Humanized single domain antibody binding to an epitope tag

Abstract

The present invention provides humanized versions of single domain antibodies that specifically bind to the epitope tag of SRLEEELRRRLTE (SEQ ID NO: 2), which can be used to bind, detect, immobilize, isolate or purify proteins comprising said epitope tag. Furthermore, the present invention relates to nucleic acids encoding said humanized antibodies and vectors comprising the nucleic acids, as well as host cells comprising the nucleic acids or vectors described herein.

Description

Technical Field

[0001] This invention provides a humanized form of a single-domain antibody that specifically binds to an epitope tag of SRLEEELRRRLTE (SEQ ID NO: 2), which can be used to bind to, detect, immobilize, isolate, or purify proteins containing said epitope tag. Furthermore, this invention relates to nucleic acids encoding said humanized antibodies and vectors containing said nucleic acids, as well as host cells containing the nucleic acids or vectors described herein. Background Technology

[0002] In 2019, Götzke et al. published an article, 'The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications' (Götzke et al., Nat Commun. Sep 27, 2019; 10(1):4403. doi: 10.1038 / s41467-019-12301-7). ALFA ® The tag is a rationally designed peptide with a unique sequence that forms a stable α-helix in solution, is electrically neutral, and highly soluble under physiological conditions. It can fuse with binding partners in different orientations (N- or C-terminus or between two domains of the fusion protein) without loss of structure and function, and can be detected with a high-affinity (two-digit pM Kd) single-domain antibody (VHH). The authors have identified the antibody from immunized alpacas using a selection method based on the separation of target-specific B cells (CELLINE). ALFA ® A detailed description of the / NbALFA epitope tagging system can be found in PCT patent publication WO 2020 / 053239A1. Therefore, ALFA... ® Tags and NbALFA single-domain antibodies specific to these tags have great potential in bioscience applications.

[0003] NbALFA is derived from immunized alpacas, and alpaca-derived single-domain antibodies (VHHs) typically exhibit low homology to human sequences. However, for ALFA... ® The application of the / NbALFA tool in the human body is expected to achieve higher sequence homology with the corresponding human sequences. Summary of the Invention

[0004] This invention relates to a single-domain antibody that specifically binds to an epitope tag containing the sequence SRLEEELRRRLTE (SEQ ID NO: 2), wherein the antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94, a CDR2 sequence as shown in SEQ ID NO: 97, and a CDR3 sequence as shown in SEQ ID NO: 98; and wherein the antibody further comprises the following amino acid residues at positions 47, 48, and 50 corresponding to sequence positions 47, 48, and 50 of SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; and wherein the antibody comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 118-125.

[0005] The present invention also relates to a protein comprising the amino acid sequence of the antibody of the present invention.

[0006] The present invention also relates to a complex comprising a protein containing the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or has no amino acid; and the antibody of the present invention.

[0007] This invention also relates to a nucleic acid that encodes the antibody of this invention.

[0008] The present invention also relates to a vector comprising the nucleic acid of the present invention.

[0009] The present invention also relates to a host cell that contains the nucleic acid of the present invention or the vector of the present invention or an antibody expressing the present invention.

[0010] The present invention also relates to the use of the antibody of the present invention for binding, detecting, immobilizing, separating or purifying proteins comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or has no amino acid.

[0011] The present invention also relates to a method for binding to or detecting a protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or no amino acid, the method comprising contacting the protein with an antibody of the present invention and optionally detecting the antibody or a label contained in the antibody.

[0012] The present invention also relates to a method for producing the antibody of the present invention, comprising culturing the host cells of the present invention under conditions that allow the expression of the antibody. Attached Figure Description

[0013] Figure 1 Affinity of humanized NbALFA variants generated via CDR transplantation

[0014] A. Affinity measurement of the peptide of SEQ ID NO: 2 by virgin alpaca-derived NbALFA (wtNbALFA). B. Affinity measurement of the peptide of SEQ ID NO: 2 by variant 3. C. Affinity measurement of the peptide of SEQ ID NO: 2 by variant 4. D. Affinity measurement of the peptide of SEQ ID NO: 2 by variant 5. E. Affinity measurement of the peptide of SEQ ID NO: 2 by variant 6. F. Affinity measurement of the peptide of SEQ ID NO: 2 by variant 7. Affinity was determined using the biolayer interferometry method as described in Method 1 of Example.

[0015] Figure 2 Affinity of further engineered humanized NbALFA variants

[0016] A, Affinity measurement of variant 8 to the peptide of SEQ ID NO: 2. B, Affinity measurement of variant 9 to the peptide of SEQ ID NO: 2. C, Affinity measurement of variant 10 to the peptide of SEQ ID NO: 2. D, Affinity measurement of variant 11 to the peptide of SEQ ID NO: 2. E, Affinity measurement of variant 12 to the peptide of SEQ ID NO: 2. F, Affinity measurement of variant 13 to the peptide of SEQ ID NO: 2. G, Affinity measurement of variant 14 to the peptide of SEQ ID NO: 2. H, Affinity measurement of variant 15 to the peptide of SEQ ID NO: 2. Affinity was determined using the biolayer interferometry method as described in Method 1 of Example.

[0017] Figure 3 SpA-assisted purification of humanized NbALFA variants

[0018] SpA purification using optimized variants. The variants were incorporated into cell culture supernatant for spiking experiments, followed by SpA chromatography using a centrifuge column.

[0019] Figure 4 Direct comparison of NbALFA with three humanized NbALFA variants

[0020] A. Purification of the NbALFA variant. Fractions are shown by SDS-PAGE and Coomassie staining analysis. The analyzed fractions correspond to 2.5 μL of culture supernatant and unbound material, and 1 μL of the original eluent after concentration to 1 mL. All protein pools after SEC were normalized to 2 μg per lane. Note that US2023 / 0093123A1-Seq35 is the only NbALFA variant showing two distinct protein species that can be clearly separated by size exclusion chromatography (SEC).

[0021] B. Direct comparison of culture supernatants. Culture supernatants from all four parallel expression cultures were analyzed by SDS-PAGE and Coomassie staining. Fractions analyzed corresponded to 5 μL of each culture supernatant.

[0022] C. Direct comparison of the original eluent. The original eluent obtained from ALP3 agarose resin was concentrated using a centrifugal column and analyzed by SDS-PAGE and Coomassie staining. The fractions analyzed corresponded to 1 μL of concentrated original eluent.

[0023] D. Preparative size exclusion chromatography. The original eluent obtained from ALP3 agarose resin was concentrated to a final volume of 1 mL and separated on a Superdex 75 Increase column equilibrated with PBS. The figure shows the chromatograms measured at 280 nm with the same scaling factor. Only for US2023 / 0093123A1-Seq35, two distinct peaks with significant contributions were separated. These correspond to the glycosylated (peak 1) and non-glycosylated (peak 2) classes.

[0024] Figure 5 Germplasm identity of human IGHV with antibodies containing 339 VH domains was obtained. Antibodies were classified according to their infix terms, and the phylogenetic identity of each subgroup was plotted. The V-segment identity of some humanized VHHs presented in this paper is indicated by dashed lines. Detailed Implementation

[0025] Humanized antibodies have the advantage of reduced immunogenicity because they are engineered to be very similar to human antibodies, thus minimizing the risk of triggering an immune response when administered to patients. Therefore, humanized antibodies are advantageous for in vivo applications (e.g., imaging techniques). Furthermore, antibodies binding to epitope tags are gaining increasing attention in various therapeutic applications (Mitwasi et al., Sci. Rep. 2020; doi:10.1038 / s41598-020-59082-4).

[0026] Therefore, the inventors of this application have recognized the need in the art to provide humanized single-domain antibodies that can bind to the peptide of SEQ ID NO: 2 with high affinity. Consequently, the inventors of this invention aim to humanize NbALFA antibodies to fill this gap in the art.

[0027] Antibody humanization can be performed on both traditional VH / VL-based molecules and their single-domain counterparts. A common approach is to graft the sequence transfer vector (CDR) onto a humanized receptor framework to preserve the spatial orientation of the original variable region from which the donor CDR originated. For VHH region humanization, the CDR can be grafted into a compatible human light chain variable region framework. However, sequence grafting can have unpredictable and undesirable effects on antibody conformation (Fernández-Quintero et al., Prot. Eng. Des. and Sel., 2019; doi: 10.1093 / protein / gzaa004). Therefore, providing humanized antibodies with binding affinity at least comparable to the original antibody remains challenging.

[0028] When the inventors of this invention attempted to humanize wt NbALFA single-domain antibodies, they initially generated seven humanized variants (variants 1-7) by transplanting CDRs into different humanized receptor frameworks. However, the resulting single-domain antibodies either failed to be expressed, or they completely lost or significantly impaired their affinity for the target antigen (see Example 1).

[0029] It has been recognized that CDR transplantation does not directly produce a humanized form of high-affinity NbALFA. The inventors of this application have analyzed the antibody structure in detail and tested the effects of retaining residues that may be associated with high-affinity binding. Therefore, they unexpectedly discovered that, in addition to CDR transplantation, several key residues of wt NbALFA should be retained to obtain humanized forms of NbALFA that retain their high affinity for the peptide of SEQ ID NO: 2 or, surprisingly, they possess even improved affinity (see Example 2). Furthermore, the inventors of this application have surprisingly discovered that the humanized forms of NbALFA thus generated can even have improved thermal stability (see Example 4).

[0030] Not wanting to be bound by theory, the inventors of this application believe that the arginine residue at position 47 of wtNbALFA is crucial for the interaction between the antibody and the C-terminus of the peptide of SEQ ID NO: 2. Therefore, retaining Arg 47 of the original Camelidae VHH framework is considered to improve binding behavior. Furthermore, not wanting to be bound by theory, the inventors of this application believe that the methionine residue at position 50 is crucial for the formation of the hydrophobic pocket, which, after binding, will be masked by the peptide of SEQ ID NO: 2, thereby providing free energy to the interaction between the antibody and antigen. Therefore, retaining Met 50 of the original Camelidae VHH framework is also considered to improve binding behavior. Moreover, not wanting to be bound by theory, the inventors of this application believe that the arginine residue at position 48 of wtNbALFA is also important for the binding of the peptide of SEQ ID NO: 2. Therefore, retaining Arg 48 of the original Camelidae VHH framework is also considered to improve binding behavior. Arg 47, Arg 48, and Met 50 at these three positions are therefore considered key residues and should be retained in the humanized form of NbALFA. In fact, variants 8-15 tested in Example 2 all contain these three key residues, and all variants 8-15 have binding affinity at least equivalent to wt NbALFA.

[0031] Not wanting to be bound by theory, the inventors of this application further believe that His 99 and / or Val 100 may be involved in NbALFA binding. They believe these two amino acid residues affect the conformation of CDR3.

[0032] Another residue considered to improve binding to the peptide of SEQ ID NO: 2 is Tyr 40 of wt NbALFA. Not wishing to be bound by theory, the inventors of this application believe that Tyr 40, found in wt NbALFA, forms a hydrogen bond with the R11' side chain on the peptide. Retaining Tyr 40 in the humanized form of NbALFA can further improve antigen binding. However, as shown in variant 8, which contains Phe at position 40, binding affinity comparable to wt NbALFA can also be achieved without the Tyr residue at position 40. However, as shown in variant 9, replacing Phe at position 40 with Tyr can further improve the binding affinity to the peptide of SEQ ID NO: 2.

[0033] On the other hand, the inventors of this application have surprisingly discovered that Met 61 and Glu 64, as determined by Kabat to form part of CDR2, are not essential for binding to the peptide of SEQ ID NO: 2. These residues can be mutated to their human counterparts, thereby, for example, further humanizing the antibody, while retaining the high binding affinity to the peptide of SEQ ID NO: 2.

[0034] Following this principle, the inventors of this application have constructed a humanized form of an NbALFA single-domain antibody with reduced immunogenicity potential, which is preferably used in vivo in human patients (e.g., for diagnostic or therapeutic applications).

[0035] Furthermore, the inventors of this invention have surprisingly discovered that, in addition to their reduced immunogenicity, some humanized forms of NbALFA can bind to Staphylococcus aureus protein A (SpA; see Example 3). Antibodies that can bind to SpA have the advantage of facilitating antibody GMP production. Therefore, the binding affinity to SpA is another advantage of some of the humanized antibodies of this invention. Surprisingly, the inventors of this application have found that introducing mutations into CDR2 as determined according to IMGT, particularly mutations of Ala 60→Thr, and / or introducing mutations at positions falling within CDR2 as determined according to Kabat, particularly mutations of Gln 67→Lys, both preserve the high binding affinity to the peptide of SEQ ID NO: 2 and promote SpA binding.

[0036] Therefore, the present invention relates to a single-domain antibody that specifically binds to an epitope containing the sequence SRLEEELRRRLTE (SEQ ID NO: 2), wherein the antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to that sequence, a CDR2 sequence as shown in SEQ ID NO: 97 or a sequence having one or two mutations relative to that sequence, and a CDR3 sequence as shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to that sequence. Preferably, the antibody further comprises at least one of the following amino acid residues at positions 47, 48, and / or 50 corresponding to sequence positions in SEQ ID NO: 1: Arg at position 47, Arg at position 48, and / or Met at position 50.

[0037] The term "antibody" generally refers to a protein-binding molecule that has immunoglobulin-like function. Typical examples of antibodies are, but are not limited to, immunoglobulins, as well as their derivatives or functional fragments that retain their binding specificity. Techniques for producing antibodies are well known in the art. The term "antibody" also includes different classes (i.e., IgA, IgG, IgM, IgD, IgE, IgY, etc.) and subclasses (e.g., IgG1, IgG2, etc.) of immunoglobulins (Ig), even those recombined in a heterologous host using techniques known to those skilled in the art. Exemplary examples of antibodies are full-length immunoglobulins, Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), diabody antibodies, or domain antibodies. Domain antibodies can be single-domain antibodies, single variable-domain antibodies, or immunoglobulin single variable-domain antibodies having only one variable domain (which can be VH or VL) that specifically binds to an antigen or epitope without dependence on other V regions or domains. Particularly preferred single-domain antibodies are those with only the VHH domain of heavy-chain antibodies. Such immunoglobulin single variable domains can comprise not only isolated antibody single variable domain peptides, but also larger peptides comprising or composed of one or more monomers of the antibody single variable domain peptide sequence. It is understood that single-domain antibodies can comprise a VHH domain and a fusion partner, such as a protein or peptide, which can be a tag. Therefore, the definition of the term "antibody" also includes embodiments such as chimeric antibodies, single-chain antibodies, and humanized antibodies. The term "antibody" can also include antibody fragments.

[0038] When used in this article, immunoglobulins can be diglycosylated proteins composed of two heavy chains, such as camel IgG (hcIgG) or shark IgNAR, which consists of only heavy chains. Immunoglobulins used in this article can also be tetraglycosylated proteins composed of two light chains (L) each approximately 25 kDa and two heavy chains (H) each approximately 50 kDa.

[0039] The terms "Fab," "Fab region," "Fab section," or "Fab fragment" are understood to define a type of Fab containing V H C H 1. V L and C L A polypeptide of an immunoglobulin domain. Fab can refer to the region alone, or the region in the context of an antibody molecule according to the invention, as well as a full-length immunoglobulin or an immunoglobulin fragment. Typically, the Fab region contains the complete light chain of the antibody. The Fab region can be used to define an “arm” of an immunoglobulin molecule. It contains the epitope-binding portion of the Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by partial papain digestion. The “F(ab’)2 moiety” is a proteolytic fragment of an immunoglobulin partially digested with pepsin. The “Fab’ moiety” is the product obtained by reducing the disulfide bonds of the F(ab’)2 moiety. The terms “Fab,” “Fab region,” “Fab moiety,” or “Fab fragment” as used herein may further include a hinge region defining the C-terminus of the antibody arm (see above). This hinge region corresponds to the region found in the C-terminus of a full-length immunoglobulin. H The C-terminal hinge region of the 1st domain is where the arms of the antibody molecule can be considered to form a "Y". The term "hinge region" is used in this art because immunoglobulins have a certain degree of flexibility in this region.

