaining the folding of the antigen-binding domain compared to the folding of the antigen-binding protein when not fused to another protein.

[0008] In a specific embodiment of the present invention, the fusion can be direct fusion or fusion via a linker or linker peptide, and the fusion site is perfectly designed to obtain a rigid and inflexible fusion protein. The linker preferably has 10, 9, 8, 7, 6, 5, 4, or 3 amino acid residues, more preferably 2, and even more preferably 1, or is a direct fusion (no linker). In another embodiment, the antigen-binding chimeric protein is fused at at least one accessible site in an exposed region, preferably a beta turn (β turn) or loop, of the antigen-binding domain. The antigen-binding chimeric protein in which a scaffold protein is linked to the antigen-binding domain at one or more accessible or exposed sites on the surface of the antigen-binding domain is further characterized in that the accessible or exposed site is other than an antigen-binding loop or a CDR loop, in order to maintain its antigen-binding functionality. In one embodiment, the antigen-binding chimeric protein comprises an antigen-binding domain consisting of at least seven antiparallel β-strands and at least three β-turns connecting the β-strands, as defined according to the IMGT® International Reference Nomenclature System (Lefranc, 2014; Figure 25). In a specific embodiment, the antigen-binding domain of the antigen-binding chimeric protein comprises an immunoglobulin (Ig) domain or an Ig fold. In a specific embodiment, the Ig domain of the antigen-binding chimeric protein is derived from a VHH, or more preferably from an immunoglobulin single variable domain (ISVD) or a nanobody.

[0009] In a specific embodiment, the exposed region of the antigen-binding domain of the antigen-binding chimeric protein specifically corresponds to β-turn AB, CC', C"D, DE, or EF according to the IMGT nomenclature (Figure 25, revised version based on LeFranc, 2014). Thus, a scaffold protein is inserted inside the antigen-binding domain into the first β-turn connecting β-strand A and β-strand B of the antigen-binding domain, or the β-turn connecting β-strand C and β-strand C' of the antigen-binding domain, or the β-turn connecting β-strand C" and β-strand D of the antigen-binding domain, or the β-turn connecting β-strand D and β-strand E of the antigen-binding domain, or the β-turn connecting β-strand E and β-strand F of the antigen-binding domain (note that the β-turn is defined according to the revised IMGT based on LeFranc, 2014). In a specific embodiment, the antigen-binding chimeric protein is generated by fusing a scaffold protein to an accessible site in an exposed region of the antigen-binding domain, including the β-turn AB connecting β-strand A and β-strand B of the domain.

[0010] In another embodiment of the invention, the scaffold protein used to generate the antigen-binding chimeric protein is a circularly permuted protein, and more specifically, a circular permutation can be introduced between the N-terminus and C-terminus of the scaffold protein. In some embodiments, the circularly permuted scaffold protein is cleaved at another accessible site of the scaffold protein to provide a fusion site for an accessible site of an Ig domain.

[0011] Another embodiment relates to an antigen-binding chimeric protein in which the scaffold protein is a monomeric protein. In an alternative embodiment, the scaffold protein has a symmetric structure, such as a protein that forms a multimer or oligomer, or a protein that is part of or forms an icosahedral structure, such as a virus-like particle (VLP). In a specific embodiment, the antigen-binding chimeric protein has a multimeric scaffold, and an Ig domain is linked or fused to the scaffold protein at an accessible site via each of the monomers of the multimeric scaffold. In another embodiment, the antigen-binding domain and the scaffold protein of the antigen-binding chimeric protein are further linked via a disulfide bond covalently formed between two cysteine ​​residues present in either the antigen-binding domain or the scaffold protein. Another embodiment of the present invention relates to an antigen-binding chimeric protein in which the scaffold protein has a total molecular weight of at least 30 kDa.

[0012] A specific embodiment of the present invention relates to an antigen-binding chimeric protein in which a scaffold protein comprises an antigen-binding domain. In particular, the scaffold protein comprises an immunoglobulin domain, more particularly a VHH, ISVD, or nanobody. Alternatively, the scaffold protein comprises an immunoglobulin-like domain, more particularly a monobody. More specifically, an antigen-binding chimeric protein is provided in which the antigen-binding domain and the scaffold comprising the antigen-binding domain maintain functionality to specifically bind to their antigen target. In a specific embodiment, the scaffold comprising the antigen-binding domain binds to a target different from that of the antigen-binding domain fused to the scaffold protein. In an alternative embodiment, the antigen target of the antigen-binding domain of the scaffold protein is the same as the antigen target of the antigen-binding protein domain fused to the scaffold protein. More specifically, the antigen target is the same protein in both antigen-binding domains of the antigen-binding chimeric protein, but their epitopes on the antigen target are different.

[0013] In another embodiment, the scaffold protein of the antigen-binding chimeric protein is a labeled protein. In a specific embodiment, the label is a detectable label. In another embodiment, the antigen-binding domain of the antigen-binding chimeric protein is a labeled antigen-binding domain. More specifically, the label fused or linked to and / or provided by the antigen-binding domain or scaffold protein allows for in vivo and / or non-covalent detection or labeling of the antigen target of the antigen-binding domain of the novel antigen-binding chimeric protein. In a specific embodiment, the labeled antigen-binding chimeric protein may contain a toxic label and is applicable for therapeutic use.

[0014] Another aspect of the present invention relates to a nucleic acid molecule encoding any of the above-described chimeric antigen-binding proteins. Alternatively, in one embodiment, a chimeric gene is provided comprising at least a promoter, the nucleic acid molecule encoding the chimeric antigen-binding protein, and a 3'-terminal region comprising a transcription termination signal. Another embodiment relates to an expression cassette encoding the chimeric antigen-binding protein or comprising the nucleic acid molecule or chimeric gene encoding the chimeric antigen-binding protein. Another embodiment relates to a vector comprising the expression cassette or nucleic acid molecule encoding the chimeric antigen-binding protein of the present invention. In certain embodiments, the vector is suitable for expression in E. coli, or for yeast, phage, bacterial, or viral (surface) display. In another embodiment, a host cell comprising the chimeric antigen-binding protein of the present invention is disclosed. Alternatively, a host cell co-expressing the chimeric antigen-binding protein and its target antigen is disclosed.

[0015] Another aspect of the present invention relates to a complex comprising the above-described chimeric antigen-binding protein and its target protein, wherein the target protein specifically binds to the chimeric antigen-binding protein, more particularly to the antigen-binding domain of the chimeric antigen-binding protein, even more particularly to the Ig domain of the chimeric antigen-binding protein, or even more particularly to the CDRs of the Ig domain.

[0016] Another embodiment provides a composition comprising said chimeric antigen-binding protein, or more specifically a pharmaceutical composition of said chimeric antigen-binding protein.

[0017] Another embodiment of the present invention relates to a composition comprising a complex formed from a first antigen-binding chimeric protein of the present invention and a second antigen-binding chimeric protein, wherein the antigen-binding domain of the second antigen-binding chimeric protein specifically binds to or recognizes a scaffold protein of the first antigen-binding chimeric protein.

[0018] Another aspect relates to the use of the chimeric antigen-binding protein of the present invention, or the use of the nucleic acid molecule, chimeric gene, expression cassette, vector, complex, or composition for structural analysis of a target or antigen protein. In particular, the invention relates to the use of the chimeric antigen-binding protein when the target protein is a protein bound to the chimeric antigen-binding protein. Specifically, one embodiment relates to the use of the chimeric antigen-binding protein in structural analysis, including single-particle cryo-EM or crystallography.

[0019] Another embodiment provides the use of said antigen-binding chimeric protein, wherein the scaffold protein or the antigen-binding domain is labeled, as a diagnostic tool, more particularly for in vivo imaging.

[0020] An alternative embodiment provides said antigen-binding chimeric protein as described above or the nucleic acid molecule, expression cassette, vector, complex or composition provided herein for use in a pharmaceutical.

[0021] A specific embodiment relates to a virus-like particle (VLP) comprising the antigen-binding chimeric protein of the invention.

[0022] A final aspect of the present invention is a method for determining the three-dimensional structure of a target or antigen protein or a protein of interest, comprising: (i) providing a composition comprising an antigen-binding chimeric protein of the present invention or a complex of the antigen-binding chimeric protein of the present invention and a target protein, and forming a complex in which the target protein specifically binds to the antigen-binding chimeric protein or the composition of the antigen-binding chimeric protein; Alternatively, providing a composite of the present invention; (ii) displaying the mixture or complex under conditions suitable for structural analysis and determining the three-dimensional structure of the antigen or target protein at high resolution.

[0023] The following drawings are schematic and non-limiting, in which the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. [Brief explanation of the drawings]

[0024] [Figure 1-1] Comparison of flexible fusion proteins and rigid antigen-binding chimeric proteins. (A) Flexible fusion or linker fusions in which only one direct fusion or linker is used to fuse the antigen-binding domain to the N- or C-terminus of the scaffold protein. [Figure 1-2] Comparison of flexible fusion proteins and rigid antigen-binding chimeric proteins. (B) Rigid fusions of antigen-binding domains and scaffold proteins, in which the antigen-binding domain is fused to the scaffold protein via at least two direct fusions or linkers connecting the antigen-binding domain to the scaffold. [Figure 2]Engineering principles for antigen-binding chimeric proteins in which a circularly permuted variant of a scaffold protein is inserted into the first β-turn connecting β-strands A and B of the nanobody. This scheme shows how a nanobody (Nb) can be grafted onto a larger scaffold protein via two peptide linkers or two short linkers connecting the antigen-binding domain to the scaffold. The scissors mark indicates which exposed turns of the nanobody and scaffold should be incised. The dashed line indicates how the Nb and the rest of the scaffold should be concatenated using peptide bonds or short peptide linkers to create the antigen-binding chimeric protein. The CDRs, framework residues, and β-turn regions of the Nb are defined according to IMGT (Figure 25, revised version based on Lefranc, 2014). [Figure 3] Model of a 58 kD GFP-binding chimeric protein in which a circularly permuted HopQ mutant is inserted into the first β-turn connecting β-strands A and B of a GFP-specific nanobody. (A) Model of an antigen-binding chimeric protein generated by fusing a GFP-specific nanobody (top) with a circularly permuted HopQ adhesin domain (bottom) from Helicobacter pylori (H. pylori) via two peptide bonds or linkers connecting the nanobody to the scaffold. (B) A circularly permuted gene encoding the adhesin domain of H. pylori G27 type 1 HopQ (bottom, PDB5LP2, SEQ ID NO: 19, cHOPQ) was inserted into the first β-turn (β-turn AB) connecting β-strand A to β-strand B of a GFP-specific nanobody (top, SEQ ID NO: 1). (C) Amino acid sequence of the resulting antigen-binding chimeric protein (MbNb207 cHopQ, SEQ ID NO: 20). Nanobody-derived sequences are shown in bold. The sequence derived from HopQ is underlined. The peptide linking the N- and C-termini of HopQ to create the circular permutation is italicized. The C-terminal tag contains 6xHis and EPEA. [Figure 4]Flow cytometry analysis of the display of the antigen-binding chimeric protein MbNb207 cHopQ on the surface of EBY100 yeast cells. Single-parameter histograms show the relative fluorescence intensity of EBY100 yeast cells (top) transformed with a pCTCON2 derivative encoding the antigen-binding chimeric protein MbNb207 cHopQ (SEQ ID NO: 22) fused to Aga2p and ACP, compared to untransformed EBY100 yeast cells (bottom). Transformed and untransformed yeast cells were orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM). [Figure 5] Flow cytometry analysis of the functionality of the antigen-binding chimeric protein MbNb207 cHopQ displayed on the surface of EBY100 yeast cells. Dot plots show the relative fluorescence intensity of individual EBY100 yeast cells (top) transformed with pCTCON2 derivatives encoding MbNb207 cHopQ (SEQ ID NO: 22) fused to Aga2p and ACP compared to untransformed EBY100 yeast cells (bottom). Transformed and untransformed yeast cells were orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. [Figure 6]Size-exclusion chromatography and SDS-PAGE analysis of the complex formation between the antigen-binding chimeric protein MbNb207 cHopQ and GFP. Analytical gel filtration experiments were performed on a Superdex 75 PG column (16 / 90, GE Healthcare) to confirm the formation of a stable complex between MbNb207 cHopQ (SEQ ID NO: 20) and green fluorescent protein (GFP) (Scholz et al., 2000). (A) SEC analysis of complex formation. Gray line: Gel filtration pattern measured by UV absorption at 280 nm for a sample containing GFP alone. Black dashed line: Gel filtration pattern measured by UV absorption at 280 nm for a sample containing MbNb207 cHopQ and GFP at a 1:4 molar ratio. Black line: Gel filtration pattern measured by UV absorption at 488 nm for a sample containing MbNb207 cHopQ and GFP at a 1:4 molar ratio. (B) SDS-PAGE analysis of the stable complex formation between the antigen-binding chimeric protein MbNb207 cHopQ and GFP. A sample containing MbNb207 cHopQ and GFP at a molar ratio of 1:4 was applied to a Superdex 75 PG column, and the corresponding fractions were analyzed by SDS-PAGE. [Figure 7] X-ray crystal structure of a 58 kD GFP-binding chimeric protein in which a circularly permuted HopQ mutant is inserted into the first β-turn connecting β-strands A and B of a GFP-specific nanobody. (A) Arrangement of 10 GFP-binding chimeric proteins (illustrated) in the asymmetric unit. The 58 kD antigen-binding chimeric protein MbNb207 cHopQ (SEQ ID NO: 20) was crystallized in space group P1, with 10 molecules in the asymmetric unit. The adhesin domain of HopQ used as the scaffold (PDB5LP2) is shown in red, and the GFP-specific Nb is shown in green. (B) The peptide linking the nanobody to the scaffold (molecule A) is clearly depicted in a 2Fo-Fc electron density map displayed at 1.0 σ. (C) Structural alignment of all antigen-binding chimeric proteins MbNb207 cHopQ contained in the asymmetric unit. The RMSD between different molecules in the asymmetric unit is 0.3-2.7 Å. [Figure 8]Yeast display vectors for optimizing the composition and length of the linker peptide connecting the scaffold protein HopQ to the nanobody. (A) Schematic diagram of the display vector. LS: appS4, an artificial secretion signal of yeast α-factor that directs extracellular secretion in yeast (Rakestraw et al., 2009). N: β-strand A of the anti-GFP nanobody (residues 1-13 of SEQ ID NO: 1). Nanobody: β-strands B-G of the anti-GFP nanobody (residues 16-126 of SEQ ID NO: 1). Aga2: Aga2p adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond with the Aga1p protein (Chao et al., 2006). ACP: Acyl carrier protein for orthogonal labeling to monitor the expression level of the displayed antigen-binding chimeric protein (Johnsson et al., 2005). (B) Sequence diversity of the displayed antigen-binding chimeric proteins (SEQ ID NOs: 34-37). The AppS4 leader sequence is shown in standard typeface, the megabody cHopQNbGFP207 and random linker are shown in bold, (X)1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition, and the flexible (GGGS)n polypeptide linker is shown in italics. The Aga2p protein sequence is underlined, the ACP sequence is double underlined, and finally the cMyc tag is shown. (C) Four pools of antigen-binding chimeric protein sequences (each representing 25% of the library) encoding a total of 184,000 AA sequence variants were generated by introducing short peptide linkers of variable length (1 or 2 amino acids) and mixed composition using an equimolar mixture of two forward PCR primers (SEQ ID NO: 126, SEQ ID NO: 127) and two reverse PCR primers (SEQ ID NO: 128, SEQ ID NO: 129). [Figure 9]Model of a 100 kDa antigen-binding chimeric protein, constructed by in vitro selection, in which a circularly permuted YgjK mutant was inserted into the first exposed β-turn (connecting β-strands A and B) of a GFP-specific nanobody. (A) Model of an antigen-binding chimeric protein composed of a fusion of a GFP-specific nanobody (top) and Escherichia coli K12YgjK (bottom). (B) A circularly permuted gene encoding E. coli K12YgjK (PDB3W7S, SEQ ID NO: 38) was fused, and the YgjK protein was inserted into the first β-turn (β-turn AB) connecting β-strand A to β-strand B of a GFP-specific nanobody (top, SEQ ID NO: 1) using short peptide linkers of variable length (1 or 2 amino acids) and mixed composition. (C) Amino acid sequences of the 100 kDa antigen-binding chimeric proteins (SEQ ID NOs: 39-42). The NbGFP207 sequence is shown in bold, the circularly permuted linker is shown in italics, the Ygjk sequence is underlined, and (X)1-2, shown in wavy underline, are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition. The C-terminal tag contains 6xhis and EPEA. [Figure 10]A 58 kD antigen-binding chimeric protein was constructed by inserting a circularly permuted HopQ mutant into the first β-turn connecting β-strands A and B of a GFP-specific nanobody, further enhancing its rigidity with an artificial disulfide bond linking the nanobody to the scaffold. To further enhance the rigidity of an antigen-binding chimeric protein, the adhesin domain of H. pylori G27 type 1 HopQ (see below, PDB5LP2, SEQ ID NO: 19) was inserted into the first β-turn (AB β-turn) connecting β-strand A to β-strand B of a GFP-specific nanobody (SEQ ID NO: 1), site-directed mutagenesis was used to engineer an additional disulfide bond linking the nanobody to the scaffold. (A) Model of the mutant MbNb207 cHopQ (SEQ ID NO: 48) antigen-binding chimeric protein, which contains an artificial disulfide bridge between C357 and C425. (B) Model of a mutant of MbNb207 cHopQ (SEQ ID NO: 49) containing an artificial disulfide bridge between C358 and C488. (C) Model of a mutant of MbNb207 cHopQ (SEQ ID NO: 50) containing an artificial disulfide bridge between C359 and C490. (D) Model of a mutant of MbNb207 cHopQ (SEQ ID NO: 51) containing an artificial disulfide bridge between C15 and C534. [Figure 11]This is the engineering principle for antigen-binding chimeric proteins, in which a scaffold protein is inserted into the first exposed β-turn (connecting β-strands A and B) of a nanobody via three peptide bonds or a short linker, which connects the immunoglobulin to the scaffold. The antigen-binding domain can be fused to the scaffold protein via a three-point direct fusion or a three-point fusion via a linker at one or more accessible sites within the exposed region or β-turn, and / or at accessible sites at the end of the protein. In this scheme, the β-turn AB and the C-terminus of the nanobody are used to link the Ig domain to the scaffold via three peptide bonds or a short linker. The scissors mark indicates which exposed turns of the nanobody and scaffold should be incised. The dashed line indicates how the Nb and the rest of the scaffold should be concatenated using peptide bonds or short peptide linkers to generate such an antigen-binding chimeric protein. The CDRs, framework residues, and β-turn region of the Nb are defined according to IMGT (Figure 25, revised version based on Lefranc, 2014). [Figure 12] Model of an antigen-binding chimeric protein in which the Cu++-binding protein azurin (Azurin) is inserted into the first β-turn connecting β-strands A and B of a GFP-specific nanobody via three peptide bonds or a short linker linking the immunoglobulin to the scaffold. (A) Model of an antigen-binding chimeric protein composed of a fusion of a GFP-specific nanobody (top, SEQ ID NO: 1) and Pseudomonas aeruginosa azurin (bottom, PDB2TSA, SEQ ID NO: 52) via three peptide linkers. The copper ions are shown in blue. (B) The first β-strand of the nanobody (β-strand A), the N-terminal portion of the scaffold protein (azurin), the C-terminal portion of the immunoglobulin, and the C-terminal portion of the scaffold (azurin) are arranged in this order. (C) Amino acid sequences of the resulting antigen-binding chimeric proteins (SEQ ID NOs: 53-60). The NbGFP207 sequence is shown in bold, the azurin sequence is underlined, (X)1-2 is a short peptide linker of variable length (1 or 2 amino acids) and mixed composition, and the C-terminal tag contains 6xHis and EPEA. [Figure 13] Model of a dimeric antigen-binding chimeric protein with two-fold rotational symmetry, in which the inverting glycosidase scaffold protein SusB is inserted into the first exposed β-turn (connecting β-strands A and B) of a GFP-specific nanobody via three peptide bonds or linkers connecting the immunoglobulin to the scaffold. (A) Model of an antigen-binding chimeric protein composed of a fusion of a GFP-specific nanobody (top left and top right, SEQ ID NO: 1) and the inverting glycosidase of Bacteroides thetaiotaomicron SusB (bottom, PDB3WFA, SEQ ID NO: 69) via three peptide linkers. SusB is an obligate dimer with two-fold rotational symmetry. (B) The first β-strand of the nanobody (β-strand A), the N-terminal portion of the scaffold protein (SusB), the C-terminal portion of the immunoglobulin, and the C-terminal portion of the scaffold are arranged in this order. (C) Amino acid sequence of the resulting antigen-binding chimeric protein. The NbGFP207 sequence is shown in bold, (X)1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition, and the susB sequence is underlined. The C-terminal tag contains 6xhis and EPEA. [Figure 14]Rigid display of nanobodies on icosahedral VLPs derived from bacteriophage PP7. (A) Schematic of an icosahedral VLP (inner molecule) rigidly displaying 90 copies of nanobodies (outer molecule) in a regular, rigid array. The wild-type icosahedral PP7 virus has a T=3 shell composed of 180 identical copies of the PP7 coat protein, but two coat proteins can be fused N- to C-terminally to generate a VLP composed of 90 covalent coat protein dimers (O'Rourke et al., 2015). (B) Model of an antigen-binding chimeric protein consisting of a GFP-specific nanobody (top, SEQ ID NO: 1) linked to a circularly permuted mutant of the covalent coat protein dimer of Pseudomonas aeruginosa bacteriophage PP7 (bottom, PDB1DWN). (C) Linkage scheme of the nanobody and the permuted coat protein dimer of PP7. A circularly permuted gene encoding this covalent coat protein dimer of PP7 (SEQ ID NOs: 3-6) was inserted into the first β-turn connecting β-strand A of the Nanobody to β-strand B. (D) Amino acid sequence of the antigen-binding chimeric coat protein. Residues derived from the Nanobody are shown in bold. (X) 1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition. The sequence corresponding to the PP7 coat protein is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 15]Rigid nanobody display on an icosahedral VLP derived from bacteriophage MS2. (A) Schematic of an icosahedral VLP (inner molecule) rigidly displaying 90 copies of a nanobody (outer molecule) in a regular, rigid array. While wild-type MS2 virus forms an icosahedral shell composed of 180 identical copies of the MS2 coat protein, two coat proteins can be fused N- to C-terminally to generate a VLP composed of 90 covalent coat protein dimers (O'Rourke et al., 2015). (B) Model of an antigen-binding chimeric protein consisting of a lysozyme-specific nanobody (top, SEQ ID NO: 7) linked to a circularly permuted mutant of the covalent coat protein dimer of E. coli bacteriophage MS2 (bottom, PDB2MS2). (C) Linkage scheme of the nanobody and the permuted coat protein dimer of MS2. A circularly permuted gene encoding this covalent coat protein dimer of MS2 was inserted into the first β-turn connecting β-strand A to β-strand B of the Nanobody (SEQ ID NO: 9). (D) Amino acid sequence of the antigen-binding chimeric coat protein. Residues derived from the Nanobody are shown in bold. (X) 1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition. The sequence corresponding to the MS2 coat protein is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 16]Model of an antigen-binding chimeric protein in which an acyl carrier protein is inserted into the first β-turn connecting β-strands A and B of a GFP-specific nanobody via two peptide bonds or short linkers linking the antigen-binding domain to the ACP. (A) Three-dimensional model of an antigen-binding chimeric protein composed of a fusion of a GFP-specific nanobody (top, SEQ ID NO: 1) and an E. coli-derived acyl carrier protein (bottom, PDB1T8K, SEQ ID NO: 86) via two peptide linkers. In this particular antigen-binding chimeric protein, circular permutation of the scaffold protein was not required because the N- and C-termini of the wild-type ACP are close enough to each other to construct two short polypeptide bonds linking the nanobody to the ACP. The reactive serine is indicated in the lower left. (B) The first β-strand of the nanobody (β-strand A), the ACP, and the C-terminal portion of the nanobody are arranged in that order. (C) Amino acid sequence of the resulting antigen-binding chimeric protein. The NbGFP207 sequence is shown in bold, (X)1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition, and the ACP sequence is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 17] Engineering principles for antigen-binding chimeric proteins in which a nanobody is inserted into another nanobody via three peptide bonds or short linkers connecting both immunoglobulin domains. This scheme shows how a nanobody (Nb) can be fused to another nanobody via three peptide bonds or three short linkers connecting the antigen-binding domains. The scissors mark indicates which exposed turn of the nanobody to be linked should be incised. The dashed line indicates how the remaining parts of both nanobodies should be concatenated using peptide bonds or short peptide linkers to create these antigen-binding chimeric proteins. The CDRs, framework residues, and β-turn regions of the Nb are defined according to IMGT (Figure 25, revised version based on Lefranc, 2014). [Figure 18]Model of the Nano2bodies antigen-binding chimeric protein. (A) Three-dimensional model of the antigen-binding chimeric protein consisting of a fusion of a GFP-specific nanobody (nanobody B, SEQ ID NO: 1) to a lysozyme-binding nanobody (nanobody A, SEQ ID NO: 7) via three peptide linkers according to Figure 17. (B) Linkage scheme of nanobodies A and B. (C) Amino acid sequence of the resulting antigen-binding chimeric protein (SEQ ID NO: 14). β-strand A of the GFP-binding nanobody is double underlined, the NbGFP207 sequence is in bold, and the lysozyme-binding nanobody sequence is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 19] Model of a Nano2body antigen-binding chimeric protein. (A) Three-dimensional model of an antigen-binding chimeric protein consisting of a fusion of a GFP-specific Nanobody (Nanobody B, SEQ ID NO: 1) to a FedF-binding Nanobody (Nanobody A, SEQ ID NO: 17) via three peptide linkers according to Figure 17. (B) Linkage scheme of Nanobody A and Nanobody B. (C) Amino acid sequence of the resulting antigen-binding chimeric protein (SEQ ID NO: 18). β-strand A of the GFP-binding Nanobody is double underlined, the NbGFP207 sequence is in bold, and the FedF-binding Nanobody sequence is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 20]Representative vectors used for generating and selecting antigen-binding chimeric proteins derived from nanobody immune libraries by phage or yeast display. The vector shown in this figure encodes antigen-binding chimeric proteins in which a nanobody is fused to a large (optionally circularly permuted) scaffold protein via two peptide bonds or two short linkers connecting the antigen-binding domain to the scaffold. LS: leader sequence. pIII: binding protein of the filamentous phage M13. Aga2: Aga2p protein adhesion subunit protruding from the yeast cell surface. ACP: acyl carrier protein for orthogonal labeling to monitor the expression level of the displayed antigen-binding chimeric protein. Large antigen-binding chimeric protein display libraries can be constructed by cloning the C-terminal portions (including β-strands B to G) from a collection of genes encoding antigen-binding domains. In the case of nanobodies, such genes can be cloned, for example, from immunized llamas or obtained from synthetic libraries. [Figure 21] Sequence analysis of GFP-specific antigen-binding chimeric proteins derived from a nanobody immune library and selected by yeast display followed by FACS. Multiple amino acid sequence alignment of nine GFP-specific antigen-binding chimeric proteins (SEQ ID NOs: 95 to 103) selected by yeast display followed by FACS from an antigen-binding chimeric protein library constructed starting from nanobodies cloned from the blood sample of a GFP-immunized llama and then fused to HopQ. Only the C-terminal parts of the nanobodies, including β-strands B to G, are shown. [Figure 22]Model of a 58 kD GFP-binding chimeric protein in which a circularly permuted HopQ mutant is inserted into the β-turn connecting β-strands C and C' of a GFP-specific nanobody. (A) Engineering principle for grafting a scaffold onto a nanobody via the β-turn connecting β-strands C and C'. The scissors mark indicates which exposed turns of the nanobody and scaffold should be incised. The dashed lines indicate how the nanobody and the rest of the scaffold should be concatenated using peptide bonds or short peptide linkers to generate these antigen-binding chimeric proteins. The CDRs, framework residues, and β-turn regions of the Nb are defined according to IMGT (Figure 25, revised version based on Lefranc, 2014). (B) Three-dimensional model of an antigen-binding chimeric protein composed of a fusion of a GFP-specific nanobody (top) to HopQ (bottom) via two peptide linkers. (C) The third β-strand (β-strand C) of the nanobody, the circularized variant of the scaffold, and the β-strands C' to G of the GFP-specific nanobody are arranged in this order. (D) The amino acid sequence of the resulting antigen-binding chimeric protein. The NbGFP207 sequence is shown in bold, (X)1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition, the circularly permuted linker is shown in italics, and the HopQ sequence is underlined. [Figure 23]Model of a 58 kD GFP-binding chimeric protein in which a circularly permuted HopQ mutant is inserted into the first β-turn connecting β-strands A and B of the monobody NS1. (A) Model of an antigen-binding chimeric protein constructed by fusing the K-Ras-specific monobody NS1 (top) with a circularly permuted H. pylori HopQ adhesin domain mutant (bottom) via two peptide bonds or linkers connecting the nanobody to the scaffold. (B) A circularly permuted gene encoding the adhesin domain of H. pylori G27 type 1 HopQ (bottom, PDB5LP2, SEQ ID NO: 19) was inserted into the first β-turn (β-turn AB) connecting β-strand A to β-strand B of the K-RAS-specific monobody (top, SEQ ID NO: 112). (C) Amino acid sequence of an antigen-binding chimeric protein constructed from NS1 and HopQ. The sequence derived from the nanobody is shown in bold. The sequence derived from HopQ is underlined. The peptides linking the N- and C-termini of HopQ to generate circular permutations are shown in italics, and (X)1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition. [Figure 24] Kinetic characterization of the interaction of the antigen-binding chimeric proteins MbNb207 cHopQ and MbNb207 cYgjkE2 with GFP analyzed by Octet. Real-time kinetic analysis of the interaction of the GFP-binding nanobody NbGFP207 (SEQ ID NO: 1), the antigen-binding chimeric proteins MbNb207 cHopQ (SEQ ID NO: 20), and MbNb207 cYgjkE2 (SEQ ID NO: 141) with GFP analyzed by biolayer interferometry. Biotinylated GFP (0.75 μg / mL) was captured using a streptavidin-coated Octet® biosensor and examined for association with several concentrations (ranging from 60 to 2.22 nM) of NbGFP207 (A), MbNb207 cHopQ (B), and MbNb207 cYgjkE2 (C). The measured responses (black line) were fitted to a monophasic 1:1 binding model (red line). Assays were performed in 10 mM Tris-HCl, 140 mM NaCl, pH 7.3, 0.05% Tween 20, and 1 mg / mL BSA. (D) Kinetic calculations are shown as the mean standard error (s.e.m.) from n = 3 independent experiments. [Figure 25] Immunoglobulin variable region structure and topology from the revised IMGT based on LeFranc (2014). (A) 3D structural ribbon diagram with IMGT strand and loop definitions (Franc et al., 2003). (B) Bilayer IMGT Collier de Perles including hydrogen bonds. The bilayer IMGT Collier de Perles shows the GFCC'C" strand (which forms a sheet at the VH / VL interface of IG) in the foreground and the ABED strand in the background. The IMGT Collier de Perles including hydrogen bonds (green lines online, shown here only for the GFCC'C" sheet) are generated from experimental 3D structure data using the IMGT / Collier-de-Perles tool integrated into the IMGT / 3Dstructure-DB database (Kaas et al., 2004; Ehrenmann et al., 2010; Ehrenmann and LeFranc, 2011). [Figure 26] Schematic diagram of antigen-binding chimeric proteins that bind to and elongate other antigen-binding chimeric proteins. (A) MbNb60 c7HopQmut2 (SEQ ID NO: 134) or MbNb60 c7HopQmut3 (SEQ ID NO: 134) binds to and elongates another MbNb207 cHopQ (SEQ ID NO: 20), resulting in an extended 116 kDa antigen-binding chimeric protein. (B) MbNb60 cYgjK (SEQ ID NO: 135) binds to and elongates MbNb207 cHopQ (SEQ ID NO: 20), resulting in an extended 168 kDa antigen-binding chimeric protein. [Figure 27] This is the crystal structure of Nb60 complexed with MbNb207 cHopQ, revealing that the Nb60 epitope is present on the scaffold protein contained in cHopQ. [Figure 28] Schematic diagram of an antigen-binding chimeric protein in which an antigen-binding domain is linked to a scaffold protein using a collection of chemical linker units. [Figure 29]Kinetic characterization of the interaction of MbNb207 cHopQ with an antigen-binding chimeric protein designated polybody was performed using Octet. Real-time kinetic analysis of the binding of HopQ-specific nanobodies Nb60 (SEQ ID NO: 132), HopQ-specific MbNb60 cHopQmut2 (SEQ ID NO: 133), HopQ-specific MbNb60 cHopQmut3 (SEQ ID NO: 134), and HopQ-specific MbNb60 cYgjkE2 (SEQ ID NO: 135) to MbNb207 cHopQ (SEQ ID NO: 20) was performed using biolayer interferometry. Biotinylated MbNb207 cHopQ (0.25 μg / mL) was captured using a streptavidin-coated Octet® biosensor. Binding and dissociation isotherms were monitored for Nb60 (A), MbNb60 cHopQmut2 (B), MbNb60 cHopQmut3 (C), and MbNb60 cYgjkE2 (D) at several concentrations (ranging from 1.67 to 500 nM). All assays were performed in 10 mM Tris-HCl, 140 mM NaCl, pH 7.3, 0.05% Tween 20, and 1 mg / mL BSA. [Figure 30]Flow cytometry analysis of the functionality of MbNb207 cHopQ mutants displayed on the surface of EBY100 yeast cells. MbNb207 cHopQ (SEQ ID NO: 22) and representative examples of MbNb207 cHopQ mutants selected by yeast display (Table 2) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. Dot plots of the relative fluorescence intensity of individual EBY100 yeast cells transformed with the MP1331_A9 megabody mutant (A) and a pCTCON2 derivative encoding MbNb207 cHopQ (B). The mean fluorescence intensity (MFI) of the relative CoA-647 fluorescence (megabody display level) and relative GFP fluorescence (GFP binding) for each yeast clone were calculated using Prism7 software (GraphPad). (C) Graph of calculated mean fluorescence intensity (MFI) values ​​of relative CoA-647 and GFP fluorescence for MbNb207 cHopQ (SEQ ID NO: 22) and each MbNb207 cHopQ mutant (Table 2). [Figure 31] MbNb207 c7HopQ and four yeast-display-selected MbNb207 c7HopQ mutants were expressed in E. coli, purified by nickel affinity chromatography, and characterized by size-exclusion chromatography. MbNb207 c7HopQ mutants containing a 1-1 amino acid peptide linker, selected by yeast display, were expressed in E. coli and subjected to preparative gel filtration on a Superdex 200 PG column (10 / 300, GE Healthcare). The MbNb207 c7HopQ (sequence number 136) clone corresponds to the wild-type linked mutant, and MbNb207 c7HopQA5 (sequence number 137), MbNb207 c7HopQA12 (sequence number 138), MbNb207 c7HopQB7 (sequence number 139), and MbNb207 c7HopQG10 (sequence number 140) correspond to the MP1331_A5, MP1331_A12, MP1331_B7, and MP1331_G10 clones in Table 2, respectively. [Figure 32]GFP binding analysis by ELISA of MbNb207 c7HopQ and four mutants selected by yeast display. MbNb207 c7HopQ (SEQ ID NO: 136), four mutants selected by yeast display (MbNb207 c7HopQA5, MbNb207 c7HopQA12, MbNb207 c7HopQB7, and MbNb207 c7HopQG10) (SEQ ID NOs: 137-140), and MbNb38 cHopQ (SEQ ID NO: 131) were purified by size exclusion and incubated in GFP-immobilized or uncoated wells. The EPEA tag at the C-terminus of the megabody was detected using a biotinylated anti-EPEA (Capture Select C-tag) antibody mixed with streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). Data are shown as the mean standard error of the sample mean (sem) from experiments performed in triplicate (n=3). [Figure 33-1] Flow cytometry analysis of the functionality of MbNb207 cYgjk mutants displayed on the surface of EBY100 yeast cells. Representative examples of MbNb207 cYgjk mutants selected by yeast display (Table 3) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. (A) Dot plots of the relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the MP1333_A2 megabody mutants. The mean fluorescence intensity (MFI) of the relative CoA-647 fluorescence (megabody display level) and relative GFP fluorescence (GFP binding) for each yeast clone were calculated using Prism7 software (GraphPad). [Figure 33-2]Flow cytometry analysis of the functionality of MbNb207 cYgjk mutants displayed on the surface of EBY100 yeast cells. Representative examples of MbNb207 cYgjk mutants selected by yeast display (Table 3) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. (B) Graph of calculated mean fluorescence intensity (MFI) values ​​for relative CoA-647 and GFP fluorescence for each MbNb207 cYgjk mutant (Table 3). [Figure 34] GFP binding analysis by ELISA of four MbNb207 cYgjk mutants selected by yeast display. Periplasmic extracts containing different MbNb207 cYgjk mutants (SEQ ID NOs: 141-144), MbNb207 cYgjkE2, MbNb207 cYgjkA2, MbNb207 cYgjkC4, and MbNb207 cYgjkF5, were compared with MbNb38 cHopQ (SEQ ID NO: 131). All samples were incubated in GFP-immobilized or uncoated wells. The EPEA tag at the C-terminus of the megabody was detected using a biotinylated anti-EPEA (Capture select C-tag) antibody mixed with streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). [Figure 35]Size-exclusion characterization of MbNb207 cYgjkE2 mutants expressed in E. coli and purified by nickel affinity chromatography. (A) Analysis of MbNb207 cYgjkE2 (SEQ ID NO: 141) expressed in the periplasm of E. coli and purified by nickel affinity chromatography on a preparative Superdex 200 PG gel filtration column (16 / 26, GE Healthcare). (B) SDS-PAGE analysis of the purity of the MbNb207 cYgjkE2 megabody in fraction "Peak 1" from the size-exclusion experiment described in (A). A sample of purified MbNb207 cYgjkE2 (left lane: "Peak 1") was applied to an 8% SDS-PAGE gel, and the molecular weight was confirmed to be approximately 100 kDa by comparison with molecular weight markers (right lane: M). [Figure 36-1] Functional and biophysical characterization of MbNb207 cHopQ variants further rigidified by artificial disulfide bonds linking the nanobody to the scaffold. GFP binding analysis of 10 MbNb207 cHopQ variants was analyzed by ELISA. (A) MbNb207 cHopQ (SEQ ID NO: 20) and four MbNb207 cHopQCys1-4 variants (SEQ ID NOs: 48-51, see Figure 10) were purified by size exclusion and incubated in GFP-immobilized or uncoated wells. [Figure 36-2]Functional and biophysical characterization of MbNb207 cHopQ variants further rigidified by artificial disulfide bonds linking the nanobody to the scaffold. GFP binding analysis of 10 MbNb207 cHopQ variants was performed by ELISA. (B) MbNb207 c7HopQ (SEQ ID NO: 136) and four MbNb207 c7HopQCys5-10 variants (SEQ ID NOs: 145-150) were purified by size exclusion and incubated in GFP-immobilized or uncoated wells. The EPEA tag at the C-terminus of the megabody was detected using a biotinylated anti-EPEA (CaptureSelect C-tag) antibody mixed with streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). Data are presented as the mean standard error of the sample mean (sem) from experiments performed in triplicate (n=3). [Figure 36-3] Functional and biophysical characterization of MbNb207 cHopQ variants further rigidified by artificial disulfide bonds linking the nanobody to the scaffold. GFP binding analysis of 10 MbNb207 cHopQ variants was analyzed by ELISA. (C) Thermal stability analysis of MbNb207 cHopQ variants was performed by thermal shift assay (TSA). Graph of normalized melting point profiles for each variant: MbNb207 c7HopQ (SEQ ID NO: 136), MbNb207 cHopQCys4 (SEQ ID NO: 51), MbNb207 c7HopQCys5 (SEQ ID NO: 145), MbNb207 c7HopQCys6 (SEQ ID NO: 146), and MbNb207 c7HopQCys10 (SEQ ID NO: 150). Calculated melting temperatures (Tm) are shown in Table 4. [Figure 37-1]Flow cytometry analysis of the functionality of MbNb207 Azurin mutants displayed on the surface of EBY100 yeast cells. Representative examples of MbNb207 Azurin mutants selected by yeast display (Table 5) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. (A) Dot plots of the relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the MP1304_B2 megabody mutants. The mean fluorescence intensity (MFI) of the relative CoA-647 fluorescence (megabody display level) and relative GFP fluorescence (GFP binding) for each yeast clone were calculated using Prism7 software (GraphPad). [Figure 37-2] Flow cytometry analysis of the functionality of MbNb207 Azurin variants displayed on the surface of EBY100 yeast cells. Representative examples of MbNb207 Azurin variants selected by yeast display (Table 5) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. (B) Graph of calculated mean fluorescence intensity (MFI) values ​​for relative CoA-647 and GFP fluorescence for each MbNb207 Azurin variant (Table 5). [Figure 38]GFP binding analysis by ELISA of eight MbNb207 Azurin mutants selected by yeast display. Eight periplasmic extracts containing different MbNb207 Azurin mutants (SEQ ID NOs: 151-158) and one extract containing MbNb38 cHopQ (SEQ ID NO: 131) were incubated in GFP-immobilized or uncoated wells. The EPEA tag at the C-terminus of the megabody was detected using a biotinylated anti-EPEA (CaptureSelect C-tag) antibody mixed with streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). Data are shown as the standard error of the mean (sem) from three experiments performed in triplicate (n = 3). [Figure 39] GFP binding analysis by ELISA. Periplasmic extracts containing different MbNb207 cPP7x2L chimeric proteins (SEQ ID NOs: 3–6) were incubated in wells with or without GFP (0.1 μg / well). The EPEA tag present at the C-terminus of the chimeric cPP7 dimer was detected using a biotinylated anti-EPEA (CaptureSelect C-tag) antibody mixed with a streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). [Figure 40]Rigid display of nanobodies on icosahedral VLPs derived from bacteriophage AP205. (A) Schematic of an icosahedral VLP (inner molecule) rigidly displaying 90 copies of the nanobody (outer molecule) in a regular, rigid array. (B) Model of an antigen-binding chimeric protein consisting of a GFP-specific nanobody (top, SEQ ID NO: 1) linked to a covalent coat protein dimer of AP205 (bottom, PDB5FS4), an icosahedral RNA bacteriophage of Acinetobacter bacteria. (C) Linkage scheme between the nanobody and AP205 coat protein. (D) Amino acid sequence of the antigen-binding chimeric coat protein (SEQ ID NO: 167). Residues derived from the nanobody are shown in bold. X represents a short, random peptide linker of one amino acid. The sequence corresponding to the AP205 coat protein is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 41] GFP binding analysis of 372 clones by ELISA. Periplasmic extracts (indicated by MP number) containing MbNb207 AP205x2XX dimers (SEQ ID NO: 167) were incubated in wells with immobilized GFP (0.1 μg / well) or uncoated wells. The EPEA tag present at the C-terminus of the chimeric AP205 dimer was detected using a biotinylated anti-EPEA (CaptureSelect C-tag) antibody mixed with a streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). [Figure 42]Rigid display of nanobodies into dimeric antigen-binding chimeric proteins. (A) Model of a dimeric antigen-binding chimeric protein with β-strand A exchange. AP205 monomers assemble into dimers. To obtain a functional antigen-binding chimera, β-strand A of a first monomeric GFP-binding nanobody must bind to β-strands B-G of a second monomeric GFP-binding nanobody, and β-strands B-G of the second monomeric GFP-binding nanobody must bind to β-strand A of the first monomeric GFP-binding nanobody, resulting in assembly into a dimeric antigen-binding chimeric protein. (B) Model (rotated 90°) of the same dimeric antigen-binding chimeric protein in which two GFP-specific nanobodies (top, SEQ ID NO: 1) are each linked to an AP205 coat protein monomer. (C) Linkage scheme between nanobodies and AP205 coat protein. (D) Amino acid sequence of the antigen-binding chimeric coat protein (SEQ ID NO: 173). Residues derived from the nanobody are shown in bold. X is a short random peptide linker of 1 amino acid. The sequence corresponding to the AP205 coat protein is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 43-1] GFP binding analysis of 372 clones by ELISA. Periplasmic extracts (indicated by MP number) containing MbNb207 AP205XX dimers (SEQ ID NO: 173) were incubated in wells with or without GFP (0.1 μg / well). The EPEA tag at the C-terminus of the AP205 dimer was detected using a biotinylated anti-EPEA antibody (Capture select C tag) mixed with a streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). [Figure 43-2]GFP binding analysis of 372 clones by ELISA. Periplasmic extracts (indicated by MP number) containing MbNb207 AP205XX dimers (SEQ ID NO: 173) were incubated in wells with or without GFP (0.1 μg / well). The EPEA tag at the C-terminus of the AP205 dimer was detected using a biotinylated anti-EPEA antibody (Capture select C tag) mixed with a streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). [Figure 44] Flow cytometry analysis of the functionality of MbNb207 ACP nanotool (NanoTool) variants displayed on the surface of EBY100 yeast cells. Representative examples of MbNb207 ACP nanotool variants selected by yeast display (Table 9) were displayed on EBY100 cells as Aga2p-ACP fusions. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. (A) Dot plots of the relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the MP1302_D10 nanotool variants. The mean fluorescence intensity (MFI) of the relative CoA-647 fluorescence (nanotool display level) and relative GFP fluorescence (GFP binding) for each yeast clone were calculated using Prism7 software (GraphPad). (B) Graph of calculated mean fluorescence intensity (MFI) values ​​for relative CoA-647 and GFP fluorescence for each MbNb207 ACP nanotool variant (Table 9). [Figure 45]GFP binding analysis by ELISA of six MbNb207 ACP nanotool mutants selected by yeast display. Periplasmic extracts containing the six MbNb207 ACP (SEQ ID NOs: 178-183) mutants and MbNb38 cHopQ (SEQ ID NO: 131) were incubated in GFP-immobilized or uncoated wells. The EPEA tag at the C-terminus of the megabody was detected using a biotinylated anti-EPEA (Capture Select C-tag) antibody mixed with a streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). Data are shown as the standard error of the mean (sem) from three experiments performed in triplicate (n = 3). [Figure 46] Model of the chimeric antigen-binding protein Nano2body. (A) Three-dimensional model of an energetically more efficient antigen-binding chimeric protein consisting of a fusion of a GFP-specific Nanobody (Nanobody B, SEQ ID NO: 1) with a FedF-binding Nanobody (Nanobody A, SEQ ID NO: 17) via three peptide linkers according to Figure 17. (B) Linkage scheme of Nanobody A and Nanobody B. (C) Amino acid sequence of the resulting antigen-binding chimeric protein (SEQ ID NOs: 184-185). β-strand A of the GFP-binding Nanobody is double underlined, the NbGFP207 sequence is in bold, and the FedF-binding Nanobody sequence is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 47] GFP and FedF binding analysis by ELISA of representative clones. Semi-purified Nano2body samples (SEQ ID NOs: 186-191) were incubated in wells immobilized with GFP (0.5 μg / well), FedF (0.5 μg / well), or uncoated wells. The EPEA tag at the C-terminus of Nano2body was detected using a biotinylated anti-EPEA antibody (Capture select C tag) mixed with streptavidin-alkali conjugate. Absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). [Figure 48-1] Flow cytometry analysis of the functionality of cHopQNbGFP207CC' megabody mutants displayed on the surface of EBY100 yeast cells. Representative examples of cHopQNbGFP207XCC' megabody mutants selected by yeast display (Table 11) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. (A) Dot plots of the relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the MP1327_D5 megabody mutant. The mean fluorescence intensity (MFI) of the relative CoA-647 fluorescence (megabody display level) and relative GFP fluorescence (GFP binding) for each yeast clone were calculated using Prism7 software (GraphPad). [Figure 48-2] Flow cytometry analysis of the functionality of cHopQNbGFP207CC' megabody mutants displayed on the surface of EBY100 yeast cells. Representative examples of cHopQNbGFP207XCC' megabody mutants selected by yeast display (Table 11) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-647 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM GFP. (B) Graph of calculated mean fluorescence intensity (MFI) values ​​for relative CoA-647 and GFP fluorescence for each cHopQNbGFP207CC' megabody mutant (Table 11). [Figure 49-1]Flow cytometry analysis of the functionality of MbNS1 cHopQ mutants displayed on the surface of EBY100 yeast cells. Representative examples of MbNS1 cHopQ mutants selected by yeast display (Table 12) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-488 (2 μM) using SFP synthase (1 μM) and incubated with 100 nM Dylight-647-labeled K-RAS. (A) Dot plots of the relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the MP1326_A11 megabody mutant. The mean fluorescence intensity (MFI) of relative CoA-488 fluorescence (megabody display level) and relative Dylight-647 fluorescence (K-Ras binding) for each yeast clone was calculated using Prism7 software (GraphPad). [Figure 49-2] Flow cytometry analysis of the functionality of MbNS1 cHopQ mutants displayed on the surface of EBY100 yeast cells. Representative examples of MbNS1 cHopQ mutants selected by yeast display (Table 12) were displayed on EBY100 cells as fusions of Aga2p and ACP. Individual yeast clones were separately induced and orthogonally stained with CoA-488 (2 μM) using SFP synthase (1 μM) and incubated with K-RAS labeled with 100 nM Dylight-647. (B) Graph of calculated mean fluorescence intensity (MFI) values ​​for relative CoA-488 and Dylight-647 fluorescence for each MbNS1 cHopQ mutant (Table 12). [Figure 50]Rigid display of nanobodies on the dodecin Rv1498A from M. tuberculosis. (A) Schematic of the dodecin Rv1498A (inner molecule) rigidly displaying 12 copies of the nanobody (outer molecule) in a regular, rigid array. (B) Three-dimensional model of an antigen-binding chimeric protein composed of a fusion of a GFP-specific nanobody (top, SEQ ID NO: 1) with an Rv1498A monomer (bottom, SEQ ID NO: 192) via two peptide linkers. In this particular antigen-binding chimeric protein, the N- and C-termini of wild-type Rv1498A are close enough to each other to construct two short polypeptide bonds linking the nanobody to Rv1498A, so circular permutation of the scaffold protein was not required. (C) The first β-strand of the nanobody (β-strand A), followed by Rv1498A, and finally the C-terminal portion of the nanobody are arranged in that order. (D) Amino acid sequence of the resulting antigen-binding chimeric protein. The NbGFP207 sequence is shown in bold, (X)1 is a short peptide linker of mixed composition, and the Rv1498A sequence is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 51] GFP-binding analysis by ELISA of six selected MbNb207 Dodecin variants. Periplasmic extracts containing the six MbNb207 Dodecin variants (Table 13) and the NbGFP207 nanobody (SEQ ID NO: 1) were incubated in GFP-immobilized or uncoated wells. The EPEA tag present at the C-terminus of the megabody was detected using a biotinylated anti-EPEA (Capture select C-tag) antibody mixed with a streptavidin-alkali conjugate. The absorbance at 405 nm (OD405) was measured after incubation with 4-nitrophenyl phosphate disodium salt hexahydrate substrate (DNPP). Data are shown as the mean standard error (sem) of the sample mean from three experiments performed in triplicate (n = 3). [Figure 52]Rigid display of nanobodies on disulfide-bridged homodimers derived from Burkholderia cenocepacia. (A) Schematic diagram of a homodimer of 4QYB (inner molecule, SEQ ID NO: 200) rigidly fused to a GFP-specific nanobody (outer molecule, SEQ ID NO: 1) via two peptide linkers. (B) The first β-strand of the nanobody (β-strand A), 4QYB, and the C-terminal portion of the nanobody are arranged in this order. (C) Amino acid sequence of the resulting antigen-binding chimeric protein. The NbGFP207 sequence is shown in bold; (X)1-2 are short peptide linkers of variable length (1 or 2 amino acids) and mixed composition; the 4QYB sequence is underlined. The C-terminal tag contains 6xHis and EPEA. [Figure 53] Ligand binding of β2AR-wt in the presence or absence of MbNb80 cHopQ. Radioligand displacement assays tested various ligands competing with [3H]-dihydroalprenolol ([3H]-DHA) for binding to β2AR-wt in the presence of MbNb80 cHopQ (black up-pointing triangles) and compared their binding to β2AR-wt in the presence of Nb80 (black squares), β2AR-wt alone (gray circles), or the unrelated MbNb207 cHopQ (gray triangles). Competition assays were performed at the β2AR-wt receptor using the natural agonist epinephrine (A) and the agonist (-)-isoproterenol (B) as competing ligands, respectively. Curves were fitted to a competitive binding model by nonlinear regression using standard settings in Graphpad Prism. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will be described below with reference to certain drawings and with reference to specific embodiments. However, the present invention is not limited to the following description, but only by the claims. Reference signs in the claims should not be construed as limiting the scope. Of course, not all aspects or advantages may be achieved in accordance with a particular embodiment of the invention. Thus, for example, one skilled in the art will understand that the present invention can be embodied or practiced in a manner that achieves or optimizes one advantage or group of advantages taught herein, without necessarily achieving other aspects or advantages that may be taught or suggested herein.

[0026] The present invention, both in terms of organization and method of operation, together with its features and advantages, can be best understood by reference to the following detailed description taken in light of the accompanying drawings. Aspects and advantages of the present invention will be more clearly understood with reference to the embodiments described below. References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various contexts throughout this specification may, but do not necessarily, refer to the same embodiment. Similarly, it will be appreciated that in describing representative embodiments of the present invention, various features of the invention may be grouped together in a single embodiment, drawing, or description for the purpose of streamlining the disclosure and facilitating understanding of one or more of the various inventive aspects. However, this method of disclosure should not be interpreted to imply that the claimed invention requires more features than are expressly recited in each claim. Rather, as will be apparent from the following claims, inventive aspects lie in fewer than all features of a single embodiment disclosed preceding the claims.

[0027] definition The use of an indefinite or definite article, e.g., "a," "an," or "the," when referring to a singular noun, includes a plural of that noun unless specifically stated otherwise. The use of the term "comprising" in the specification and claims does not exclude other elements or steps. Furthermore, the terms first, second, third, etc., used in the specification and claims are used to distinguish between similar elements and do not necessarily denote a chronological or temporal order. It will be understood that such terms are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein may be performed in sequences other than those described or illustrated herein. The following terms or definitions are provided solely to facilitate understanding of the invention. Unless otherwise defined herein, all terms used herein have the same meaning as those used by one skilled in the art of the invention. For definitions and terminology in the art, practitioners are particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). The definitions set forth herein should not be construed narrower than understood by one of ordinary skill in the art.

[0028] As used herein, "about" when referring to a measurable value, such as an amount, length of time, etc., means including a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% of the specified value, provided that such variation is appropriate for performing the disclosed method. As used herein, "similar" is synonymous with similar, analogous, equivalent, corresponding, and like, and means having the same or common characteristics and / or showing quantitatively equivalent results, i.e., a maximum variation of 20%, 10%, more preferably 5%, or even more preferably 1% or less.

[0029] As used herein, the terms "nucleotide sequence," "DNA sequence," or "nucleic acid molecule" refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA and RNA. It also encompasses known modifications, such as methylation and "cap" substitution of one or more naturally occurring nucleotides with an analog. A "nucleic acid construct" refers to a nucleic acid sequence engineered to contain one or more functional units that do not occur together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from λ phage), and viral genomes containing non-naturally occurring nucleic acid sequences.

[0030] A "coding sequence" is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of a coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. A coding sequence may include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and may also contain introns in certain circumstances.

[0031] As used herein, a "promoter region of a gene" refers to a functional DNA sequence unit operably linked to a coding sequence, sufficient to promote transcription of the coding sequence when optionally subjected to appropriate inducing conditions. "Operably linked" means that the components thus expressed are in a relationship permitting their intended function. A promoter sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence occurs under conditions compatible with the promoter sequence. As used herein, a "gene" includes both the promoter region and the coding sequence of a gene. This term refers to genomic sequences (including possible introns) as well as cDNA derived from spliced ​​messengers and operably linked to a promoter sequence. The term "terminator" or "transcription termination signal" refers to a DNA sequence at the end of a transcription unit, a control sequence that directs 3' processing and polyadenylation of the primary transcript and the termination of transcription. Terminators can be derived from the native gene, various other plant genes, or T-DNA. The additional terminator may be derived, for example, from the nopaline synthase or octopine synthase gene, or from another plant gene, or, less preferably, from any other eukaryotic gene.

[0032] "Chimeric gene" or "chimeric construct" or "chimeric gene construct" means a recombinant nucleic acid sequence in which a nucleic acid sequence encoding an mRNA is operably linked or associated with a promoter or regulatory nucleic acid sequence such that transcription or expression of the associated nucleic acid coding sequence can be regulated by the regulatory nucleic acid sequence. The regulatory nucleic acid sequence of a chimeric gene is not operably linked to the associated nucleic acid sequence as it occurs in nature.

[0033] An "expression cassette" includes any nucleic acid construct capable of directing expression of a gene / coding sequence of interest operably linked to the promoter of the expression cassette. Expression cassettes are generally DNA constructs that preferably contain (in the 5' to 3' transcription direction) a promoter region, a polynucleotide sequence, homolog, variant, or fragment thereof operably linked to a transcription initiation region, and a termination sequence including an RNA polymerase stop signal and a polyadenylation signal. Naturally, all of these regions must be functional in the biological cell, such as a prokaryotic or eukaryotic cell, to be transformed. The promoter region, including the transcription initiation region, which preferably includes an RNA polymerase binding site, and the polyadenylation signal, may originate from the biological cell to be transformed, or may originate from an alternative source, provided that such regions are functional in the biological cell. A "vector" can be constructed from such a cassette.

[0034] As used herein, the terms "vector," "vector construct," "expression vector," or "transfer vector" refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked, and include any vector known to those of skill in the art, including any suitable type, including, but not limited to, plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus, AAV, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary to express the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Expression vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors with an origin of replication that functions in the host cell). Other vectors can integrate into the genome of a host cell after introduction, thereby replicating along with the host genome. Suitable vectors contain regulatory sequences such as promoter, enhancer, and terminator sequences as needed for a particular host organism (e.g., bacterial cells, yeast cells). Cloning vectors are generally used to construct and amplify a given desired DNA fragment, and may not contain functional sequences necessary for the expression of the desired DNA fragment. The construction of expression vectors used to transfect prokaryotic cells is also well known in the art and can be carried out by standard techniques (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 4 for definitions and terms in the art). th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016)).