[0040] An “Fv” or “Fv fragment” consists only of the VL and VH domains of an immunoglobulin “single arm.” Therefore, an “Fv” is the smallest antibody fragment containing a complete antigen recognition and binding site. A “double-stranded” Fv fragment consists of a tightly, non-covalently associated dimer of variable domains from one heavy chain and one light chain. Single-stranded Fvs (scFvs) include the VH and VL domains of an immunoglobulin, which are present in a single polypeptide chain covalently linked to each other by a flexible peptide linker. Typically, in scFv fragments, the variable domains of the light and heavy chains are associated in a dimer structure similar to that of double-stranded Fvs. In single-stranded Fv fragments, either the light chain variable domain can be positioned at the N-terminus of the single polypeptide chain, followed by a linker and a heavy chain variable domain positioned at the C-terminus of the polypeptide chain, or conversely, the heavy chain variable domain can be positioned at the N-terminus, the light chain variable domain at the C-terminus, and the peptide linker positioned between them. Peptide linkers can be any flexible linker known in the art, such as linkers composed of glycine and serine residues. Domain association between the VH and VL domains can be further stabilized by introducing disulfide bonds into conserved framework regions (see Reiter et al. Stabilization of the Fvfragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions, Biochemistry 1994, 33, 6551-5459). These scFv fragments are also referred to as disulfide-stabilized scFv fragments (ds-scFv).

[0041] This document uses the term "Fc region" or "Fc fragment" to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The Fc region mediates the effector functions of antibodies, such as the complement system and the activation of immune effector cells (e.g., NK cells) with Fc receptors. In human IgG molecules, the Fc region is generated by cleavage of the N-terminus of Cys226 by papain. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its C-terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the preparation or purification of the antibody molecule, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody molecule. Therefore, complete antibody compositions can include antibody groups with all K447 residues removed, antibody groups without K447 residues removed, and antibody groups including mixtures of antibodies with or without K447 residues. Suitable natural sequence Fc regions for the antibodies used in this invention include mammalian (e.g., human or mouse) IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. The Fc region contains two or three constant domains, depending on the type of antibody. In embodiments where the immunoglobulin is IgG, the Fc region has a CH2 domain and a CH3 domain.

[0042] Single-domain antibodies are a class of antibodies whose complementation-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally occurring light-chain-free antibodies, single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain scaffolds of those not derived from antibodies. Single-domain antibodies can be any existing or future single-domain antibodies in the art. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, or cattle. The single-domain antibodies used herein can be derived from naturally occurring antibodies called light-chain-free heavy-chain antibodies. Such single-domain antibodies are disclosed, for example, in WO 94 / 04678. For clarity, the variable domain derived from a naturally occurring light-chain-free heavy-chain antibody is referred to herein as VHH to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in species of the Camelidae family, such as camels, dromedary camels, llamas, alpacas, llamas, and guanacos. Other species besides camels can also produce naturally occurring heavy chain antibodies without light chains. As an illustrative example, sharks are known to produce naturally occurring heavy chain antibodies without light chains (often named IgNAR), which also contain a VHH domain. Furthermore, some species have been genetically modified to artificially produce naturally occurring heavy chain antibodies without light chains, such as some mouse strains. Additionally, VHHs can be obtained from synthetic libraries. All such VHHs are within the scope of this invention. The single-domain antibodies used herein also include humanized forms of single-domain antibodies, such as humanized forms with VHH regions.

[0043] VHHs, according to the present invention and as known to those skilled in the art, are preferably derived from the heavy chain variable domains of naturally occurring, light-chain-free immunoglobulins, such as those derived from Camelidae as described in WO 94 / 04678 (hereinafter referred to as VHH domains or nanobodies). VHH molecules are approximately 10 times smaller than IgG molecules. They are single polypeptides and are very stable, resistant to extreme pH and temperature conditions. Moreover, they exhibit high resistance to protease activity, a property not possessed by conventional antibodies. Furthermore, in vitro expression of VHHs or expression in prokaryotic or eukaryotic organisms suitable for recombinant protein expression yields high-yield, correctly folded functional VHHs.

[0044] The term "variable" refers to the portion of an immunoglobulin domain that exhibits sequence variability and is relevant to determining the specificity and binding affinity of a particular antibody (i.e., a "variable domain"). This variability is not uniformly distributed throughout the variable domain of an antibody; it is concentrated in subdomains of the variable regions of the heavy and light chains. These subdomains are called "hypervariable regions," "HVRs," or "HVs," or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRs). The naturally occurring variable domains of the heavy and light chains each contain four FRs, primarily adopting a β-sheet configuration linked by three hypervariable regions that form a loop connecting the β-sheet structure and, in some cases, a portion of the β-sheet structure. The hypervariable regions in each chain are tightly bound together by FRs and, together with hypervariable regions from other chains, promote the formation of antigen-binding sites (see Kabat et al., see below). Typically, naturally occurring immunoglobulins comprise six CDRs (see below); three in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3). Among naturally occurring immunoglobulins, H3 and L3 exhibit the greatest diversity among the six CDRs, with H3 in particular considered to play a unique role in conferring precise specificity to immunoglobulins. However, naturally occurring light-chain-free immunoglobulins comprise three CDRs in the VHH region. The constant domains are not directly involved in antigen binding but exhibit various effector functions, such as antibody-dependent, cell-mediated cytotoxicity, and complement activation.

[0045] Unless otherwise expressly or implicitly stated, the CDR sequences of this disclosure follow the hybrid Kabat / IMGT definition. Heavy chain CDR1 follows the hybrid Kabat / IMGT definition, with the start point of heavy chain CDR1 following the IMGT definition and the end point following the Kabat definition. As an illustrative example, the CDR1 of wtNbALFA according to IMGT is GVTISALNAMA (SEQ ID NO: 127), while the CDR1 of wtNbALFA according to Kabat is ALNAMAMG (SEQ ID NO: 128). According to the hybrid Kabat / IMGT definition preferred in the context of this disclosure, the CDR1 sequence of wtNbALFA is GVTISALNAMAMG (SEQ ID NO: 94). Heavy chain CDR2 and CDR3 are defined according to the IMGT definition. Light chain CDR sequences are also defined according to the IMGT definition. The reason for the hybrid definition of heavy chain CDR1 is to include a longer sequence to ensure that CDR1 transplantation includes all important residues. For the definition of CDR, see, for example, Dondelinger et al., 'Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / ResidueDefinition', 2018; doi: 10.3389 / fimmu.2018.02278. Other criteria exist for defining CDR, such as those defined according to Maass 2007 (Journal of Immunological Methods 324 (2007) 13-25). Another criterion for characterizing antigen-binding sites is the highly variable ring described by Chothia (see, for example, Chothia et al., (1992); J. MoI. Biol. 227:799-817; and Tomlinson et al., (1995) EMBO J. 14:4628-4638).

[0046] Consistent with the CDR definitions described herein, unless otherwise expressly or implicitly stated, the FR regions used herein follow a hybrid Kabat / IMGT definition. For heavy chain variable regions, FR1, FR3, and FR4 are defined according to the IMGT definition. For FR2 of the heavy chain variable region, the start point is defined according to the Kabat definition, and the end point is defined according to the IMGT definition. Thus, as an illustrative example, FR1 of wtNbALFA is preferably defined by amino acid positions 1-25 of SEQ ID NO: 1, FR2 of wtNbALFA is preferably defined by amino acid positions 39-53 of SEQ ID NO: 1, FR3 of wtNbALFA is preferably defined by amino acid positions 61-98 of SEQ ID NO: 1, and FR4 of wtNbALFA is preferably defined by amino acid positions 112-122 of SEQ ID NO: 1.

[0047] Each VHH, VH, and VL has three CDRs and four FRs, arranged in the following order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and / or light chains contain binding domains that interact with antigenic epitopes. The term "immunoglobulin" can refer to a protein that may have two heavy chains but no light chains, such as antibodies without light chains, or their antigen-binding moieties. Immunoglobulins may also include at least two heavy chains (H) and two light chains (L) linked by disulfide bonds, or their antigen-binding moieties.

[0048] The antibodies according to the present invention can be isolated antibody molecules. As used herein, the term "isolated antibody molecule" refers to an antibody molecule that has been identified and isolated and / or recovered from components of its natural environment. Contaminating components of its natural environment are substances that can interfere with the use of the antibody and may include enzymes, hormones, and other protein- or non-protein-based solutes. In some embodiments, the antibody molecule is purified to a degree greater than 95% by weight, such as greater than 99% by weight, according to the Lowry method. In some embodiments, the antibody molecule is purified to a degree sufficient to obtain at least 15 N-terminal residues or an internal amino acid sequence using a spinneret sequencer. In some embodiments, the antibody is purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, more preferably, silver staining. In some embodiments, the isolated antibody molecule may be present in a heterologous host cell containing one or more components not present in the antibody's natural environment. Isolated antibodies are typically prepared by at least one purification step.

[0049] When an antibody or antibody molecule / fragment recognizes a target antigen in a complex mixture of proteins and / or macromolecules, the antibody or antibody molecule / fragment is said to "specifically" bind to the antigen. Typically, an antibody can specifically interact with and / or bind to its target, but essentially does not bind to another (preferably unrelated) epitope or antigen. If antibodies cross-competitively bind to the same epitope, the antibody is said to "bind to the same epitope," meaning that only one antibody can bind to the epitope at a given time point; that is, one antibody prevents the binding or regulatory effect of other antibodies. However, antibodies or antibody molecule / fragment that specifically bind to a target can cross-react with similar structures, such as closely related variants of the target to which they specifically bind.

[0050] Typically, bindings considered specific can also have high affinity, for example, when the binding affinity is higher than 10. -6 M (with K) D (Calculation) Time. Specifically, the binding affinity can be approximately 10. -8 Up to 10 -11 M (K D ), or about 10 -9 Up to 10 -11 M or even higher. Therefore, it possesses an affinity in the picomolar range (Kd = 9.9 × 10⁻⁶). -10 M to 10 -12 Antibody molecules of type M are also covered in this invention. If necessary, non-specific binding at the binding site can be reduced by changing the binding conditions, without substantially affecting specific binding.

[0051] The term "epitaxy," also known as "antigenic determinant," refers to the portion of an antigen that an antibody specifically binds to to form a complex. Therefore, the term "epitaxy" includes any molecular or protein determinant capable of specifically binding to immunoglobulins or T-cell receptors. The binding site (antibody binding site) of the antibody molecule described herein can specifically bind to / interact with a conformational epitope or a sequential epitope that is unique to the target structure. Epitope determinants typically consist of chemically active surface components of a molecule (such as amino acids or sugar side chains) and usually possess specific three-dimensional structural features and specific charge characteristics. Epitope determinants include chemically active surface components of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may possess specific three-dimensional structural features and / or specific charge characteristics. For polypeptide antigens, conformational epitopes or discontinuous epitopes are characterized by the presence of two or more discontinuous amino acid residues that are separated from each other in the primary sequence, but assemble into a consistent structure on the molecular surface when the polypeptide folds into its native protein / antigen (Sela, M., Science (1969) 166, 1365-1374; Laver, WG, et al., Cell (1990) 61, 553-556). The two or more discontinuous amino acid residues constituting an epitope can be present on a separate segment of one or more polypeptide chains. When the polypeptide chain folds into a three-dimensional structure, these residues aggregate on the molecular surface to form the epitope. In contrast, continuous or linear epitopes consist of two or more discontinuous amino acid residues that are present in a single linear segment of the polypeptide chain. As an exemplary example, a “context-dependent” CD3 epitope refers to the conformation of the epitope. This environment-dependent epitope, located on the CD3ε chain, forms its correct conformation only if it is embedded within the remainder of the ε chain and maintained in the correct position through heterodimerization of the ε chain with the CD3γ or δ chain. In contrast, environment-independent CD3 epitopes can be polypeptides or functional fragments of the N-terminus of CD3ε consisting of 1-27 amino acid residues. Typically, epitopes can be naturally linear or discontinuous. Therefore, the term “conformational epitope” as used herein refers to a discontinuous epitope formed by the spatial relationship between antigenic amino acids, rather than a continuous sequence of amino acids. The term “epitaph” also includes the antigenic determinants of haptens, which are small molecules that, when bound to larger substances (e.g., proteins), can act as antigens by displaying one or more immunologically recognized epitopes.

[0052] As used herein, an "eptopic tag" refers to a segment of amino acids for which a specific antibody or antibody-like function is generated. Such epitope tags allow for the specific recognition and / or tracing of tagged peptides or proteins present in living organisms or cultured cells. Detection of tagged molecules can be achieved using a variety of different techniques. Examples of these techniques include immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, electron microscopy, ELISA, Western blot, and affinity chromatography. The epitope tag adds a known epitope (antibody binding site) to the test peptide for binding to known, typically high-affinity, antibodies. Epitope tags can also be used to isolate and / or purify tagged molecules, for example, via drop-down applications.

[0053] As used in this article, the term "mutation" refers to an alteration in the sequence of a biomolecule, such as the amino acid sequence of a protein or the nucleic acid sequence of DNA or RNA. Mutations include deletions, insertions, and substitutions, and these can be applied to single nucleotides / amino acids as well as large segments of nucleotide / amino acid sequences.

[0054] As described herein, the antibody of the present invention contains at least one of the following amino acid residues at positions 47, 48 and / or 50 corresponding to sequence positions SEQ ID NO: 1: Arg at position 47, Arg at position 48 and / or Met at position 50.

[0055] Preferably, the antibody of the present invention contains an Arg residue at position 47 corresponding to SEQ ID NO: 1. Preferably, the antibody of the present invention contains an Arg residue at position 48 corresponding to SEQ ID NO: 1. Preferably, the antibody of the present invention contains a Met residue at position 50 corresponding to SEQ ID NO: 1. Any of these residues is considered to facilitate the binding of NbALFA to the target of SEQ ID NO: 2.

[0056] Preferably, the antibody of the present invention comprises at least two of the following amino acid residues at positions 47, 48, and / or 50 corresponding to sequence positions SEQ ID NO: 1: Arg at position 47, Arg at position 48, and / or Met at position 50. More preferably, the antibody of the present invention preferably comprises the following amino acid residues at positions 47, 48, and 50 corresponding to sequence positions SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50.

[0057] In addition to positions 47, 48, and 50, retaining the Tyr residue at position 40 is also considered to improve the affinity of the antibody of the present invention for SEQ ID NO: 2. Meanwhile, single-domain antibodies without the Tyr residue at position 40 can also exhibit binding affinity to SEQ ID NO: 2 comparable to wtNbALFA, such as variant 8, while other variants containing Tyr at said position can show further improved affinity. Therefore, the antibody of the present invention can preferably contain the Tyr residue at position 40 corresponding to sequence position 40 of SEQ ID NO: 1. Therefore, the antibody of the present invention preferably contains the following amino acid residues at positions 40, 47, 48, and 50 corresponding to sequence positions SEQ ID NO: 1: Tyr at position 40, Arg at position 47, Arg at position 48, and Met at position 50.

[0058] Furthermore, it is assumed that the His residue at position 99 and / or the Val residue at position 100 can positively influence the binding affinity of the antibody of the present invention to SEQ ID NO: 2. Therefore, the antibody of the present invention preferably contains a His residue at position 99 corresponding to sequence position 99 of SEQ ID NO: 1. Therefore, the antibody of the present invention preferably contains a Val residue at position 100 corresponding to sequence position 100 of SEQ ID NO: 1. Therefore, the antibody of the present invention preferably contains a His residue at position 99 corresponding to sequence position 99 of SEQ ID NO: 1 and a Val residue at position 100 corresponding to sequence position 100 of SEQ ID NO: 1. Therefore, the antibody of the present invention preferably contains the following amino acid residues at positions 47, 48, 50, 99 and 100 corresponding to sequence positions 47, 48, 50, 99 and 100 of SEQ ID NO: 1: Arg at position 47, Arg at position 48, Met at position 50, His at position 99 and Val at position 100. Therefore, the antibody of the present invention preferably contains the following amino acid residues at positions 40, 47, 48, 50, 99 and 100 corresponding to SEQ ID NO: 1: Tyr at position 40, Arg at position 47, Arg at position 48, Met at position 50, His at position 99, and Val at position 100.

[0059] In the antibody of the present invention, other natural amino acids of wtNbALFA can be retained. Therefore, the antibody of the present invention may contain a Gln residue at position 5 corresponding to sequence position 5 of SEQ ID NO: 1. However, it is not desirable to be bound by theory, as it is considered that the Gln at position 5 has little effect on binding the peptide of SEQ ID NO: 2. In a preferred embodiment, Val is contained at position 5 corresponding to SEQ ID NO: 1. In addition, the antibody of the present invention preferably contains a Gln residue at position 117 corresponding to sequence position 1 of SEQ ID NO: 1. However, it is not desirable to be bound by theory, as it is considered that the Gln at position 117 has little effect on binding the peptide of SEQ ID NO: 2. However, it is considered that Gln 117 increases solubility. Therefore, the antibody of the present invention preferably contains a Val residue at position 5 corresponding to sequence position 5 of SEQ ID NO: 1 and a Gln residue at position 117 corresponding to sequence position 1 of SEQ ID NO: 1. Preferably, the antibody of the present invention comprises the following amino acid residues at positions 5, 47, 48, 50, and 117 corresponding to sequence positions SEQ ID NO: 1: Val at position 5, Arg at position 47, Arg at position 48, Met at position 50, and Gln at position 117. Preferably, the antibody of the present invention comprises the following amino acid residues at positions 5, 47, 48, 50, 99, 100, and 117 corresponding to sequence positions SEQ ID NO: 1: Val at position 5, Arg at position 47, Arg at position 48, Met at position 50, His at position 99, Val at position 100, and Gln at position 117. Preferably, the antibody of the present invention comprises the following amino acid residues at positions 5, 40, 47, 48, 50, 99, 100, and 117 corresponding to sequence positions of SEQ ID NO: 1: Val at position 5, Tyr at position 40, Arg at position 47, Arg at position 48, Met at position 50, His at position 99, Val at position 100, and Gln at position 117. In wt NbALFA, Met61 and Glu64 form part of CDR2 as determined by Kabat. However, the inventors of this application have surprisingly discovered that these two residues are not essential for the binding of the peptide of SEQ ID NO: 2. As shown in variants 13 and 15, a high binding affinity for the peptide of SEQ ID NO: 2 can be retained by mutating these two residues to the human IGHV3-23 derived sequence counterparts Tyr61 and / or Asp64. The antibody of the present invention may also comprise the Tyr residue at position 61 corresponding to sequence position of SEQ ID NO: 1. The antibody of the present invention may also contain an Asp residue at position 64 corresponding to sequence position 64 of SEQ ID NO: 1.The antibody of the present invention may also contain a Tyr residue at position 61 corresponding to sequence position 61 of SEQ ID NO: 1 and an Asp residue at position 64 corresponding to sequence position 64 of SEQ ID NO: 1.

[0060] When used in conjunction with proteins or peptides, the term "amino acid" or "amino acid residue" typically refers to an α-aminocarboxylic acid having the generally accepted definition in the art, such as those selected from the following amino acids: L-alanine (Ala or A); L-arginine (Arg or R); L-asparagine (Asn or N); L-aspartic acid (Asp or D); L-cysteine ​​(Cys or C); L-glutamine (Gln or Q); L-glutamic acid (Glu or E); glycine (Gly or G); L-histidine (His or H); L-isoleucine (Ile or I); L-leucine (Leu or L); L-lysine (Lys or K); L-methionine (Met or M); L - Phenylalanine (Phe or F); L-proline (Pro or P); L-serine (Ser or S); L-threonine (Thr or T); L-tryptophan (Trp or W); L-tyrosine (Tyr or Y); and L-valine (Val or V), but modified, synthetic, or rare amino acids may also be used as needed, such as taurine, ornithine, selenocysteine, homocysteine, hydroxyproline, thioproline, iodotyrosine, 3-nitrotyrosine, ornithine, citrulline, canavalialine, 5-hydroxytryptophan, carnosine, cycloleucine, 3,4-dihydroxyphenylalanine, N-acetylcysteine, proline, allylglycine, or aziridine-2-carboxylic acid. Generally, amino acids can be grouped into those with nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); those with negatively charged side chains (e.g., Asp, Glu); those with positively charged side chains (e.g., Arg, His, Lys); or those with uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0061] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or identical to any one of SEQ ID NO: 118-125. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity to any one of SEQ ID NO: 118-125. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity to any one of SEQ ID NO: 118-125. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity to any one of SEQ ID NO: 118-125. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity to any one of SEQ ID NO: 118-125. Preferably, the antibody of the present invention comprises or is composed of an amino acid sequence selected from the following:

[0062]

[0063] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 118. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 118. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 118. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 118. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 118. Preferably, the antibody of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 118.

[0064] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 119. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 119. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 119. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 119. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 119. Preferably, the antibody of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 119.

[0065] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 120. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 120. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 120. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 120. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 120. Preferably, the antibody of the present invention comprises or is composed of the amino acid sequence shown in SEQ ID NO: 120.

[0066] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 121. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 121. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 121. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 121. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 121. Preferably, the antibody of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 121.

[0067] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 122. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 122. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 122. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 122. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 122. Preferably, the antibody of the present invention comprises or is composed of the amino acid sequence shown in SEQ ID NO: 122.

[0068] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 123. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 123. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 123. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 123. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 123. Preferably, the antibody of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 123.

[0069] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 124. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 124. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 124. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 124. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 124. Preferably, the antibody of the present invention comprises or is composed of the amino acid sequence shown in SEQ ID NO: 124.

[0070] The single-domain antibody of the present invention preferably comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with or identical to SEQ ID NO: 125. Preferably, the antibody of the present invention comprises a sequence having at least 90% sequence identity with SEQ ID NO: 125. Preferably, the antibody of the present invention comprises a sequence having at least 95% sequence identity with SEQ ID NO: 125. Preferably, the antibody of the present invention comprises a sequence having at least 98% sequence identity with SEQ ID NO: 125. Preferably, the antibody of the present invention comprises a sequence having at least 99% sequence identity with SEQ ID NO: 125. Preferably, the antibody of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 125.

[0071] The “percentage (%) sequence identity” of amino acid sequences disclosed herein is defined as the percentage of amino acid residues in a candidate sequence that pair identically with a reference sequence (i.e., the antibody molecule of this disclosure) after alignment and the introduction of gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be performed in various ways within the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to achieve maximum alignment of the full length of the compared sequence. This also applies to the nucleotide sequences disclosed herein.

[0072] As disclosed herein, the antibody of the present invention may comprise a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 94) or a sequence having one or two mutations relative to that sequence, a CDR2 sequence VSERGNX1, wherein X1 is A or T (SEQ ID NO: 97) or a sequence having one or two mutations relative to that sequence, and a CDR3 sequence HVLEDRVDSFHDY (SEQ ID NO: 98) or a sequence having one or two mutations relative to that sequence. Preferably, the antibody of the present invention may comprise a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 94) or a sequence having one mutation relative to that sequence, a CDR2 sequence VSERGNX1, wherein X1 is A or T (SEQ ID NO: 97) or a sequence having one mutation relative to that sequence, and a CDR3 sequence HVLEDRVDSFHDY (SEQ ID NO: 98) or a sequence having one mutation relative to that sequence. More preferably, the antibody of the present invention may comprise the CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 94), the CDR2 sequence VSERGNX1, wherein X1 is A or T (SEQ ID NO: 97), and the CDR3 sequence HVLEDRVDSFHDY (SEQ ID NO: 98).

[0073] The antibody of the present invention preferably comprises a CDR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to any of SEQ ID NO: 95 or 96. Such an antibody of the present invention may comprise the CDR2 sequence VSERGNA (SEQ ID NO: 95) or a sequence having one or two mutations relative to that sequence, or the CDR2 sequence VSERGNT (SEQ ID NO: 96) or a sequence having one or two mutations relative to that sequence. Preferably, the antibody of the present invention comprises the CDR2 sequence of SEQ ID NO: 95 or a sequence having one mutation relative to that sequence, or the CDR2 sequence of SEQ ID NO: 96 or a sequence having one mutation relative to that sequence. Preferably, the antibody of the present invention comprises the CDR2 sequence of SEQ ID NO: 95 or SEQ ID NO: 96.

[0074] The antibody of the present invention may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to that sequence, the CDR2 sequence shown in SEQ ID NO: 95 or a sequence having one or two mutations relative to that sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to that sequence. Preferably, the antibody of the present invention may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one mutation relative to that sequence, the CDR2 sequence shown in SEQ ID NO: 95 or a sequence having one mutation relative to that sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one mutation relative to that sequence. More preferably, the antibody of the present invention may comprise the CDR1 sequence shown in SEQ ID NO: 94, the CDR2 sequence shown in SEQ ID NO: 95, and the CDR3 sequence shown in SEQ ID NO: 98.

[0075] The antibody of the present invention may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one or two mutations relative to that sequence, the CDR2 sequence shown in SEQ ID NO: 96 or a sequence having one or two mutations relative to that sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one or two mutations relative to that sequence. Preferably, the antibody of the present invention may comprise the CDR1 sequence shown in SEQ ID NO: 94 or a sequence having one mutation relative to that sequence, the CDR2 sequence shown in SEQ ID NO: 96 or a sequence having one mutation relative to that sequence, and the CDR3 sequence shown in SEQ ID NO: 98 or a sequence having one mutation relative to that sequence. More preferably, the antibody of the present invention may comprise the CDR1 sequence shown in SEQ ID NO: 94, the CDR2 sequence shown in SEQ ID NO: 96, and the CDR3 sequence shown in SEQ ID NO: 98.

[0076] It should be understood that this antibody is a single-domain antibody. It should also be understood that the antibody of the present invention, wherein one or two mutations are introduced into one, two, or all three CDR sequences, preferably still specifically binds the peptide of SEQ ID NO: 2 with a binding affinity at least equivalent to wtNbALFA.

[0077] The antibody of the present invention preferably contains an FR1 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or is identical to the sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:99).

[0078] The antibody of the present invention preferably contains an FR2 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or is identical to the sequence of WX1RQAPGKRREMVAA (SEQ ID NO: 102), wherein X1 is F or Y.

[0079] The antibody of the present invention preferably comprises an FR3 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or is identical to the sequence of X1YRX2SVX3GRFTISRDNX4KX5X6X7YLQMNSLRAEDTAVYYC (SEQ ID NO: 108), wherein X1 is M or Y, X2 is D or E, X3 is Q or K, X4 is A or S, X5 is N or R, X6 is M or T, and X7 is V or L.

[0080] The antibody of the present invention preferably contains an FR4 sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or is identical to the sequence of WGQGTQVTVSS (SEQ ID NO: 109).

[0081] Therefore, the antibody of the present invention preferably comprises FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences of SEQ ID NO:99, 102, 108 and 109, respectively.

[0082] The antibody of the present invention preferably comprises an FR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to any one of SEQ ID NO: 100 or 101. Such an antibody of the present invention may comprise an FR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to WFRQAPGKRREMVAA (SEQ ID NO: 100). Such an antibody of the present invention may also comprise an FR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to WYRQAPGKRREMVAA (SEQ ID NO: 101).

[0083] The antibodies of the present invention preferably comprise an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to any one of SEQ ID NO: 103-107. Such antibodies of the present invention may comprise an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence MYRESVQGRFTISRDNAKRMVYLQMNSLRAEDTAVYYC (SEQ ID NO: 103). Such antibodies of the present invention may comprise an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence MYRESVKGRFTISRDNAKRMVYLQMNSLRAEDTAVYYC (SEQ ID NO: 104). The antibody of the present invention may comprise an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence YYRDSVKGRFTISRDNAKRMVYLQMNSLRAEDTAVYYC (SEQ ID NO: 105). The antibody of the present invention may comprise an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence MYRESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 106). The antibody of the present invention may comprise an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence YYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 107).

[0084] The antibodies of the present invention preferably comprise FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 100, 103 and 109, respectively.

[0085] The antibodies of the present invention preferably comprise FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 103 and 109, respectively.

[0086] The antibodies of the present invention preferably comprise FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 104 and 109, respectively.

[0087] The antibodies of the present invention preferably comprise FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 105 and 109, respectively.

[0088] The antibodies of the present invention preferably comprise FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 106 and 109, respectively.

[0089] The antibody of the present invention preferably comprises FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences of SEQ ID NO: 99, 101, 107 and 109, respectively.

[0090] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 97 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

[0091] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 95 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 100, 103 and 109.

[0092] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 95 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 103 and 109.

[0093] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 96 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 103 and 109.

[0094] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 95 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 104 and 109.

[0095] The antibodies of the present invention preferably comprise the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 96 and 98, and further comprise the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 104 and 109.

[0096] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 96 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 105 and 109.

[0097] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 95 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 106 and 109.

[0098] The antibody of the present invention preferably comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 96 and 98, and further comprises the FR1, FR2, FR3 and FR4 sequences, each of which has at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or is identical to the sequences shown in SEQ ID NO: 99, 101, 107 and 109.

[0099] Preferably, the frame sequence of the antibody is defined by amino acid positions 1-25, 39-53, 61-98 and 112-122 corresponding to SEQ ID NO: 1.

[0100] Preferably, the FR1, FR2, FR3 and / or FR4 regions of the antibody of the present invention correspond to amino acid positions 1-25, 39-53, 61-98 and 112-122 of SEQ ID NO: 1.

[0101] The antibody of the present invention is preferably humanized.

[0102] As used in this article, "humanization" refers to the process of modifying non-human antibodies, typically derived from animals, to maximize the identity of their amino acid sequence with the closest human equivalent. This modification involves genetic engineering to minimize potential adverse effects when the antibody is administered to humans while preserving its ability to recognize and bind specific antigens. Typically, the degree of antibody humanization is defined as the percentage of identity between the V-segment or frame region and its human counterpart.

[0103] Both heavy and light chain variable regions are known to contain three complementarity-determining regions (CDRs) that vary in response to the epitope in question and determine binding affinity. These CDRs are flanked by four framework regions (FRs), which are relatively conserved in a given species and are presumed to provide scaffolding for the CDRs. When preparing non-human antibodies targeting specific epitopes, the variable regions can be “remodeled” or “humanized” by grafting CDRs derived from non-human antibodies onto the FRs of the human antibody to be modified. Sato, K., et al., (1993) Cancer Res 53:851-856; Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, CA, et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, SD, et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, PR, et al., (1991) Bio / Technology 9:266-271; Co, MS, et al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) ProcNatl Acad Sci USA 89:4285-4289; and Co, MS et al., (1992) J Immunol 148:1149-1154 reported the application of this method in various antibodies.

[0104] In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs derived from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, and these CDRs are also referred to as one or more CDRs “derived” from the original antibody.

[0105] In the context of this invention, and particularly in the context of the humanized form of wtNbALFA, the term "humanized" preferably refers to an antibody that has a higher degree of frame sequence identity with a given human pedigree sequence compared to wtNbALFA. The given human pedigree sequence is preferably a human pedigree sequence with the highest sequence identity to the frame region of wtNbALFA. More preferably, the given human pedigree sequence is the V segment IGHV3-23*4 shown in SEQ ID NO: 110 and the J segment IGHJ6*01 shown in SEQ ID NO: 126.

[0106] The term "germ sequence" refers to the amino acid sequence of a naturally occurring protein that represents an unaltered genetic sequence found in the reproductive cells (sperm or egg cells) of an organism. This sequence serves as a baseline genetic information, and the term "germ sequence" is used to distinguish proteins derived from the original genetic composition from proteins containing any modifications (i.e., mutations) introduced for a specific purpose into that protein's sequence.

[0107] As used herein, “frame identity” refers to the percentage of identity between the germline coding frames of the closest hominid (e.g., IGHV3-23 or one of its allotypes) and the compared humanized or wtNbALFA frames. Frames are defined as described herein (SEQ ID NO: 99-109). Alignments are computed using the Needleman and Wunsch global alignment algorithm, which can be used with pairwise sequence alignment tools (…). https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / Access was performed. As a MATRIX, a Blosum62 matrix was used, with a gap open value of 10, a gap extended value of 0.5, an end gap penalty of false, an end gap open value of 0, and an end gap extended value of 0.5. These values ​​represent the standard operating parameters of the tool. After running the algorithm, identity values ​​were given. As used in this paper, “overall frame identity” refers to the weighted average frame identity, calculated as the average identity of FR1 to FR4, normalized by the sequence lengths of each region (i.e., FR1, FR2, FR3, and FR4 are 25, 15, 38, and 11 amino acids, respectively; see Table 3).

[0108] The antibodies of the present invention preferably comprise a sequence having at least about 71%, at least about 75%, or preferably at least about 80% overall frame identity with the closest human phylogenetic sequence. The antibodies of the present invention may even comprise a sequence having at least about 85%, or at least about 90% overall frame identity with the closest human phylogenetic sequence. The antibodies of the present invention preferably comprise a sequence having at least about 71%, at least about 75%, or preferably at least about 80% overall frame identity with the frame regions of IGHV3-23*4 (for FR1, FR2, and FR3) shown in SEQ ID NO: 110 and IGHJ6*01 (for FR4) shown in SEQ ID NO: 126. The antibodies of the present invention may even contain sequences that have at least about 85% or at least about 90% overall frame identity with the frame regions of IGHV3-23*4 (for FR1, FR2 and FR3) shown in SEQ ID NO: 110 and IGHJ6*01 (for FR4) shown in SEQ ID NO: 126.