[0035] "Host cells" can be prokaryotic or eukaryotic cells. These cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished by any technique known in the art, including, but not limited to, standard bacterial transformation, calcium phosphate co-precipitation, electroporation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polycation-mediated transfection, or viral transfection. All standard techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). In this context, a recombinant host cell is a host cell that has been genetically modified to contain an isolated DNA molecule, nucleic acid molecule, or expression construct or vector of the present invention. DNA can be introduced by any means known in the art appropriate to the particular cell type, including, but not limited to, transformation, lipofection, electroporation, or viral transfection. DNA constructs capable of expressing the chimeric proteins of the present invention can be readily generated by techniques known in the art, such as cloning, hybridization screening, and polymerase chain reaction (PCR). In addition to standard techniques for cloning, DNA isolation, amplification, and purification, and enzymatic reactions utilizing DNA ligases, DNA polymerases, restriction endonucleases, etc., various isolation techniques are known to those skilled in the art and are widely used. Many standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993), and Ausubel et al. (2016). Exemplary host cells that can be used in the present invention include, but are not limited to, bacterial cells, yeast cells, plant cells, and animal cells. Bacterial host cells suitable for use in the present invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Animal host cells suitable for use in the present invention include insect cells and mammalian cells, most particularly those derived from Chinese hamster ovarian (e.g., CHO) and human cell lines such as HeLa.Yeast host cells suitable for use in the present invention include species within the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, and Zygosaccharomyces. Saccharomyces cerevisiae, S. carlsbergensis, and K. lactis are the most widely used yeast hosts, with convenient fungal hosts. Host cells can be provided in suspension or flask culture, tissue culture, organ culture, etc. Alternatively, host cells can be transgenic animals.

[0036] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids. The terms also include post-translational modifications of polypeptides, such as glycosylation, phosphorylation, and acetylation. Based on the amino acid sequence and modifications, the atomic or molecular mass or atomic or molecular weight of a polypeptide is expressed in (kilo)daltons (kDa). "Recombinant polypeptide" refers to a polypeptide made using recombinant techniques, i.e., by expression of a recombinant or synthetic polynucleotide. When a chimeric polypeptide or a biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium; i.e., culture medium preferably represents less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%. "Isolated" refers to material that is substantially or essentially free from components that normally accompany it in its natural state. For example, an "isolated polypeptide" refers to a polypeptide that has been purified from molecules that naturally flank it, e.g., an antigen-binding chimeric protein that has been removed from molecules that flank the polypeptide and are present in the production host. Isolated chimeric proteins can be produced by amino acid chemical synthesis or by recombinant production. The term "heterologous protein" can mean that the protein is not derived from the same species or strain as that used to display or express the protein.

[0037] A "homologue" or "homologues" of a protein includes peptides, oligopeptides, polypeptides, proteins, and enzymes that contain amino acid substitutions, deletions, and / or insertions relative to the unmodified protein and that have biological and functional activity similar to that of the original unmodified protein. As used herein, the term "amino acid identity" refers to the degree to which two sequences are identical on an amino acid basis within a comparison window. Thus, "percent sequence identity" is calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met, sometimes designated by single-letter codes herein) occur in both sequences, determining the number of matched positions, dividing this number by the total number of positions within the comparison window (i.e., window size), and multiplying the resulting number by 100 to determine the percentage sequence identity. As used herein, a "substitution" or "mutation" results from the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid or nucleotide sequence of a parent protein or fragment thereof. Of course, a protein or fragment thereof may also contain conservative amino acid substitutions that have substantially no effect on the activity of the protein.

[0038] The term "wild-type" refers to a gene or gene product isolated from a natural source. A wild-type gene is that gene found most frequently in a population and is therefore arbitrarily referred to as the "normal" or "wild-type" form of the gene. On the other hand, the terms "altered," "mutant," or "variant" refer to a gene or gene product that exhibits modified sequence, post-translational modifications, and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. However, naturally occurring mutants can also be isolated and are identified by the fact that they have altered properties when compared to the wild-type gene or gene product.

[0039] A "protein domain" is a distinct functional and / or structural unit within a protein. Typically, a protein domain is involved in a specific function or interaction and contributes to the overall role of the protein. Domains can occur in a variety of biological contexts, and similar domains may occur in proteins with different functions.

[0040] Before a protein folds into its three-dimensional tertiary structure, protein secondary structure elements (SSEs) typically form spontaneously as intermediates. The two most common secondary structure elements in proteins are alpha helices and beta (β) sheets, although beta turns and omega loops also exist. β sheets consist of beta strands (also called β strands) laterally connected by at least two or three main-chain hydrogen bonds, typically forming a twisted, folded sheet. β strands are extensions of the polypeptide chain, typically 3–10 amino acids long, along the main chain in an extended conformation. β turns are a type of irregular secondary structure in proteins that alters the direction of the polypeptide chain. Beta turns (β turns, β-turns, β bends, tight turns, reverse turns) are very common motifs in proteins and polypeptides, primarily serving to connect β strands. For the IMGT® definition of β turns in variable domains, see LeFranc (2014) and also Figure 25. A β-turn is generally composed of four amino acid residues (referred to as i, i+1, i+2, and i+3) and is defined in two ways: either by an intrachain hydrogen bond between the CO of residue i and the NH of residue i+3, or by a distance of less than 7 Å between the Cα atoms of residues i and i+3. The hydrogen-bonding criterion is best for routine use, in part because it can be divided into four distinct classes.

[0041] The term "circularly permuted protein" or "circularly permuted protein" refers to a protein in which the order of amino acids in its amino acid sequence has been altered relative to the wild-type protein sequence, resulting in a protein structure with a different linkage order but a similar overall three-dimensional (3D) shape. Circularly permuted proteins are similar to the mathematical realization of circular permutation, in that the sequence of a first portion of the wild-type protein (adjacent to its N-terminus) is related to the sequence of a second portion of the circularly permuted protein (near its C-terminus), as described, for example, in Bliven and Prlic (2012). Circularly permuting a protein relative to its wild-type protein is achieved by genetically or artificially manipulating the protein sequence to "link" the N- and C-termini of the wild-type protein, splitting the protein sequence at a different site, and creating new N- and C-termini for the protein. The circularly permuted scaffold protein of the present invention maintains or is similar to the folding of the wild-type protein by linking the N-terminus and C-terminus of the wild-type protein sequence and cleaving or splitting the sequence at an accessible or exposed site (preferentially a β-turn or loop) of the scaffold protein. The link between the N-terminus and C-terminus of the circularly permuted scaffold protein may be via a peptide bond, a peptide linker may be introduced, or peptide regions near the original N-terminus and C-terminus of the wild-type protein may be deleted and the remaining amino acids may be peptide-bonded.

[0042] As used herein, the term "fused to" is used interchangeably with "linked to," "conjugated to," and "ligated to," and specifically refers to "genetic fusion," e.g., by recombinant DNA techniques, and "chemical and / or enzymatic linkage" that results in a stable covalent linkage.

[0043] The terms "chimeric polypeptide," "chimeric protein," "chimera," "fusion polypeptide," "fusion protein," or "non-naturally occurring protein" are used interchangeably herein to refer to a protein comprising at least two distinct polypeptide components that may or may not be derived from the same protein. This term also refers to non-naturally occurring molecules, meaning they are man-made. The term "fused to" and other grammatically equivalent terms, such as "covalently linked," "linked," "coupled," "ligated," and "conjugated," when referring to chimeric polypeptides (as defined herein) refer to any chemical or recombinant mechanism that links two or more polypeptide components. Fusion of two or more polypeptide components can be a direct fusion of sequences or an indirect fusion, for example, via an amino acid sequence or linker sequence or chemical linker. Fusion of two polypeptides or antigen-binding domains and a scaffold protein, as described herein, can also refer to a non-covalent fusion achieved by chemical linkage. For example, both the C-terminus of the A-strand and the N-terminus of the B-strand of the antigen-binding domain can be linked to complementary chemical units or chemical units capable of binding to binding pockets linked or fused to exposed or accessible sites on a portion or full-length scaffold protein (as shown in Figure 28) (circularly permuted).

[0044] As used herein, the terms "protein complex," "complex," or "associated protein" refer to a group of two or more associated polymers, at least one of which is a protein. As used herein, "protein complex" generally refers to an association of polymers that can form under physiological conditions. The individual members of a protein complex are connected by noncovalent interactions. A protein complex can be a noncovalent interaction of only proteins, in which case it is referred to as a protein-protein complex, such as a noncovalent interaction between two protein molecules, three protein molecules, or four protein molecules. More specifically, it is a complex of an antigen-binding chimeric protein and an antigen itself. As used herein, a "protein complex" can also be a noncovalent interaction of at least one protein molecule and at least another polymer, such as a nucleic acid, in which case it is referred to as a protein-nucleic acid complex, such as a noncovalent interaction between one protein molecule and one nucleic acid molecule, two protein molecules and one nucleic acid molecule, or two protein molecules and two nucleic acids. Naturally, a protein complex can be a multimer. The association of a protein complex can result in the formation of a homomultimer or heteromultimer complex. Furthermore, interactions can be stable or transient. The terms "multimer," "multimeric complex," or "multimeric protein" include multiple identical or heterogeneous polypeptide monomers. Polypeptides can self-assemble to form multimeric assemblies (i.e., dimers, trimers, hexamers, pentamers, octamers, etc.) formed from the self-assembly of multiple single polypeptide monomers (i.e., "homomultimeric assemblies"). As used herein, "multiple" means two or more. Multimeric assemblies can contain three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more polypeptide monomers. Multimeric assemblies can be used for a variety of purposes and provide a means for developing a wide range of protein "nanomaterials." In addition to finite, cage-like or shell-like protein assemblies, assemblies can also be designed by selecting appropriate target symmetry architectures. The monomeric and / or multimeric assemblies of the present invention can also be used in the design of higher-order assemblies, providing the attendant benefits of hierarchical assembly.The resulting multimeric assemblies are highly ordered materials with excellent rigidity and monodispersity that can form the basis for evolvable functional materials and custom-designed molecular machines with a wide range of applications.

[0045] As used herein, the terms "quantifying," "measuring," "assessing," and "assaying" are used interchangeably and include qualitative and quantitative measurements.

[0046] The term "appropriate conditions" refers specifically to environmental factors such as temperature, motion, other components, and / or "buffer conditions," which specifically refer to the composition of the solution in which the assay is performed, including buffer solutions and / or solutes, such as pH buffers, water, saline, physiological salt solutions, glycerol, preservatives, etc., recognized by those skilled in the art as being appropriate for optimal assay performance.

[0047] "Binding" refers to any interaction, whether direct or indirect. A direct interaction refers to contact between binding partners. An indirect interaction refers to any interaction in which the interacting partners interact in a complex of three or more molecules. The interaction can be fully indirect, via one or more bridging molecules, or partially indirect, where there is direct contact between the partners but is stabilized by one or more additional interactions. Generally, binding domains can be immunoglobulin-based or immunoglobulin-like, or can be based on domains present in proteins such as, but not limited to, microbial proteins, protease inhibitors, toxins, fibronectin, lipocalin, single-stranded antiparallel coiled-coil proteins, or repeat motif proteins. The term "specifically binds," as used herein with respect to an antigen-binding domain, immunoglobulin domain, immunoglobulin-like domain, or antibody domain, refers to a binding domain that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample, also referred to as an "antigen-binding domain" or "antigen-binding protein." For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from more than one species. However, such species cross-reactivity does not alter the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used to describe the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction is dependent on a specific structure (e.g., an antigenic determinant or epitope) present in the chemical species, e.g., an antigen-binding protein recognizes and binds to a specific protein structure rather than proteins in general. If an antigen-binding protein is specific for epitope "A," the presence of a molecule containing epitope A (i.e., free, unlabeled A) in a reaction containing labeled "A" and the antigen-binding protein will reduce the amount of labeled A binding to the antigen-binding protein.As used herein, the term "specificity" refers to the ability of a binding domain, particularly an antigen-binding domain, an immunoglobulin domain or immunoglobulin-like domain, or an immunoglobulin fragment such as a VHH or nanobody, to bind preferentially to one antigen over another, but does not necessarily imply high affinity. Other examples of antigen-binding proteins include synthetic binding proteins, antibody mimetics, or, more specifically, monobodies (see, for example, Sha et al., 2017 for a review). Such monobodies are defined as fibronectin type III domain-containing binding proteins that specifically bind to target proteins, similar to camelid single-domain antibodies (VHHs), due to their overall fold resembling the immunoglobulin fold. Monobodies are the most widely used non-antibody scaffolds, exhibiting an immunoglobulin-like fold due to their fibronectin type III domains. Similar to antibody variable domains, seven antiparallel β-strands are interconnected via an FN3 loop or β-turn on one side of the domain, corresponding to the antigen-binding region similar to the CDRs of an antibody, and via a β-turn on the other side, which can serve as an accessible site for fusing a scaffold protein, to form the antigen-binding chimeric proteins of the present invention. As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope can comprise three amino acids in a spatial conformation unique to the epitope. Typically, an epitope consists of at least four, five, six, or seven such amino acids, and more commonly, at least eight, nine, or ten such amino acids. Methods for determining the spatial conformation of amino acids are known in the art, including, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, the term "conformational epitope" refers to an epitope that comprises amino acids in a spatial conformation that is unique to the folded three-dimensional conformation of a polypeptide. Generally, conformational epitopes are composed of amino acids that are discontinuous in the linear sequence but that cluster together in the folded structure of the protein.Alternatively, a conformational epitope may consist of a linear sequence of amino acids that assumes a conformation specific to the folded, three-dimensional conformation of a polypeptide (not present in the denatured state). In a protein complex, a conformational epitope may consist of amino acids that are discontinuous in the linear sequence of one or more polypeptides, but that assemble when the individual polypeptides fold and associate into a unique quaternary structure. Similarly, a conformational epitope, as used herein, may consist of a linear sequence of amino acids of one or more polypeptides that assemble into a conformation specific to the quaternary structure. The term "conformation" or "conformational state" of a protein generally refers to the range of structures that a protein can adopt at any given time. As will be appreciated by those skilled in the art, determinants of a conformation or conformational state include the primary structure of the protein, as represented by the amino acid sequence of the protein (including modified amino acids), and the environment surrounding the protein. The conformation or conformational state of a protein also refers to structural features such as protein secondary structure (e.g., alpha helices, beta sheets, among others), tertiary structure (e.g., the three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interactions of the polypeptide chain with other protein subunits). Post-translational and other modifications of the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, among others, can affect protein conformation. Furthermore, environmental factors, such as the pH, salt concentration, ionic strength, and osmolality of the surrounding solution, among others, as well as interactions with other proteins and cofactors, can also affect protein conformation. The conformational state of a protein can be determined by functional assays measuring activity or binding to other molecules, or by physical methods, such as X-ray crystallography, NMR, or spin labeling, among others.For a general discussion of protein conformation and conformational states, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

[0048] The term "affinity" as used herein generally refers to the degree to which a ligand binds to a target protein (as defined in detail herein), shifting the equilibrium between the target protein and the ligand, resulting in the formation of a complex. Thus, for example, when an antigen-binding chimeric polypeptide and a ligand are used together at relatively equal concentrations, the high-affinity ligand will bind to the antigen-binding chimeric polypeptide, shifting the equilibrium so that the resulting complex is at a higher concentration. The dissociation constant Kd is widely used to express the affinity between a ligand and a target protein. Generally, the dissociation constant is 10 -5 The dissociation constant is less than 10 -6 Preferably, it is less than 10 -7 More preferably, the dissociation constant is less than 10 -8 Most preferably, the affinity is less than M. Other ways to express the affinity of a ligand to its target protein are the association constant (Ka), the inhibition constant (Ki), or the half-maximal inhibitory concentration (IC 50 ) or half maximum effective concentration (EC 50 It will be appreciated that within the scope of the present invention, the term "affinity" is used in the context of an antigen-binding chimeric protein comprising an Ig domain that binds to a (conformational) epitope of a target protein, more particularly an antigen-binding chimeric protein Ig domain that maintains its "functionality" to bind to its target via the CDR regions of said Ig domain.

[0049] Thus, the term "functional antigen-binding protein" or "conformation-selective antigen-binding domain," as used herein in the context of the present invention, refers to an Ig domain of said chimeric antigen-binding protein that is optionally conformation-selectively functional in binding to its target protein. A binding domain that specifically binds to a particular conformation of a target protein refers to a binding domain that binds to the target in a predetermined subset of conformations with higher affinity than to other possible conformations of the target. As will be apparent to those skilled in the art, a binding domain that selectively binds to a particular conformation of a target will stabilize or maintain the target in this particular conformation. For example, an active state conformation-selective binding domain will preferentially bind to a target in an active conformational state and will not bind or will bind to a lesser extent to a target in an inactive conformational state, thereby increasing affinity for the active conformational state, or vice versa. The terms "specifically bind," "selectively bind," "preferentially bind," and their grammatical equivalents are used interchangeably herein. The terms "conformation-specific" or "conformation-selective" are also used interchangeably herein.

[0050] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule containing an immunoglobulin (Ig) domain that specifically binds to an antigen. An antibody can be an intact immunoglobulin from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. Antibodies are generally tetramers of immunoglobulin molecules. As used herein, the term "immunoglobulin (Ig) domain" refers to the globular region of an antibody chain or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by maintaining the immunoglobulin fold (referred to herein as the Ig fold) characteristic of antibody molecules, which consists of a bilayer sandwich of approximately 7-9 antiparallel β-strands arranged in two β-sheets, optionally stabilized by conserved disulfide bonds. The term "immunoglobulin (Ig) domain" includes "immunoglobulin constant domains" and "immunoglobulin variable domains" (abbreviated "IVDs"), the latter of which refers to immunoglobulin domains essentially composed of four "framework regions", referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, between which are located three "complementarity-determining regions" or "CDRs", referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3", respectively. The general structure or sequence of an immunoglobulin variable domain can be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain (IVD) contains the antigen-binding site and thereby confers antigen specificity to the antibody.According to the IMGT classification, immunoglobulin variable domains or V-domains contain approximately 100 AA and consist of nine antiparallel β-strands (A, B, C, C', C", D, E, F, and G) connected by β-turns (AB, CC', C", D, DE, and EF) and three loops (or CDRs) (BC, C'C", and FG), forming a two-sheet sandwich [ABED][GFCC'C"] (Figure 25, revised version based on Lefranc, 2014). The sheets are packed tightly together by hydrophobic interactions to form a hydrophobic core and are interconnected by a disulfide bridge between the first highly conserved cysteine ​​(Cys1) in β-strand B (of the first sheet) and the second equally conserved cysteine ​​(Cys2) in β-strand F (of the second sheet). The unique numbering system of the IMGT® definition used in the present invention provides a precise and unambiguous CDR-IMGT division, in contrast to the CDRs described in the literature. For other numbering systems, see, for example, Kabat (Kabat et al., 1991) or Chothia (Chothia and Lesk, 1987). In V domains, CDR1-IMGT comprises positions 27-38, CDR2-IMGT comprises positions 56-65, and CDR3-IMGT comprises positions 105-117 (Lefranc, 2014). The "exposed region" or "exposed loop" of an Ig domain of the present invention refers to a region or polypeptide chain exposed on the surface of the protein. In Ig domains, the exposed region or loop is preferably a β-turn, most preferably a β-turn as defined by Lefranc (2014). Although CDRs are also considered "loops" according to the IMGT definition, they are not considered to be preferred candidates for the "exposed region" of the present invention with an accessible site for fusing a scaffold, since fusing a scaffold to the CDRs would most likely result in a loss of antigen binding, making it impossible to obtain a functional antigen-binding chimeric protein.

[0051] The term "immunoglobulin domain" of the present invention also includes an "immunoglobulin single variable domain" (abbreviated as "ISVD"), which is synonymous with the term "single variable domain," and refers to a molecule in which an antigen-binding site is present in and formed by a single immunoglobulin domain. This means an immunoglobulin single variable domain that is different from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Generally, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site. In light of the above definition, in the case of the antigen-binding domain of a conventional four-chain antibody (e.g., an IgG, IgM, IgA, IgD, or IgE molecule known in the art), or an Fab fragment, F(ab')2 fragment, Fv fragment such as a disulfide-linked Fv fragment or scFv fragment derived from such a conventional four-chain antibody, or a diabody (all known in the art), binding to each epitope of an antigen is not mediated by one (single) immunoglobulin domain, but by a pair (associated) immunoglobulin domain such as a light chain variable domain and a heavy chain variable domain; i.e., the VH-VL pair of immunoglobulin domains binds to each epitope of the antigen together, and therefore these antigen-binding domains are not normally considered to be immunoglobulin single variable domains. In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without being paired with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain, and therefore the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs.Thus, the single variable domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, so long as it is possible to form a single antigen-binding unit (i.e., a functional antigen-binding unit essentially composed of the single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form the functional antigen-binding unit). In one embodiment of the present invention, the immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g., a VH sequence), and more specifically, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy-chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), a nanobody (as defined herein, including but not limited to a VHH), or any other single variable domain or any suitable fragment of any one of them. In particular, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx NV. For a general description of Nanobodies, see the detailed description below and the prior art cited herein, e.g., WO2008 / 020079.

[0052] Immunoglobulin domains as referred to herein also include "VHH domains," which are also referred to as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, and were originally described as antigen-binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al (1993) Nature 363:446-448). The term "VHH domain" was chosen to distinguish these variable domains from the heavy chain variable domains (referred to herein as "VH domains") present in conventional four-chain antibodies and the light chain variable domains (referred to herein as "VL domains") present in conventional four-chain antibodies.For a detailed description of VHHs and nanobodies, see the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001) and the following patent applications cited as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 in the name of Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 in the name of Unilever; WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016 and WO03 / 055527 to VIB; WO03 / 050531 to Algonomics NV and Ablynx NV; WO01 / 90190 to the National Research Council of Canada; WO03 / 025020 (=EP1433793) to the Institute of Antibodies; and Ablynx See WO04 / 041867, WO04 / 041862, WO04 / 041865, WO04 / 041863, WO04 / 062551, WO05 / 044858, WO06 / 40153, WO06 / 079372, WO06 / 122786, WO06 / 122787 and WO06 / 122825 in the name of Ablynx NV, as well as other published patent applications in the name of Ablynx NV. As described in these documents, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) can be characterized by the presence of one or more "hallmark residues" in one or more of the framework sequences. For a detailed description of Nanobodies, including humanization and / or camelization and other modifications, parts or fragments, derivatives or "nanobody fusions" of Nanobodies, multivalent constructs (including some non-limiting examples of linker sequences), and various modifications to extend the half-life of Nanobodies and formulations thereof, see, e.g., WO08 / 101985 and WO08 / 142164.

[0053] "Domain antibodies", also known as "Dabs", "domain antibodies" and "dAbs" (the terms "domain antibody" and "dAb" are trademarks used by the GlaxoSmithKline group of companies), are described, for example, in EP 0368684, Ward et al. (Nature 341:544-546, 1989), Holt et al. (Tends in Biotechnology 21:484-490, 2003) and WO 03 / 002609, and further in, for example, WO 04 / 068820, WO 06 / 030220, WO 06 / 003388 and other published patent applications in the name of Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domain of four-chain antibodies from mammals other than camelids, in particular humans. To bind to an epitope as a single antigen-binding domain, i.e., without pairing with a VL or VH domain, respectively, it is necessary to specifically select for such antigen-binding properties, for example, by using a library of human single VH or VL domain sequences. Domain antibodies, like VHHs, have a molecular weight of about 13 to about 16 kDa, and if derived from a fully human sequence, do not need to be humanized for use in human therapeutics, for example. Single variable domains derived from certain species of sharks can also be used (e.g., so-called "IgNAR domains"; see, for example, WO05 / 18629).

[0054] Immunoglobulin single variable domains such as domain antibodies (including VHH domains and humanized VHH domains) and nanobodies are in vivo mature macromolecules when produced, but can be further affinity matured by introducing one or more mutations into the amino acid sequence of one or more CDRs so that the affinity of the resulting immunoglobulin single variable domain for its respective antigen is improved compared to the respective parent molecules. The affinity-matured immunoglobulin single variable domain molecules of the invention can be produced by methods known in the art, for example, as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas et al. (Proc. Nat. Acad. Sci. USA 91:3809-3813, 1994), Shier et al. (Gene 169:147-155, 1995), Yelton et al. (Immunol. 155:1994-2004, 1995), Jackson et al. (J. Immunol. 154:3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226:889 896, 1992), and Johnson and Hawkins (Affinity maturation of antiodies using phage display, Oxford University Press, 1996). The process of designing / selecting and / or producing a polypeptide starting from an immunoglobulin single variable domain, such as a domain antibody or nanobody, is also referred to herein as "formatting" said immunoglobulin single variable domain, and the immunoglobulin single variable domains that make up a polypeptide are referred to as being "formatted" or "included in the format of" said polypeptide. Examples of ways in which immunoglobulin single variable domains can be formatted, and examples of such formats, for example to avoid glycosylation, will be clear to those skilled in the art from the disclosure herein.

[0055] Immunoglobulin single variable domains, such as domain antibodies (including VHH domains) and nanobodies, can be humanized, i.e., the degree of sequence identity with the closest human germline sequence can be increased. In particular, humanized immunoglobulin single variable domains, such as nanobodies (including VHH domains), can be immunoglobulin single variable domains as generally defined in the preceding paragraph, but with the presence of at least one amino acid residue (particularly at least one framework residue) that is and / or corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions can be identified by comparing the sequences of the framework regions of a native VHH sequence with the corresponding framework sequences of one or more closely related human VH sequences. One or more (or a combination of) the potentially useful humanizing substitutions thus determined can then be introduced into the VHH sequence (using any method known per se, as described in detail herein), and the resulting humanized VHH sequence can be tested for affinity for the target, stability, ease and level of expression, and / or other desirable properties. Thus, by reasonable trial and error, one skilled in the art can determine other suitable humanizing substitutions (or suitable combinations thereof). Furthermore, based on the above, (the framework regions of) immunoglobulin single variable domains such as nanobodies (including VHH domains) can be partially or fully humanized. It should be noted that immunoglobulin single variable domains and, in the broader sense, the antigen-binding chimeric proteins of the present invention are not limited to any particular biological source or any particular production method.For example, immunoglobulin single variable domains, and in particular, antigen-binding chimeric proteins of the present invention, can generally be produced by (1) isolating a VHH domain of a naturally occurring heavy chain antibody and further manipulating the sequence to obtain an antigen-binding chimeric protein; (2) expressing a nucleotide sequence encoding the naturally occurring VHH domain in a format fused to said scaffold protein of the antigen-binding chimeric protein; (3) "humanizing" the naturally occurring VHH domain and / or scaffold protein, or by using a nucleotide sequence encoding such a humanized VHH domain and / or scaffold protein and / or antigen-binding chimeric protein. (4) by "mutating" a naturally occurring VHH domain to weaken its binding to a pre-existing antibody, or by carefully manipulating the scaffold protein fusion site to obtain an antigen-binding chimeric protein of the invention that has weakened binding to a pre-existing antibody compared to a naturally occurring VHH; (5) by "camelizing" a naturally occurring VH domain from any animal species, particularly a mammalian species such as human, for subsequent fusion with the scaffold protein; or (6) by using synthetic or semi-synthetic techniques for producing proteins, polypeptides or other amino acid sequences known per se. To generate a polypeptide with weakened binding to a pre-existing antibody (see, for example, WO2012 / 175741 and WO2015 / 173325), appropriate mutations, particularly substitutions, can be introduced during the humanization process, for example, at at least one of positions 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108. Alternatively, positions sensitive to binding to a pre-existing antibody may be sterically shielded by designing a fusion with a scaffold protein to interfere with binding to the antigen-binding chimeric protein.

[0056] As an alternative to immunoglobulin domains, the Ig superfamily or "Ig-like domains" are also present in many proteins, and indeed constitute domains very similar in sequence and structure to immunoglobulin domains and Ig folds, respectively. However, to distinguish them from the domains of immunoglobulin antibodies themselves, Ig-like domains are used. Ig folds are known to be particularly effective at interacting with other proteins rather than being special in antigen recognition, and are widely used. Immunoglobulin-like domains can be classified into V, C1, C2, and I according to their sequence patterns. Monobodies, for example, contain immunoglobulin-like domains.

[0057] As used herein, the term "detectable label," "label," or "tag" refers to a detectable label or tag that allows for the detection, visualization, and / or isolation, purification, and / or immobilization of the isolated or purified (poly)peptides described herein, and is intended to include any label / tag known in the art for these purposes. Particularly preferred are affinity tags such as chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6xHis or His6), Strep-tag®, Strep-tag II®, and Twin-Strep-tag®; solubilization tags such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags such as FLAG-tag; epitope tags such as V5-tag, myc-tag, and HA-tag; fluorescent labels or tags (i.e., fluorochromes / fluorophores) such as fluorescent proteins (e.g., GFP, YFP, RFP, etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin, and cyanine); luminescent labels or tags such as luciferase; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, β-galactosidase, urease, or glucose oxidase). Combinations of any of the above labels or tags are also included. The antigen-binding chimeric protein may be fused or linked to, for example, a half-life extension module, or may function as a half-life extension module itself. Such modules are known to those skilled in the art and include, for example, albumin, albumin-binding domains, immunoglobulin Fc regions / domains, immunoglobulin-binding domains, FcRn-binding motifs, and polymers. Particularly preferred polymers include polyethylene glycol (PEG), hydroxyethyl starch (HES), hyaluronic acid, polysialic acid, and PEG-mimetic peptide sequences. Modifications to prevent aggregation of isolated (poly)peptides are also known to those skilled in the art, such as substituting one or more hydrophobic amino acids, preferably substituting one or more hydrophilic amino acids for surface-exposed hydrophobic amino acids.In one embodiment, the isolated (poly)peptide or immunogenic variant thereof or immunogenic fragment of either of these has up to 10, 9, 8, 7, 6, 5, 4, 3 or 2, preferably 5, 4, 3 or 2, hydrophobic amino acids, preferably surface-exposed hydrophobic amino acids, substituted with hydrophilic amino acids, preferably such substitutions do not impair other properties of the isolated (poly)peptide, such as its immunogenicity or antigen-binding functionality.