[0109] As used in this paper, “V-segment identity” refers to the percentage of identity between the germline codes FR1, CDR1, FR2, CDR2, and FR3 of the closest human lineage (e.g., IGHV3-23 or one of its allotypes) and the compared humanized or wtNbALFA FR1, CDR1, FR2, CDR2, and FR3. For this purpose, the IMGT DomainGapAlign tool (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) was used. The domain type was set to “V”, the species was selected as “Any” or “Homo sapiens (human)”, and the Smith-Waterman score was set to greater than 0. An E value of 200 was selected, the query gap penalty was set to -5, and the reference gap penalty was set to -20. These values ​​represent the standard operating parameters of this tool. The target antibody sequence was inserted in FASTA format. The tool calculated the closest germline sequence and provided a “V-segment identity percentage” value for each sequence. The “V-region identity percentage” of the most closely related person sequences is referred to as V-region identity in this paper.

[0110] The antibody of the present invention preferably comprises a sequence having at least about 60%, at least about 65%, or preferably at least about 70% V segment identity with the closest human ancestral sequence. The antibody of the present invention preferably comprises a sequence having at least about 60%, at least about 65%, or preferably at least about 70% V segment identity with IGHV3-23*4 shown in SEQ ID NO:110.

[0111] Preferably, the antibody of the present invention has an affinity for the epitope tag shown in SEQ ID NO: 2, which is at least equivalent to the affinity of the antibody in SEQ ID NO: 1. As used herein, "at least equivalent" preferably means a K0 of a given antibody. D Not higher than the reference antibody (e.g., the antibody of SEQ ID NO: 1) K. D Approximately 2.5 times. It is preferable to use the same method to measure the K of both the given antibody and the reference antibody. D Any suitable determination of K D All of these methods can be used in this field. Determining K D Known methods include, but are not limited to, ELISA, surface plasmon resonance (SPR), or biolayer interferometry (BLI). Preferably, affinity is measured by biolayer interferometry, more preferably by methods substantially as described in Method 1.

[0112] Preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K is not higher than wtNbALFA for the epitope tag D Approximately 2.5 times. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K is not higher than wtNbALFA for the epitope tag D Approximately twice as much. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K is not higher than wtNbALFA for the epitope tag D Approximately 1.5 times. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K is not higher than wtNbALFA for the epitope tag D Approximately 1.2 times that.

[0113] The antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K is not higher than wtNbALFA for the epitope tag D Preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K of the epitope tag is lower than wtNbALFA D Preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K of the epitope tag compared to wtNbALFA D The antibody is at least about 1.5 times lower than the K-type epitope tag shown in SEQ ID NO: 2. Preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. DK of the epitope tag compared to wtNbALFA D At least about 2.0 times lower. Preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D K of the epitope tag compared to wtNbALFA D It is at least about 2.5 times lower.

[0114] The antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 100 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 50 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 40 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 30 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 20 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 15 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 10 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 5 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA. D The antibody of the present invention is approximately 2.5 times higher to approximately 4 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The K value of the epitope tag can be compared with wtNbALFA.D The difference ranges from approximately 2.5 times higher to approximately 3 times lower.

[0115] Preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO:121 against the epitope tag K D Approximately 2.5 times. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 121 against the epitope tag K D Approximately twice as much. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 121 against the epitope tag K D Approximately 1.5 times. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies not exceeding SEQ ID NO: 121 against the epitope tag K D Approximately 1.2 times. The antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D An antibody not exceeding the K-value of the epitope tag of SEQ ID NO: 121 D .

[0116] The antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 100 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 50 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 40 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 30 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag.D The antibody of the present invention is approximately 2.5 times higher to approximately 20 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 15 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 10 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 5 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 4 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 121 can be compared to the K of the epitope tag. D The difference ranges from approximately 2.5 times higher to approximately 3 times lower.

[0117] Preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibodies with a K-value not exceeding SEQ ID NO:123 against the epitope tag D Approximately 2.5 times. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibody K-values ​​not higher than SEQ ID NO: 123 against the epitope tag D Approximately twice as much. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibody K-values ​​not higher than SEQ ID NO: 123 against the epitope tag D Approximately 1.5 times. More preferably, the antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibody K-values ​​not higher than SEQ ID NO: 123 against the epitope tag D Approximately 1.2 times. The antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D Antibody K-values ​​not higher than SEQ ID NO: 123 against the epitope tag D .

[0118] The antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 100 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 50 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 40 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 30 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 20 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 15 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 10 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 5 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The antibody of the present invention is approximately 2.5 times higher to approximately 4 times lower than the K-type epitope tag shown in SEQ ID NO: 2. D The antibody shown in SEQ ID NO: 123 can be compared to the K of the epitope tag. D The difference ranges from approximately 2.5 times higher to approximately 3 times lower.

[0119] The antibody of the present invention targets the K-type epitope tag shown in SEQ ID NO: 2. D It can be approximately 5×10 -10 M or lower, preferably 3×10 -10 M or lower, which is preferably determined by biolayer interferometry, preferably by the method described substantially as in method 1. The K of the antibody of the present invention against the epitope tag shown in SEQ ID NO: 2 D It could also be approximately 2×10 -10 M or lower, or 2×10 -10 M or lower, preferably determined by biological layer interferometry, preferably determined by the method described substantially as in method 1.

[0120] Compared with the antibody of SEQ ID NO: 1, the antibody of the present invention preferably has a higher Tm.

[0121] As used herein, the term "Tm" refers to a temperature at which half of all molecules denature and the other half retain their conformation. For example, a Tm of 60°C means that at this temperature, 50% of the antibodies present in the sample remain undenatured, while the other 50% are denatured. Tm can be measured substantially as described in Method 2.

[0122] The antibodies of the present invention preferably have a Tm of at least about 62°C, more preferably at least about 63°C. The antibodies of the present invention may also have a Tm of at least about 64°C, at least about 65°C, or even at least about 66°C. Preferably, the Tm is measured substantially as described in Method 2.

[0123] The antibody of the present invention preferably contains a protein A binding site.

[0124] As used herein, staphylococcal protein A (SpA) or protein A refers to a molecule derived from the cell wall of the bacterium *Staphylococcus aureus* that has a strong affinity for immunoglobulins, particularly human IgG. The unique ability of SpA to specifically interact with the Fc region of antibodies offers enormous potential for a wide range of biotechnological applications, including antibody purification, fixation, and detection in diagnostic and therapeutic contexts. SpA affinity purification is currently the most commonly used affinity system for antibody purification.

[0125] In the context of this application, up to 13 amino acid positions are considered potentially involved in SpA recognition: residues corresponding to sequence positions 15, 17, 19, 60, 62, 67, 68, 69, 71, 73, 84, 86, and 87 of SEQ ID NO: 1. Specifically, residues Gly 15, Ser 17, Arg 19, Thr 60, Tyr 62, Lys 67, Gly 68, Arg 69, Thr 71, Ser 73, Gln 84, Asn 86, and Ser 87 are important for SpA binding. Residues Gly 15, Ser 17, Arg 19, Tyr 62, Gly 68, Arg 69, Thr 71, and Gln 84 are already present in wt NbALFA. Residues Ser 73, Asn 86, and Ser 87 are not included in wtALFA, but are included in all receptor frames used to generate CDR variants 1-7. However, according to the invention, position 60 is located within the CDR2 region, while position 67 is located within the CDR2 region if determined according to Kabat. Since wtNbALFA contains residues Ala60 and Gln 67, these two residues need to be mutated to improve SpA binding. Although both residues are located within the CDR2 region or, if determined according to Kabat, within the CDR2 region, the inventors of this application have surprisingly found that introducing the mutations Ala60→Thr and / or Gln 67→Lys preserves a high binding affinity for the epitope tag of SEQ ID NO: 2, as shown in variants 10-13 and 15. However, introducing the mutations Ala 60→Thr and / or Gln 67→Lys, as shown in Examples 3 and Table 4, can promote SpA binding.

[0126] The antibody of the present invention may contain a Thr residue at position 60 corresponding to sequence position SEQ ID NO: 1. The antibody of the present invention may contain a Lys residue at position 67 corresponding to sequence position SEQ ID NO: 1.

[0127] The antibody of the present invention may contain the following amino acid residues at positions 60 and 67 corresponding to SEQ ID NO: 1: Thr at position 60 and Lys at position 67.

[0128] The antibody of the present invention may contain a Gly residue at position 15. The antibody of the present invention may contain a Ser residue at position 17. The antibody of the present invention may contain an Arg residue at position 19. The antibody of the present invention may contain a Tyr residue at position 62. The antibody of the present invention may contain a Gly residue at position 68. The antibody of the present invention may contain an Arg residue at position 69. The antibody of the present invention may contain a Thr residue at position 71. The antibody of the present invention may contain a Ser residue at position 73. The antibody of the present invention may contain a Gln residue at position 84. The antibody of the present invention may contain an Asn residue at position 86. The antibody of the present invention may contain a Ser residue at position 87.

[0129] The antibody of the present invention contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or preferably all of the following amino acid residues at positions 15, 17, 19, 62, 68, 69, 71, 73, 84, 86, and 87 corresponding to SEQ ID NO: 1: Gly at position 15, Ser at position 17, Arg at position 19, Tyr at position 62, Gly at position 68, Arg at position 69, Thr at position 71, Ser at position 73, Gln at position 84, Asn at position 86, and Ser at position 87.

[0130] The antibody of the present invention may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or preferably all of the following amino acid residues at positions 15, 17, 19, 60, 62, 67, 68, 69, 71, 73, 84, 86, and 87 of the sequence corresponding to SEQ ID NO: 1: Gly at position 15, Ser at position 17, Arg at position 19, Thr at position 60, Tyr at position 62, Lys at position 67, Gly at position 68, Arg at position 69, Thr at position 71, Ser at position 73, Gln at position 84, Asn at position 86, and Ser at position 87.

[0131] The antibodies of the present invention preferably do not contain NxT / S motifs, such as NXT or NKT motifs. The NxT / S motifs are classic glycosylation sites. Glycosylation is generally undesirable due to its heterogeneity, making glycosylated antibodies less attractive for in vivo applications such as therapeutic uses. The reference antibody of SEQ ID NO: 129 contains the NKT motif at amino acid positions 78-80. As shown in Example 5, glycosylation was observed in SEQ ID NO: 129, while no glycosylation was observed in the wild-type and humanized NbALFA forms that do not contain NxT / S motifs.

[0132] The antibody of this invention can be conjugated to a detectable marker.

[0133] The term "detectable label" generally refers to any suitable chemical substance or enzyme that directly or indirectly produces a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. For example, fluorescent or radioactive labels can be conjugated to antibodies to produce fluorescence or X-rays as a detectable signal. Alkaline phosphatase, horseradish peroxidase, and β-galactosidase are examples of enzyme labels (and concurrent optical labels) that catalyze the formation of colorimetric reaction products. A detectable label is a detectable entity that can be used to detect a target, for example, in microscopy, immunohistochemistry, flow cytometry, or in vivo applications (e.g., in vivo imaging). Preferably, the label does not adversely affect the properties of the antibody conjugated to the label. Many types of detectable labels exist, including fluorescent labels, chromophore labels, isotope labels, metal labels, and radioactive labels. The presence of the peptide of SEQ ID NO: 2 can be detected by contacting the fusion protein with the antibody of the present invention conjugated with the detectable label and detecting the signal of the detectable label. Non-exhaustive examples of suitable chromophore labeling include alkaline phosphatases or peroxidases exposed to TMB (3,3',5,5'-tetramethylbenzidine), DAB (3,3',4,4'-diaminobenzidine), and 4CN (4-chloro-1-naphthol); ABTS (2,2'-azino-bis[3-ethylbenzothiazoline]sulfonic acid), OPD (o-phenylenediamine), and BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium). Non-exhaustive examples of isotope labeling include 13C, 15N, 19F, 27Al, 11B, 127I, or various lanthanide isotopes. Non-exhaustive examples of metal labeling include Au, Pd, Pb, Pt, Ag, Hg, and Os. Labeling can be direct labeling, i.e., directly detectable labeling. Alternatively, the marker can be an indirect marker, i.e., an affinity tag (or epitope tag) that can specifically bind to another specific binding partner conjugated to another detectable marker (e.g., a fluorescent or chromophore tag). Examples of suitable epitope tags include, but are not limited to, FLAG-tags, Strep-tags, Myc-tags, HA-tags, VSV-G-tags, HSV-tags, V5-tags, SPOT-tags, BC2-tags, and EPEA-tags. The antigen can also be a protein, such as glutathione S-transferase (GST), maltose-binding protein (MBP), chitin-binding protein (CBP), or thioredoxin.

[0134] According to other embodiments of the invention, the detectable marker may be a fluorescent marker. Examples of fluorescent markers are phycoerythrin, allophycocyanin (APC), Brilliant Violet 421, Alexa Fluor 488, coumarin, or rhodamine, to name just a few. Detection of a fluorescent marker refers to detecting the emitted light when the fluorescent marker is excited. Non-exhaustive examples of suitable fluorescent markers include green emitters (Atto488, Alexa488, Cy2, etc.), orange emitters (Atto542, Alexa555, Cy3, etc.), red-far-red emitters (Alexa633, Atto 647N, Cy5, etc.), infrared emitters (Atto700, LiCor IRDye700, LiCor IRDye800, etc.), and ultraviolet-absorbing fluorescent dyes (Atto390 or Alexa405). Fluorescent markers may also be fluorescent proteins, such as GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, or near-infrared fluorescent proteins.

[0135] According to a further embodiment of the invention, the detectable mark may be an affinity label.

[0136] The term "affinity tag" refers to an amino acid segment that can specifically bind to another specific binding partner conjugated to another detectable tag (such as a fluorescent or chromophore tag). In this case, the detectable tag attached to the antibody of the present invention can be detected by contacting the antibody with a specific binding partner that specifically binds to the detectable tag conjugated to the antibody of the present invention. The specific binding partner can be labeled with another detectable tag, which is preferably a tag distinguishable from the first detectable tag (such as a fluorescent or chromophore tag). However, the specific binding partner can be a structure that can be recognized by another specific (labeled) binding reagent. For example, the specific binding partner that binds to the detectable tag conjugated to the antibody of the present invention can be a (primary) antibody that can be specifically recognized by a (secondary) antibody carrying a detectable tag that is preferably distinguishable from the first detectable tag (such as a fluorescent tag). Therefore, the method for detecting fusion proteins can include the step of contacting the fusion protein and the antibody of the present invention with a specific binding partner of the detectable tag contained in the antibody of the present invention. When a second detectable tag is present, the method can include the step of detecting the first and / or the second detectable tag.

[0137] According to a method in which the detectable label conjugated to the antibody of the present invention is an affinity tag, a method for detecting fusion proteins may include, in a first step, contacting a sample containing or suspected to contain a peptide-containing fusion protein with an antibody of the present invention. In a second step, a (secondary) specific binding partner may be contacted with a sample containing a fusion protein bound to the antibody of the present invention. In cases where the (secondary) specific binding partner is not conjugated to a detectable label or the detectable label is an affinity tag, the method may further include the step of contacting an additional specific binding partner, such as an additional antibody that specifically binds to the (secondary) specific binding partner or its detectable label. The additional specific binding partner may contain a detectable label, such as a fluorescent label that can be used to detect fusion proteins. The presence, amount, and / or location of a labeled peptide or protein can be detected or determined by measuring or observing a report signal obtained from the detectable label contained in the (secondary) specific binding partner or the additional specific binding partner.

[0138] This invention relates to proteins comprising the amino acid sequence of the antibody of the present invention. The protein may be a fusion protein comprising the antibody of the present invention and another protein.

[0139] As used herein, the term "protein" includes one or more polypeptides. As used herein, the term "fusion protein" refers to a polypeptide or protein comprising two or more subunits. At least one of the subunits is preferably a protein or polypeptide. In a fusion protein, these subunits may be linked by covalent or non-covalent linkages. Preferably, a fusion protein is a translational fusion between two or more subunits. Translational fusions can be generated by genetically engineering the coding nucleotide sequence of one subunit with the coding nucleotide sequence of another subunit within the reading frame. The subunits may be dispersed by a linker. If one or more subunits are part of a protein (complex) composed of more than one polypeptide chain, the term "fusion protein" may also refer to a protein comprising a fusion sequence and all other polypeptide chains of that protein (complex).

[0140] As used in this article, the term "peptide" generally refers to a peptide having at least about 30, at least about 40, or at least about 50 amino acids.

[0141] As used herein, the term "peptide" refers to a linear series of amino acids, preferably linked together by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The term "amino acid" as used herein refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics, preferably protein-forming amino acids. "Protein-forming amino acids" are amino acids that can be biosynthetically introduced into proteins during translation. Currently, there are 22 known genetically encoded (protein-forming) amino acids, 20 standard genetic codes, and 2 additional amino acids that can be introduced through specific translation mechanisms. As used herein, a "peptide" preferably contains no more than about 50 amino acids.

[0142] This invention relates to a complex comprising a protein and an antibody of the present invention, said protein containing the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and X13 is P or A or S or K or D or E or has no amino acid. Therefore, the antibody of the present invention can complex with an epitope to which it specifically binds. Such an epitope can be an epitope tag as described herein or a peptide shown in SEQ ID NO: 2-93. Preferably, said protein comprises the amino acid sequence of SEQ ID NO: 2.

[0143] This invention also relates to nucleic acids encoding antibodies of the present invention as described herein. The nucleic acid molecule can allow for antibody expression. It may include sequence elements containing information about transcriptional and / or translational regulation, and these sequences may be “operably linked” to a nucleotide sequence encoding the protein. Operable linking is a link in which the regulatory sequence elements and the sequence to be expressed are linked in a manner that enables gene expression. The precise nature of the regulatory regions necessary for gene expression varies by species, but typically these regions include promoters, which in prokaryotes contain the promoter itself, i.e., the DNA element that directs transcription initiation, and the DNA element that will emit a translation initiation signal when transcribed into RNA. Such promoter regions typically include 5' non-coding sequences involved in transcription and translation initiation, such as the -35 / -10 box and Shine-Dalgarno element in prokaryotes, or the TATA box, CAAT sequence, and 5'-capping element in eukaryotes. These regions may also include enhancers or repressor elements, as well as translation signals and leader sequences for targeting the native protein to specific compartments of the host cell.

[0144] The nucleic acid encoding the antibody of this invention can be DNA.

[0145] Alternatively, the nucleic acid encoding the antibody of the present invention can be RNA, such as mRNA.

[0146] This invention relates to vectors comprising nucleic acids encoding the antibodies of the present invention. Therefore, the nucleic acid molecules of the present invention can be vectors or part of any other type of cloning or expression vector. In addition to the regulatory sequences and nucleic acid sequences encoding the peptides or proteins described herein, such vectors may include replication and control sequences derived from a species compatible with the host cells used for expression, as well as selection markers that confer selectable phenotypes to transformed or transfected cells. A large number of suitable cloning vectors are known in the art and are commercially available, such as plasmids, phage particles, bacteriophages, baculoviruses, granules, or artificial chromosomes.

[0147] This invention also relates to host cells comprising the nucleic acids or vectors of this invention, or host cells expressing the antibodies of this invention. Therefore, the cloning or expression of the nucleic acid molecules or vectors of this invention can be performed, at least in part, using host cells transformed with said nucleic acids or vectors, or using host cells into which said nucleic acid molecules or vectors have already been transferred by other methods, including transduction or transfection. DNA transfer can be performed using standard techniques.

[0148] The host cells of this invention can be prokaryotic or eukaryotic. Prokaryotic host cells include bacterial cells, such as *Escherichia coli* or *Bacillus subtilis*. Eukaryotic cells can be microbial cells, such as yeast cells (e.g., *Saccharomyces cerevisiae*, *Pichia pastoris*), plant cells, or animal cells, such as insect cells (e.g., SF9 or High5 insect cells), avian cells, or mammalian cells. Mammalian cells can be derived from any species, such as mice, rats, rabbits, monkeys, apes, or humans. In some embodiments, the host cell is a non-human host cell. The host cell can be an immortalized mammalian cell line (e.g., HeLa cells or CHO cells) or a primary mammalian cell. The host cell of this invention is preferably an isolated host cell.

[0149] The present invention also relates to a method for producing the antibodies of the present invention. Any known and established method, including the use of established expression systems and / or recombinant cell culture techniques, can be used to produce the antibodies according to the present invention. The method can be carried out in vivo, and the antibodies can be produced, for example, in a bacterial or eukaryotic host organism and then isolated from that host organism or its culture. Proteins can also be produced in vitro, for example, using an in vitro translation system.

[0150] When antibodies are produced in vivo, the nucleic acid encoding the antibody is introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA technology (as outlined above). For this purpose, host cells are first transformed with a cloning vector containing a nucleic acid molecule encoding the antibody as described herein, using established standard methods. The host cells are then cultured under conditions that allow for the expression of heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the antibody can be recovered from the cells or the culture medium.

[0151] Therefore, a method for producing the antibodies of the present invention may include the step of culturing host cells as disclosed herein under conditions that allow for the expression of the antibodies of the present invention. The method may further include recovering and / or isolating the antibodies from the cells or culture medium.

[0152] The antibodies of this invention can be produced in genetically modified organisms (such as goats or plants). Antibodies can also be produced through chemical synthesis.

[0153] This invention relates to the use of the antibody of the present invention for binding, detecting, immobilizing, separating, or purifying proteins, said proteins comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G, S, P, or T; X2 is R, G, or P; X5 is E or Q; X7 is L or I; X12 is S, T, P, A, D, or E; and X13 is P, A, S, K, D, or E, or no amino acid. Preferably, the protein comprises the amino acid sequence of SEQ ID NO: 2.

[0154] This invention also relates to the antibody of this invention, used for binding, detecting, immobilizing, separating, or purifying proteins, said proteins comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or has no amino acid. Preferably, said protein comprises the amino acid sequence of SEQ ID NO: 2.

[0155] The present invention also relates to a method for binding to or detecting a protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and X13 is P or A or S or K or D or E or no amino acid, the method comprising contacting the protein with an antibody of the present invention and optionally detecting the antibody or a label contained in the antibody. Preferably, the protein comprises the amino acid sequence of SEQ ID NO: 2.

[0156] SEQ ID NO: 3 defines the core structure of a peptide that can be bound by the antibody of the present invention, which may further include up to two additional amino acids at the N-terminus and up to two additional amino acids at the C-terminus. These additional amino acids at the ends of the core structure of the peptide generally do not necessarily affect the secondary structure of the peptide or the specific binding of the peptide to a peptide-specific antibody, but can instead act as linker structures in the fusion protein. Therefore, the type and number of additional amino acids can depend on the position of the peptide in the fusion protein and can vary depending on whether the peptide is located at the N-terminus, C-terminus, or somewhere in between. Thus, the peptide sequence may contain amino acids Xa-Xb at the N-terminus of X1, where Xa is D, S, G, M, P, or no amino acid, and Xb is S, D, P, M, R, G, or no amino acid. The peptide may also contain amino acids Xy-Xz at the C-terminus of X13, where Xy is G, S, P, D, A, E, K, or no amino acid, and Xz is S, P, or no amino acid.

[0157] Detection can be optical detection, isotope detection, or detection by electron microscopy. The method includes contacting a protein containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof (such as any one of SEQ ID NO: 3-93) with the antibody of the present invention, preferably under conditions that allow the formation of a complex between the protein and the antibody. The antibody of the present invention may carry a detectable label as defined herein. When the detectable label is a fluorescent label, chromophore label, isotope label, or metal label attached thereto, it should be understood that the antibody preferably has a defined number of labels attached thereto. The method may include the step of detecting the detectable label. The method may include expressing a peptide-containing fusion protein prior to contacting the fusion protein with the antibody of the present invention.

[0158] As used herein, the term "detection" includes direct detection of a target (i.e., detection of a target via a signal derived from the target) and indirect detection of a target (i.e., detection of a target via a signal not directly derived from the target, such as a signal derived from another molecule attached to the target). The term "detection" as used herein also includes qualitative and quantitative detection. The term "detection" can refer to determining the presence, subcellular location, or amount of a given molecule or structure, such as a protein comprising the amino acid sequence of SEQ ID NO:2 or a variant sequence thereof, such as any one of SEQ ID NO:3-93. Such a protein to be detected, located, and / or quantified can be detected at an intracellular location within its host cell, such as in the nucleus, cell membrane, or another cellular compartment. The protein to be detected and / or quantified can also be detected in a solution containing the protein, such as cell lysates obtained from host cells or tissues containing host cells.

[0159] As used herein, the term "optics" preferably refers to visible light, but is not usually limited to this. The term may also refer to the infrared, ultraviolet, and other bands of the electromagnetic spectrum.

[0160] As used herein, “isotope detection” refers to the detection of a molecule in which one or more atoms have been replaced by another isotope that typically has detectable differences (i.e., “labeling”). Isotope labels can be detected by a variety of methods, such as by their mass (e.g., by mass spectrometry, matrix-assisted laser desorption / ionization (MALDI), desorption electrospray ionization (DESI), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) or secondary ion mass spectrometry (SIMS), vibrational modes (e.g., by infrared spectroscopy), gyromagnetic ratio (e.g., by nuclear magnetic resonance), or radioactive decay (e.g., autoradiography of an ionization chamber or gel).

[0161] Electron microscopy involves the detection methods that utilize electron microscopes. Types of electron microscopes include transmission electron microscopy (TEM), scanning electron microscopy (SEM), reflection electron microscopy (REM), scanning transmission electron microscopy (STEM), and related optical and electron microscopy (CLEM).

[0162] In the first step of the detection method, the antibody of the present invention, which specifically binds to the peptide contained in the fusion protein, can be contacted with a sample containing the fusion protein. The sample can be host cells, tissues, a solution of cell lysate containing host cells, or any other sample containing the fusion protein, such as a supernatant obtained after centrifugation of a liquid containing host cells, wherein the host cells are capable of secreting the target polypeptide into the liquid or another sample (such as body fluids).

[0163] The contact step described above is preferably performed under conditions that allow for specific interaction between the antibody and a protein containing a sequence that the antibody specifically binds to. Such conditions are well known to those skilled in the art. A washing step typically follows the contact step between the antibody and its antigen, and those skilled in the art know how and when to apply said washing step. Upon contact with the sample, the antibody will specifically interact with the fusion protein. This interaction can be detected, monitored, and quantified by measuring or observing a report signal obtained from the detectable label. For example, if the detectable label is a fluorescent label, fluorescence can be measured and observed upon excitation.

[0164] The advantage of a two-step detection method employing two types of binding couplers is that the tag-specific interaction is separated from the actual detection step. This allows the antibody of the present invention to remain unchanged because it does not need to contain an additional detectable portion. Compared to antibodies containing additional detectable tags, this approach can enhance the specificity or affinity of the antibody in some cases, as the detectable tag can affect the interaction between the peptide contained in the fusion protein of the present invention and the antibody of the present invention. Therefore, in some cases, the reliability and efficiency of the detection method can be enhanced. Furthermore, if only the presence and amount of protein need to be determined, the antibody of the present invention can be simply used as a capture antibody rather than as a capture and detection antibody, thus separating the capture and detection steps. Therefore, a separation or enrichment step can be performed after the first step using the antibody of the present invention to produce the captured target protein. The separated and / or enriched protein can then be subjected to a detection step, thereby improving the reliability of the obtained quantification and simplifying the operation of the detection step.

[0165] Suitable biophysical or biomolecular assays for qualitative detection of epitope tag / antibody interactions include any suitable methods known in the art. Such methods include, but are not limited to, methods for qualitative or quantitative assays, such as those for enzyme-linked immunosorbent assays (ELISA), ELISPOT assays, Western blotting, or immunoassays. Preferably, when using any of the aforementioned labels, markers, or linkers, these methods include, for example, optical, radiometric, or chromatographic methods, more preferably, fluorescent detection methods, radiometric detection methods, Coomassie blue staining, silver staining or other protein staining methods, electron microscopy methods, methods of staining tissue sections by immunohistochemistry or by direct or indirect immunofluorescence, etc. Such methods may be used in conjunction with antibodies, or may involve the use of additional tools, such as secondary binding couplers that specifically bind a portion of the fusion protein, the antibody of the present invention, or the complex.

[0166] In some implementations, the subcellular localization of the tagged target peptide or protein can also be determined. For example, different subcellular structures, such as intermediate filamentous networks or important parts of the replication mechanism, can be observed and monitored.

[0167] The antibodies of this invention, which are intracellular antibodies, can also be used for the detection of fusion proteins. As used herein, "intracellular antibody" refers to an antibody located within a cell to bind intracellular proteins. Due to the lack of a reliable mechanism for bringing antibodies from the extracellular environment into living cells, this typically requires antibody expression within the target cell. After expression, the antibody may be retained in the cytoplasm, or it may have a nuclear localization signal, or it may undergo transmembrane cotranslational transport into the lumen of the endoplasmic reticulum, provided it is retained in that compartment via a KDEL sequence. The detectable marker conjugated to the intracellular antibody can be a protein marker that can be expressed as a fusion protein along with the intracellular antibody. Ideally, such a marker can be optically detectable, for example, by fluorescence. Therefore, the detectable marker can be a fluorescent protein.

[0168] In the context of this invention, the term "fixation" refers to the attachment of the antibody of this invention to a solid support, which can be any type of carrier material that can be used to fix affinity ligands (e.g., antibodies or portions thereof), and can refer to materials in particulate form (e.g., beads or granules commonly used in extraction columns) or sheet form (e.g., membranes or filters, glass or plastic slides, microtiter plates, dipsticks, capillary filling devices, etc.), which can be flat, wrinkled, or hollow fibers or tubular. Suitable and well-known matrices (not an exhaustive list) are: silica (porous amorphous silica), agarose or polyacrylamide supports, or macroporous polymers. Examples include dextran, collagen, polystyrene, polypropylene, polyvinyl chloride, polyacrylamide, methacrylate, cellulose, calcium alginate, controlled-porosity glass, aluminum, titanium and porous ceramics, synthetic polymers and copolymers, latex, silica, agarose, metals, glass, and carbon. Alternatively, the solid surface may include part of a mass-dependent sensor, such as a surface plasmon resonance detector. The solid support can also be magnetic beads, polymer beads, or chromatographic stationary phase.

[0169] The antibodies of the present invention used for isolation and / or purification can be used in solution or immobilized. To immobilize the antibody, it can be bound to a sample carrier, solid support, or matrix. This immobilization step can be performed before or after the antibody binds to the peptide contained in the fusion protein. Methods for immobilizing antibodies and their portions are well known to those skilled in the art, and any method that allows immobilization without impairing binding properties can be used.

[0170] If the antibody of the present invention is not immobilized on a solid support, the method may include the further step of separating the complex, said separation being, for example, by using a specific binding partner of the complex, said specific binding partner being, for example, a secondary antibody that is specific to the complex or to the antibody or to a detectable label (such as an affinity tag conjugated to an antibody). The secondary binding partner may be in solution or may be immobilized or immobilized on a solid support.

[0171] In an optional further step following the capture step, the solid support containing the fixed antibody bound to the protein is washed to remove unbound and non-specifically bound components.

[0172] Optionally, in a further step, the protein can be eluted to obtain a separated protein. Elution of the protein bound to the immobilized antibody can be achieved by methods known in the art. For example, the protein can be eluted by competitive elution with an epitope peptide described herein in a separated form. This separated form of epitope peptide will then compete with the protein for binding to the immobilized tag-specific antibody. If the separated peptide is added in excess, the binding equilibrium will shift towards the binding of the immobilized antibody to the separated epitope tag. This results in the release of the protein. The epitope peptide used for elution can be the same as the epitope peptide contained in the protein. The epitope peptide used for elution can also be a different peptide from the epitope peptide contained in the protein. If the epitope peptide used for elution is a different epitope peptide, it is preferable that the epitope peptide used for elution has a higher affinity for the antibody than the epitope peptide contained in the protein. Additional steps for further purification of the released polypeptide, such as steps well known to those skilled in the art, can optionally be added.

[0173] Proteins can also remain immobilized on solid supports (e.g., (magnetic) beads) and be further processed in downstream applications (e.g., mass spectrometry) without elution steps.

[0174] The protein may contain a linker with a cleavage site that can be cleaved by a suitable method (e.g., a protease) to remove the peptide. Therefore, the polypeptide of the protein can be released from the immobilized antibody and obtained in its native form. For this embodiment, the nucleic acid sequence encoding the protein should contain not only a sequence encoding an epitope tag but also a sequence encoding a linker with a cleavage site, such as a cleavage site recognized by a protease. The release step by enzymatic cleavage can replace the elution step or be performed after the elution step.