[0058] For purposes of the present invention, a "patient" or "subject" refers to any organism, such as a vertebrate, particularly any mammal, including humans and other mammals, including, for example, rodents, rabbits, cows, sheep, horses, dogs, cats, llamas, pigs, or non-human primates (e.g., monkeys). Rodents include mice, rats, hamsters, guinea pigs, or chinchillas. In one embodiment, the subject is a human, rat, or non-human primate. Preferably, the subject is a human. In one embodiment, the subject is one who has or is suspected of having a disease or disorder, and is also referred to herein as a "patient."

[0059] As used herein, the term "prevent" can refer to halting / inhibiting the onset of a disease or disorder (e.g., by prophylactic treatment). The term can also refer to delaying the onset of a disease or disorder, reducing the frequency of its symptoms, or reducing the severity of accompanying symptoms (e.g., by prophylactic treatment). The terms "treatment" or "therapeutic" or "treating" can be used interchangeably and are defined as a therapeutic intervention that delays, interrupts, deterrents, suppresses, arrests, reduces, or reverses the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily imply the complete elimination of all signs, symptoms, condition, or disorder associated with a disease.

[0060] Detailed Description This application proposes a novel concept for the design of rigidly fused chimeric proteins containing antigen-binding domains. Novel antigen-binding chimeras are obtained by creating fusions of antigen-binding domains with scaffold proteins. The scaffold proteins disrupt the topology of the antigen-binding protein, but surprisingly, the fusions retain their typical fold and function to specifically bind antigens or target proteins in the same way as the unfused antigen-binding domains. This concept is based on specific examples in which the unique properties of immunoglobulin or immunoglobulin-like domains are exploited by fusing them to scaffold proteins to create novel and unique antigen-binding chimeras that exhibit a more rigid and inflexible linkage. The classical method for linking polypeptide components that are generally not linked in their native state is by linking their respective amino (N)- and carboxyl (C)-termini directly or via peptide bonds to form a single, contiguous polypeptide. These fusions are often achieved via flexible linkers, or at least flexibly linked, i.e., the fusion partners are not positioned or conformed in a stable manner relative to each other. As shown in Figure 1, simple linear concatemerization, linking proteins via their N- and C-termini, facilitates fusion but is unstable and prone to degradation, making it unsuitable for therapeutic applications in some cases. In contrast, the rigid chimeric / fusion proteins described herein have one or more fusion or junction points within the primary topology of two or more proteins, with at least one inflexible fusion point (Figure 1). Essentially, the present invention encompasses antigen-binding chimeric proteins in which several fusions are engineered to prevent one protein from rotating or bending relative to its fusion partner. The presence of several fusions within the same chimera results in improved rigidity in the novel chimeras of the present invention, a result of perfectly engineered fusion sites that allow the fusions to maintain their antigen-binding domain folding and function in binding to their antigen targets. Protein rigidity is actually inherent in the (tertiary) structure of the protein (in this case, the novel chimera). It has been reported that enhanced rigidity can be achieved by altering the topology of known protein folds (King et al., 2015).Thus, the rigidity of fusions created with the antigen-binding chimeric proteins of the present invention provides sufficient rigidity to "orient" or "fix" the target, but generally this rigidity will remain lower than the rigidity of the target or antigen itself. The fact that the rigid antigen-binding chimeric proteins of the present invention continue to maintain their antigen-binding functionality is a surprising result, as disruption of the primary topology was thought to alter domain or protein folding, significantly altering the tertiary topology and antigen binding. This disruption of the primary topology has been demonstrated herein to open new avenues in the field of antibodies and their target proteins, as it does not affect antigen binding. The present invention relates to the novel combination of high-affinity and / or conformation-selective antigen binding with the provision of a unique next-generation fusion technology to enable noncovalent targeting of proteins. This novel type of antigen-binding chimeric protein lends itself to several useful applications, depending on the type of scaffold protein used to generate the chimera. By overcoming the frequent problem of small proteins being unable to obtain high-resolution structures due to particle size limitations, preferential orientation, and fragment alignment restrictions, this technology offers many advantages, is easily applicable to structural biology, and facilitates cryo-EM and X-ray crystallography. With the availability of rigid chaperone tools today, it is entirely feasible to use these tools to develop more robust and improved therapeutic and diagnostic molecules through structure-based drug design and structure-based screening of novel compounds. This next-generation fusion technology is expected to lead to breakthroughs in structural biology. Indeed, when these tools are used for conformation-selective recognition of antigens or targets, they can be applied in binding modes that stabilize targets in active conformations, more specifically, functional conformations such as agonist, partial agonist, or biased agonist conformations. Furthermore, several fusion protein drugs, including Enbrel® (tumor necrosis factor / Fc-IgG1) and Nplate® (thrombopoietin / Fc-IgG1), are known and have been approved by the FDA.The antigen-binding chimeras of the present invention may offer a unique solution to address a well-known obstacle in the therapeutic development of single-domain antibodies: binding to existing antibodies. Furthermore, the antigen-binding chimeric proteins of the present invention also offer novel solutions in the field of biologics and protectants in the crop protection industry. It is clear that the development of camelid-derived binding domains specific for essential molecules of pests and pathogens would provide a highly specific mode of action while minimizing risks to wildlife, honeybees, producers, and consumers. Such protectants developed from the antigen-binding chimeras of the present invention offer other advantages while maintaining their cost-effective large-scale production methods.

[0061] The chimeric antigen-binding proteins of the present invention can also utilize antigen-binding domains based on Nbs, which have been described to specifically stabilize druggable signaling conformations to enable the screening of pathway-selective agonists.

[0062] In view of such rapid technological advances in biotechnology, it is expected that the present invention will promote the creation of novel protein therapeutic drugs and lead to improvements in the performance of current protein pharmaceuticals.

[0063] In a first aspect, the present invention relates to an antigen-binding chimeric protein in which an antigen-binding domain is fused to a scaffold protein, where the scaffold protein is linked to the antigen-binding domain by fusing at least one or more accessible amino acid sites in the antigen-binding domain fold, thereby disrupting the topology of the antigen-binding domain. The antigen-binding chimeric protein is further characterized by maintaining its antigen-binding functionality in its native or wild-type form, as compared to an antigen-binding domain not fused to the scaffold protein. Thus, in one embodiment, the antigen-binding chimeric protein is a conformationally selective binding domain. One embodiment provides an antigen-binding chimeric protein in which the antigen-binding domain is fused to a scaffold protein such that the scaffold protein "disrupts / blocks" the antigen-binding domain in its topology. Generally, the "topology" of a protein refers to the orientation of regular secondary structures relative to each other in three-dimensional space. Protein folds are primarily determined by polypeptide chain topology (Orengo et al., 1994). Thus, at the most basic level, "primary topology" is defined as the interconnection of secondary structural elements (SSEs) involved in protein fold recognition motifs and, therefore, secondary and tertiary protein / domain folding. In other words, from the perspective of protein structure, true topology, or primary topology, is the interconnection of SSEs; that is, if you imagine that you can take the N- and C-termini of a protein chain and pull it straight, the topology remains the same regardless of the protein fold. In this case, the protein fold is referred to as a tertiary topology, by analogy with the primary and tertiary structures of proteins (see also Martin, 2000). Thus, by introducing a scaffold protein fusion, the antigen-binding domain of the antigen-binding chimeric protein of the present invention is disrupted from its primary topology, yet, surprisingly, the antigen-binding domain maintains its tertiary structure and thus its functional antigen-binding ability.

[0064] The term "scaffold protein" refers to any type of protein whose structure allows it to be fused to another protein, particularly an antigen-binding domain, as described herein. Such a "scaffold," "junction," or "fusion partner" protein preferably has at least one exposed region in its tertiary structure to provide at least one accessible site for cleavage as a fusion point for the antigen-binding domain. By using a scaffold polypeptide to bind to an antigen-binding domain, the antigen-binding chimeric protein is placed in a docked configuration, which can increase mass, provide symmetry, and / or provide a label, and / or add additional antigen-binding sites, and / or extend half-life, and / or reduce immunogenicity, and / or improve or add functionality to the antigen-binding domain. Thus, depending on the type of scaffold protein used, different purposes for the resulting antigen-binding chimeric protein are expected. The type and nature of the scaffold protein are not critical in that any protein can be used, and scaffold proteins fused to the antigen-binding domain, such as the antigen-binding chimeric proteins of the present invention, may be useful in a variety of applications depending on their structure, size, function, or presence. Because the structure of the scaffold protein affects the final chimeric structure, those skilled in the art should utilize known structural information about the scaffold protein and consider reasonable predictions when selecting a scaffold. Examples of scaffold proteins are provided in the Examples section of this application, but proteins that can be used as scaffold proteins to create the antigen-binding chimeric proteins of the present invention are not limited, and such proteins include, in particular, enzymes, membrane proteins, receptors, adaptor proteins, chaperones, transcription factors, nuclear proteins, and antigen-binding proteins themselves (e.g., nanobodies, in particular). In a preferred embodiment, the three-dimensional structure of the scaffold protein is known or can be predicted by those skilled in the art, so that accessible sites for fusing an antigen-binding domain can be determined by those skilled in the art.

[0065] The novel chimeric proteins are fused in a unique manner so that the junctions are flexible, loose, and do not form weak links / regions within the chimeric protein structure. A convenient method for linking or fusing two polypeptides is to express a fusion protein from a recombinant nucleic acid molecule in which a first polynucleotide encoding a first polypeptide is operably linked to a second polynucleotide encoding a second polypeptide, using conventional methods. Meanwhile, the recombinant nucleic acid molecules of the present invention also take into consideration disruption of the topology of the antigen-binding domain by the scaffold when designing a gene fusion to express the antigen-binding chimeric protein. Thus, in one embodiment, the antigen-binding chimeric protein is encoded by a chimeric gene formed by recombining a portion of a gene encoding the antigen-binding domain with a portion of a gene encoding a scaffold protein, and the encoded scaffold protein disrupts the primary topology of the encoded antigen-binding domain at one or more accessible sites of the domain via at least two or more direct fusions or fusions via encoded peptide linkers. Thus, polynucleotides encoding the polypeptides to be fused are fragmented or recombined to provide chimeric antigen-binding proteins that provide a rigid, inflexible link, connection, or fusion between the proteins. Novel chimeras are generated by fusing a scaffold protein to an antigen-binding domain in such a way that the primary topology of the antigen-binding domain is disrupted; i.e., the amino acid sequence of the antigen-binding domain is disrupted at accessible sites and joined to accessible amino acids of the scaffold protein, optionally disrupting this amino acid sequence as well. The junction is formed intramolecularly, i.e., internally within the amino acid sequence (see Examples and Figures). Thus, the recombinant fusions of the present invention are not simply N- or C-terminally fused, but rather chimeras that contain at least one internal fusion site, either directly or via a linker peptide.When using a circularly permuted scaffold to prepare an antigen-binding chimeric protein, the amino acid sequence of the scaffold protein is altered by linking the N-terminus and C-terminus, then cleaving or separating the amino acid sequence at another site in the scaffold protein sequence that corresponds to an accessible site in its tertiary structure, and fusing it with the amino acid sequence of the antigen-binding domain portion. The linking of the N-terminus and C-terminus to obtain circular permutation may be by direct fusion, via a linker peptide, or by peptide-bonding the ends after short deletion of regions near the N-terminus and C-terminus.

[0066] The terms "accessible site," "fusion site," "fusion point," "linking site," or "exposed site" are used interchangeably herein and refer to structurally accessible amino acid sites in a protein sequence, preferably on the surface of the protein or exposed on the surface. Those skilled in the art will be able to determine these sites. The antigen-binding site of an antigen-binding domain often refers to exposed regions, such as the CDRs of an Ig domain. However, disruption of these sites to fuse the antigen-binding domain to a scaffold protein may reduce antigen-binding ability, making them unsuitable for the antigen-binding chimeric proteins of the present invention. Therefore, "accessible site" and "exposed region" as used herein, such as a "loop" or "β-turn," refer to sites and regions other than the antigen-binding site or region, and therefore other than the CDRs. The N-terminus or C-terminus of a protein are also often "loose" ends of the protein's three-dimensional structure and therefore accessible from the surface. The N- or C-terminus of a protein can also be considered an accessible site in the chimera of the present invention, provided that at least one other accessible site in the antigen-binding domain is used for fusion, resulting in a disruption / insertion at this accessible site, disrupting the topology and providing rigidity to the novel chimera. That is, accessible sites can include the amino- and / or carboxy-terminal sites of a protein, but chimeras cannot be formed solely by fusion from accessible sites consisting of the N- or C-termini. At least one site in the antigen-binding domain is used for fusion with a scaffold protein, disrupting the topology of a conventional, known domain fold. Thus, in one embodiment, the at least one accessible site, if present, is other than the N- and / or C-terminal site of the domain and / or does not include the N- or C-terminal site of the domain. In a specific embodiment, the at least one site is other than the N- or C-terminal amino acid of the domain.In another embodiment, when at least two or more sites are used for fusion to the scaffold protein, the accessible site can be at the N- or C-terminal site of the antigen-binding domain. The scaffold protein is also fused via an accessible site visible from its tertiary structure, in which case in one embodiment, the at least one site is other than the N- or C-terminus of the scaffold protein, and in an alternative embodiment, the at least one site is the N- or C-terminus of the scaffold.

[0067] In certain embodiments, the antigen-binding chimera comprises an N-terminal fragment of the scaffold protein fused to a split in the exposed region of the antigen-binding domain and a C-terminal fragment of the scaffold protein fused to the C-terminus of the antigen-binding domain.

[0068] In certain embodiments of the present invention, fusion can be direct or via a linker peptide, with the fusion site perfectly designed to result in a rigid, inflexible fusion protein. In addition to the location of the accessible site selected, the length and type of linker peptide also contribute to the rigidity of the resulting chimeric protein. Within the context of the present invention, the polypeptides constituting the antigen-binding chimeric protein can be fused to each other directly, via a peptide bond, or indirectly, where the two polypeptides are linked via a short peptide linker. Preferred "linker molecules," "linkers," or "short polypeptide linkers" are peptides up to 10 amino acids in length, more typically 4 amino acids in length; although 3 amino acids are common, 2 amino acids are preferred, and 1 amino acid is even more preferred, to provide the desired rigidity at the fusion junction at the accessible site. Non-limiting examples of suitable linker sequences are provided in the Examples section; however, these can be selected randomly; linkers have been successfully selected to maintain a consistent distance between structural domains while preserving the individual functions (e.g., antigen binding) of the fusion partners. In embodiments involving the use of rigid linkers, these are generally known to exhibit unique conformations due to their alpha helical structure or the inclusion of multiple proline residues. Flexible linkers may also be suitable, preferably limited to 1-4 amino acids in length, but in many situations, rigid linkers separate functional domains more efficiently than flexible linkers.

[0069] In one embodiment, the accessible sites of the antigen-binding domain are located in exposed regions of the domain fold, which can be considered as stretches of amino acids with low rigidity and are located primarily on the surface and structural ridges of the protein, preferably as loops or β-turns in the protein structure.

[0070] In a specific embodiment, the antigen-binding chimeric protein comprises an antigen-binding domain consisting of at least seven antiparallel β-strands arranged primarily as a β-sheet and at least three β-turns, also considered exposed regions. In one embodiment, the antigen-binding domain consisting of at least seven antiparallel β-strands and at least three β-turns comprises an immunoglobulin-like domain, particularly the antigen-binding domain of a monobody. Monobodies are synthetic binding proteins constructed using the fibronectin type III domain (FN3) as a molecular scaffold. The native FN3 scaffold consists of 94 amino acids and has a molecular weight of approximately 10 kDa, similar to the size of a single variable domain of an antibody. These are based on the structure of human fibronectin, more specifically, its tenth extracellular type III domain. This domain has a structure similar to that of an antibody variable domain, with seven β-strands and three exposed loops on each side. In another specific embodiment, the antigen-binding chimeric protein comprises an antigen-binding domain that is an immunoglobulin (Ig) domain. Immunoglobulin (Ig) domains are protein domains consisting of a bilayer sandwich of seven to nine antiparallel β-strands arranged in two β-sheets with a Greek key topology, consisting of approximately 125 amino acids. The variable (V) and constant (C) subunits of immunoglobulins differ in the number and regularity of the β-strands forming the bilayer. The C domain is composed of seven β-strands arranged with four strands forming one β-sheet and three strands forming the second β-sheet, while the V domain contains nine β-strands instead of seven. The remaining two strands of the V domain are inserted into one edge of the β-sheet and constitute a second hypervariable region directly involved in the formation of the antigen recognition site, making them functionally important. By disrupting the topology of the antigen-binding domain to obtain the antigen-binding chimeric proteins of the present invention, the resulting chimeric proteins unexpectedly maintained their immunoglobulin or immunoglobulin-like fold and their functionality in binding to target proteins or antigens was also maintained.

[0071] With respect to proteins containing an antigen-binding domain or an Ig domain to which a scaffold protein is fused, the antigen or target of the antigen-binding or Ig domain may be of any type. Therefore, the type of target is not critical to the present invention, and any epitope that specifically binds to the antigen-binding site or CDR of the antigen-binding domain or Ig or Ig-like domain, respectively, can be considered a valid target protein. The target can be, by way of non-limiting example, a monomeric protein, another macromolecular structure, a multimer, a protein complex, or a transient protein-protein interaction. The target can have any functionality, including, but not limited to, enzymes, membrane proteins such as GPCRs, ion channels, and, in particular, nuclear receptors and other receptor proteins, that can be targeted by the antigen-binding domain. Alternatively, the source of the target can be of any origin, including, but not limited to, those derived from bacteria, viruses, or insects, in addition to humans, mammals, or animals.

[0072] In one embodiment, the antigen of the antigen-binding domain or Ig / Ig-like domain is other than the scaffold protein fused to the antigen-binding domain or Ig / Ig-like domain to form the antigen-binding chimeric protein. When a scaffold protein is used to fuse or link to the antigen-binding domain or Ig domain as described herein, it is more preferred that the novel antigen-binding chimeric protein does not specifically bind to the scaffold protein present in its monomeric native or fused form. Alternatively, the present application discloses a composition of antigen-binding chimeric proteins, comprising a first antigen-binding chimeric protein and a second antigen-binding chimeric protein described herein, wherein the antigen-binding domain of the second antigen-binding chimeric protein specifically binds to the scaffold protein of the first antigen-binding chimeric protein. To avoid aggregation or chain reaction of the antigen-binding chimeric protein by binding to its own scaffold, the scaffold of the second antigen-binding chimeric protein is different from the scaffold protein of the first antigen-binding chimeric protein. "Different" for the purposes of the present invention in this application means that the antigen-binding domain of the second antigen-binding chimeric protein no longer binds to said scaffold protein portion of the second antigen-binding chimeric protein as a result of amino acid mutations, deletions, insertions or substitutions or modifications of the scaffold protein of the second antigen-binding chimeric protein. Another embodiment relates to the above compositions of antigen-binding chimeric proteins in a complex bound to its antigen or target protein.

[0073] In an alternative embodiment, the antigen-binding chimeric protein is expressed as a rigid fusion protein comprising i) a conserved N-terminal amino acid sequence of an immunoglobulin or Ig-like domain, ii) a scaffold protein, and iii) an immunoglobulin domain sequence lacking the conserved N-terminal amino acid sequence of i), wherein i) and iii) are concatemerized with the scaffold protein of ii). In a preferred embodiment, the rigid fusion protein comprises a conserved N-terminal amino acid sequence that is the conserved N-terminal domain of the FR1 region, which comprises a conserved consensus sequence consisting of residues as set forth in SEQ ID NO: 1, or a homologous sequence thereof 11 to 15 residues in length (e.g., at the end of the N-terminal portion of residues 11 to 15 of SEQ ID NO: 1, i.e., near the first β-turn).

[0074] In another embodiment, the antigen-binding domain of the antigen-binding chimeric protein comprises a helical secondary structure. In particular, the antigen-binding domain may be an alphabody, which is a portion of an antigen-binding domain that serves as an antibody mimetic, or a DARPin, which is an antibody mimetic containing an α-helix that can be used for fusion as described herein to create an antigen-binding chimeric protein, even though it has a structure very different from that of immunoglobulins. However, because the antigen-binding properties of antibody mimetics are synthetically created or predicted binding sites and do not exist in nature, they may be less accessible than, for example, the in vivo mature antigen-binding site of a single-domain antibody. Furthermore, compared to fusions that contain β-turns to disrupt the topology, methods for creating rigid fusions using the helical structure or coiled-coil structure may be more complicated.

[0075] The simplest example of an "exposed region" of an Ig domain is an exposed loop, preferably a β-turn, which is an exposed loop located at the ridge of a β-sheet sandwich three-dimensional structure.

[0076] In other embodiments, the exposed region of the antigen-binding domain comprises a β-turn as defined by IMGT (Lefranc, 2014), and the scaffold protein is inserted into or fused to an exposed region of an immunoglobulin (variable) domain that is: a. the first β-turn connecting β-strands A and B of the antigen-binding domain, or b. a β-turn connecting β-strands C and C' of the antigen-binding domain, or c. a β-turn connecting β-strands C" and D of the antigen-binding domain, or d. a β-turn connecting β-strands D and E of the antigen-binding domain, or e. a β-turn connecting β-strands E and F of the antigen-binding domain. In a preferred embodiment, the accessible site is located in the exposed region of an AB β-turn connecting the A and B β-strands of an Ig domain. Alternatively, the accessible site is located in the exposed region defined by a CC' β-turn connecting the C and C' β-strands of an Ig domain. Another embodiment comprises an exposed region with an accessible site in a C"D β-turn or an EF β-turn.

[0077] In fact, these are surface loops that connect β-strands A and B, C and C', C" and D, or E and F, respectively, to form a typical sandwich β-sheet and provide the immunoglobulin fold. Accessible sites are most preferably located in exposed regions, loops, or β-turns so that the CDRs of an Ig domain maintain their ability to bind to the epitope of the target protein. The CDRs themselves can also be considered exposed regions, and therefore theoretically accessible sites. However, an antigen-binding chimeric protein will only be functional, and therefore antigen-binding, if it is able to bind the target protein, which is not possible when amino acids of the CDRs are used as accessible sites for fusion.

[0078] In another embodiment, the antigen-binding domain comprises an immunoglobulin-like domain, for example, more particularly a monobody immunoglobulin-like domain, and the scaffold protein is inserted in the first β-turn connecting β-strands A and B of the Ig-like domain of a monobody, or in the β-turn connecting β-strands C and D of the Ig-like domain, or in the β-turn connecting β-strands E and F of the Ig-like domain, as defined according to the structures annotated in Koide et al. (2012), similar to the options described above for Ig domains. This nomenclature has been adopted based on the VHH Ig-fold annotation.

[0079] In another embodiment, the scaffold protein comprises a circular permutation. In a preferred embodiment, the circular permutation of the scaffold protein is introduced at the N-terminus and / or C-terminus of the scaffold protein, most preferably between the N-terminus and C-terminus of the scaffold protein. Another embodiment provides a scaffold protein comprising at least two antiparallel β-strands.

[0080] In one embodiment, a fusion protein is obtained that links an immunoglobulin or antigen-binding domain to a scaffold (via two peptide bonds or two short linkers) by fusing it to a circularly permuted scaffold protein at a truncated, accessible site in its sequence that corresponds to an exposed region of the structure, and disrupting the primary topology of the Ig domain or antigen-binding domain at a truncated, accessible site in its sequence that corresponds to an AB β-turn, provided that the exposed or accessible site is other than the N- or C-terminus. Thus, in certain embodiments where circular permutations of the scaffold protein are introduced at the N- and C-termini (as shown in Figure 2), the antigen-binding protein fragment and scaffold protein sequence as a whole can be recombinantly fused together (as shown in Figure 8). In a specific embodiment, the rigidity of the chimera is enhanced by further creating reinforcing disulfide bridges formed by cysteine ​​residues located within the antigen-binding domain or Ig domain, preferably at the ends of β-strand A and β-strand G, near the accessible sites of the AB β-turn. In one embodiment, the antigen-binding domain and the scaffold are further linked by a disulfide bond to improve the rigidity of the antigen-binding chimeric protein. These sites are most preferably the last amino acids of the Aβ strand (e.g., around residues 11-15 of SEQ ID NO: 1), which are replaced with cysteine, and one of the last amino acids of the Ig domain is also mutated to cysteine ​​(see Figure 10).

[0081] In another embodiment, the fusion protein linking an immunoglobulin to a scaffold (via two peptide bonds or two short linkers) is obtained by fusing the scaffold protein with a circularly permuted protein at its accessible truncated site in an exposed region of the structure (other than the N- or C-terminus) located in its sequence, disrupting the Ig domain topology at the accessible truncated site in the CC' β-turn. Thus, in one particular embodiment where circular permutation of a scaffold protein is introduced by linking the N- and C-termini (as shown in Figure 22), the Ig protein fragment and scaffold protein sequence as a whole can be recombinantly fused together (as shown in Figure 8).

[0082] In another embodiment, the scaffold proteins described herein are obligate dimers or multimers (e.g., 12-mers, which may be homo- or hetero-oligomers) and / or coat proteins, virus-like particle proteins, or fragments thereof. The coat proteins or virus-like particle proteins form multimers that self-assemble into a symmetrical structure on which the fused Ig domains are displayed.

[0083] In another embodiment, a fusion or chimeric protein is obtained by fusing the scaffold protein at its N-terminal amino acid and disrupting the antigen-binding domain or Ig / Ig-like domain topology at an accessible site in the ABβ-turn, linking the immunoglobulin to the scaffold (via three peptide bonds or three short linkers). The scaffold protein also requires a structurally accessible site in its sequence that can be cleaved to reconnect with the ABβ-turn site, allowing fusion to β-strand B of the Ig domain. The second accessible site in the Ig domain can be provided, for example and without limitation, by its C-terminal amino acid, which is then fused to the remainder of the scaffold protein (see Figure 11). Thus, in certain embodiments where the scaffold protein is fused to two different accessible sites in the antigen-binding domain or Ig / Ig-like domain, recombinant fusion of the antigen-binding domain or Ig domain protein fragment and the scaffold protein sequence as two fragments is required. In a specific embodiment, the chimeric fusion protein, which links an immunoglobulin to a scaffold via three peptide bonds or short linkers, is further enhanced in rigidity by creating reinforcing disulfide bridges formed by cysteine ​​residues located within the Ig domain. In a specific embodiment, the scaffold protein is an obligate dimer or multimer for symmetry, or can be a coat protein or virus-like particle or fragment thereof.

[0084] In a specific embodiment, a fusion protein linking an immunoglobulin to a scaffold (via three peptide bonds or a short linker) comprises two scaffold proteins forming a heterodimer. The fusion protein is obtained by fusing a first circularly permuted scaffold protein at its accessible cleavage site in an exposed region of its structure (other than the N- or C-terminus) located in its sequence, disrupting the Ig domain topology at the accessible cleavage site in the AB β-turn, and then fusing the same accessible site at its C-terminus and reconnecting it to β-strand B to restore the Ig domain. A second accessible site in the Ig domain is provided by its C-terminal amino acid, and is finally fused to the N-terminus of a second scaffold protein, which dimerizes with the first scaffold protein and enhances its rigidity. In a specific embodiment, circular permutations of the scaffold protein are introduced at the N- and C-termini. Thus, in a specific embodiment where circular permutations are introduced at the N- and C-termini of a first scaffold protein, the circularly permuted sequence of the first scaffold protein can be recombinantly fused together with an Ig protein fragment inserted between the N- and C-terminal portions of the Ig sequence (cleaved inside the ABβ turn), and the N-terminus of the second scaffold protein is fused to the C-terminus of the Ig domain sequence, such that the multimeric scaffold protein is linked or fused to the Ig domain at an accessible site via each of the monomers of the multimeric scaffold.