[0175] The peptide sequence contained in the protein preferably comprises the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO: 4), said protein being a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated, or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention, wherein X1 is S or T, X2 is R or G, X5 is E or Q, X12 is T or D or E, and wherein X13 is A or D or E or has no amino acid. The peptide may comprise the sequence SRLEEELRRRLTE (SEQ ID NO: 2) or a variant thereof, wherein, compared to SEQ ID NO: 3, said variant has 1 to 5 mutations selected from the following: S1→T, R2→G, E5→Q, T12→D and T12→E, E13→A, E13→D, and deletion of E13. Compared to SEQ ID NO: 3, the variant may have the following mutations: (a) S1→T and E13→A; (b) R2→G; (c) R2→G and E5→Q; (d) R2→G, E5→Q and E13→A; (e) R2→G, E5→Q and T12→D and E13→A; (f) R2→G, E5→Q and T12→E and E13→A; (g) T12→D and E13→A; (h) T12→E and E13→A; (i) and E13→A; (j) and E13→D; or (k) deletion of E13. The peptide containing sequence SEQ ID NO: 4 as its core structure may contain amino acids Xa-Xb at the N-terminus of X1, where Xa is S, G, M, P, or no amino acid, and Xb is R, G, S, P, M, or no amino acid. Xa-Xb can be selected from P, MP, GR, PG, PS, SP, GP, SP, M, and MS, preferably P or MP. Such peptides may also contain amino acids Xy-Xz at the C-terminus of X13, wherein Xy is P, D, A, or no amino acid, and Xz is P, S, or no amino acid. Xy-Xz can be selected from no amino acid, P, DP, A, and AS, preferably no amino acid or P. The peptide may comprise combinations of Xa-Xb and Xy-Xz selected from: (a) MP and P; (b) P and P; and (c) P and no amino acid.

[0176] The peptide sequence contained in the protein preferably includes the sequence SRLEEELRRRLTE (SEQ ID NO: 2), said protein being a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, separated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention.

[0177] The peptide sequence contained in the protein may include sequences selected from those of a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention:

[0178] (a) MPSRLEEELRRRLTEP (SEQ ID NO: 5);

[0179] (b) PSRLEEELRRRLTEP (SEQ ID NO: 6);

[0180] (c) PSRLEEELRRRLTE (SEQ ID NO: 7);

[0181] (d) GRRSRLEEELRRRLTA (SEQ ID NO: 8);

[0182] (e) PGSRLEEELRRRLTAP (SEQ ID NO: 9);

[0183] (f) PSTRLEEELRRRLTAP (SEQ ID NO: 10);

[0184] (g) SPSRLEEELRRRLTAP (SEQ ID NO: 11);

[0185] (h) SPSRLEEELRRRLDAP (SEQ ID NO: 12);

[0186] (i) SPSRLEEELRRRLEAP (SEQ ID NO: 13);

[0187] (j) SPSRLEEELRRRLTDP (SEQ ID NO: 14);

[0188] (k) SPSRLEEELRRRLTEP (SEQ ID NO: 15);

[0189] (l) SPSRLEEELRRRLTADP (SEQ ID NO: 16);

[0190] (m) SPSGLEEELRRRLTEP (SEQ ID NO: 17);

[0191] (n) GPSRLEEELRRRLT (SEQ ID NO: 18);

[0192] (o) GPSRLEEELRRRLTA (SEQ ID NO: 19);

[0193] (p) GPSRLEEELRRRLTAA (SEQ ID NO: 20);

[0194] (q) GPSRLEEELRRRLTAAS (SEQ ID NO: 21);

[0195] (r) SPSGLEQELRRRLTAP (SEQ ID NO: 22);

[0196] (s) SPSGLEQELRRRLDAP (SEQ ID NO: 23);

[0197] (t) SPSGLEQELRRRLEAP (SEQ ID NO: 24);

[0198] (u) SPSGLEQELRRRLTEP (SEQ ID NO: 25);

[0199] (v) GPSRLEEELRRRLTAP (SEQ ID NO: 26);

[0200] (w) GPSRLEEELRRRLTEP (SEQ ID NO: 27);

[0201] (x) GPSRLEEELRRRLTE (SEQ ID NO: 28);

[0202] (y) MSRLEEELRRRLTEP (SEQ ID NO: 29); and

[0203] (z) MSSRLEEELRRRLTEP (SEQ ID NO: 30).

[0204] The peptide sequence contained in the protein may include the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 31), said protein being a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention, wherein X1 is G or S or P, X2 is R or G, X5 is E or Q, X7 is L or I, X12 is S or T or P or A, and X13 is P or A or S or has no amino acids.

[0205] The peptide sequence contained in the protein may include the sequence GRLEEELRRRLS (SEQ ID NO:32) or a variant thereof, said protein being a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention, wherein, compared with SEQ ID NO: 32, said variant has 1 to 6 mutations selected from the following: G1→S, G1→P, R2→G, E5→Q, L7→I, S12→T, S12→P, S12→A, addition of P13, addition of A13 and addition of S13. Compared to SEQ ID NO: 32, the variant may have the following mutations: (a) G1→S, R2→G, E5→Q, and addition of P13; (b) R2→G, E5→Q, S12→T, and addition of A13; (c) G1→P, R2→G, E5→Q, S12→T, and addition of A13; (d) G1→S, R2→G, E5→Q, S12→T, and addition of P13; (e) G1→S, R2→G, S12→T, and addition of A13; (f) G1→S, R2→G, E5→Q, and S12→T; (g) G1→S, R2→G, E5→Q, S12→T, and addition of A13; (h) G1→S and S12→P, and addition of P13; (i) G1→S, R2→G, E5→Q, S12→T and addition of P13; (j) E5→Q, L7→I and addition of P13; (k) addition of P13; or (l) S12→A. A peptide containing the sequence SEQ ID NO: 31 as its core structure may contain amino acids Xa-Xb at the N-terminus of X1, wherein Xa is M, S, P, D, G, or no amino acid, and Xb is S, D, P, or no amino acid. Xa-Xb may be selected from MS, SD, PD, PS, DS, SP, and GP, preferably MS. Such a peptide may also contain amino acids Xy-Xz at the C-terminus of X13, wherein Xy is G, P, A, E, K, S, or no amino acid, and Xz is P, S, or no amino acid. Xy-Xz may be selected from no amino acid, G, P, A, EP, AS, K, and S, preferably no amino acid. The peptide may comprise a combination of Xa-Xb and Xy-Xz, which is MS and free of amino acids.

[0206] The peptide sequence contained in the protein may include sequences selected from those of a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention:

[0207] (a) GRLEEELRRRLS (SEQ ID NO: 32);

[0208] (b) MSGRLEEELRRRLSP (SEQ ID NO: 33);

[0209] (c) SDSGLEQELRRRLSPG (SEQ ID NO: 34);

[0210] (d) PDGGLEQELRRRLTAP (SEQ ID NO: 35);

[0211] (e) PSGGLEQELRRRLTAP (SEQ ID NO: 36);

[0212] (f) DSPGLEQELRRRLTAP (SEQ ID NO: 37);

[0213] (g) PDSGLEQELRRRLTPA (SEQ ID NO: 38);

[0214] (h) SPSGLEEELRRRLTAEP (SEQ ID NO: 39);

[0215] (i) GPSGLEQELRRRLT (SEQ ID NO: 40);

[0216] (j) GPSGLEQELRRRLTAAS (SEQ ID NO: 41);

[0217] (k) SPSRLEEELRRRLPSK (SEQ ID NO: 42);

[0218] (l) SPSGLEQELRRRLTPS (SEQ ID NO: 43);

[0219] (m) SPGRLEQEIRRRLSPS (SEQ ID NO: 44);

[0220] (n) PSGRLEEELRRRLSPS (SEQ ID NO: 45);

[0221] (o) PSGRLEEELRRRLS (SEQ ID NO: 46);

[0222] (p) PSGRLEEELRRRLA (SEQ ID NO: 47); and

[0223] (q) PSGRLEEELRRRLSP (SEQ ID NO: 48).

[0224] The peptide sequence contained in the protein may include a peptide with the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO: 49) as a core structure, said protein being a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated, or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention, wherein X1 is S, G, or P; X2 is R, G, or P; X5 is E or Q; X12 is S, T, D, or E; and X13 is P, A, D, or no amino acid. The peptide containing the sequence SEQ ID NO: 49 as a core structure may contain amino acids Xa-Xb at the N-terminus of X1, wherein Xa is P, D, S, G, or no amino acid, and Xb is D, S, P, or no amino acid. Such a peptide may also contain amino acids Xy-Xz at the C-terminus of X13, wherein Xy is G, P, E, D, S, or no amino acid, and Xz is P or no amino acid.

[0225] The peptide sequence contained in the protein may have a core structure of sequence SEQ ID NO: 49 and may contain sequences selected from the following, wherein the protein is a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention:

[0226] (a) PDSGLEQELRRRLSPG (SEQ ID NO: 50);

[0227] (b) PDSGLEQELRRRLTAP (SEQ ID NO: 51);

[0228] (c) PSSGLEQELRRRLTAP (SEQ ID NO: 52);

[0229] (d) DPSGLEQELRRRLTAP (SEQ ID NO: 53);

[0230] (e) DSGPLEQELRRRLTAP (SEQ ID NO: 54);

[0231] (f) SPSRLEEELRRRLTAEP (SEQ ID NO: 55);

[0232] (g) SPSGLEEELRRRLTAP (SEQ ID NO: 56);

[0233] (h) SPSGLEEELRRRLDAP (SEQ ID NO: 57);

[0234] (i) SPSGLEEELRRRLEAP (SEQ ID NO: 58);

[0235] (j) SPSGLEEELRRRLTDP (SEQ ID NO: 59);

[0236] (k) SPSGLEEELRRRLTADP (SEQ ID NO: 60);

[0237] (l) GPSGLEQELRRRLTA (SEQ ID NO: 61);

[0238] (m) SPSGLEQELRRRLTDP (SEQ ID NO: 62);

[0239] (n) SPSGLEQELRRRLTADP (SEQ ID NO: 63);

[0240] (o) SPSGLEQELRRRLTAEP (SEQ ID NO: 64);

[0241] (p) DSPGLEQELRRRLTAP (SEQ ID NO: 37); and

[0242] (q) SPSGLEQELRRRLSPS (SEQ ID NO: 65).

[0243] The peptide sequence contained in the protein may include a peptide with the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-RL-X12-X13 (SEQ ID NO: 66) as a core structure. The protein is a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, separated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention, wherein X1 is G or S, X2 is R or G or A or E, X3 is L or V, X4 is E or Q, X5 is E or Q, X6 is E or Q, X7 is L or I or V, X8 is R or A or Q or E, X9 is R or A or Q or E, X12 is S or T or L or no amino acid, and X13 is K or P or S or no amino acid. The peptide containing the sequence SEQ ID NO: 66 as its core structure may contain amino acids Xa-Xb at the N-terminus of X1, wherein Xa is D, S, G, M, or no amino acid, and Xb is S, D, P, M, or no amino acid. This peptide may also contain amino acids Xy-Xz at the C-terminus of X13, wherein Xy is G, S, P, or no amino acid, and Xz is S or no amino acid.

[0244] The peptide sequence contained in the protein may have a core structure of sequence SEQ ID NO: 66 and may contain sequences selected from the following, wherein the protein is a protein contained in the complex of the present invention, or a protein to be bound, detected, immobilized, isolated or purified in the use of the present invention, or a protein to be bound or detected in the binding or detection method of the present invention:

[0245] (a) DSGRLEEELRRRLSKG (SEQ ID NO: 67);

[0246] (b) DSGRLEEELRRRLSPG (SEQ ID NO: 68);

[0247] (c) SDSGLEELRRRLSPG (SEQ ID NO: 69);

[0248] (d) SDSGVEEELRRRLSPG (SEQ ID NO: 70);

[0249] (f) SDSAVEEELRRRLSPG (SEQ ID NO: 71);

[0250] (g) SDSGLQEELRRRLSPG (SEQ ID NO: 72);

[0251] (h) SDSGLEEQLRRRLSPG (SEQ ID NO: 73);

[0252] (i) SDSGLEEEIRRRLSPG (SEQ ID NO: 74);

[0253] (j) SDSGLEEEVRRRLSPG (SEQ ID NO: 75);

[0254] (k) DSGELEEELRRRLSPG (SEQ ID NO: 76);

[0255] (l) DSGRLEQELRRRLSPG (SEQ ID NO: 77);

[0256] (m) DSGRLEEEIRRRLSPG (SEQ ID NO: 78);

[0257] (n) DSGRLEQEIRRRLSPG (SEQ ID NO: 79);

[0258] (o) DSGRLEQEIARRLSPG (SEQ ID NO: 80);

[0259] (p) DSGRLEQEIQRRLSPG (SEQ ID NO: 81);

[0260] (q) DSGRLEQEIERRLSPG (SEQ ID NO: 82);

[0261] (r) DSGRLEQEIRARLSPG (SEQ ID NO: 83);

[0262] (s) DSGRLEQEIRQRLSPG (SEQ ID NO: 84);

[0263] (t) DSGRLEQEIRERLSPG (SEQ ID NO: 85);

[0264] (u) GPSRLEEELRRRL (SEQ ID NO: 86);

[0265] (v) MSGLEQELRRRLTPS (SEQ ID NO: 87);

[0266] (w) MSGRLEEELRRRLSPS (SEQ ID NO: 88);

[0267] (x) SPSAVEEELRRRLSPS (SEQ ID NO: 89);

[0268] (y) GPSAVEEELRRRLS (SEQ ID NO: 90);

[0269] (z) MPSGLEQELRRRLTPS (SEQ ID NO: 91);

[0270] (aa) MSSGLEQELRRRLTPS (SEQ ID NO: 92); and

[0271] (bb) MPSGRLEEELRRRLSPS (SEQ ID NO: 93).

[0272] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used in this article include plural references and vice versa.

[0273] Unless otherwise stated, the term "at least" preceding a series of elements shall be understood to refer to each element in the series.

[0274] Those skilled in the art will recognize, or can determine by conventional experiments alone, many equivalents of the specific embodiments of the invention described herein. This invention is intended to cover such equivalents.

[0275] The term “and / or” as used herein includes the meaning of “and,” “or,” and “all or any other combination of elements connected by the term.”

[0276] As used herein, the terms "about" or "approximately" mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, it also includes specific numbers, such as about 20 including 20.

[0277] Throughout this specification and claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" shall be understood to mean including the integers or steps or sets of integers or steps stated herein, but not excluding any other integers or steps or sets of integers or steps. When used herein, the term "comprising" may be replaced by the terms "containing," "including," or, as sometimes used herein, "having."

[0278] When used herein, “consisting of…” excludes any element, step, or component not specified in the elements of the claim. When used herein, “consisting substantially of…” does not exclude materials or steps that do not substantially affect the essential or novel features of the claim.

[0279] In each case herein, any one of the terms “comprising,” “substantially consisting of,” and “consisting of” may be replaced by any of the other two terms. For example, the term “comprising” is intended to provide explicit support for “substantially consisting of” and “consisting of,” the term “substantially consisting of” is intended to provide explicit support for “comprising” and “consisting of,” and the term “consisting of” is intended to provide explicit support for “substantially consisting of” and “comprising.” The possibility of the terms being interchangeable should not be construed as meaning that these terms are synonymous.

[0280] The invention is further characterized by the following items.

[0281] Project 1: A single-domain antibody that specifically binds to an epitope tag containing the sequence SRLEEELRRRLTE (SEQ ID NO:2), wherein the antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94, a CDR2 sequence as shown in SEQ ID NO: 97, and a CDR3 sequence as shown in SEQ ID NO: 98; wherein the antibody further comprises the following amino acid residues at positions 47, 48, and 50 corresponding to sequence positions 47, 48, and 50 of SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; and wherein the antibody comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 118-125.

[0282] Project 2: The antibody described in Project 1, wherein the antibody is humanized.

[0283] Item 3: The antibody described in any one of the preceding items, wherein the antibody comprises a sequence having at least about 70% V segment identity with the closest human pedigree sequence.

[0284] Project 4: The antibody described in Project 3, wherein the human lineage sequence is IGHV3-23*4 as shown in SEQ ID NO: 110.

[0285] Item 5: The antibody according to any one of the preceding items, wherein the antibody has an affinity for the epitope tag shown in SEQ ID NO: 2 that is at least equivalent to the affinity of the antibody in SEQ ID NO: 1, wherein "at least equivalent" preferably means that the K of the antibody... D Not higher than SEQ ID NO: 1 K D 2.5 times that of K D The measurement is preferably performed by biological layer interferometry, and more preferably by the method described substantially as in Method 1.

[0286] Item 6: The antibody according to any one of the preceding items, wherein the antibody has approximately 5 × 10⁻⁶ epitope tags as shown in SEQ ID NO: 2. -10 M or lower, preferably 3×10 -10 M or lower K D The K D The measurement is preferably performed by biological layer interferometry, and more preferably by the method described substantially as in Method 1.

[0287] Item 7: The antibody described in any one of the preceding items, wherein the antibody has a higher Tm compared to the antibody of SEQ ID NO: 1.

[0288] Item 8: The antibody of any one of the preceding items, wherein the antibody has a Tm of at least about 63°C, wherein the Tm is preferably measured substantially as described in Method 2.

[0289] Item 9: The antibody according to any one of the preceding items, wherein the antibody contains a His residue at position 99 corresponding to sequence position 99 of SEQ ID NO: 1.

[0290] Item 10: The antibody according to any one of the preceding items, wherein the antibody contains a Val residue at the position corresponding to sequence position 100 of SEQ ID NO: 1.

[0291] Item 11: The antibody according to any one of the preceding items, wherein the antibody contains a Val residue at position 5 corresponding to sequence position 5 of SEQ ID NO: 1.

[0292] Item 12: The antibody of any one of the preceding items, wherein the antibody contains a Tyr residue at position 40 corresponding to sequence position 40 of SEQ ID NO: 1.

[0293] Item 13: The antibody according to any one of the preceding items, wherein the antibody contains a Gln residue at the position corresponding to sequence position 117 of SEQ ID NO: 1.

[0294] Item 14: The antibody according to any one of the preceding items, wherein the antibody comprises an FR1 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence of SEQ ID NO: 99.

[0295] Item 15: The antibody according to any one of the preceding items, wherein the antibody comprises an FR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence of SEQ ID NO: 102.

[0296] Item 16: The antibody according to any one of the preceding items, wherein the antibody comprises an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence of SEQ ID NO:108.

[0297] Item 17: The antibody according to any one of the preceding items, wherein the antibody comprises an FR4 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to the sequence of SEQ ID NO: 109.

[0298] Item 18: The antibody according to any one of the preceding items, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with or identical to the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

[0299] Item 19: The antibody according to any one of the preceding items, wherein the frame sequence of the antibody is defined by amino acid positions 1-25, 39-53, 61-98 and 112-122 corresponding to SEQ ID NO: 1.

[0300] Item 20: The antibody described in any one of the preceding items, wherein the antibody comprises a protein A binding site.

[0301] Item 21: The antibody according to any one of the preceding items, wherein the antibody contains a Thr residue at the position corresponding to sequence position 60 of SEQ ID NO: 1.

[0302] Item 22: The antibody according to any one of the preceding items, wherein the antibody contains a Lys residue at position 67 corresponding to sequence position 67 of SEQ ID NO: 1.

[0303] Item 23: The antibody of any one of the preceding items, wherein the antibody comprises the following amino acid residues at positions 60 and 67 corresponding to sequence positions SEQ ID NO: 1: Thr at position 60 and Lys at position 67.

[0304] Item 24: The antibody of any one of the preceding items, wherein the antibody contains a Tyr residue at position 61 corresponding to sequence position 61 of SEQ ID NO: 1.

[0305] Item 25: The antibody of any one of the preceding items, wherein the antibody contains an Asp residue at position 64 corresponding to sequence position 64 of SEQ ID NO: 1.

[0306] Item 25a: The antibody of any one of the preceding items, wherein the antibody contains a Tyr residue at position 61 corresponding to SEQ ID NO: 1 and an Asp residue at position 64 corresponding to SEQ ID NO: 1.

[0307] Item 26: An antibody according to any one of the preceding items, wherein the antibody comprises the CDR2 sequence shown in SEQ ID NO: 95 or 96.

[0308] Item 26a: An antibody according to any one of the preceding items, wherein the antibody comprises the CDR2 sequence shown in SEQ ID NO: 95.

[0309] Item 26b: An antibody according to any one of the preceding items, wherein the antibody comprises the CDR2 sequence shown in SEQ ID NO: 96.

[0310] Item 27: An antibody according to any one of the preceding items, wherein the antibody comprises an FR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to any one of SEQ ID NO: 100, 101.

[0311] Item 28: An antibody according to any one of the preceding items, wherein the antibody comprises an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with or identical to any one of SEQ ID NO: 103-107.

[0312] Item 29: An antibody according to any one of the preceding items, wherein the antibody comprises having at least 95% sequence identity with any one of SEQ ID NO: 118-125.

[0313] Item 30: An antibody according to any one of the preceding items, wherein the antibody comprises the sequence shown in any one of SEQ ID NO: 118-125.

[0314] Item 30a: The antibody described in any one of the preceding items, wherein the antibody does not contain an NxT / S motif.

[0315] Item 31: The antibody of any one of the preceding items, wherein the antibody is conjugated to a detectable label.

[0316] Item 32: The antibody of any one of the preceding items, wherein the detectable label is a fluorescent label.

[0317] Item 33: The antibody of any one of the preceding items, wherein the detectable label is an affinity tag.

[0318] Item 34: A protein comprising the amino acid sequence of any one of items 1-33.

[0319] Item 35: A complex comprising

[0320] a. A protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or has no amino acid; and

[0321] b. The antibody as described in any one of items 1-33.

[0322] Item 36: A nucleic acid that encodes an antibody as described in any one of items 1-33.

[0323] Item 37: The nucleic acid described in Item 36, wherein the nucleic acid is DNA.

[0324] Item 38: The nucleic acid described in Item 36, wherein the nucleic acid is RNA.

[0325] Item 39: A vector comprising any one of items 34-36.

[0326] Item 40: A host cell comprising the nucleic acid described in any one of Items 36-38 or the vector described in Item 39 or the antibody expressing any one of Items 1-33.

[0327] Item 41: Use of the antibody described in any one of Items 1-33 for binding, detecting, immobilizing, separating or purifying a protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or has no amino acid.

[0328] Item 42: The use described in Item 41, wherein the amino acid sequence comprises the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO: 4), wherein X1 is S or T, X2 is R or G, X5 is E or Q, X12 is T or D or E, and wherein X13 is A or D or E or no amino acid.

[0329] Item 43: The use described in Item 41 or 42, wherein the amino acid sequence comprises the sequence SRLEEELRRRLTE (SEQ ID NO: 2).

[0330] Item 44: Use of any one of items 41 to 43, wherein the amino acid sequence comprises a sequence selected from:

[0331] (a) MPSRLEEELRRRLTEP (SEQ ID NO: 5);

[0332] (b) PSRLEEELRRRLTEP (SEQ ID NO: 6);

[0333] (c) PSRLEEELRRRLTE (SEQ ID NO: 7);

[0334] (d) GRRSRLEEELRRRLTA (SEQ ID NO: 8);

[0335] (e) PGSRLEEELRRRLTAP (SEQ ID NO: 9);

[0336] (f) PSTRLEEELRRRLTAP (SEQ ID NO: 10);

[0337] (g) SPSRLEEELRRRLTAP (SEQ ID NO: 11);

[0338] (h) SPSRLEEELRRRLDAP (SEQ ID NO: 12);

[0339] (i) SPSRLEEELRRRLEAP (SEQ ID NO: 13);

[0340] (j) SPSRLEEELRRRLTDP (SEQ ID NO: 14);

[0341] (k) SPSRLEEELRRRLTEP (SEQ ID NO: 15);

[0342] (l) SPSRLEEELRRRLTADP (SEQ ID NO: 16);

[0343] (m) SPSGLEEELRRRLTEP (SEQ ID NO: 17);

[0344] (n) GPSRLEEELRRRLT (SEQ ID NO: 18);

[0345] (o) GPSRLEEELRRRLTA (SEQ ID NO: 19);

[0346] (p) GPSRLEEELRRRLTAA (SEQ ID NO: 20);

[0347] (q) GPSRLEEELRRRLTAAS (SEQ ID NO: 21);

[0348] (r) SPSGLEQELRRRLTAP (SEQ ID NO: 22);

[0349] (s) SPSGLEQELRRRLDAP (SEQ ID NO: 23);

[0350] (t) SPSGLEQELRRRLEAP (SEQ ID NO: 24);

[0351] (u) SPSGLEQELRRRLTEP (SEQ ID NO: 25);

[0352] (v) GPSRLEEELRRRLTAP (SEQ ID NO: 26);

[0353] (w) GPSRLEEELRRRLTEP (SEQ ID NO: 27);

[0354] (x) GPSRLEEELRRRLTE (SEQ ID NO: 28);

[0355] (y) MSRLEEELRRRLTEP (SEQ ID NO: 29); and

[0356] (z) MSSRLEEELRRRLTEP (SEQ ID NO: 30).

[0357] Item 45: The use described in Item 41, wherein the amino acid sequence comprises the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 31), wherein X1 is G or S or P, X2 is R or G, X5 is E or Q, X7 is L or I, X12 is S or T or P or A, and X13 is P or A or S or no amino acid.

[0358] Item 46: The use described in Item 41 or 45, wherein the amino acid sequence comprises the sequence GRLEEELRRRLS (SEQ ID NO: 32) or a variant thereof, wherein, compared to (SEQ ID NO: 32), the variant has 1 to 6 mutations selected from the following: G1→S, G1→P, R2→G, E5→Q, L7→I, S12→T, S12→P, S12→A, addition of P13, addition of A13, and addition of S13.

[0359] Item 47: Use as described in any one of items 41 and 45-46, wherein the amino acid sequence comprises a sequence selected from:

[0360] (a) GRLEEELRRRLS (SEQ ID NO: 32);

[0361] (b) MSGRLEEEELRRRLSP (SEQ ID NO: 33);

[0362] (c) SDSGLEQELRRRLSPG (SEQ ID NO: 34);

[0363] (d) PDGGLEQELRRRLTAP (SEQ ID NO: 35);

[0364] (e) PSGGLEQELRRRLTAP (SEQ ID NO: 36);

[0365] (f) DSPGLEQELRRRLTAP (SEQ ID NO: 37);

[0366] (g) PDSGLEQELRRRLTPA (SEQ ID NO: 38);

[0367] (h) SPSGLEEELRRRLTAEP (SEQ ID NO: 39);

[0368] (i) GPSGLEQELRRRLT (SEQ ID NO: 40);

[0369] (j) GPSGLEQELRRRLTAAS (SEQ ID NO: 41);

[0370] (k) SPSRLEEELRRRLPSK (SEQ ID NO: 42);

[0371] (l) SPSGLEQELRRRRLTPS (SEQ ID NO: 43);

[0372] (m) SPGRLEQEIRRRLSPS (SEQ ID NO: 44);

[0373] (n) PSGRLEEELRRRLSPS (SEQ ID NO: 45);

[0374] (o) PSGRLEEELRRRLS (SEQ ID NO: 46);

[0375] (p) PSGRLEEELRRRLA (SEQ ID NO: 47); and

[0376] (q) PSGRLEEELRRRLSP (SEQ ID NO: 48).

[0377] Item 48: A method for binding or detecting a protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or no amino acid, the method comprising contacting the protein with an antibody as described in any one of Items 1-33, and optionally detecting the antibody or a label contained in the antibody.

[0378] Item 49: A method for producing an antibody according to any one of items 1-33, comprising culturing the host cells of item 40 under conditions that allow the expression of the antibody.

[0379] Item 50: The method according to Item 49 further includes recovering and / or isolating the antibody from the host cell or from the culture medium.

[0380] Example

[0381] Example 1: CDR transplantation and humanization of NbALFA

[0382] In the first step, the CDR of NbALFA was grafted onto multiple receptor frameworks. As described herein, the CDR was broadly defined using a combination of the Kabat and IMGT numbering schemes. For CDR grafting, seven variants of the humanized VHH receptor framework were selected. Variants 1 to 7 of the humanized NbALFA antibody are shown in SEQ ID NO: 111 to 117.

[0383] Genes encoding variants 1 through 7 were cloned into expression vectors and subsequently produced in *E. coli* using methods well known to those skilled in the art. Antibody variants were purified by IMAC chromatography, and antigen binding was subsequently tested using biolayer interferometry.

[0384] Variants 1 and 2 failed to produce, and variants 3 through 6 did not show binding to the epitope tag of SEQ ID NO: 2. Variant 7 showed binding to the epitope tag of SEQ ID NO: 2, but antigen recognition was significantly impaired, manifested by a rapid dissociation rate and low K+. D (See Table 1.)

[0385] Table 1: Binding affinity of humanized NbALFA variants

[0386] .

[0387] Example 2: Further Engineering of Humanized Variants

[0388] Based on extensive structural analysis, eight additional variants of humanized NbALFA were derived. Variants 8 through 15 are shown in SEQ ID NOs: 118 through 125, and Table 2 shows the affinity, binding / dissociation rate, thermal stability, aggregation behavior, and IGHV3-23 framework identity of these variants. Surprisingly, all variants showed binding affinity to SEQ ID NO: 2 at least comparable to that of wtNbALFA. Notably, variants 9, 10, 12, 13, 14, and 15 exhibited wild-type affinity, while variant 11 even showed an increased affinity for SEQ ID NO: 2.

[0389] Table 2: Variants for Affinity Optimization

[0390] .

[0391] Table 3: Frame identity and V-segment identity of humanized variants

[0392] .

[0393] Example 3: Determination of SpA binding

[0394] To further investigate the newly generated variant, SpA affinity was determined using humanized NbALFA. SpA binding ability provides a direct purification method for this humanized antibody under GMP conditions, avoiding the need to develop new purification procedures. Therefore, SpA binding and purification using SpA columns were investigated (see [link to study]). Figure 3 (Table 4). Spike assays revealed quantitative binding of variants 10, 12, 13, and 15 to the column, as no VHH was present in the flow-through. These four variants were undetectable in both wash fractions and eluted in both elution steps. However, variants 11 and 14 were bound to the column but remained detectable in the flow-through fraction. Significant "bleeding" was detected in the wash fraction, and elution did not increase the presence of VHH in the fraction, making these two variants unsuitable for SpA-based purification.

[0395] Table 4: Determination of SpA binding

[0396] .

[0397] Example 4: Determination of thermal stability

[0398] The thermal stability of the humanized variants of NbALFA was determined. Table 5 shows the Tm values ​​representing thermal stability. Notably, despite humanization, variant 8 exhibits a Tm value comparable to wtNbALFA, while variants 9-15 even surprisingly exhibit higher thermal stability than wtNbALFA.

[0399] Table 5: Determination of thermal stability

[0400] .

[0401] Table 6: VHH sequence of humanized NbALFA nanobodies

[0402]

[0403]

[0404] .

[0405] Table 7: CDR and framework region sequences of the humanized NbALFA nanobody of the present invention

[0406] .

[0407] Example 5: Direct comparison of NbALFA with three humanized NbALFA variants

[0408] The purpose of this experiment was to compare wtNbALFA (SEQ ID NO: 1) with three humanized NbALFA variants (SEQ ID NO: 123 (variant 13), SEQ ID NO: 121 (variant 11) and SEQ ID NO: 35 of US2023 / 0093123A1 (“US2023 / 0093123A1-Seq35”, SEQ ID NO: 129 of this disclosure) regarding their relative expression levels and glycosylation patterns when expressed in the mammalian expression system (HEK293 / ExPi293).

[0409] For direct parallel comparison, all four NbALFA variants were expressed, purified, and analyzed in parallel using the same settings.

[0410] Experiments showed that, compared with wtNbALFA, humanized variants 13 and 11 exhibited slightly higher expression levels and yields (Table 8).

[0411] Table 8: Comparison of yields obtained from four NbALFA variants

[0412] .

[0413] When passed through SDS-PAGE gel ( Figure 4 AC) and size exclusion chromatography ( Figure 4 During the analysis of (D), all three variants showed very uniform behavior.