[0085] In a specific embodiment, the scaffold protein of a fusion protein linking an antigen-binding domain to a scaffold (via three peptide bonds or a short linker) comprises a second antigen-binding domain. The fusion protein is obtained by fusing a scaffold protein comprising a (second) antigen-binding domain at its N-terminal amino acid and disrupting the (first) antigen-binding domain topology at a cleaved accessible site in the ABβ-turn. In a specific embodiment, the scaffold protein comprising the (second) antigen-binding domain comprises an Ig domain, specifically providing a structurally accessible site within its sequence near the C-terminus of cleaved β-strand G for relinking to the ABβ-turn site that allows fusion of the (first) antigen-binding domain with β-strand B. In this case, the second accessible site of the (first) antigen-binding domain is provided by its C-terminal amino acid, which is ultimately fused to the remainder of the scaffold protein comprising the (second) antigen-binding Ig domain (see Figure 17). Thus, in this particular embodiment, a scaffold protein sequence comprising a (second) antigen-binding domain must be recombinantly fused to the (first) antigen-binding protein fragment as two fragments. The two antigen-binding domains fused to each other as described in this embodiment are disclosed in one particular embodiment as two identical antigen-binding domains, or as different antigen-binding domains that bind different epitopes of the same target protein, or as two antigen-binding domains that target different proteins. However, the epitope bound to the CDR of the antigen-binding protein that is an Ig domain should not be present in other Ig domains that are part of the resulting antigen-binding chimera, or in the newly formed antigen-binding chimera itself. In a particular embodiment, the Ig domains are two nanobodies, and in a more particular embodiment, they are two identical Nbs, or different Nbs that bind different epitopes of the same target protein, or two Nbs that target different proteins. However, the epitope bound to the Nb should not be present in other Nbs that are part of the resulting antigen-binding chimera, or in the newly formed antigen-binding chimera itself.

[0086] In certain embodiments, the scaffold proteins of the invention are monomeric proteins. In other embodiments, the scaffold proteins are proteins that provide symmetry, such as multimeric scaffold proteins, coat proteins, or scaffold proteins that self-assemble into virus-like particles. Multimeric scaffold proteins provide symmetry through oligomerization, which may be hetero- or homo-oligomerization, obligate, persistent, or transient. The resulting symmetry may be of any type, including, but not limited to, cyclic, cubic, dihedral, hexahedral, octahedral, icosahedral, etc. In particular, the term "icosahedron" refers to a type of symmetry derived from an icosahedron, a polyhedral geometric shape with 20 faces. The terms "virus-like particle (VLP)," "icosahedral VLP protein," "coat protein," or "protein that forms an icosahedral VLP" refer to a protein derived from a virus, but which is non-infectious, and capable of self-assembling into a VLP, a multimeric structure that provides (icosahedral) symmetry. For example, bacteriophages have an icosahedral head structure. Furthermore, any icosahedral virus for which a reverse genetics system for generating viral particles has been established can be readily used to display chimeric surface proteins containing all or part of a heterologous protein. Chimeric surface proteins include viral surface or coat proteins fused to heterologous antigen-binding domains, such as those provided by the present invention. To serve as a scaffold, viral coat proteins can be fused via a linker sequence or peptide bond. In some cases, viral coat proteins are partially deleted to facilitate the accommodation of linkers and / or antigen-binding domains fused to the linkers. Whether a deletion impairs the ability of a viral protein to re-coat particles can be assessed by incubating recombinantly expressed viral proteins in the presence of viral particles and observing the formation of re-coated viral particles.

[0087] Another aspect of the present invention relates to novel chimeric antigen-binding proteins in which an antigen-binding domain is fused to a scaffold protein, which disrupts the topology of the domain and has a total mass or molecular weight of at least 30 kDa, such that the mass addition resulting from binding of the chimera to the target of the antigen-binding domain will be significant and sufficient to allow three-dimensional structural analysis of the target when non-covalently bound to the chimera. In another embodiment, the total mass or molecular weight of the scaffold protein is at least 10 kDa, at least 20 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa, at least 50 kDa, or at least 60 kDa. This particular size or mass increase will result in a reduced loss of signal-to-noise ratio in images. Second, the chimera will serve as a structural guide by providing sufficient features for accurate image alignment of small or difficult-to-crystallize proteins to reach sufficiently high resolution using cryo-EM and X-ray crystallography.

[0088] Another aspect of the present invention relates to novel chimeric antigen-binding proteins in which an antigen-binding domain is fused to a scaffold protein, which disrupts the topology of the domain and further comprises an antigen-binding domain. In a specific embodiment, the scaffold protein comprises an Ig domain that is a VHH, nanobody, or antibody itself. Thus, the fusion to obtain the novel chimeric antigen-binding protein results in an antigen-binding chimeric protein with at least two antigen-binding sites, and the fusion of the antigen-binding moieties to each other results in a rigid chimera that maintains their function of binding to their respective targets. The at least two antigen-binding sites can target the same or different epitopes of a single target, thereby increasing affinity and / or efficacy. Alternatively, the two antigen-binding sites of the chimera comprising the two antigen-binding domains fused according to the present invention can bind to different target proteins, allowing for targeting of two proteins using a single rigid chimera. This unique feature of chimeras offers a solution for therapeutic uses of bispecific antibodies when a rigid structure is required, for example, for targets that are otherwise difficult to reach due to their close proximity.

[0089] Antigen-binding domains and / or scaffold proteins comprising antigen-binding domains are also within the scope of the present invention. The antigen-binding domains are in a "multivalent" form, formed by linking two or more (monovalent) antigen-binding domains, such as Ig domains, together by chemical conjugation or recombinant DNA techniques. Non-limiting examples of multivalent constructs include "bivalent" constructs, "trivalent" constructs, and "tetravalent" constructs. The immunoglobulin domains contained within a multivalent construct may be the same or different. In particular, the immunoglobulin domains of the present invention or the Ig domains constituting the scaffold proteins of the present invention are in a "multispecific" form, formed by linking two or more immunoglobulin domains, at least one of which has a different specificity. Non-limiting examples of multispecific constructs include "bispecific" constructs, "trispecific" constructs, and "tetraspecific" constructs. More specifically, any multivalent or multispecific immunoglobulin domain (as defined herein) of the invention may suitably be specific for two or more different epitopes on the same antigen, e.g., two or more different epitopes of a target, or may be specific for two or more different antigens, e.g., an epitope of the target and an epitope of a natural binding partner of the target. In particular, a monovalent immunoglobulin domain of the invention is selected to bind to the target with a lower affinity than that conferred by a multivalent or multispecific immunoglobulin single variable domain of the invention. In a specific embodiment, such multivalent or multispecific Ig domains of the invention can be fused to each other by disrupting the topology of at least one of the Ig domains and a scaffold protein. Alternatively, a multivalent or multispecific Ig domain of the invention may be conventionally fused via its N-terminus and / or C-terminus and then applied as a whole and fused to another Ig domain and / or another scaffold by disrupting the Ig domain to which it is fused or by disrupting the Ig domain of the multivalent or multispecific Ig domain itself.

[0090] Another embodiment provides scaffold proteins that are non-immunoglobulins, but are proteins known to bind to other types of proteins and therefore may also be useful for therapeutic targeting.

[0091] In alternative aspects, the antigen-binding chimeric proteins of the invention comprise modified amino acids. Another embodiment relates to scaffold proteins present in modified form and / or comprising (or fused to) other moieties. Another embodiment relates to the antigen-binding domain of said antigen-binding chimeric proteins present in modified form and / or comprising other moieties. Examples of modifications and examples of amino acid residues within the protein domains of the invention (i.e., on the protein backbone, but preferably on the side chains) that can be modified, methods and techniques that can be used to introduce such modifications, as well as the potential uses and advantages of such modifications, will be apparent to those skilled in the art. For example, such modifications can include methods of introducing one or more functional groups, residues, or moieties into or onto the binding agent (e.g., by covalent linkage or another suitable method). Examples of such functional groups and techniques for introducing them will be clear to those skilled in the art and may generally include all functional groups and techniques mentioned in the art, as well as functional groups and techniques known per se for modifying pharmaceutical proteins, particularly antibodies or antibody fragments (including ScFvs and single-domain antibodies); see, for example, Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). As will also be clear to those skilled in the art, such functional groups may be linked, for example, directly (e.g., covalently) to the scaffold protein, or may be linked, optionally via a suitable linker or spacer.

[0092] When an antigen-binding chimeric protein has potential therapeutic value, one of the most widely used techniques for extending the half-life and / or reducing the immunogenicity of a pharmaceutical protein involves the attachment of a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or its derivatives (e.g., methoxypoly(ethylene glycol) or mPEG). Generally, any suitable type of PEGylation can be used, such as those used in the art for antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFvs); see, for example, Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev., 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003), and WO04060965. Various protein PEGylation reagents are also commercially available, for example, from Nektar Therapeutics, USA. Site-specific PEGylation via cysteine ​​residues is particularly preferred (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003)). For example, for this purpose, PEG can be attached to a cysteine ​​residue naturally present in the scaffold protein, the scaffold protein can be modified to appropriately introduce one or more cysteine ​​residues for PEG attachment, or an amino acid sequence containing one or more cysteine ​​residues for PEG attachment can be fused to the N-terminus and / or C-terminus of the scaffold, all of which are achieved using protein engineering techniques known to those skilled in the art. For the scaffold protein of the novel antigen-binding chimeric protein of the present invention, it is preferred to use PEG with a molecular weight of more than 5,000, e.g., more than 10,000 and less than 200,000, e.g., less than 100,000, e.g., in the range of 20,000 to 80,000. Other modifications, although generally less preferred, include N-linked or O-linked glycosylation, which typically occur as part of co- and / or post-translational modifications depending on the host cell used to express the antigen-binding chimeric protein of the invention.Other techniques for extending the half-life of the chimeric antigen-binding protein include constructing a bifunctional construct (e.g., one antigen-binding domain for Target 1 and one antigen-binding domain for a serum protein such as albumin, located inside a scaffold protein), or constructing a fusion of the chimeric antigen-binding protein with a peptide (e.g., a peptide for a serum protein such as albumin) via or as a scaffold protein.

[0093] Another aspect of the present invention relates to novel chimeric antigen-binding proteins in which an antigen-binding domain is fused to a scaffold protein, which disrupts the topology of the domain, and the scaffold protein is a modified protein, i.e., a labeled protein. Alternatively, the antigen-binding domain is a labeled protein. Further modifications include the introduction of one or more detectable labels or other signal-generating groups or moieties depending on the intended use of the labeled chimeric antigen-binding protein. Suitable labels and their attachment, use, and detection techniques will be apparent to those skilled in the art and include, but are not limited to, fluorescent labels (e.g., IRDye800, VivoTag800, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine, as well as fluorescent metals such as Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels, or bioluminescent labels (e.g., luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, dioxetane, or GFP and its analogs), radioisotopes, metals, and metal chelates. or metal cations, or other metals or metal cations particularly suitable for use in in vivo, in vitro, or in situ diagnostics and imaging, as well as chromophores and enzymes (e.g., maleate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase). Other suitable labels will be apparent to those skilled in the art and include, for example, moieties detectable using NMR or ESR spectroscopy.Such labeled antigen-binding chimeric proteins of the invention can be used, for example, in vitro, in vivo, or in situ assays (including immunoassays known per se, such as ELISA, RIA, EIA, and other "sandwich assays"), as well as for in vivo diagnostic and imaging purposes, depending on the choice of the particular label. As will be apparent to those skilled in the art, further modifications include the introduction of chelating groups, for example, for chelating one of the metals or metal cations listed above. Suitable chelating groups include, but are not limited to, 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA), 2,2'-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), or ethylenediaminetetraacetic acid (EDTA). Yet another modification includes the introduction of a functional group that is one half of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group is conjugated to the other half of the binding pair. A conjugate can be used to link the antigen-binding chimeric protein to another protein, polypeptide, or compound, i.e., by forming a binding pair. For example, the antigen-binding chimeric protein of the present invention can be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such a conjugated antigen-binding chimeric protein can be used as a reporter in a diagnostic system in which a detectable signal-generating agent is conjugated to avidin or streptavidin. Such a binding pair can also be used to bind the antigen-binding chimeric protein of the present invention to a carrier, including a carrier suitable for pharmaceutical purposes. A non-limiting example is the liposome formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such a binding pair can also be used to link a therapeutically active agent to the antigen-binding chimeric protein of the present invention.Furthermore, the use of toxic labels or radionuclides as payloads is also within the scope of the antigen-binding chimeric proteins. Finally, the use of "labels" or "tags" linked or fused to the scaffold protein offers the advantage of non-covalently labeling the target protein of the Ig domain.

[0094] Another aspect of the present invention relates to a nucleic acid molecule encoding the antigen-binding chimeric protein of the present invention, which comprises a coding sequence for the antigen-binding domain and the scaffold protein and / or a fragment thereof, and the topological separation of the domain is reflected in the fact that the domain sequence comprises an insertion of a scaffold protein sequence (or a circularly permuted sequence or a fragment thereof) such that the N-terminal antigen-binding domain fragment and the C-terminal antigen-binding domain fragment are separated within the nucleic acid molecule by the scaffold protein sequence or a fragment thereof.

[0095] Another embodiment describes a chimeric gene comprising at least a promoter, the nucleic acid molecule encoding the chimeric antigen-binding protein, and a 3'-terminal region comprising a transcription termination signal. Another embodiment relates to an expression cassette encoding the chimeric antigen-binding protein of the invention, or an expression cassette comprising the nucleic acid molecule or chimeric gene encoding the chimeric antigen-binding protein. The expression cassette, in some embodiments, is applied in a universal format as an immune library comprising a large set of Ig domains for selection for optimal binders of a target.

[0096] Other embodiments relate to vectors comprising the expression cassettes or nucleic acid molecules encoding the chimeric antigen-binding proteins of the invention. In certain embodiments, the chimeric antigen-binding proteins can be produced by E. coli expression vectors and purified in the presence or absence of their target.

[0097] An alternative embodiment relates to a host cell comprising an antigen-binding chimeric protein of the invention, or a nucleic acid molecule or expression cassette or vector encoding the antigen-binding chimeric protein of the invention. In certain embodiments, the host cell further co-expresses an antigen or target protein that specifically binds to the antigen-binding domain of the antigen-binding chimeric protein.

[0098] Further embodiments disclose the use of said host cells, or membrane preparations isolated from said cells, or proteins isolated from said cells for ligand screening, drug screening, protein capture and purification, or biophysical studies.

[0099] The present invention, which provides such vectors, further includes the option of high-throughput cloning in a universal fusion vector. The universal vectors are described in other embodiments, in which the vectors are particularly suitable for surface display in yeast, phage, bacteria, or viruses. Furthermore, the vectors can be used to select and screen immune libraries containing such universal vectors or expression cassettes with a large set of different Ig domains, fusing the same N-terminus of the conserved Ig domains and scaffold proteins to the remaining Ig domain sequences provided by the library. Thus, sequence variability in the library generated for screening novel antigen-binding chimeric proteins for specific targets is provided by differences in Ig domain sequences, more specifically, by differences in the CDR regions of the Ig domain library.

[0100] Another embodiment of the present invention relates to a method for producing a chimeric antigen-binding protein of the present invention, comprising the steps of: (a) culturing a host comprising a vector, expression cassette, chimeric gene, or nucleic acid sequence of the present invention under conditions that induce expression of the chimeric antigen-binding protein; and (b) optionally recovering the expressed polypeptide.

[0101] In one embodiment, the vectors of the present invention are suitable for use in a method involving displaying a collection of chimeric antigen-binding proteins, preferably an immune library, on the extracellular surface of a cell population. Surface display methods are reviewed in Hoogenboom, (2005; Nature Biotechnol 23, 1105-16) and include bacterial display, yeast display, and (bacteri)phage display. The cell population is preferably yeast cells. Each of the various yeast surface display methods provides a means for firmly linking each antigen-binding chimeric protein encoded in the library to the extracellular surface of yeast cells that have been transformed with a plasmid encoding the protein. While most previously described yeast display methods use the yeast Saccharomyces cerevisiae, other yeast species, such as Pichia pastoris, may also be used. More specifically, in certain embodiments, the yeast strain is from a genus selected from the group consisting of Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida. In certain embodiments, the yeast species is selected from the group consisting of S. cerevisiae, Pichia pastoris, Hansenula polymorpha, S. pombe, Kluyveromyces lactis, Yarrowia lipolytica, and C. albicans. Most fusion proteins for yeast expression are based on GPI (glycosylphosphatidylinositol)-anchored proteins, which play a key role in the surface expression of cell surface proteins and are essential for yeast viability.One example of such a protein, α-agglutinin, consists of a core subunit encoded by AGA1 linked via a disulfide bridge to a small binding subunit encoded by AGA2. Proteins encoded by nucleic acid libraries can be introduced into the N-terminal region of AGA1 or the C- or N-terminal regions of AGA2. Either fusion pattern will result in display of the polypeptide on the yeast cell surface.

[0102] The vectors disclosed herein may also be suitable for surface display of proteins in prokaryotic host cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as species of the Enterobacteriaceae family, including Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as B. subtilis and B. licheniformis (e.g., B. licheniformis, as disclosed in publication DD 266,710 dated April 12, 1989). Examples of suitable E. coli cloning hosts include Bacilli such as Bacillus sp. 41P, Pseudomonas sp. such as P. aeruginosa, and Streptomyces sp. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and not limiting. When the host cell is a prokaryotic cell, examples of suitable cell surface proteins include suitable bacterial outer membrane proteins. Such outer membrane proteins include fimbriae and flagella, lipoproteins, ice nucleation proteins, and autotransporters. Examples of bacterial proteins used for heterologous protein display include LamB (Charbit et al., EMBO J, 5(11):3029-37(1986)), OmpA (Freudl, Gene, 82(2):229-36(1989)), and intimin (Wentzel et al., J Biol Chem, 274(30):21037-43, (1999)).Other examples of outer membrane proteins include, but are not limited to, FliC, pullulanase, OprF, OprI, PhoE, MisL, and cytolysin. Extensive lists of bacterial membrane proteins used for surface display are described in detail in Lee et al., Trends Biotechnol, 21(1):45-52 (2003), Jose, Appl Microbiol Biotechnol, 69(6):607-14 (2006), and Daugherty, Curr Opin Struct Biol, 17(4):474-80 (2007).

[0103] Furthermore, to enable thorough screening and selection, vectors can be applied to yeast and / or phage display systems, followed by FACS and panning, respectively. A preferred selection method is achieved by combining the display of antigen-binding chimeras on yeast cells with the resolution of, for example, fluorescence-activated cell sorting (FACS). In yeast display, each antigen-binding protein is displayed on the surface of a single cell as a fusion with, for example, ~50,000 copies of the Aga2p protein. For FACS selection, each antigen-binding protein is labeled with a different fluorescent dye to determine the selection procedure. Next, the yeast library displaying the antigen-binding chimeras can be stained with a mixture of the fluorescent proteins used. Subsequently, two-color FACS can be used to analyze the properties of each antigen-binding chimera displayed on specific yeast cells, and separate cell populations can be analyzed. Yeast cells displaying antigen-binding chimeras that are highly suitable for targeting the target protein can be sorted along a diagonal line using two-color FACS. For example, when screening for antigen-binding chimeric proteins that specifically target transient protein-protein interactions or when conformation-selective binding is desired, it is most preferable to use a vector for such selection methods. Similarly, a phage display vector can be used to display antigen-binding chimeras on bacteriophage, followed by panning. Display can be performed, for example, on M13 particles by fusing the antigen-binding chimera with phage coat protein III within the gene vector (Hoogenboom, 2000; Immunology Today. 5699:371-378). To select antigen-binding proteins that specifically bind to a given conformation and / or transient protein-protein interactions, for example, only one of the interacting protomers is immobilized on a solid phase. Then, bioselection by panning of the phage-displayed antigen-binding chimeras is performed in the presence of an excess of the remaining soluble promoter. Optionally, panning rounds can be initiated with crosslinked complexes or proteins immobilized on a solid phase.

[0104] Another aspect of the present invention relates to a complex comprising the antigen-binding chimeric protein or antigen-binding chimeric protein composition and an antigen or target protein, wherein the target protein specifically binds to the antigen-binding chimeric protein or antigen-binding chimeric protein composition. More specifically, the target protein binds to the antigen-binding domain of the antigen-binding chimeric protein, and even more specifically, in embodiments where the antigen-binding domain is an Ig domain, the target protein binds to the CDR of the Ig domain of the antigen-binding chimeric protein. One embodiment discloses a complex described herein, wherein the antigen-binding domain is a conformation-selective binding domain. More specifically, the antigen-binding domain stabilizes the target protein in a functional conformation. More specifically, the functional conformation may specifically include an agonist conformation, a partial agonist conformation, or a biased agonist conformation. Alternatively, conjugates of the invention are disclosed wherein the antigen-binding domain stabilizes the target protein in a functional conformation, wherein said functional conformation is an inactive conformation, or wherein said functional conformation comprises an inverse agonist conformation.

[0105] Another aspect of the present invention relates to a composition comprising the antigen-binding chimeric protein. The "composition" of the present invention can be provided in the form of a kit comprising a first container containing the lyophilized antigen-binding chimeric protein and a second container containing a solution for resuspending the lyophilized protein. One or more lyoprotectants, such as sucrose, dextran, sorbitol, and amino acids, can be added to the protein powder to stabilize the protein during lyophilization. Alternatively, the composition can be provided as a single container containing a suspension or solution of the antigen-binding chimeric protein. Either solution can contain one or more excipients. The solution is typically aqueous. Thus, purified water can form the primary excipient. For example, proteins are typically diluted with water for injection (WFI) to achieve the desired final concentration. The solution typically contains a buffer. Therefore, other excipients include buffers and pH adjusters, such as sodium citrate, sodium dihydrogen phosphate monohydrate, and sodium hydroxide. In some cases, a thickener, such as xanthan gum, can be added as another excipient. Surfactants may also be added, especially non-ionic surfactants such as polysorbate 80. Other excipients include sucrose, sorbitol, inorganic salts, amino acids and vitamins.

[0106] The present invention also relates to "pharmaceutical compositions" containing one or more compounds of the present invention, particularly antigen-binding chimeric proteins, and a pharmaceutically acceptable carrier or diluent. These pharmaceutical compositions can be used to achieve a desired pharmacological effect by administering them to a patient in need thereof. The present invention includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of the present invention or a salt thereof. A pharmaceutically effective amount of a compound is preferably an amount that produces an effect or influences the particular condition being treated. In general, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to the amount necessary to achieve one or more desired results. Those skilled in the art will understand that potency, and thus an "effective amount," may vary depending on the type and structure of the compound of the present invention. One of ordinary skill in the art can readily assess the potency of the compound. "Pharmaceutically acceptable" means a material that is not biologically or otherwise harmful; i.e., such a material can be administered to an individual in conjunction with the compound without producing undesired biological effects or adversely interacting with any of the other components of the pharmaceutical composition containing it. Preferably, the pharmaceutically acceptable carrier is one that is relatively non-toxic and harmless to the patient at concentrations consistent with the effective activity of the active ingredient, so that any side effects attributable to the carrier do not impair the beneficial effects of the active ingredient. Suitable carriers or additives generally include one or more of the compounds included in the following non-exhaustive list: large molecular weight, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M.F. et al. ("Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311), Strickley, R.G. ("Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999) - Part-1" PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349), and Nema, S. et al. ("Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51(4), 166-171).

[0107] As used herein, the term "excipient" includes any substance that may be present in a pharmaceutical composition other than the active ingredient, including salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, stabilizers, flavoring or coloring agents, etc. "Diluents," particularly "pharmaceutically acceptable solvents," include solvents such as water, saline, physiological salt solution, glycerol, ethanol, etc. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, etc., may be added to such solvents.

[0108] The antigen-binding chimeric proteins of the invention and pharmaceutically acceptable carriers can be administered using any effective conventional dosage form, including immediate-release, delayed-release, and time-release formulations, with pharmaceutically acceptable carriers well known in the art, and can be administered by any suitable route, such as any of the routes commonly known to those of ordinary skill in the art. For therapeutic use, the pharmaceutical compositions of the invention can be administered to any patient according to standard techniques.

[0109] For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, which can be prepared according to methods known in the art for the manufacture of pharmaceutical compositions. Solid unit dose preparations can be capsules, which can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. In another embodiment, the compounds of the present invention can be tableted using conventional tablet bases such as lactose, sucrose, and cornstarch, along with binders (e.g., gum arabic, cornstarch, or gelatin), disintegrants (e.g., potato starch, alginic acid, cornstarch, guar gum, tragacanth gum, and gum arabic) to aid in tablet disintegration and dissolution after administration, lubricants (e.g., talc, stearic acid, magnesium stearate, calcium stearate, or zinc stearate) to improve the flowability of the tablet granulation and prevent the tablet material from adhering to the surfaces of the tablet die punch, and dyes, coloring agents, and flavoring agents (e.g., peppermint, oil of wintergreen, or cherry flavor) to improve the aesthetic qualities of the tablet and increase patient acceptance. Suitable excipients for use in oral liquid dosage forms include dicalcium phosphate and diluents such as water and alcohols (e.g., ethanol, benzyl alcohol, and polyethylene alcohols), with or without pharmaceutically acceptable surfactants, suspending agents, or emulsifying agents. Various other materials may be used as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Dispersible powders and granules are suitable for preparing aqueous suspensions. In such cases, the active ingredient is provided in a mixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are as described above. Other excipients, such as the above-mentioned sweetening agents, flavoring agents, and coloring agents, may also be added.

[0110] The pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions. Such suspensions can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and arabic gum). The dispersing or wetting agent may be a natural phosphatide such as lecithin, a condensation product of an alkylene oxide and a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide and a long-chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The sterile injectable preparations may be sterile injectable solutions or suspensions prepared in a non-toxic, parenterally acceptable diluent or solvent. Diluents and solvents that can be used include, for example, water, Ringer's solution, isotonic sodium chloride solution, and isotonic glucose solution. Furthermore, sterile fixed oils have traditionally been used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid can also be used in injection preparations.

[0111] Another aspect relates to the use of the antigen-binding chimeric protein of the present invention, or the use of a nucleic acid molecule, chimeric gene, expression cassette, vector, complex, or composition in the structural analysis of a target protein. In particular, the present invention relates to the use of the antigen-binding chimeric protein in the structural analysis of a target protein, where the target protein is a protein that specifically binds to the antigen-binding chimeric protein. As used herein, the term "structurally analyzing" or "structural analysis" refers to determining the atomic arrangement or coordinates of a protein, often performed by biophysical methods such as X-ray crystallography or cryo-electron microscopy (cryo-EM). Specifically, one embodiment relates to the use in structural analysis, including single-particle cryo-EM or crystal structure analysis. The use of the antigen-binding chimeric protein of the present invention in structural biology is highly advantageous for use as a crystallization aid, i.e., to act as a crystal contact and increase symmetry, and also as a rigid tool in cryo-EM, which is very useful for analyzing large structures. Primarily, it is highly advantageous for reducing the size barrier currently being considered and for increasing symmetry.

[0112] The use of cryo-EM for structure determination offers several advantages over traditional approaches such as X-ray crystallography. In particular, cryo-EM does not impose stringent requirements on the purity, homogeneity, and quality of the samples analyzed. Importantly, cryo-EM can be applied to targets that do not form suitable crystals for structure determination. Purified or unpurified proteins, either alone or in complex with other proteinaceous molecules such as antigen-binding chimeric proteins or non-proteinaceous molecules such as nucleic acids, can be dropped onto carbon grids for imaging by cryo-EM. Supported membrane grids are typically flash-frozen in liquid ethane, preserving the particles in suspension in a frozen, hydrated state. Larger particles can be vitrified by cryofixation. Vitrified samples can be cut into thin sections (typically 40–200 nm thick) using a cryo-ultramicrotome, and the sections can be mounted on electron microscope grids for imaging. The quality of the data obtained from the images can be improved by using collimated illumination and better microscope alignment to achieve a high resolution of ~3.3 Å. At such high resolutions, ab initio modeling of all-atom structures is possible. However, if atomic-resolution structural data for the selected target protein or closely related target protein and a selected heterologous protein or close homologue are available for constrained comparative modeling, lower-resolution imaging may be sufficient. To further improve data quality, the Fourier transform of carbon film images recorded under the same conditions as those used for imaging can be adjusted to 1 / 3 Å. -1 The microscope can be carefully aligned so that there are visible contrast transfer function (CTF) rings above σ. Software such as CTFFIND can then be used to determine the out-of-focus value for each micrograph.

[0113] Another aspect of the present invention is a method for determining the three-dimensional structure of a target protein or a protein of interest, comprising: (i) providing an antigen-binding chimeric protein or composition of the invention and a targeted protein to form a complex, wherein the target protein is specifically bound to the antigen-binding chimeric protein or composition; or preparing a composite of the present invention; (ii) displaying the mixture or complex under conditions suitable for structural analysis and determining the three-dimensional structure of the target protein at high resolution.

[0114] In a specific embodiment, the structural analysis is performed by X-ray crystallography. In another embodiment, the three-dimensional analysis includes cryo-EM. More specifically, an example of a cryo-EM analysis technique is as follows: A sample (e.g., a selected megabody protein in complex with a target of interest) is dropped onto a selected, glow-discharged grid (e.g., a carbon-coated copper grid, C-Flat, 1.2 / 1.3 200 mesh: Electron Microscopy Sciences; a gold R1.2 / 1.3 300 mesh UltraAuFoil grid: Quantifoil), blotted, and then plunge-frozen in liquid ethane (Vitrobot Mark IV (FEI) or other selected plunger). Data for one grid are acquired using a 300 kV electron microscope (e.g., a Krios 300 kV microscope with a selected phase plate inserted) equipped with a selected detector (e.g., a Falcon 3EC direct detector). Micrographs are acquired in electron counting mode at an appropriate magnification appropriate for the expected megabody-antigen complex size. Acquired micrographs are manually inspected before further image processing. Drift correction, electron beam-induced motion, dose weighting, CTF fitting, and phase shift estimation are applied using the software of choice (e.g., RELION, SPHIRE package). Particles are extracted using the software of choice and used for 2D classification. 2D classes are manually inspected to remove false positives. Particles are binned according to the data acquisition settings. An initial 3D reference model is created by applying appropriate low-pass filters, and multiple 3D classes (e.g., six) are generated. The original particles are used for 3D refinement (using a soft mask, if necessary). Fourier shell correlation (FSC) = 0.143 is used as a criterion to estimate the reconstruction resolution. Local resolution can be calculated by running MonoRes in Scipion. Reconstructed cryo-EM maps can be analyzed using UCSF Chimera and Coot software. UCSF Chimera can be used to initially fit the design model, which can then be analyzed using the software of choice (UCSF Chimera, PyMOL, or Coot).

[0115] Another advantage of the method of the present invention is that structural analysis requires highly purified proteins, whereas conventional methods require only highly purified proteins. The use of chimeric antigen-binding proteins relaxes the purity requirements. Such antigen-binding proteins, particularly nanobodies, will specifically extract the target protein by binding to its epitope within the complex mixture. The target protein can then be trapped, frozen, and analyzed by cryo-EM.

[0116] In an alternative embodiment, the method is also suitable for three-dimensional analysis in which the target protein is a transient protein-protein complex. Furthermore, the chimeric antigen-binding molecule can be applied in methods to determine the three-dimensional structure of a target in order to stabilize the target as a transient protein-protein interaction to enable its structural analysis.

[0117] Another embodiment relates to a method for selecting or screening a panel of chimeric antigen-binding proteins that bind to different epitopes of the same target protein, comprising the steps of: (i) designing an immune library of chimeric antigen-binding proteins that bind to the target protein; and (ii) selecting chimeric antigen-binding proteins by surface yeast display, phage display, or bacteriophage display to obtain a panel of chimeric antigen-binding proteins that include proteins that bind to several epitopes of the target, thereby enabling analysis of several conformations of the target protein as separate images, for example by cryo-EM.

[0118] In another embodiment, the method and the antigen-binding chimeric protein of the present invention are used in structure-based drug design and structure-based drug screening. The iterative process of structure-based drug design often involves multiple cycles before an optimized lead enters Phase I clinical trials. The first cycle involves cloning, purifying, and determining the structure of the target protein or nucleic acid. The structure is determined by one of three major methods: X-ray crystallography, NMR, or homology modeling. A computer algorithm is used to place compounds or compound fragments from a database into selected regions of the structure. The antigen-binding chimeric protein of the present invention can be used to fix or stabilize a predetermined structural conformation of the target. The selected compounds are scored and ranked based on their steric and electrostatic interactions with the target site, and the best compounds are tested in biochemical assays. In the second cycle, structure determination of the target in complex with promising leads from the first cycle that demonstrate in vitro inhibition at least at the micromolar level identifies sites on the compound that can be optimized for increased potency. Again, the antigen-binding chimeric protein of the present invention can be used to facilitate structural analysis of the target in a predetermined conformational state. Other cycles include the synthesis of optimized leads, structural determination of new target-lead complexes, and further optimization of the lead compounds. After several cycles of the drug design process, optimized compounds typically show significant improvements in binding and often in specificity for the target. Library screening leads to hit compounds, which then lead to the development of lead compounds, for which medicinal chemistry is essential for structural information and structure-activity relationship analysis.

[0119] In another embodiment, since the antigen-binding domain of the antigen-binding chimeric protein used in the method of the invention comprises a Nanobody Ig domain, the method has the additional advantage of only providing an average image of the correctly folded target protein, since the use of Nanobodies to select for displayed antigen-binding chimeras primarily identifies binders to conformational epitopes.

[0120] Another embodiment is a method for identifying a (conformation-selective) compound, comprising: i) providing a target protein and an antigen-binding chimeric protein of the present invention that specifically binds to the target protein; ii) providing a test compound; iii) evaluating the selective binding of the test compound to the target protein.

[0121] According to a particularly preferred embodiment, the method for identifying conformationally selective compounds is carried out by a ligand binding or competition assay, more preferably by a radioligand binding or competition assay. Most preferably, the method for identifying conformationally selective compounds is carried out by a comparative assay, more particularly a comparative ligand competition assay, and even more particularly a comparative radioligand competition assay, as described in detail in the Examples section.

[0122] The test compound can be any small molecule or a macromolecule such as a protein, sugar, nucleic acid, or lipid. Typically, the test compound will be a small molecule, peptide, antibody, or fragment thereof. Of course, in some cases, the test compound can be a library of test compounds. High-throughput screening assays for therapeutic compounds, such as agonists, antagonists, or inverse agonists and / or modulators, are also contemplated by the present invention. For high-throughput purposes, compound libraries or combinatorial libraries, such as allosteric compound libraries, peptide libraries, antibody libraries, fragment-based libraries, synthetic compound libraries, natural compound libraries, and phage display libraries, can be used. Methods for generating and screening such libraries are known to those skilled in the art. The test compound may optionally be covalently or noncovalently linked to a detectable label. Suitable detectable labels, their attachment, use, and detection techniques are readily apparent to those skilled in the art and include, but are not limited to, any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels include magnetic beads (e.g., Dynabeads), fluorescent dyes (e.g., all Alexa Fluor dyes, fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), and radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), and colorimetric labels (e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads). Means for detecting such labels are also well known to those skilled in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected by detecting the emitted light intensity using a photodetector. Enzyme labels are generally detected by adding a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, while colorimetric labels are detected simply by visualizing the colored label. Other suitable detectable labels are described above in connection with the first aspect of the invention relating to the chimeric polypeptides of the invention. Thus, according to a specific embodiment, the test compound used in any of the above screening methods is selected from the group comprising polypeptides, peptides, small molecules, natural products, peptidomimetics, nucleic acids, lipids, lipopeptides, carbohydrates, antibodies or any fragments derived therefrom (e.g. Fab, Fab' and F(ab')2, Fd, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (dsFv), fragments comprising a VL or VH domain, heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies, variable domains derived from camelid heavy chain antibodies (VHH or nanobodies), variable domains of novel antigen receptors derived from shark antibodies (VNAR)), protein scaffolds such as alphabodies, protein A, protein G, molecules designed from ankyrin repeat domains (DARPins), fibronectin type III repeats, anticalins, knottins, artificially engineered CH2 domains (nanobodies), as defined above. In one preferred embodiment, high-throughput screening methods involve preparing a combinatorial chemical or peptide library containing a large number of potential therapeutic ligands. Such "combinatorial libraries" or "compound libraries" are then screened in one or more assays, such as those described herein, to identify library members (particular chemical species or subclasses) that exhibit a desired characteristic activity.A "compound library" is a collection of stored compounds that is typically used ultimately in high-throughput screening. A "combinatorial library" is a diverse collection of compounds generated by chemical or biological synthesis by combining multiple chemical "building blocks." The creation and screening of combinatorial libraries is well known to those skilled in the art. Compounds thus identified can serve as traditional "lead compounds" or can themselves be used as potential or actual therapeutic agents.

[0123] Another aspect provides the use of the antigen-binding chimeric protein, wherein the scaffold protein is a labeled protein, as a diagnostic tool, or more specifically for in vivo imaging.

[0124] The final aspect provides the antigen-binding chimeric protein or nucleic acid, vector, complex, or composition for use as a medicine. As used herein, the term "medicine" refers to a substance / composition used in therapy, i.e., for the prevention or treatment of a disease or disorder. According to the present invention, the term "disease" or "disorder" refers to any pathological condition, in particular a disease or disorder as defined herein.

[0125] While specific embodiments, specific structures, and materials and / or molecules have been described above for the artificial cells and methods of the present invention, it will be appreciated that various changes or modifications may be made in form and content without departing from the scope and spirit of the present invention. The following examples are provided to more fully illustrate specific embodiments and should not be construed as limiting the present application. The present application is limited only by the claims. [Example]

[0126] overview We have designed rigid antigen-binding chimeric proteins consisting of an antigen-binding domain and a scaffold protein, with the antigen-binding domain linked to the scaffold protein via two or three short linkers or two or three direct bonds. Depending on the properties of the scaffold, these rigid antigen-binding chimeric proteins can be used for a variety of purposes.

[0127] As an example, the "antigen-binding chimeric proteins" described herein, also referred to as "megabodies" (Mb or Mgb), are composed of immunoglobulin or immunoglobulin-like domains, including nanobodies, VHHs, or monobodies, grafted onto a protein scaffold, particularly a large monomeric scaffold. These antigen-binding chimeras, or megabodies as specifically used herein, provide a means for determining the protein structure of (smaller) proteins, making them useful for several applications, including X-ray crystallography and cryo-EM. Mb functions as a next-generation crystallization chaperone by reducing the conformational flexibility of the target, expanding the surface area amenable to forming crystal contacts, and providing additional topological information. Furthermore, as an innovative auxiliary tool, these chimeras have reduced the size barrier for obtaining high-resolution structures using cryo-EM. By mixing a specific megabody-antigen-binding chimeric protein with its target, their specific interaction results in an additional "mass," adding defined features to particles embedded in glassy ice on a grid, facilitating precise image alignment and improving the resolution of three-dimensional reconstructions. The use of such chimeric antigen-binding proteins, such as Mb, selectively binds conformational epitopes in a one-to-one ratio, facilitating particle sorting, improving the structural / conformational uniformity of sorted particles, and improving the resolution of 3D reconstructions. Importantly, the use / combination of different sets of Mb that are similar to each other but bind different epitopes on the same protein allows for the generation of different particles from the same macromolecular complex.

[0128] As a proof-of-concept for this approach, a circularly permuted mutant (cHopQ) of the gene encoding the adhesive domain of HopQ (a periplasmic protein derived from Helicobacter pylori, PDB5LP2) was inserted into the first exposed β-turn (connecting β-strands A and B) of a GFP-specific nanobody (see Figure 2) (Example 1). This chimera was expressed as a secreted protein in the periplasm of Escherichia coli and purified to homogeneity in milligram quantities (Example 2). The binding of this chimeric antibody to GFP was then confirmed, and its structure was analyzed by X-ray crystallography (Example 3). Using the same scaffold, we also constructed antigen-binding chimeric proteins that stabilize protein complexes (Example 4), bind to GPCRs (Example 5), and bind to ion channels (Examples 6 and 22). Furthermore, in Example 5, conformation-selective stabilization was achieved, maintaining all functional properties of the Nb or antigen-binding domain that constitute the megabody. We also used in vitro evolution techniques to engineer other functional HopQ-based megabodies via two short polypeptide bonds of varying linker length and composition (Example 7). To demonstrate that other large scaffold proteins can also be used to generate megabodies, we inserted a circular permutation mutant of the gene encoding YgjK (PDB3W7S), an 86 kDA periplasmic protein from Escherichia coli, into the first β-turn connecting β-strands A and B of a GFP-specific nanobody via two short polypeptide bonds of varying length and composition (Example 8). We also demonstrated that a disulfide bond can be constructed between one of the linker peptides and the C-terminus of the nanobody to rigidify the megabody (Example 9). We also generated other rigid antibody chimeras by linking Nb to the scaffold using more complex linkage schemes. Using in vitro evolution, we fused a GFP-specific nanobody to azurin (PDB2TSA) via three short polypeptide bonds of varying length and composition. Azurin is a copper-containing single-domain protein used in anomalous scattering phasing in X-ray crystallography (Example 10).The resulting antigen-binding chimeric proteins (referred to herein as megabodies) were found to enable structural studies of proteins or complexes by X-ray crystallography and single-particle cryo-EM. Similarly, antigen-binding domains can be rigidly linked to multimeric scaffolds while maintaining structural symmetry, resulting in multimeric antigen-binding chimeric proteins or multimeric megabodies with structural symmetry. To demonstrate this principle, we grafted a lysozyme-binding nanobody onto the inverting glycosidase homodimer SusB (PDB3WFA) from Bacteroides thetaiotaomicron via three short polypeptide linkers connecting the nanobody to the scaffold (Example 11). Such homodimeric nanobody structures with two-fold rotational symmetry can be used to exploit symmetry constraints in cryo-EM or X-ray crystallography, facilitating high-resolution protein structure determination. In another example, we generated virus-like particles (VLPs) displaying nanobodies from antigen-binding chimeric proteins composed of Nb rigidly fused to the coat protein of PP7 or the naturally occurring circularly permuted PP7, AP205. PP7 is an icosahedral bacteriophage of Pseudomonas aeruginosa. PP7-derived VLPs encapsulate the mRNA responsible for their synthesis, thereby establishing the genotype / phenotype relationship required for affinity-selected sequences of antigen-specific Nb. Therefore, we applied in vitro evolution techniques to design robust VLPs displaying 90 Nb in a highly symmetric arrangement on their surface (Examples 12–15). These nanobody-displaying VLPs are excellent tools for analyzing the structures of small proteins by cryo-EM.

[0129] Antigen-binding domains, such as immunoglobulins, can also be rigidly grafted onto scaffolds that can be labeled with fluorophores, dyes, ions, or metals for use in diagnostics, imaging, or other biophysical applications. In Example 10, a GFP-specific nanobody was fused to azurin (PDB2TSA), a copper-containing single-domain protein used in anomalous scattering phasing in X-ray crystallography. Additionally, a GFP-binding nanobody was rigidly grafted onto an acyl carrier protein (ACP) (Example 16). ACPs are proteins that can be orthogonally labeled with covalently attached fluorophores in a one-step enzymatic reaction.

[0130] Immunoglobulin domains can also be rigidly fused to the same or different Ig domains (or different therapeutic scaffolds) via three short peptide linkers to generate bivalent or bispecific chimeric antigen-binding proteins, respectively (Nano2body, N2b, Examples 17 and 18). Bivalent or bispecific chimeric antigen-binding proteins can be used as chaperones in X-ray crystallography or cryo-EM, as well as have numerous applications in biophysical applications, imaging, diagnostics, and therapy.

[0131] Because β-strand A is contained within a highly conserved N-terminal sequence common to all Nbs (Harmsen et al., 2000), the comprehensive in vivo matured nanobody repertoire (including chimeric antigen-binding proteins such as those referred to herein as megabodies, nanotools, or nano2bodies) can be conveniently cloned into a rigid chimeric antigen-binding protein library and screened for binders by standard methods. Functional chimeric antigen-binding proteins are then selected by phage, yeast, or viral display (Example 19).

[0132] We also prepared rigid antigen-binding chimeric proteins by inserting a circularly permuted mutant of the gene encoding the adhesive domain of HopQ (a periplasmic protein derived from Helicobacter pylori; the circularly permuted mutant is referred to herein as "cHopQ") into the second exposed β-turn (connecting β-strands C and C') of a GFP-specific nanobody (Example 20). We also prepared antigen-binding chimeric proteins composed of synthetic immunoglobulin-like antigen-binding proteins such as monobodies (Example 21).

[0133] Furthermore, some of the designed and constructed antigen-binding chimeric proteins were applied to the structural analysis of intractable membrane-bound complexes such as GPCRs, ion channels, and receptor tyrosine kinases (Example 22).

[0134] In another example, we demonstrated that defined compositions of antigen-binding chimeric proteins, or "polybodies," can be formed to further increase scaffold size by generating antigen-binding chimeric proteins or megabodies derived from megabodies that bind to the cHopQ scaffold protein (Example 23).

[0135] Example 24 demonstrates that multimeric antigen-binding chimeric proteins can be generated from dodecin protein inserted into the first β-strand AB of Nb. Finally, disulfide-bridged homodimers were also tested as scaffold proteins to generate antigen-binding chimeric proteins of different formats with increased molecular weight and symmetry (Example 25).

[0136] [Example 1] Design and construction of a 58 kD antigen-binding chimeric protein in which a cHopQ scaffold is inserted into the first β-turn connecting β-strands A and B of a GFP-specific nanobody.

[0137] As a first proof-of-concept for obtaining rigid antigen-binding chimeric proteins such as megabodies, rigid megabodies were generated by grafting nanobodies onto a large scaffold protein via two peptide bonds linking the nanobody to the scaffold according to Figure 2 .

[0138] The 58 kDa megabody described in this example is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked according to Figures 2 and 3. The immunoglobulin domain used in this example is a GFP-binding nanobody as shown in SEQ ID NO: 1. The scaffold protein is an adhesin domain called HopQ from the Helicobacter pylori G27 strain (PDB: 5LP2, SEQ ID NO: 19) (Javaheri et al., 2016). The N-terminus and C-terminus of HopQ were linked to create a circularly permuted mutant of HopQ called cHopQ, and the sequence was truncated at another position within the sequence. Mb Nb207 cHopQ To design the construct, all parts were linked together by peptide bonds from the amino (N) to the carboxy (C) terminus in the following order: β-strand A of the anti-GFP nanobody (SEQ ID NO: 1; residues 1-13), the C-terminal part of HopQ (SEQ ID NO: 19; residues 192-414), a short peptide linker (SEQ ID NO: 21) connecting the C- and N-termini of HopQ to generate the circularly permuted scaffold protein cHopQ, the N-terminal part of HopQ (residues 14-186, SEQ ID NO: 19), β-strand G of the GFP-binding nanobody (SEQ ID NO: 1; residues 16-126), a 6xHis tag, and an EPEA tag (US9518084B2; SEQ ID NO: 209) (SEQ ID NO: 20).

[0139] Mb as a correctly folded functional protein Nb207 cHopQ To demonstrate that megabody (SEQ ID NO: 20) can be expressed, this protein was displayed on the yeast surface (Boder, 1997), and specific binding of this megabody-displaying yeast cells to the cognate antigen (GFP) was examined by flow cytometry. Nb207 cHopQ To display Mb on yeast, standard methods were used to fused Mb to a number of accessory peptides and proteins, including the appS4 leader sequence (Rakestraw, 2009), which directs extracellular secretion in yeast. Nb207 cHopQAn open reading frame encoding the Aga2p adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond with the Aga1p protein, a flexible peptide linker, an acyl carrier protein (Johnsson, 2005) for orthogonal fluorescent staining of the displayed fusion protein, and a cMyc tag was constructed. This open reading frame was inserted into the pCTCON2 vector (Chao, 2006) under the transcriptional control of the galactose-inducible GAL1 / 10 promoter and transformed into the yeast strain EBY100.

[0140] EBY100 yeast cells transfected with this plasmid were grown and induced overnight in galactose-rich medium, and then expressing Mb Nb207 cHopQ Expression and secretion of the -Aga2p-ACP fusion was induced. For orthogonal staining of ACP, cells were incubated for 1 h in the presence of a fluorescently labeled CoA analog (coA-647, 2 μM) and catalytic amounts of SFP synthase (1 μM). The growth conditions were changed from glucose-rich to galactose-rich media, which induced the attachment of Mb to the yeast surface. Nb207 cHopQ The expression of megabodies was confirmed to be induced (Figure 4). The surface display level can be easily analyzed quantitatively by flow cytometry. In these experiments, induced yeast cells were washed and subjected to flow cytometry. The Mb display level of each cell was measured by comparing the CoA647 fluorescence level with that of yeast cells that did not display megabodies but were similarly orthogonally stained. Distinguishable yeast cells with high CoA647 fluorescence signal levels were identified as Mb-displaying yeast cells. Nb207 cHopQ Since the megabody was only detected in the culture medium expressing the β-actin, it was determined that the megabody could be efficiently displayed on the yeast surface and stained orthogonally (Figure 4).

[0141] To analyze the functionality of the displayed megabodies, their binding to the cognate antigen (GFP) was tested by flow cytometry. EBY100 yeast cells were induced as described above and orthogonally fluorescently stained with CoA647 to express Mb. Nb207 cHopQThe display of the -Aga2p-ACP fusion was monitored. These orthogonally stained yeast cells were then incubated for 1 hour in the presence of 100 nM GFP (Scholz et al., 2000). After washing the cells, the amount of GFP bound was measured as the amount of Mb binding to the yeast surface. Nb207 cHopQ should be linearly correlated with the expression level of the presented Mb Nb207 cHopQ In fact, two-dimensional flow cytometry analysis confirmed that GFP (high GFP fluorescence level) only bound to yeast cells with significant levels of megabody display (high CoA647 fluorescence level) (Figure 5). On the other hand, similarly stained cells, but with Mb Nb207 cHopQ GFP does not bind to wild-type yeast cells that do not express Mb. These experiments conclude that Mb is a well-folded, functional, antigen-binding (GFP-binding) chimeric protein. Nb207 cHopQ can be expressed on the yeast surface.

[0142] [Example 2] Expression, purification and characterization of a 58 kD antigen-binding chimeric protein in which the cHopQ scaffold is inserted into the first β-turn connecting β-strands AB of a GFP-specific nanobody.

[0143] Next, this 58kDa Mb Nb207 cHopQ We expressed Mb in the periplasm of Escherichia coli, purified it to homogeneity, and investigated its properties. Nb207 cHopQTo express megabodies such as (SEQ ID NO: 20) in the periplasm of E. coli, standard methods were used to generate a cloning vector (designated pMESD2) that allows the expression of any desired megabody by inserting (a circular permutation mutant of) HopQ into the first β-turn connecting the highly conserved β-strands A and B of any nanobody. This vector is a derivative of pMES4 (Pardon, 2014) and contains the following polypeptides: the DsbA leader sequence, which directs secretion of the megabody into the periplasm of E. coli; the NbA sequence, which corresponds to the highly conserved FR1 portion of the nanobody; GFP It contains an open reading frame encoding β-strand A of 207, a circularly permuted variant of HopQ called cHopQ, a 6xHis tag, an EPEA tag, and finally an amber stop codon. The C-terminal portion of any nanobody (β-strand B to β-strand G) can be cloned into this vector as a SapI fragment.

[0144] Mb Nb207 cHopQ The recombinant protein was expressed in the periplasm of E. coli and purified to homogeneity using Nb derived from β-strands B to G. GFP A DNA fragment encoding 207 (nucleotides 52 to 378 of SEQ ID NO: 121) was amplified by PCR (using primers SEQ ID NO: 122 and SEQ ID NO: 123) and expressed in the periplasm of E. coli under the transcriptional control of the Plac promoter with a His tag and an EPEA tag. Nb207 cHopQ The gene was cloned as a SapI fragment into the pMESD2 vector, which expresses the gene.

[0145] WK6 bacterial cells (WK6 is a - The non-suppressed strain was grown in 6 L of TB medium at 37°C and induced with IPTG when the cells reached logarithmic growth phase. Nb207 cHopQ Periplasmic expression of Mb was continued overnight at 28°C. Cells were harvested by centrifugation and recombinant Mb was isolated using the osmotic shock method. Nb207 cHopQThe recombinant megabody was released from the periplasm (Pardon et al., 2014). The recombinant megabody was then separated from the cytoplasm by centrifugation and recovered from the clarified supernatant using a HisTrap FF 5 mL pre-packed column. The protein was then eluted from the NiNTA resin by applying 500 mM imidazole and concentrated by centrifugation using a 3 kDa NMWL filter (Nominal Molecular Weight Limit). The concentrated sample was then applied to a Superdex 200PG16 / 90 size-exclusion column, and 24 mg of recombinant megabody was recovered as a soluble protein with an apparent molecular weight of approximately 60 kDa.

[0146] Next, purified recombinant Mb Nb207 cHopQ The functional properties of the megabody were analyzed by size-exclusion chromatography (SEC). Megabodies were incubated with a four-fold molar amount of GFP for 30 min at 4°C and then applied to a Superdex 75PG16 / 90 column. Purified GFP alone was applied separately to the same size-exclusion column (Figure 6). Elution of the various proteins was monitored by measuring UV absorbance at 280 nm (absorption by any protein) and 488 nm (absorption by GFP). The elution spectrum shown in Figure 6 reveals that the mixture containing megabodies and excess GFP elutes in two symmetrical peaks (blue and red absorbance profiles). The first peak (highest molecular weight) absorbs at 488 nm, indicating that it contains GFP. The second peak elutes with the same elution volume of GFP alone (green absorbance profile) and also absorbs at 488 nm. SDS-polyacrylamide gel electrophoresis of the corresponding elution fractions confirms that the first elution peak of the mixture containing megabodies and excess GFP contains megabodies and GFP, while the second peak contains only GFP. Taking all these data together, purified recombinant Mb Nb207 cHopQ It can be seen that Mb forms a complex with GFP and is difficult to separate by size exclusion chromatography. Nb207 cHopQReal-time kinetic analysis of the specific binding of Mb to GFP was performed by biolayer interferometry. Streptavidin-coated Octet® biosensors were used to capture biotinylated GFP, and various concentrations of Mb were detected. Nb207 cHopQ As demonstrated by the data shown in Figure 24, Mb Nb207 cHopQ The affinity for GFP when using Nb GFP The affinity was similar to that when 207 was used alone (Fig. 24B).

[0147] [Example 3] Structure determination by X-ray crystallography of a 58 kD antigen-binding chimeric protein in which a cHopQ scaffold was inserted into the first β-turn connecting β-strands AB of a GFP-specific nanobody.

[0148] As described in Examples 1 and 2, the 58 kDa chimeric Mb Nb207 cHopQ We were able to express and purify the megabody, and decided to crystallize it and analyze its structure by X-ray crystallography.

[0149] Mb Nb207 cHopQ The complex was purified by SEC (Figure 6) as described in Example 2, concentrated to 48 mg / mL, and subjected to a number of commercially available sparse-matrix crystallization screens (JSCG / Proplex / PEGion / Wizard12 / Morpheus) using a 0.1 μL drop of 0.1 μL of mother liquor. Small crystals obtained under JSCG screen A2 conditions (0.1 M sodium citrate, pH 5.5, 20% w / v PEG3000) were used in a seeding optimization approach. Well-diffracting crystals were obtained in 0.2 M ammonium citrate, 17% PEG3350, 10% glycerol, 48 mg / mL Mb207 seeded from JSCG A2 crystals. Data were acquired at a Diamond (UK) I3 source, and the structure was refined to 2.6 Å resolution (Table 1).

[0150] [Table 1]

[0151] The megabody was crystallized in P1, with 10 molecules per asymmetric unit. The RMSD between different molecules in the asymmetric unit ranged from 0.3 to 2.7 Å, indicating that the nanobody was rigidly linked to the scaffold via two peptide bonds connecting the nanobody to the scaffold (Figure 7).

[0152] [Example 4] Expression and purification of a 58 kD antigen-binding chimeric protein in which a cHopQ scaffold was inserted into the first β-turn connecting β-strands AB of a nanobody that stabilizes the protein complex.

[0153] As a second example of an antigen-binding chimeric protein, we expressed and purified a 58 kD antigen-binding chimeric protein in which the cHopQ scaffold was inserted into the first β-turn connecting β-strands A and B of Nb35, which binds to the interface between the Gβ subunit and the Gα subunit of the β2-adrenergic receptor-Gs protein complex (Rasmussen et al., 2011a).

[0154] Mb Nb35 cHopQThe 58 kDa megabody, designated as , is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked according to Figures 2 and 3. The immunoglobulin domain used in this example is a nanobody that binds to the interface between the Gβ and Gα subunits of the β2-adrenergic receptor-Gs protein complex (Rasmussen et al., 2011a), with CDR1 interacting primarily with Gβ and the long CDR3 loop interacting with both the Gβ and Gα subunits as shown in SEQ ID NO:24. All parts were linked together by peptide bonds from the amino to the carboxy terminus in the following order: β-strand A of the anti-GFP nanobody (residues 1-13 of SEQ ID NO: 1), the C-terminal part of HopQ (residues 192-414 of SEQ ID NO: 19), a short peptide linker (SEQ ID NO: 21) connecting the C- and N-termini of HopQ to generate a circular permutation of the scaffold protein, the N-terminal part of HopQ (residues 14-186 of SEQ ID NO: 19), strands B to G of the nanobody (residues 16-128 of SEQ ID NO: 24), and the 6xHis / EPEA tag (SEQ ID NO: 25).

[0155] Mb Nb35 cHopQ In order to express Nb35 in the periplasm of E. coli and purify the recombinant protein to homogeneity, a DNA fragment encoding Nb35 derived from β-strands B to G was amplified by PCR (using primers SEQ ID NO: 122 and SEQ ID NO: 123) and cloned as a SapI fragment into the pMESD2 vector, which expresses the His-tagged and EPEA-tagged cHopQNb35 megabody (SEQ ID NO: 25) under the transcriptional control of the PLac promoter in the periplasm of E. coli.

[0156] Mb Nb35 cHopQ cHopQNb35 was also expressed in the periplasm of E. coli and purified to homogeneity as described in Example 2. Furthermore, purified cHopQNb35 selectively binds to the interface between the Gβ and Gα subunits of the β2-adrenergic receptor-Gs protein complex.

[0157] [Example 5] Expression and purification of a 58 kD antigen-binding chimeric protein in which the cHopQ scaffold was inserted into the first β-turn connecting β-strands A and B of a GPCR-specific nanobody.

[0158] As another example, we expressed and purified another 58 kD chimeric antigen-binding protein in which cHopQ was inserted into the first β-turn connecting β-strands A and B of Nb80, which binds to the human β2-adrenergic receptor and exhibits G protein-like behavior (Rasmussen et al., 2011b). Alternative examples of such nanobodies are described, for example, in WO 2012 / 007593 (see Tables 1 and 2), WO 2012 / 175643 (see Tables 2 and 3), WO 2014 / 122183 (see Tables 1 and 2), and WO 2015 / 110449 (see Tables 2 and 3). Accordingly, the present invention also encompasses all chimeric antigen-binding proteins designed and produced as described herein based on the sequences of the Nbs described in these cited patent applications (as set out in the tables). Alternatively, since the CDRs of the Nb are sufficient to obtain specific stabilization of the desired GPCR complex protein as described, the present invention also includes antigen-binding chimeric proteins designed and constructed as described herein, in which the CDRs of the antigen-binding domain of the antigen-binding chimeric protein that binds to the GPCR complex are based on CDRs derived from a Nb specific for the GPCR complex.