[0414] In contrast, the yield of US2023 / 0093123A1-Seq35 was significantly lower than that of all other NbALFA variants analyzed in this experiment (Table 8). US2023 / 0093123A1-Seq35 was also the only variant to show two distinct species on SDS gels. Figure 4 (A + C), they can be clearly separated by size exclusion chromatography ( Figure 4Based on its more dispersed appearance on the SDS gel, the corresponding upper band (corresponding to SEC peak 1) can be attributed to the type of glycosylation. The type corresponding to the lower band observed on the SDS gel had an SEC elution volume of approximately 14 mL, consistent with other NbALFA variants that did not show any second type when analyzed by SDS-PAGE in this experiment. The small peaks of humanized variants 13 and 11 observed at an elution volume of ~12.5 mL most likely correspond to transient dimer forms, which are frequently observed when analyzing single-domain antibodies by SEC.

[0415] The glycosylation observed in US2023 / 0093123A1-Seq35 is unique among the NbALFA variants analyzed that share a common glycosylation site (protein sequence EVQLQESGGGLVQAGGSLRLSCTASGVTISALNAMAMGWYRQAPGKRRVMVAAVSERGNTMYRESVKGRFTVSRDFT). NKT This is consistent with the fact that VYLQMNSLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS,NXT-type shared glycosylation sites (bolded and underlined (SEQ ID NO: 129)).

[0416] Example 6: Comparison of thermal stability between humanized NbALFA variants, wild-type NbALFA, and another NbALFA variant

[0417] The thermostability of variant 13 (SEQ ID NO: 123), wild-type NbALFA (SEQ ID NO: 1), and SEQ ID NO: 35 of US2023 / 0093123A1 (SEQ ID NO: 129) after production in mammalian cells was investigated. Generally, higher thermostability of VHH was observed in mammalian production systems compared to prokaryotic expression systems (Tables 5-5). Although variant 13 showed significantly higher thermostability compared to wild-type NbALFA, SEQ ID NO: 35 of US2023 / 0093123A1 showed only a small improvement relative to its parental counterpart. In direct comparisons, SEQ ID NO: 35 of US2023 / 0093123A1 showed significantly lower thermostability compared to variant 13.

[0418] Table 9: Determination of the thermal stability of VHH derived from mammalian production systems

[0419] .

[0420] Example 7: Comparison of human lineage homology with antibodies that have obtained International Nonproprietary Names (INNs)

[0421] During early clinical trials, antibodies are typically assigned a formal generic name, the International Nonproprietary Name (INN). Prior to 2017, these names included infixes indicating the origin of the antibody sequence. These infixes included -u- (human), -xi- (chimeric), -zu- (humanized), and -xizu- (chimeric / humanized) (Source: Wilkinson and Hale, 2022 (DOI: 10.1080 / 19420862.2022.2123299)). Although this nomenclature has been discontinued and replaced by a naming convention that focuses less on sequence origin and more on sequence engineering modifications, the original infixes can still be used to determine which antibodies are officially recognized as humanized. Supplementary Table 2 of the Wilkinson and Hale 2022 publication (DOI: 10.1080 / 19420862.2022.2123299) lists the sequences of antibodies that have obtained INNs. These sequences were screened to include only antibodies belonging to the "mAb" type, displaying the "IgG" form, and belonging to the "huIgG1" isotype category. The "protein 1" sequence, i.e., the antibody heavy chain, was extracted, and the constant domain was removed. Finally, among the remaining 339 VH sequences, they were divided into five categories: -u- (human), -xi- (chimeric), -zu- (humanized), -xizu- (chimeric / humanized), and unclassified (without any of the aforementioned infixes). For all these sequences, the highest human V segment identity with the IGHV lineage was determined and plotted as a violin plot. Figure 5 ).

[0422] Descriptive statistics were performed on these phylogenetic homology values ​​to obtain mean and percentile values ​​related to the infix. This allows for comparison of humanized NbALFA antibodies with other humanized molecules known in the art (Table 10).

[0423] Table 10: Descriptive statistics on the identity of infix terms and V-segments in 339 VH sequences.

[0424] .

[0425] Antibodies containing the suffix -xi-mAb are defined as chimeric. In these molecules, the variable region is transplanted onto a human constant domain (e.g., mouse VH, human CH1-CH3). In the analyzed dataset, the lowest human germline identity of this non-humanized antibody was 57.6%, which is only slightly lower than the human V segment identity of wtNbALFA (59.6%). Therefore, even by chimeric standards, wtNbALFA is far from human germline. SEQ ID NO: 35 of US2023 / 0093123 shows 66.7% human V segment identity, very close to the median (67.85%) of chimeric antibodies analyzed in this dataset. Therefore, SEQ ID NO: 35 of US2023 / 0093123 cannot be considered a humanized antibody, but clearly belongs to the category of chimeric antibodies.

[0426] The suffix -xi-zu-mab is used for chimeric / humanized hybrid antibodies. Here, VH is humanized, while the VL sequence remains unchanged compared to the parent animal-derived sequence. Since this paper only analyzes the VH sequence (because VH and VHH have high homology), this class is also considered to represent "humanized antibodies". SEQ ID NO: 35 of wtNbALFA and US2023 / 0093123 failed to reach the minimum of 68.0%, while variant 11 (73.7%) was located within the first quartile of the sequence. Even variants 13 (76.8%) and 15 (80.8%) were found within the second quartile, less than one standard deviation from the mean.

[0427] Humanized antibodies with the suffix "-zu-mab" exhibit V-segment identity starting at 70.4%. Variant 11 (73.7%) and Variant 13 (76.8%) fall in the first quartile of this category, while wtNbALFA (59.6%) and SEQ ID NO: 35 of US2023 / 0093123 (66.7%) are again significantly lower than the observed minimum. Furthermore, Variant 15 (80.8%) even reaches the second quartile while being less than one standard deviation from the mean V-segment identity of humanized antibodies. Therefore, all three antibodies are classified as similar to humanized antibodies that have obtained the INN classification in terms of V-segment identity. Even the candidate with the lowest degree of humanization presented in this application—Variant 8 (72.7%)—exhibits a higher V-segment identity than observed in the "-zu-mab" class of antibodies.

[0428] Human-derived antibodies possess the infix -u-mab. Therefore, the range of V-segment identity can be viewed as a mutational range from which high-frequency somatic mutations can be used to generate diverse human antibodies. The observed minimum V-segment identity was 78.6%, with variant 15 even occupying the first quartile of this infix category, highlighting its properties as a humanized antibody.

[0429] Unclassified sequences are named using an alternative naming scheme that does not include species- or humanization-dependent infixes. While this does not allow for the classification of antibodies as humanized or human antibodies, it is clear that the V region identity of wtNbALFA is significantly lower than the minimum observed for antibodies in this class. Although US2023 / 0093123A1-Seq35 (66.7%) is only slightly above the minimum threshold of 66.3%, all humanized antibodies presented in this application occupy the first quartile of the sequence.

[0430] Example 8: Method

[0431] Method 1: Affinity Test

[0432] To determine the affinity of the humanized NbALFA variant for the epitope tag of SEQ ID NO: 2, ForteBio's Octet RED96 or Octet HTX was used. The measurement principle was based on biolayer interferometry (BLI). The SAX2.0 biosensor was immersed in kinetic buffer (KB) for 10 minutes, provided by the instrument manufacturer. First, a baseline was measured in KB for 60 or 120 seconds, followed by a 240-second loading step with 83 ng / ml of the biotinylated peptide of SEQ ID NO: 2. After loading, free streptavidin on the biosensor was quenched during a 120-second incubation step in 100 μg / ml biocytidine solution. Subsequently, baselines were measured in KB from 60 to 600 seconds, followed by VHH association. VHH was applied at a maximum concentration of 10 nM and serially diluted 1:1 in KB. As a reference, only KB measurements were performed. Binding was performed for 900 seconds, followed by dissociation for 1500 seconds in the KB. The signal from the reference well was subtracted from the signals from all other biosensors, and the signals from these processed data were aligned with the average of the second baseline. For inter-step correction, the data were aligned with the dissociation step, and Savitzky-Golay filtering was applied to all curves. Binding and dissociation were globally fitted using a 1:1 Langmuir binding model.

[0433] Method 2: Thermal stability analysis

[0434] Thermal stability was investigated using a Prometheus Tycho from NanoTemper. 10 μl of VHH sample was loaded into a capillary. After loading the capillary into the instrument, a thermal gradient from 35 °C to 95 °C at a heating rate of 30 °C / min was applied to all samples. During this process, the intrinsic fluorescence of the proteins was measured at 350 nm and 330 nm. This ratio was plotted against temperature, and the first derivative was calculated. The minimum and maximum values ​​correspond to the different TM values ​​observed during denaturation.

[0435] Method 3: SEC Analysis

[0436] Aggregation analysis was performed via SEC using an Agilent Infinity II HPLC system and a Biozen 1.8 µm dSEC-2, 200 Å LC column (300 x 4.6 mm). The flow rate was adjusted to 0.25 mL / min, generating a pressure of approximately 255 bar. The mobile phase consisted of 0.2 M potassium phosphate, 250 mM KCl, pH 6.2, and 5% acetonitrile. Each run was 20 minutes, with a 2–3 minute wash interval between analyses. 10 μL of VHH protein was loaded and detected by absorbance at 280 nm.

[0437] Method 4: SpA binding assay

[0438] 45 μg of purified VHH was diluted in 200 μL of CHO cell culture supernatant. A Cytiva SpA HPSpinTrap column was equilibrated once with 600 μL of PBS, followed by loading 200 μL of sample and incubating according to the manufacturer's instructions. Two washes were then performed with 600 μL of PBS, followed by two elutions with 400 μL of 100 mM glycine (pH 2.7). SDS-PAGE analysis was performed on VHH in the CHO supernatant, the flow-through fraction, the fractions from the two washes, and the fractions from the two elutions.

[0439] Method 5: Protein Expression (Example 5)

[0440] In parallel experiments, each experiment involved transient transfection of 100 mL of Expi 293 cells with a similar eukaryotic expression vector encoding an NbALFA variant under CMV promoter control. For this purpose, Expi 293 cells cultured in Expi 293 medium (Gibco) were set at 3 × 10⁶ cells / mL. 6The concentration was [cells / mL]. For a 100 mL transfection volume, 50 μg of plasmid DNA was diluted in 10 mL of OptiMEM (Gibco) and then added to 50 μL of FectoPro transfection reagent (Polyplus). The mixture was incubated at room temperature for 10 minutes and then added to 90 mL of Expi 293 cells. One hour later, 50 μL of enhancer (FectoPro kit) was added. Cells were cultured at 37°C, 8% CO2, 85% humidity, and 100 rpm. After 5 days, the culture supernatant was harvested by centrifugation.

[0441] Method 6: Protein purification (Example 5)

[0442] The culture supernatant was filtered through a 0.45 μm membrane and loaded by gravity into 2 mL of agarose resin (NanoTag Biotechnologies, #N1530-ALP3) containing a low-affinity ALFA-like peptide. The resin was washed with >10 column volumes of PBS. The bound protein was eluted with pH 3 buffer (100 mM sodium citrate, 100 mM NaCl, pH 3.0) and immediately neutralized by adding 1 / 10 volume of 1M Tris-HCl at pH 8.5. The original eluent from the ALP3 agarose resin was concentrated to 1 mL using a 3 kDa cutoff centrifugal ultrafiltration tube (Amicon Ultra-4, Merck) and further purified using an Äkta Pure system (Cytiva) equipped with a Superdex 75 Increase 10 / 30 column (Cytiva) equilibrated with PBS.

[0443] Method 7: Protein Quantification (Example 5)

[0444] Protein quantification was performed using the specific extinction coefficient of the protein at 280 nm based on ultraviolet absorbance.

[0445] Method 8: Protein Analysis (Example 5)

[0446] Protein fractions were analyzed by SDS-PAGE using pre-prepared gels (Bolt 4% to 12%, Bis-Tris gel, Thermo Scientific, #NWO4127BOX). The gels were stained with Coomassie Brilliant Blue.

[0447] The embodiments exemplified herein may be practiced in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, the terminology and expressions used herein are used as descriptive rather than restrictive terms, and in using these terms and expressions, no equivalents of the shown and described features or portions thereof are intended to be excluded, but rather various modifications are recognized as being within the scope of the invention. Therefore, it should be understood that while these embodiments have been specifically disclosed by way of preferred embodiments and optional features, modifications and variations thereof can be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. Each narrower group of species and subgenus falling within the scope of the general disclosure also forms part of the invention. This includes the general description of the invention, with conditions or negative limitations that remove any subject matter from that species, regardless of whether such removed material is specifically stated herein. Furthermore, when features are described in the form of a Markush group, those skilled in the art will recognize that this disclosure is therefore also described in the form of any individual member or subgroup within that Markush group.

[0448] Equivalents: Many equivalents of the invention described herein would be recognized or determined by a person skilled in the art using no more than conventional experimentation. The following claims are intended to cover these equivalents.

[0449] It should be understood that the present invention is not limited to the specific methods, schemes, materials, reagents, and substances described herein, and therefore these can be varied. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0450] All publications referenced throughout this specification (including all patents, patent applications, scientific publications, manufacturers' instructions, user manuals, etc.) are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the invention was not prior to the disclosure described due to prior art. If any material incorporated by reference contradicts or is inconsistent with this specification, the specification shall supersede any such material.

[0451] Further embodiments will become apparent from the following claims.

Claims

1. A single-domain antibody that specifically binds to an epitope tag containing the sequence SRLEEELRRRLTE (SEQ ID NO: 2), wherein the antibody comprises a CDR1 sequence as shown in SEQ ID NO: 94, a CDR2 sequence as shown in SEQ ID NO: 97, and a CDR3 sequence as shown in SEQ ID NO: 98; The antibody further comprises the following amino acid residues at positions 47, 48, and 50 corresponding to sequence positions SEQ ID NO: 1: Arg at position 47, Arg at position 48, and Met at position 50; Furthermore, the antibody described herein comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 118-125. The antibody contains a sequence that has at least about 70% V region identity with the human ancestral sequence IGHV3-23*4 shown in SEQ ID NO:

110.

2. The antibody of claim 1, wherein the antibody is humanized.

3. The antibody of claim 1 or 2, wherein the antibody has a higher Tm compared to the antibody of SEQ ID NO:

1.

4. The antibody according to any one of the preceding claims, wherein the antibody contains a His residue at position 99 corresponding to SEQ ID NO: 1, a Val residue at position 100 corresponding to SEQ ID NO: 1, and / or a Tyr residue at position 40 corresponding to SEQ ID NO:

1.

5. The antibody according to any one of the preceding claims, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

6. The antibody according to any one of the preceding claims, wherein the antibody comprises FR1, FR2, FR3 and FR4 sequences, which are identical to the sequences shown in SEQ ID NO: 99, 102, 108 and 109, respectively.

7. The antibody according to any one of the preceding claims, wherein the antibody comprises a protein A binding site.

8. The antibody according to any one of the preceding claims, wherein the antibody comprises the CDR2 sequence shown in SEQ ID NO:

95.

9. The antibody according to any one of the preceding claims, wherein the antibody comprises the CDR2 sequence shown in SEQ ID NO:

96.

10. The antibody of claim 8, wherein the antibody contains a Tyr residue at position 61 corresponding to sequence position 61 of SEQ ID NO: 1 and an Asp residue at position 64 corresponding to sequence position 64 of SEQ ID NO:

1.

11. The antibody according to any one of the preceding claims, wherein the antibody comprises a sequence having at least 95% sequence identity with any one of SEQ ID NO: 118-125.

12. The antibody of claim 1, wherein the antibody comprises the sequence shown in any one of SEQ ID NO: 118-125.

13. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

118.

14. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

119.

15. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

120.

16. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

121.

17. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

122.

18. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

123.

19. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

124.

20. The antibody of claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO:

125.

21. A protein comprising the amino acid sequence of the antibody according to any one of claims 1-16.

22. A complex comprising a. A protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or has no amino acid; and b. The antibody according to any one of claims 1-20.

23. A nucleic acid encoding an antibody according to any one of claims 1-20.

24. A vector comprising the nucleic acid of claim 23.

25. A host cell comprising the nucleic acid of claim 23 or the vector of claim 24 or expressing the antibody of any one of claims 1-20.

26. Use of the antibody according to any one of claims 1-20 for binding, detecting, immobilizing, separating or purifying a protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or has no amino acid.

27. A method for binding to or detecting a protein comprising the amino acid sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO: 3), wherein X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and wherein X13 is P or A or S or K or D or E or no amino acid, the method comprising contacting the protein with an antibody according to any one of claims 1-20, and optionally detecting the antibody or a label contained in the antibody.

28. A method for producing an antibody according to any one of claims 1-20, comprising culturing the host cell of claim 25 under conditions that allow the expression of the antibody.

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