[0159] Mb Nb80 cHopQThe 58 kDa megabody, designated Nb80, is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked according to Figures 2 and 3. The immunoglobulin domain used in this example is Nb80 (SEQ ID NO: 26), a nanobody that binds to the cytoplasmic side of the β2-adrenergic receptor. The 8-amino acid sequence of its CDR3 enters a hydrophobic pocket formed by amino acids from TM segments 3, 5, 6, and 7 of the receptor. The 4-amino acid sequence of its CDR1 provides additional stabilizing interactions with the cytoplasmic ends of TM segments 5 and 6 (Rasmussen et al., 2011b). The CDR3 occupies a position similar to the carboxyl-terminal peptide of Gs in the β2AR-Gs protein complex (Rasmussen et al., 2011a). All parts were linked together by peptide bonds from the amino to the carboxy terminus in the following order: β-strand A of the anti-GFP nanobody (residues 1-13 of SEQ ID NO: 1), the C-terminal part of HopQ (residues 192-414 of SEQ ID NO: 19), a short peptide linker (SEQ ID NO: 21) connecting the C- and N-termini of HopQ to generate a circular permutation of the scaffold protein, the N-terminal part of HopQ (residues 14-186 of SEQ ID NO: 19), β-strands B to G of Nb80 (residues 16-120 of SEQ ID NO: 27), and the 6xHis / EPEA tag (SEQ ID NO: 27).

[0160] Mb Nb80 cHopQ To express Nb80 in the periplasm of E. coli and purify the recombinant protein to homogeneity, a DNA fragment encoding Nb80 derived from β-strands B to G was amplified by PCR (using primers SEQ ID NO: 122 and SEQ ID NO: 123) and cloned as a SapI fragment into the pMESD2 vector as described in Example 4. This plasmid expresses His-tagged and EPEA-tagged Nb80 under the transcriptional control of the Plac promoter in the periplasm of E. coli. Nb80 cHopQ (SEQ ID NO: 27) is expressed.

[0161] As with the megabody in the above example, Mb Nb80cHopQ can also be expressed in the periplasm of E. coli and purified to homogeneity as described in Example 2. Nb80 cHopQ selectively binds to and stabilizes the active conformation of β2AR. Nb80 cHopQ The pharmacological properties of the β2AR wild-type (wt) receptor in the presence of β2AR-wt alone and in the presence of an unrelated megabody, Mb Nb207 cHopQ The properties of Nb80, which binds to the human β2-adrenergic receptor and exhibits G protein-like behavior (Rasmussen et al., 2011b), were compared with those of β2AR-wt in the presence of Mb (Figure 53). Nb80 is a nanobody that selectively binds to agonist-bound β2AR and exhibits G protein-like behavior, thus stabilizing the active state conformation of the receptor in the agonist-β2AR-Nb80 complex (Rasmussen et al., 2011b). Nb80 cHopQ We investigated whether the β2AR-specific binding of Nb1200 specifically binds to GFP and maintains its affinity for β2AR using a radioligand assay. GFP 207 Mb Nb207 cHopQ was used as a negative control along with β2AR but showed no affinity for β2AR (Figure 53).

[0162] Nb80 or Mb against β2AR Nb80 cHopQTo compare the pharmacological effects of the β2AR constructs, a radioligand assay was performed as follows. To express the β2AR wild-type (wt) receptor, 1 μg of the pFastBac1-β2ARwt construct was transformed into DH10Bac™ cells using the Bac-to-Bac® Baculovirus Expression System (Invitrogen, catalog no. 10359-016) according to the manufacturer's instructions and plated onto fresh LB agar plates supplemented with 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml X-gal, and 40 μg / ml IPTG. White colonies were picked, the bacmid was purified, and the sequence of the open reading frame was confirmed by sequencing. Recombinant baculovirus was generated by transfecting 2 μg of bacmid DNA into Sf9 cells in a 6-well plate format with 8 μl of Cellfectin as the transfection reagent. The cells were incubated at 27°C, and P1 virus was harvested after 3 days. The virus was then amplified by serial passage, and P3 virus was harvested. 2–3 × 10 6Sf9 cells were infected with baculovirus at a concentration of 0.5 / ml and allowed to express the receptor for 72 hours at 27°C. Receptor expression and cell surface localization were assessed by flow cytometry using mouse anti-FLAG M2 antibody (1:100), which recognizes the FLAG peptide sequence in the N-terminal extracellular portion of the receptor, and anti-mouse DyLight405 (1:100) as the secondary antibody. Cells were centrifuged at 1000 x g for 30 minutes and membranes were prepared from these cells by resuspending the resulting pellet in TME binding buffer (75 mM Tris / HCl pH 7.4, 12.5 mM MgCl2, 1 mM EDTA) supplemented with protease inhibitors (cOmplete™ EDTA-free protease inhibitor cocktail tablets, Roche). Cells were lysed using an Ultra Turrax homogenizer at maximum speed with six 10-second bursts. The membrane-containing lysate was then centrifuged at 40,000 × g for 40 minutes at 4°C, the supernatant discarded, and the membrane pellet resuspended in TME binding buffer supplemented with 10% sucrose. Membranes were stored at -80°C until further use. Total membrane protein content was estimated using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instructions. All samples were diluted to a final concentration of 0.2 mg / ml to normalize data according to total membrane protein concentration for subsequent analysis. For radioligand competitive binding assays, 2 nM [ 3 5 μM Nb80, Mb Nb80 cHopQ , Mb Nb207 cHopQ The nanobody was incubated at a concentration of 10 s with either epinephrine (natural agonist, Sigma catalog no. E4250) or (-)-isoproterenol hydrochloride (full agonist, Sigma catalog no. I6504) in the presence or absence of the nanobody. -11 M~10 -4Ten micrograms of membranes expressing β2AR-wt were incubated with increasing concentrations of β2AR-wt in a range of 10 μM. Nonspecific binding was determined in the presence of 10 μM alprenolol. Samples were incubated for 2 hours at room temperature on a shaking platform, and receptor-bound radioligand was separated from free radioligand by filtration through Whatman GF / C unifilters (Perkin Elmer, catalog no. 6005174) using a 96-well FilterMate harvester (Perkin Elmer). After filtration, membranes retained on the filter plates were washed with ice-cold wash buffer (20 mM Tris-HCl pH 7.4), and the filters were dried for 1 hour at 50°C. After adding 35 μl of scintillation fluid (MicroScint™-O, Perkin Elmer), the radioactivity (cpm) retained on the filters was measured in a Wallac MicroBeta TriLux scintillation counter. Data represent the mean ± SE of each experiment performed in duplicate. IC50 values ​​were determined by nonlinear regression analysis using Prism (GraphPad Software, San Diego, CA).

[0163] As shown in Figure 53, Mb Nb80 cHopQ The pharmacological properties of β2AR-wt in the presence of Nb80 were very similar to those of β2AR-wt in the presence of Nb80, and were significantly different from those of β2AR-wt alone or Mb Nb207 cHopQ The results showed that the activity of Mb120, which exhibits G protein-like behavior (see Nb80), was significantly different from that of β2AR-wt in the presence of Nb120. Nb80 cHopQ In the presence of Mb, the β2AR-wt receptor exhibits increased affinity for agonists (epinephrine, isoproterenol), Nb80 cHopQ The receptor in the presence of Mb is shown to adopt an active state conformation (Rasmussen et al., 2011b). Nb80 cHopQThe increased affinity of β2AR-wt for the natural agonist epinephrine in the presence of β2AR-wt increases the IC50 of epinephrine for β2AR-wt alone by Mb Nb80 cHopQ IC50 of epinephrine against β2AR-wt in the presence of high Divide by and the apparent titer shift is

[0164]

number

[0165]

number

[0166] [Example 6] Expression and purification of a 58 kD antigen-binding chimeric protein in which a cHopQ scaffold was inserted into the first β-turn connecting β-strands AB of an ion channel-binding nanobody.

[0167] Another example is the pentameric ligand-gated ion channel GABA AWe decided to express and purify another 58 kD antigen-binding chimeric protein in which cHopQ was inserted into the first β-turn connecting β-strands A and B of Nb25, which binds to Nb25 (Miller et al., 2017).

[0168] Mb Nb25 cHopQ The 58 kDa megabody, designated Nb25, is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked according to Figures 2 and 3. The immunoglobulin domain used in this example was Nb25 (SEQ ID NO: 28), a nanobody that binds to the extracellular domain of the GABAA β3 subunit (Miller et al., 2017). All parts were linked together by peptide bonds from the amino terminus to the carboxy terminus in the following order: β-strand A of the anti-GFP nanobody (residues 1-13 of SEQ ID NO: 1), the C-terminal portion of HopQ (residues 192-414 of SEQ ID NO: 19), a short peptide linker (SEQ ID NO: 21) connecting the C-terminus and N-terminus of HopQ to generate a circular permutation of the scaffold protein, the N-terminal portion of HopQ (residues 14-186 of SEQ ID NO: 19), β-strands B to G of Nb25 (residues 16-125 of SEQ ID NO: 28), and a 6xHis / EPEA tag (SEQ ID NO: 29).

[0169] Mb Nb25 cHopQ To express Nb25 in the periplasm of E. coli and purify the recombinant protein to homogeneity, a DNA fragment encoding Nb25 derived from β-strands B to G was amplified by PCR (using primers SEQ ID NO: 122 and SEQ ID NO: 123) and cloned as a SapI fragment into the pMESD2 vector as described in Example 4. This plasmid expresses His-tagged and EPEA-tagged Nb25 under the transcriptional control of the Plac promoter in the periplasm of E. coli. Nb25 cHopQ (SEQ ID NO: 29) is expressed.

[0170] As with the megabody in the above example, Mb Nb25 cHopQcan also be expressed in the periplasm of E. coli and purified to homogeneity as described in Example 2. Nb25 cHopQ binds to the extracellular domain of the GABAA β3 subunit.

[0171] [Example 7] Design and production by in vitro selection of another 58 kD antigen-binding chimeric protein in which c7HopQ is inserted into the first β-turn connecting β-strands AB of a GFP-specific nanobody.

[0172] Because the folding ability, as well as the stability and rigidity of a megabody, can depend on the composition and length of the polypeptide linkages connecting the immunoglobulin to the scaffold, we employed in vitro evolution techniques to fine-tune specific megabody formats as needed. Starting with the megabody described in Example 1, we constructed a library available for in vitro selection that encoded similarly designed megabodies in which two short peptides of variable length and mixed amino acid composition linked the nanobody to the scaffold, according to Figure 2.

[0173] The 58 kDa megabody described in this example is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked by a short polypeptide bond according to Figure 2. The immunoglobulin used in this example is the GFP-binding nanobody shown in SEQ ID NO: 1. All portions were linked to each other by peptide bonds from the amino terminus to the carboxy terminus in the following order: anti-GFP nanobody β-strand A (residues 1-12 of SEQ ID NO: 1), a peptide linker of one or two amino acids of random composition, the C-terminal portion of HopQ (residues 193-414 of SEQ ID NO: 19), a peptide linker connecting the C-terminus and N-terminus of HopQ to generate a circular permutation of the scaffold protein (SEQ ID NO: 21), the N-terminal portion of HopQ (residues 15-186 of SEQ ID NO: 19), a peptide linker of one or two amino acids of random composition, anti-GFP nanobody β-strands B to G (residues 11-126 of SEQ ID NO: 1), a 6xHis tag, and an EPEA tag (SEQ ID NOs: 30-33).

[0174] To display and select functional variants of the megabodies described in Examples 1, 2, and 3 on yeast, with different linker compositions and lengths connecting the nanobodies to the scaffold, standard methods were used to display and select various megabodies (SEQ ID NOs: 34-37) fused to a number of accessory peptides and proteins according to Figure 8, namely, the appS4 leader sequence directing extracellular secretion in yeast (Rakestraw, 2009), β-strand A of the anti-GFP nanobody (SEQ ID NO: 1, residues 1-12), a peptide linker of one or two amino acids of random composition, the C-terminal part of HopQ (residues 193-414 of SEQ ID NO: 19), a short peptide linker (SEQ ID NO: 21) connecting the C-terminus and N-terminus of HopQ to generate a circular permutation of the scaffold protein, the N-terminal part of HopQ (residues 15-186 of SEQ ID NO: 19), a peptide linker of one or two amino acids of random composition, β-strands B to G of the anti-GFP nanobody (residues 11-126 of SEQ ID NO: 1), a flexible (GGSG) n A library of open reading frames encoding peptide linkers, the Aga2p adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via disulfide bonds with the Aga1p protein, an acyl carrier protein (Johnsson, 2005) for orthogonal fluorescent staining of the displayed fusion protein, and a myc tag was constructed. These open reading frames were inserted into the pCTCON2 vector (Chao, 2006) under the transcriptional control of the galactose-inducible GAL1 / 10 promoter to construct a yeast display library encoding 184,000 different variants of the megabody described in Examples 1, 2, and 3 (see Figure 8).

[0175] For in vitro selection, this library was transformed into the yeast strain EBY100. Transformants were grown and induced overnight in galactose-rich medium. The induced cells were orthogonally stained with coA-647 (2 μM) and incubated with 100 nM GFP using SFP synthase (1 μM). These cells were then washed and subjected to two-parameter FACS analysis to identify yeast cells that displayed high levels of specific megabodies (high CoA-647 fluorescence) and bound the GFP antigen (high GFP fluorescence). Cells displaying high levels of GFP-binding nanobodies were selected and amplified in glucose-rich medium for subsequent rounds of selection using yeast display and two-parameter FACS analysis.

[0176] After one round of selection, a representative number of cells with high fluorescence levels in the CoA-647 and GFP channels were grown as single colonies and subjected to DNA sequencing to determine the sequences of a representative number of peptide linkers linking nanobodies to scaffold proteins. Four representative clones for each linker type (1-1, 1-2, 2-1, and 2-2 amino acid short linker variant) (Table 2) were confirmed to bind to 100 nM GFP in FACS experiments (Figure 30). These results demonstrate that various short peptide linkages of antigen-binding domains and scaffold proteins can be selected from the megabody library by in vitro selection and displayed as functional antigen-binding chimeric proteins. Since functional mutants of megabodies were successfully displayed on the yeast surface (see above), we decided to express four representative megabody clones with 1-amino acid short linkers (MP1331_A5, MP1331_A12, MP1331_B7, and MP1331_G10 in Table 2) in the periplasm of E. coli, purify these chimeras to homogeneity, and characterize them. Nb207 c7HopQ Four mutants (Mb Nb207 c7HopQ A5, Mb Nb207 c7HopQ A12, Mb Nb207 c7HopQ B7, Mb Nb207c7HopQ G10) and wild-type plus a short circular permutation (c7; see Example 23) (Mb Nb207 c7HopQ ) was prepared essentially as described in Example 2. Nb207 c7HopQ (SEQ ID NO: 136) was made from the conserved N-terminal β-strand A of anti-GFP nanobody (residues 1 to 13 of SEQ ID NO: 1), the C-terminal part of HopQ (residues 192 to 411 of SEQ ID NO: 19), the N-terminal part of HopQ (residues 18 to 186 of SEQ ID NO: 19), β-strands B to G of anti-GFP nanobody (residues 16 to 126 of SEQ ID NO: 1), a 6xHis tag, and an EPEA tag.

[0177] 4 types of Mb Nb207 c7HopQ The mutants (SEQ ID NOs: 137-140) were generated from the conserved N-terminal β-strand A of the anti-GFP nanobody (residues 1-12 of SEQ ID NO: 1), a one-amino acid linker (Table 2), the C-terminal part of HopQ (residues 193-411 of SEQ ID NO: 19), the N-terminal part of HopQ (residues 18-185 of SEQ ID NO: 19), a one-amino acid linker (Table 2), β-strands B-G of the anti-GFP nanobody (residues 17-126 of SEQ ID NO: 1), and a 6xHis / EPEA tag.

[0178] These megabodies (SEQ ID NOs: 136-140) were expressed in E. coli and purified as described in Example 2. The concentrated sample was then applied to a Superdex 200PG10 / 300 size-exclusion column, and a homogeneous, soluble protein sample with an apparent molecular weight of approximately 60 kDa was eluted (Figure 31). The functional properties of the purified recombinant megabodies (SEQ ID NOs: 136-140) were analyzed by enzyme-linked immunosorbent assay (ELISA). Purified GFP was immobilized to the wells of a Maxisorp microtiter plate (Nunc) at a concentration of 0.1 μg / well in sodium bicarbonate buffer (pH 8.2). Remaining protein-binding sites in the wells were blocked with milk-PBS solution for 2 hours at room temperature. Purified megabody samples were incubated in GFP-coated and uncoated wells. After a washing step, binding of the megabodies to GFP was tested using a CaptureSelect biotinylated antibody (Life Technologies), which specifically recognizes the EPEA tag present only in megabodies. The Capture Select biotinylated antibodies were then detected using streptavidin-alkaline phosphatase (Promega). After addition of the enzyme substrate, p-nitrophenyl phosphate, the absorbance at 405 nm was measured. When immobilized with GFP, the detected signal was at least 10-fold higher for each megabody compared to the GFP-free condition. ELISA and SEC data indicate that the purified recombinant megabodies (SEQ ID NOs: 136-140) can be purified to homogeneity and can form complexes with GFP (Figures 31 and 32).

[0179] [Table 2]

[0180] [Example 8] Design and production by in vitro selection of a 100 kDa antigen-binding chimeric protein in which cYgjK was inserted into the first exposed β-turn (connecting β-strands A and B) of a GFP-specific nanobody.

[0181] Alternatively, megabodies were designed from nanobodies linked to larger scaffolds. A library encoding megabody variants in which two short peptides link the nanobody to another scaffold was constructed for in vitro selection according to Figures 2 and 9.

[0182] The designed 100 kDa megabody is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked by a short polypeptide linker according to Figure 2. The immunoglobulin used was the GFP-binding nanobody shown in SEQ ID NO: 1. The alternative scaffold protein used was YgjK (PDB3W7S, SEQ ID NO: 38), an 86 kDa periplasmic protein from E. coli. All parts were linked together by peptide bonds from the amino terminus to the carboxy terminus in the following order: anti-GFP nanobody β-strand A (residues 1-12 of SEQ ID NO: 1), a peptide linker of one or two amino acids of random composition, the C-terminal part of YgjK (residues 464-760 of SEQ ID NO: 38), a short peptide linker (SEQ ID NO: 43) connecting the C-terminus and N-terminus of YgjK to generate a circular permutation of the scaffold protein, the N-terminal part of YgjK (residues 1-461 of SEQ ID NO: 38), a peptide linker of one or two amino acids of random composition, and anti-GFP nanobody β-strands B to G (residues 17-126 of SEQ ID NO: 1) (SEQ ID NOs: 39-42).

[0183] Mb with different linker compositions and lengths connecting the nanobody to the scaffold Nb207 cYgjkQStandard methods were used to display and select functional variants of random linkers (SEQ ID NOS: 39-42) on yeast. Various megabodies (SEQ ID NOS: 44-47) were fused to a number of accessory peptides and proteins: the appS4 leader sequence (Rakestraw, 2009) that directs extracellular secretion in yeast; β-strand A of an anti-GFP nanobody (SEQ ID NOS: 1-12); a peptide linker of one or two amino acids of random composition; the C-terminal portion of YgjK (residues 464-760 of SEQ ID NOS: 38); a short peptide linker (SEQ ID NOS: 43) connecting the C- and N-termini of YgjK to generate a circular permutation of the scaffold protein; the N-terminal portion of YgjK (residues 1-461 of SEQ ID NOS: 38); a peptide linker of one or two amino acids of random composition; β-strands B to G of an anti-GFP nanobody (residues 17-126 of SEQ ID NOS: 1); and flexible (GGSG). n A library of open reading frames encoding peptide linkers, the Aga2p adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond with the Aga1p protein, an acyl carrier protein (Johnsson, 2005) for orthogonal fluorescent staining of the displayed fusion proteins, and myc tags was constructed. These open reading frames were inserted into the pCTCON2 vector (Chao, 2006) under the transcriptional control of the galactose-inducible GAL1 / 10 promoter and expressed in Mb Nb207 cYgjkQ A yeast display library encoding 184,000 different variants of the random linker (SEQ ID NOs: 39-42) was constructed.

[0184] For in vitro selection, this library was transformed into the yeast strain EBY100. Transformants were grown and induced overnight in galactose-rich medium. The induced cells were orthogonally stained with coA-647 (2 μM) and incubated with 100 nM GFP using SFP synthase (1 μM). These cells were then washed and subjected to two-parameter FACS analysis to identify yeast cells that displayed high levels of specific megabodies (high CoA-647 fluorescence) and bound the GFP antigen (high GFP fluorescence). Cells displaying high levels of GFP-binding nanobodies were selected and amplified in glucose-rich medium for subsequent rounds of selection using yeast display and two-parameter FACS analysis.

[0185] After two rounds of selection, a representative number of cells with high fluorescence levels in the CoA-647 and GFP channels were grown as single colonies and subjected to DNA sequencing to determine the sequences of a representative number of peptide linkers linking nanobodies to scaffold proteins. One or two representative clones from each linker type (length) of 1-1, 1-2, 2-1, and 2-2 amino acid short linker variants (Table 3) were confirmed to bind to 100 nM GFP in FACS experiments (Figure 33). These results demonstrate that various short peptide linkages of antigen-binding domains and scaffold proteins can be selected from the megabody library by in vitro selection and displayed as functional antigen-binding chimeric proteins. Nb207 cYgjkQ Since we were able to display functional mutants of the 1333-like protein on the yeast surface (see above), we decided to express one representative megabody clone of each amino acid short linker (MP1333_E2, MP1333_A2, MP1333_C4, and MP1333_F5 in Table 3) in the periplasm of E. coli. Nb207 cYgjkQ Four mutants of were constructed as chimeric polypeptides with the following amino acids: Mb Nb207 cYgjkE2 (SEQ ID NO: 141) was constructed from β-strand A of the anti-GFP nanobody (residues 1-12 of SEQ ID NO: 1), a Tyr1 amino acid linker, the C-terminal portion of YgjK (residues 464-760 of SEQ ID NO: 38), a short peptide linker (SEQ ID NO: 43) connecting the C-terminus and N-terminus of YgjK to generate a circular permutation of the scaffold protein, the N-terminal portion of YgjK (residues 1-461 of SEQ ID NO: 38), an Asp1 amino acid linker, β-strands B to G of the anti-GFP nanobody (residues 17-126 of SEQ ID NO: 1), and a 6xHis / EPEA tag.

[0186] Change the linker and Mb Nb207 cYgjk A2 (SEQ ID NO: 142) is a Glu1 amino acid linker, a Gly-Asp2 amino acid linker, and Mb Nb207 cYgjk C4 (SEQ ID NO: 143) is a Met-Tyr2 amino acid linker and an Asn1 amino acid linker, and Mb Nb207 cYgjk Similar constructs were obtained for the other megabodies, except that F5 (SEQ ID NO: 144) used a Trp-Thr 2 amino acid linker and a Gly-Ala 2 amino acid linker. Mb using modified pMESD2 vector Nb207 cYgjk The mutants (SEQ ID NOs: 141-144) were expressed in E. coli as described in Example 2. This new vector (designated pMESP3) contains the following polypeptides: the pelB leader sequence, which directs secretion of the megabody into the periplasm of E. coli; GFP It contains an open reading frame encoding β-strand A of 207, a circularly permuted mutant of YgjK, the C-terminal portion of any nanobody (β-strand B to β-strand G), a 6xHis tag, an EPEA tag, and finally an amber stop codon.

[0187] 4 types of Mb Nb207 cYgjkPeriplasmic extracts of each of the mutants were used to analyze the functional properties of the expressed megabodies by ELISA as described in Example 7. Comparing the signals detected in samples with and without GFP fixation (Figure 34), it was found that the functional megabodies Nb207 cYgjk Periplasmic expression of the mutants (SEQ ID NOs: 141 to 144) was clearly confirmed.

[0188] Using IMAC followed by SEC as described in Example 2, Mb corresponding to the 1-1 amino acid short linker mutant was isolated. Nb207 cYgjk E2 (SEQ ID NO: 141) was further purified to homogeneity (Figure 35). Nb207 cYgjk The binding kinetics of E2 (SEQ ID NO: 141) to GFP was measured by Octet® and Nb GFP 207 nanobody (SEQ ID NO: 1) and Mb Nb207 cHopQ (SEQ ID NO: 20) (Figure 24). Binding affinity calculations confirmed that the antigen-binding chimeric proteins generated from the circularly permuted HopQ or YgjK scaffold proteins did not compromise the binding properties of the original single-domain immunoglobulin.

[0189] [Table 3]

[0190] [Example 9] Design and construction of a 58 kDa antigen-binding chimeric protein in which c / c7HopQ is inserted into the first β-turn connecting β-strands A and B of a GFP-specific nanobody, and further rigidified by an additional disulfide linking the scaffold to the nanobody.

[0191] A more rigid antigen-binding chimeric protein was further developed by constructing additional disulfide bonds linking the antigen-binding domain (here, the immunoglobulin domain) to the scaffold according to Figure 10. Specifically, site-directed mutagenesis was used to construct the Mb domain, where two short peptides and disulfide bonds link the nanobody to the scaffold according to Figures 2 and 10.Nb207 cHopQ Mutants of the above were generated.

[0192] The 58 kDa megabody described in this example is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked by short polypeptide and disulfide bonds according to Figures 2 and 10. The immunoglobulin used in this example is the GFP-binding nanobody shown in SEQ ID NO: 1. The scaffold protein is an adhesin domain of H. pylori strain G27 called HopQ (PDB: 5LP2, SEQ ID NO: 19). All parts were linked together by peptide bonds from amino to carboxy termini in the following order: β-strand A of the anti-GFP nanobody (residues 1-13 of SEQ ID NO: 1), the C-terminal part of HopQ (residues 192-414 of SEQ ID NO: 19), a short peptide linker (SEQ ID NO: 21) connecting the C- and N-termini of HopQ to generate a circularly permuted scaffold protein (cHopQ), the N-terminal part of HopQ with a single cysteine ​​replacement (residues 14-186 of SEQ ID NO: 19), β-strands B to G of the nanobody with a single cysteine ​​replacement (residues 16-126 of SEQ ID NO: 1), and a 6xHis / EPEA tag (SEQ ID NOs: 48-50). SEQ ID NO:51 was generated from β-strand A of an anti-GFP nanobody (residues 1-13 of SEQ ID NO:1), the C-terminal portion of HopQ with one residue replaced by cysteine ​​(residues 192-414 of SEQ ID NO:1), a short peptide linker (SEQ ID NO:21) connecting the C-terminus and N-terminus of HopQ to generate a circularly permuted scaffold protein (cHopQ), the N-terminal portion of HopQ (residues 14-186 of SEQ ID NO:19), β-strands B to G of a nanobody with one residue replaced by cysteine ​​(residues 16-126 of SEQ ID NO:1), and a 6xHis / EPEA tag.

[0193] Other constructs were designed using a shorter c7HopQ circularly permuted form (see Example 23 for c7HopQ) as a scaffold protein: β-strand A of the anti-GFP nanobody (SEQ ID NO: 1; residues 1-13), the C-terminal portion of HopQ (residues 192-411, SEQ ID NO: 19), the N-terminal portion of HopQ with a single cysteine ​​replacement (residues 18-186, SEQ ID NO: 19), β-strands B through G of the nanobody with a single cysteine ​​replacement (residues 16-126, SEQ ID NO: 1), a 6xHis tag, and an EPEA tag, generating SEQ ID NOs: 146-150. SEQ ID NO: 145 was generated from β-strand A of an anti-GFP nanobody (residues 1-13 of SEQ ID NO: 1), the C-terminal portion of HopQ with one residue replaced with cysteine ​​(residues 192-411 of SEQ ID NO: 1), the N-terminal portion of HopQ (residues 18-186 of SEQ ID NO: 19), β-strands B to G of a nanobody with one residue replaced with cysteine ​​(residues 16-126 of SEQ ID NO: 1), a 6xHis tag, and EPEA.

[0194] A total of 10 megabody mutants (Mb Nb207 cHopQ Cys1 to 4 (SEQ ID NOs: 48 to 51) and Mb Nb207 c7HopQ Cys5-10 (SEQ ID NOs: 145-150)) were expressed and purified from E. coli as described in Example 2. The functionality of these purified recombinant megabodies was analyzed by ELISA as described in Example 7. Comparing the signals detected in samples with and without immobilized GFP (Figure 36), Mb purified from the periplasm of E. coli showed Nb207 cHopQ Cys1 to 4 (SEQ ID NOs: 48 to 51) and Mb Nb207 c7HopQ The functionality of Cys5 to 10 (SEQ ID NOs: 145 to 150) was clearly confirmed.

[0195] To measure the effect of the introduced additional disulfides on the thermal stability of these engineered megabody mutants, thermal shift assays (TSA) were performed, and the melting temperature (Tm) of each megabody mutant was calculated as previously described (Hunynh et al., 2015). Specifically, 0.2 mg / mL of each gel filtration-purified megabody mutant was mixed with SYPRO Orange dye (Sigma) in 140 mM NaCl and 10 mM Tris pH 7.3 buffer. The fluorescence of 20 μL samples was then measured in triplicate by real-time PCR on a Bio-Rad CFX96 machine over a temperature range of 25–100 °C. Individual Tm values ​​were calculated using the Boltzmann equation with GraphPad Prism software (Table 4). The melting temperatures were significantly higher than those of the wild-type megabody (Mb). Nb207 cHopQ C 15 -C 534 (SEQ ID NO: 51), Mb Nb207 c7HopQ C 14 -C 512 (SEQ ID NO: 145), Mb Nb207 c7HopQ C 316 -C 472 (SEQ ID NO: 147) and Mb Nb207 c7HopQ C 314 -C 472 (SEQ ID NO: 148), the temperature increases by 10.9°C, 3.43°C, 4.29°C, and 6.14°C, and it is considered that these disulfide bonds are formed to rigidify the megabody.

[0196] [Table 4]

[0197] [Example 10] Design and production of an antigen-binding chimeric protein in which a GFP-specific nanobody is grafted onto azurin by in vitro selection.

[0198] Another approach to generating rigid, antigen-binding chimeric proteins is to link immunoglobulins to scaffolds using alternative linkage schemes. Specifically, we constructed a library of megabodies that encode nanobodies grafted onto scaffold proteins via three polypeptide bonds linking the nanobodies to the scaffold, as shown in Figure 11, and used them for in vitro selection.

[0199] The rigid antigen-binding chimeric protein described in this example is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked by three short polypeptide bonds according to Figure 11. The immunoglobulin used was the GFP-binding nanobody shown in SEQ ID NO: 1. The scaffold protein used was azurin, a small copper-containing single-domain protein from Pseudomonas aeruginosa (PDB2TSA, SEQ ID NO: 52, containing an M121A mutation compared to the P. aeruginosa protein). All parts were linked together by peptide bonds from the amino terminus to the carboxy terminus in the following order: anti-GFP nanobody β-strand A (residues 1-12 of SEQ ID NO: 1), a peptide linker of one or two amino acids of random composition, the N-terminal part of azurin (residues 3-22 of SEQ ID NO: 52), a peptide linker of one or two amino acids of random composition, anti-GFP nanobody β-strands B to G (residues 17-126 of SEQ ID NO: 1), a peptide linker of one or two amino acids of random composition, the C-terminal part of azurin (residues 31-128 of SEQ ID NO: 52), 6xHis, and EPEA tag (SEQ ID NOs: 53-60).

[0200] Standard methods were used to display and select functional representatives of these metal-binding megabodies (SEQ ID NOS: 53-60) on yeast, in which three short peptides link the nanobody to the scaffold. Various megabodies (SEQ ID NOS: 61-68) fused to a number of accessory peptides and proteins were synthesized using the appS4 leader sequence (Rakestraw, 2009) that directs extracellular secretion in yeast, β-strand A of an anti-GFP nanobody (SEQ ID NOS: 1-12), a peptide linker of one or two amino acids of random composition, the N-terminal portion of azurin (residues 3-22 of SEQ ID NOS: 52), a peptide linker of one or two amino acids of random composition, strands B through G of an anti-GFP nanobody (residues 17-126 of SEQ ID NOS: 1), a peptide linker of one or two amino acids of random composition, the C-terminal portion of azurin (residues 31-128 of SEQ ID NOS: 52), and flexible (GGSG) nanobodies. n We constructed a library of open reading frames encoding a peptide linker, the Aga2p adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond with the Aga1p protein, an acyl carrier protein for orthogonal fluorescent staining of the displayed fusion protein (Johnsson, 2005), and a myc tag. These open reading frames were inserted into the pCTCON2 vector (Chao, 2006) under the control of the galactose-inducible GAL1 / 10 promoter, and a yeast display library encoding 77,280,000 different variants of a megabody in which three short peptides link the nanobody to azurin was constructed as shown in Figure 12.

[0201] The library was transformed into the yeast strain EBY100 for in vitro selection by yeast display and FACS. ++The nanobodies were grown and induced overnight in galactose-rich medium in the presence of SFP synthase (1 μM). Induced cells were orthogonally stained with coA-647 (2 μM) and incubated with 100 nM GFP. These cells were then washed and subjected to two-parameter FACS analysis to identify yeast cells that displayed high levels of the specific megabody (high CoA-647 fluorescence) and bound the antigen GFP (high GFP fluorescence). Cells displaying high levels of GFP-binding nanobodies were selected and amplified in glucose-rich medium for subsequent rounds of selection using yeast display and two-parameter FACS analysis.

[0202] After two rounds of selection, a representative number of cells with high fluorescence levels in the CoA-647 and GFP channels were grown as single colonies and subjected to DNA sequencing to determine the sequences of a representative number of peptide linkers linking nanobodies to scaffold proteins. Two representative clones (Table 5) of linker types 1-2-1, 2-2-1, 1-2-2, and 2-2-2 amino acid short linker variants were confirmed to bind to 100 nM GFP in FACS experiments (Figure 37). These results demonstrate that megabodies concatenated from single-domain immunoglobulin portions and scaffold protein portions linked by three short polypeptide bonds can be selected from a megabody library by in vitro selection and displayed as functional antigen-binding chimeric proteins.

[0203] Mb Nb207 Azurin Since functional mutants of Mb were successfully displayed on the yeast surface (see above), we decided to express these antigen-binding chimeric proteins in the periplasm of E. coli. Nb207 Azurin The mutants (Table 5) were identified as follows: Nb207 AzurinThe mutants (SEQ ID NOs: 151-158) were produced as chimeric polypeptides containing β-strand A of the anti-GFP nanobody (residues 1-12 of SEQ ID NO: 1), an amino acid linker (Table 5), the N-terminal portion of azurin (residues 3-22 of SEQ ID NO: 52), an amino acid linker (Table 5), strands B-G of the anti-GFP nanobody (residues 17-126 of SEQ ID NO: 1), an amino acid linker (Table 5), the C-terminal portion of azurin (residues 31-128 of SEQ ID NO: 52), and a 6xHis / EPEA tag.

[0204] Mb Nb207 Azurin To express megabodies, such as mutants (SEQ ID NOS: 151-158), in the periplasm of E. coli, the pMESD2 vector described in Example 2 was modified. This new vector (designated pMESP5) contains the following polypeptides: the pelB leader sequence, which directs secretion of the megabody into the periplasm of E. coli; GFP It contains 207 β-strand A, the N-terminal portion of azurin, the C-terminal portion of any nanobody (β-strand B to β-strand G), the C-terminal portion of azurin, a 6xHis tag, an EPEA tag, and finally an open reading frame encoding an amber stop codon. Nb207 Azurin The mutants (SEQ ID NOs: 151 to 158) were expressed in the periplasm of E. coli as described in Example 2. Nb207 Azurin Periplasmic extracts of each of the mutants were used to analyze the functional properties of the expressed megabodies by ELISA as described in Example 7. Comparing the signals detected in samples with and without GFP immobilization (Figure 38), the functional megabodies Nb207 Azurin The periplasmic expression of the mutants (SEQ ID NOs: 151 to 158) was clearly confirmed. This result demonstrates that a megabody concatemerized from a single-domain immunoglobulin portion and a scaffold protein portion linked by three short polypeptide bonds can be functionally expressed in the periplasm of E. coli.

[0205] [Table 5]

[0206] [Example 11] An antigen-binding chimeric protein was designed and produced by in vitro selection, in which a circularly permuted mutant of an inverting glycosidase was inserted into the first exposed β-turn of a GFP-specific nanobody, and a homodimeric megabody with structural symmetry was designed and produced by in vitro selection.

[0207] We further engineered multimeric megabodies by grafting Nb onto the subunits of a multimeric scaffold protein with structural symmetry. We constructed a library encoding megabodies in which nanobodies were grafted onto each subunit of a large homodimeric scaffold protein via three short polypeptide bonds linking the nanobodies to the scaffold, as shown in Figure 11. Rigid homodimeric megabodies with two-fold rotational symmetry were identified by yeast display and FACS.

[0208] The homodimeric megabody (191 kDa per dimer) described in this example is a chimeric polypeptide concatenated from a single-domain immunoglobulin portion and a scaffold protein portion linked by three short polypeptide linkers according to Figure 11. The immunoglobulin used was the anti-GFP nanobody shown in SEQ ID NO: 1. The scaffold protein used was SusB (PDB3WFA, SEQ ID NO: 69), a homodimeric glucan 1,4-α-glucosidase from B. thetaiotaomicron. All parts were linked together from the amino to the carboxy terminus in the following order according to Figure 13: anti-GFP Nanobody β-strand A (residues 1-14 of SEQ ID NO: 1), serine, a peptide linker of one or two amino acids of random composition, the N-terminal part of SusB (residues 1-68 of SEQ ID NO: 69), a peptide linker of one or two amino acids of random composition, anti-GFP Nanobody β-strands B to G (residues 15-125 of SEQ ID NO: 1), a peptide linker of one or two amino acids of random composition, the C-terminal part of SusB (residues 76-718 of SEQ ID NO: 69), and a 6xHis / EPEA tag (SEQ ID NOs: 70-77).

[0209] Standard methods were used to display and select functional representatives of these homodimeric megabodies (SEQ ID NOS: 70-77) in yeast, in which three short peptides link the nanobody to each subunit of the homodimeric scaffold. Various megabodies (SEQ ID NOS: 78-85) fused to a number of accessory peptides and proteins were prepared using the appS4 leader sequence (Rakestraw, 2009) that directs extracellular secretion in yeast, β-strand A of an anti-GFP nanobody (residues 1-14 of SEQ ID NOS: 1), serine, a one- or two-amino acid peptide linker of random composition, the N-terminal portion of SusB (residues 1-68 of SEQ ID NOS: 69), a one- or two-amino acid peptide linker of random composition, β-strands B through G of an anti-GFP nanobody (residues 15-125 of SEQ ID NOS: 1), a one- or two-amino acid peptide linker of random composition, the C-terminal portion of SusB (residues 76-738 of SEQ ID NOS: 69), and flexible (GGSG). nWe constructed a library of open reading frames encoding a peptide linker, the Aga2p adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond with the Aga1p protein, an acyl carrier protein for orthogonal fluorescent staining of the displayed fusion protein (Johnsson, 2005), and a myc tag. These open reading frames were inserted into the pCTCON2 vector (Chao, 2006) under the transcriptional control of the galactose-inducible GAL1 / 10 promoter, and a yeast display library encoding 77,280,000 different variants of a megabody, in which three short peptides link the nanobody to each subunit of a homodimeric scaffold, was constructed according to Figure 11.

[0210] The library was transformed into the yeast strain EBY100 for in vitro selection by yeast display and FACS. ++ The cells were grown and induced overnight in galactose-rich medium in the presence of SFP synthase (1 μM). Induced cells were orthogonally stained with coA-647 (2 μM) and incubated with 100 nM GFP. These cells were then washed and subjected to two-parameter FACS analysis to identify yeast cells that displayed high levels of specific megabodies (high CoA-647 fluorescence) and bound GFP (high GFP fluorescence). Cells displaying high levels of GFP binding were selected and amplified in glucose-rich medium for subsequent rounds of selection using yeast display and two-parameter FACS analysis.

[0211] After multiple rounds of selection, a representative number of cells with high fluorescence levels in the CoA-647 and GFP channels were grown as single colonies and subjected to DNA sequencing. The length and composition of the linker connecting the nanobody to SusB in several megabody variants that displayed high levels and bound antigen were analyzed and determined to be well expressed, secreted from cells, and able to fold into functional GFP-binding chimeric proteins.

[0212] One of these homodimeric megabodies was expressed in the periplasm of Escherichia coli and purified to homogeneity. The purified protein was crystallized and its structure was analyzed in complex with GFP.

[0213] [Example 12] Design and production by in vitro selection of nanobody-displaying icosahedral virus-like particles in which a circularly permuted mutant of the PP7 viral coat protein was inserted into the first exposed β-turn of a GFP-specific nanobody.

[0214] Three-dimensional structure determination by cryo-EM relies on averaging the information present in two-dimensional projection images of multiple copies of individual particles oriented differently relative to the incident electron beam. In the case of icosahedral viruses, each virion can be considered a particle, but the symmetry of these structures is such that each particle contains multiple identical copies of the asymmetric unit, increasing the effective number of units averaged to determine the structure many-fold. Therefore, icosahedral viruses, which rigidly arrange nanobodies according to the symmetry imposed by their coat proteins, may be the ultimate tool for analyzing the structures of small proteins and their complexes by cryo-EM.

[0215] In this example, we designed icosahedral virus-like particles (VLPs) displaying nanobodies by grafting Nb onto the coat protein of an icosahedral bacteriophage that self-assembles into VLPs. When overexpressed in Escherichia coli, 90 copies of concatemerized dimers of the coat protein of the icosahedral bacteriophage PP7 of Pseudomonas aeruginosa or 180 copies of the coat protein can self-assemble to form icosahedral VLPs (O'Rourke et al., 2015). In these VLPs, the coat proteins are intertwined in pairs, with the N-terminus of one monomer closely adjacent to the C-terminus of the other. It has also been demonstrated that peptides can be inserted into the exposed loops of these dimers and displayed on the surface of the corresponding VLPs. Therefore, we constructed a random library encoding rigid antibody chimeras composed of nanobodies grafted onto concatemerized dimers of PP7 coat protein, with two short peptides linking the nanobodies to the scaffold, as shown in Figure 2. The 3.9 MDa icosahedral virus-like particles described in this example self-assemble from chimeric polypeptides composed of directly linked single-domain immunoglobulin and scaffold protein portions according to Figure 14. The immunoglobulin used is the GFP-binding nanobody shown in SEQ ID NO: 1. The scaffold protein used is a circular permutation of a covalent dimer of the coat protein of PP7 (PDB: 1DWN, SEQ ID NO: 2). PP7 is an icosahedral RNA bacteriophage of Pseudomonas aeruginosa. All parts were linked together from the amino to the carboxy terminus in the following order: methionine encoded by the start codon, anti-GFP nanobody β-strand A (residues 1-13 of SEQ ID NO: 1), PP7 coat protein (residues 12-128 of SEQ ID NO: 2), a peptide linker of one or two amino acids of random composition, glycine, PP7 coat protein (residues 2-128 of SEQ ID NO: 2), a peptide linker of one or two amino acids of random composition, glycine, PP7 coat protein (residues 2-8 of SEQ ID NO: 2), GFP-binding nanobody β-strands B to G (residues 16-126 of SEQ ID NO: 1), and a 6xHis / EPEA tag (SEQ ID NOs: 3-6).This rigid antigen-binding chimeric protein self-assembles into an icosahedral VLP (nano-VLP) that displays 90 copies of the nanobody on its surface (Figure 14).

[0216] To select functional representatives of these artificial GFP-binding coat proteins that self-assemble into nano-VLPs, we used standard methods to construct a library of open reading frames (SEQ ID NOS: 3-6) encoding methionine, β-strand A of the anti-GFP nanobody, circularly permuted dimeric PP7 coat protein, β-strands B-G of the GFP-binding nanobody, and a 6xHis / EPEA tag. These DNA fragments were cloned as NdeI-EcoRI fragments into the pMESP vector (ref. CA12729, a derivative of pMES4, GenBank GQ907248, in which the LguI site was removed), replacing the pelB signal peptide, any Nb sequences, and all detection tags, including gene 3. This newly created library was designated pcPP72Nb. GFP It was called 207L.

[0217] To select recombinant GFP-binding PP7 coat proteins that can be recombinantly expressed and assembled in vitro to form icosahedral virus-like particles, libraries of chimeric dimers were constructed at the plasmid level. These libraries were used to transform E. coli cells. Chimeric dimers capable of assembly into nanoVLPs were expressed in E. coli and subjected to chromatographic selection. IMAC purification was performed on the library to select clones that could express chimeric dimers and assemble into VLPs. Since each assembly-competent VLP encapsulated a nucleic acid encoding the nanobody-coat protein within its shell, clones were amplified by RT-PCR of RNA derived from these nanoVLPs. Several clones expressing the antigen-binding chimeric proteins were grown as single colonies and subjected to DNA sequencing to determine the sequence of the peptide linker connecting the first PP7 coat protein to the next PP7, as well as the sequence of the circularly permuted peptide linker. Individual clones containing full-length constructs were subjected to ELISA as described in Example 7 to identify clones that bound GFP (Figure 39). These results demonstrate that a concatemerized antigen-binding chimeric protein consisting of a single-domain immunoglobulin portion linked by a polypeptide bond and a (circularly permuted) PP7 coat protein portion can be selected from the library as a functional antigen-binding chimeric protein. Representative clones of various linker mutants are shown in Table 6.

[0218] [Table 6]

[0219] [Example 13] Design and production of nanobody-displaying icosahedral virus-like particles by in vitro selection, in which a circularly permuted mutant of the MS2 viral coat protein is inserted into the first exposed β-turn of a lysozyme-specific nanobody.

[0220] The 3.9 MDa icosahedral virus-like particles described in this example self-assemble from chimeric polypeptides composed of a single-domain immunoglobulin portion and a scaffold protein portion connected by a short polypeptide linker according to Figure 15. The immunoglobulin used was a lysozyme-binding nanobody (PDB: 1MEL, SEQ ID NO: 7). The scaffold protein used was a circularly permuted covalent dimer of the coat protein of MS2 (PDB: 2MS2, SEQ ID NO: 2). MS2 is an icosahedral RNA bacteriophage of Escherichia coli. All parts were linked together from the amino to the carboxy terminus in the following order: start codon-encoded methionine, Nanobody β-strand A (anti-GFP Nanobody β-strand A, residues 1-12), a random two-amino acid circularly permuted linker, MS2 coat protein (residues 17-130 of SEQ ID NO: 8, followed by residues 2-130 of SEQ ID NO: 8 and residues 2-14 of SEQ ID NO: 8), lysozyme-binding Nanobody β-strands B to G (residues 17-133 of SEQ ID NO: 7), and a 6xHis / EPEA tag (SEQ ID NOs: 9-13). This rigid, antigen-binding chimeric protein self-assembles into an icosahedral VLP displaying 90 copies of the Nanobody on its surface (Figure 15).

[0221] To select functional representatives of these artificial lysozyme-binding coat proteins that self-assemble into nano-VLPs, we used standard methods to construct a library of open reading frames (SEQ ID NOS: 9-13) encoding methionine, β-strand A of the anti-GFP nanobody, circularly permuted dimeric MS2 coat protein, β-strands B-G of the lysozyme-binding nanobody, and a 6xHis / EPEA tag. These DNA fragments were cloned as NdeI-EcoRI fragments into the pMESP vector (ref CA12729). This newly created plasmid library was designated pcMS22cAb. Lys It was called 3L.

[0222] To select recombinant lysozyme-binding MS2 coat proteins that can be recombinantly expressed and assembled in vitro to form icosahedral virus-like particles, libraries of nanoVLPs were constructed at the plasmid level. These libraries were used to transform E. coli cells. The nanoVLPs were expressed in E. coli and subjected to chromatographic selection for VLP assembly. Because each assembly-competent VLP encapsulated the nucleic acid encoding the nanobody-coat protein within its shell, these nanoVLPs could be purified by size-exclusion chromatography after sedimentation. RT-PCR sequencing of RNA derived from these nanoVLPs revealed that selection for assembly increased gene expression. Following affinity selection using lysozyme, RNA from these nanoVLPs that specifically binds to lysozyme was isolated and amplified.

[0223] A portion of these assembly-competent lysozyme-specific nanoVLPs composed of lysozyme-binding MS2 coat proteins was purified to homogeneity and analyzed by single-particle cryo-EM in the presence and absence of lysozyme.

[0224] [Example 14] Design and production by in vitro selection of nanobody-displaying icosahedral virus-like particles in which the naturally substituted viral coat protein of AP205 is inserted into the first exposed β-turn of a GFP-specific nanobody.

[0225] The dimers described in this example self-assemble from chimeric polypeptides consisting of a single-domain immunoglobulin portion and a scaffold protein portion connected by a short polypeptide linker, as shown in Figure 40. The immunoglobulin used was a GFP-binding nanobody (SEQ ID NO: 1). The scaffold protein used was a covalent dimer of the AP205 coat protein (PDB: 5FS4, SEQ ID NO: 166). AP205 is an icosahedral RNA bacteriophage of the genus Acinetobacter. The AP205 coat protein dimer adopts the conserved Leviviridae coat protein fold, except for the N-terminal region, which forms a β-heparin in other known single-stranded RNA phages. AP205 has a similar structure at the same position formed by the N- and C-terminal β-strands, resulting in a circular permutation compared to other coat proteins. As a result of this permutation, the coat protein termini are shifted to the most surface-exposed portion of the assembled particle, explaining their increased tolerance to long N- and C-terminal fusions (Shishovs et al., 2016).

[0226] All parts were linked together in the following order from amino to carboxy terminus: methionine encoded by the start codon, Nanobody β-strand A (anti-GFP Nanobody β-strand A, residues 1-11), a one-amino acid random linker, AP205 coat protein dimer (residues 4-128 of SEQ ID NO: 166 and residues 4-126 of SEQ ID NO: 166), GFP-binding Nanobody β-strands B-G (residues 16-126 of SEQ ID NO: 1), and a 6xHis / EPEA tag (SEQ ID NO: 167). To select functional representatives of such antigen-binding chimeric proteins, a library of open reading frames encoding methionine, anti-GFP Nanobody β-strand A, dimeric AP205 coat protein, GFP-binding Nanobody β-strands B-G, and a 6xHis / EPEA tag was constructed using standard methods. These DNA fragments were cloned as NdeI-EcoRI fragments into the pMESP vector (ref CA12729). This newly created plasmid was designated pMESAP2052XXPNb GFPThe antigen-binding chimeric (AP205) protein was designated 207. To select for antigen-binding chimeric (AP205) proteins that can be recombinantly expressed and correctly assembled in vitro, libraries of antigen-binding chimeric proteins are constructed at the plasmid level. These libraries are used to transform E. coli cells. To identify chimeric proteins that correctly assemble as chimeric dimers, individual clones were expressed in E. coli and subjected to ELISA as described in Example 7 to screen for clones that bind GFP (Figure 41). GFP-binding Mb Nb207 AP205x2 Several clones expressing XX were grown as single colonies and subjected to DNA sequencing to determine the sequence of the peptide linker connecting the nanobody to the scaffold protein. These results demonstrate that antigen-binding chimeric proteins concatenated from a single-domain immunoglobulin portion and a viral AP205 coat protein portion linked by two short polypeptide bonds can be selected from the library as functional antigen-binding chimeric proteins. Representative clones of the 1-1 amino acid short linker mutants are shown in Table 7. Some of these antigen-binding chimeric proteins were found to self-assemble into icosahedral VLPs that displayed 90 copies of the nanobody on their surface.

[0227] [Table 7]

[0228] [Example 15] Design and production by in vitro selection of a dimeric antigen-binding chimeric protein consisting of two polypeptide chains in which the AP205 coat protein is inserted into the first β-turn connecting β-strands A and B of a GFP-specific nanobody.

[0229] The dimers described in this example self-assemble from chimeric polypeptides composed of a single-domain immunoglobulin portion and a scaffold protein portion linked by a short polypeptide linker according to Figure 42. The immunoglobulin used was a GFP-binding nanobody (SEQ ID NO: 1). The scaffold protein used was AP205 (PDB: 5FS4, SEQ ID NO: 166). When AP205 monomers assemble into a dimer or VLP, the N-terminus of one monomer is close to the C-terminus of the second monomer, and the C-terminus of one monomer is close to the N-terminus of the second monomer. To obtain a functional antigen-binding chimera, β-strand A of the first monomer must bind to β-strands B to G of the GFP-binding nanobody of the second monomer, and β-strands B to G of the GFP-binding nanobody of the second monomer must bind to β-strand A of the first monomer, resulting in assembly into a dimeric antigen-binding chimeric protein.

[0230] All parts were linked together from the amino to the carboxy terminus in the following order: methionine encoded by the start codon, Nanobody β-strand A (β-strand A of the anti-GFP Nanobody, residues 1-11), a one amino acid random linker, AP205 coat protein (residues 4-126 of SEQ ID NO: 166), GFP-binding Nanobody β-strands B-G (residues 16-126 of SEQ ID NO: 1), and 6xHis / EPEA (SEQ ID NO: 173).

[0231] To select functional representatives of such dimeric antigen-binding chimeric proteins, a library of open reading frames encoding methionine, β-strand A of the anti-GFP nanobody, monomeric AP205 coat protein, β-strands B-G of the GFP-binding nanobody, and a 6xHis / EPEA tag was constructed using standard methods. These DNA fragments were cloned as NdeI-EcoRI fragments into the pMESP vector. This newly created plasmid was designated pMESAP2051XXPNb GFP It was called 207.

[0232] These libraries of dimeric antigen-binding chimeric proteins are constructed at the plasmid level to select for dimeric antigen-binding chimeric proteins that can be recombinantly expressed and correctly assembled in vitro. These libraries are used to transform E. coli cells. To identify chimeric proteins that correctly assemble as dimers, individual clones are expressed in E. coli and subjected to ELISA as described in Example 7 to screen for clones that bind to GFP (Figure 43). GFP-binding Mb Nb207 AP205 Several clones expressing the XX dimer were grown as single colonies and subjected to DNA sequencing to determine the sequence of the peptide...

Claims

1. An antigen-binding chimeric protein in which a functional antigen-binding domain is fused to a scaffold protein, the antigen-binding domain is a VHH; the scaffold protein has at least one exposed region in its tertiary structure to provide a fusion point; the scaffold protein is fused to the antigen-binding domain via two or more direct fusions or fusions using a linker, and is fused to the antigen-binding domain at one or more accessible sites present in a β-turn, which is an exposed region of the antigen-binding domain; the one or more accessible sites are other than an antigen binding loop or a CDR loop; and the scaffold protein is inserted into a β turn connecting β strands A and B of the antigen-binding domain or a β turn connecting β strands C and C' of the antigen-binding domain; the β-strands are defined according to the IMGT nomenclature; the fused scaffold protein disrupts the primary topology of the antigen-binding domain at the one or more accessible sites of the antigen-binding domain; The antigen-binding chimeric protein.

2. The antigen-binding chimeric protein of claim 1 , wherein the scaffold protein is a circularly permuted protein.

3. The antigen-binding chimeric protein according to claim 1 or 2, wherein the scaffold protein is a symmetric protein such as a protein that forms a multimeric scaffold or a virus-like particle.

4. The antigen-binding chimeric protein of any one of claims 1 to 3, wherein the antigen-binding domain and the scaffold are further linked by a disulfide bond.

5. The antigen-binding chimeric protein of any one of claims 1 to 4, wherein the scaffold protein has a total molecular weight of at least 30 kDa.

6. The antigen-binding chimeric protein according to any one of claims 1 to 5, wherein the scaffold protein comprises an antigen-binding domain such as a VHH.

7. The antigen-binding chimeric protein of any one of claims 1 to 6, wherein the scaffold protein is a labeled protein.

8. A nucleic acid molecule encoding the antigen-binding chimeric protein of any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule of claim 8.

10. The vector of claim 9 for surface display in yeast, phage, bacteria or viruses.

11. A host cell comprising the antigen-binding chimeric protein of any one of claims 1 to 7.

12. The host cell of claim 11 , wherein the antigen-binding chimeric protein and the target antigen are co-expressed.

13. (i) an antigen-binding chimeric protein according to any one of claims 1 to 7; and (ii) a target protein; wherein the target protein specifically binds to the antigen-binding chimeric protein.

14. 8. A composition comprising a first antigen-binding chimeric protein and a second antigen-binding chimeric protein according to any one of claims 1 to 7, wherein an antigen-binding domain of the second antigen-binding chimeric protein specifically binds to a scaffold protein of the first antigen-binding chimeric protein.

15. A composition for structural analysis of a target protein, comprising the nucleic acid molecule of claim 8, the vector of claim 9 or 10, the host cell of claim 11 or 12, or the complex of claim 13.

16. 16. The composition of claim 15, wherein the structural analysis comprises single particle cryo-EM or crystallography.

17. 1. A method for determining the three-dimensional structure of a target molecule, comprising: (i) providing a composition of the antigen-binding chimeric protein of any one of claims 1 to 7 or the antigen-binding chimeric protein of claim 14 and a target protein to form a complex, wherein the target protein is specifically bound to the antigen-binding chimeric protein; or providing a composite according to claim 13; (ii) displaying the complex under conditions suitable for structural analysis, wherein the three-dimensional structure of the target protein is determined at high resolution; The method comprising:

18. A diagnostic composition comprising the antigen-binding chimeric protein of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the vector of claim 9 or 10, the host cell of claim 11 or 12, or the complex of claim 13.

19. A composition for in vivo imaging, comprising the antigen-binding chimeric protein according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the vector according to claim 9 or 10, the host cell according to claim 11 or 12, or the complex according to claim 13.

20. A pharmaceutical composition comprising the antigen-binding chimeric protein of any one of claims 1 to 7 or the composition of claim 14.

21. A virus-like particle comprising the antigen-binding chimeric protein of any one of claims 1 to 7 or the composition of claim 14.

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