Novel human variable domain scaffold and single domain antibody library based on same

Abstract

The present invention relates to a novel human variable domain scaffold and a single domain antibody library based on same. The scaffold according to the present invention is monomeric and stable, and maintains appropriate folding, and is sufficiently flexible to withstand various CDR loop shapes, thereby being able to accommodate various CDR sequences and shapes, and thus is very suitable for use as a scaffold for an antibody library.

Description

A novel human variable domain scaffold and a single-domain antibody library based on it

[0001] The present invention relates to a novel human variable domain scaffold and a single domain antibody library based thereon.

[0002] Monoclonal antibodies are well-established therapeutics and essential tools for biological research. They bind to their targets with high affinity and specificity and are easily engineered for desired antibody functions. However, one of the major challenges in developing therapeutic antibodies in the conventional IgG format is the need to produce large (~150 kDa) heterotetrameric proteins with multiple domains and disulfide bonds. In this regard, research into smaller antibody formats has attracted significant attention in recent years. For example, antigen-binding fragments (Fabs) and single-chain variable fragments (scFvs) can be extensively engineered for higher affinity and improved physicochemical properties, and are being utilized in a wide range of applications, including biotherapeutics.

[0003] Because antibody fragments are structurally simpler and much smaller than immunoglobulins, they can be functionally expressed in prokaryotic host cells and displayed on bacteriophages (phage display). Antibody phage display is a technology that allows the in vitro isolation of antibody fragments specific to a target antigen. Many antibody-based therapeutics have been discovered or optimized through phage display, demonstrating its utility as a technology platform for biopharmaceutical development.

[0004] The quality of an antibody library is crucial for successfully identifying binders with target specificity and desired properties. Factors determining the quality of an antibody library include its functional diversity and the characteristics of individual clones within the library (e.g., folding stability, expression level, solubility, and proper assembly into phage particles). Another critical issue in the development of antibody-based therapeutics is monomer content. The aggregation propensity of antibody proteins is primarily determined by the variable domains (VH and VL), and antibody fragments such as scFv are known to be prone to forming dimers and high-molecular-weight species.

[0005] For practical applications, it is necessary to develop variable domains that have a low aggregation tendency and are more stable under various conditions. Antibodies (HCAbs) consisting of only the first constant domain (CH1) of conventional IgG and a heavy chain without a light chain were originally discovered in the serum of Arabian camels (Camelus dromedarius) and later discovered in other camel species. The heavy chain variable domain of camelid HCAbs is V H H (variable heavy domain of HCAb) or nanobodies are highly stable, function autonomously without a light chain counterpart, and represent the smallest antibody-based target-binding proteins. Other notable features of these single variable domain antibodies (sdAbs) include high expression levels in a variety of prokaryotic and eukaryotic host cells, high solubility, and modularity due to their small molecular size and excellent stability, which allows them to be incorporated into molecular structures that require bispecific antibodies, chimeric antigen receptors, and other target-binding moieties.

[0006] Camelid V HH has high sequence homology to human VH3 and can be easily humanized to minimize immunogenicity in therapeutic applications. However, camelid animals, such as camels, llamas, and alpacas, are less accessible as immunogenic hosts than common laboratory animals such as mice and rabbits, and the production of monoclonal antibodies through animal immunization followed by library construction and phage display selection is time-consuming and expensive. A synthetic VH sdAb library based on a human variable domain scaffold could be an attractive alternative for camelid immunization.

[0007] Camelid V H A key difference between human VH and human VH3 lies in the framework 2 (FR2) region, which forms part of the VH-VL interface in conventional antibodies. The nonpolar amino acids in FR2 form hydrophobic interactions with the VL and are primarily responsible for the low solubility of the intact, non-camelid VH domain produced as sdAbs. Consequently, many human VH-based sdAbs and sdAb libraries have adopted a configuration that "camelizes" the FR2 sequence to increase solubility and reduce aggregation.

[0008] Accordingly, the researchers of the present invention developed a synthetic human VH domain antibody library by introducing mutations in key residues located in the FR2 region of the human VH3-23 domain to design a stable and aggregation-resistant human VH domain, and completed the present invention by evaluating the performance of the library by panning it against a test antigen and analyzing the affinity and physicochemical properties of the isolated target-binding clones.

[0009] One object of the present invention is to provide a novel human variable domain scaffold and a single domain antibody library based thereon.

[0010] Another object of the present invention is to provide a method for producing a single domain antibody library based on a novel human variable domain scaffold.

[0011] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0012] In addition, the terminology used in the present invention is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. It should be understood that the terms “comprise” or “have” in the present invention are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

[0013] Additionally, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0014] Furthermore, to avoid confusion due to overlapping content, the description of overlapping content has been omitted. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0015]

[0016] The present invention provides a single-domain antibody having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a library thereof, wherein FR2 is any one selected from the group consisting of SEQ ID NOs: 33 to 36. The present invention also provides a single-domain antibody and a library thereof.

[0017] Antibodies comprise a heavy chain variable region (VH) separated by hypervariable regions, also known as "complementarity determining regions" ("CDRs"), and more conserved regions, known as "framework regions" ("FRs") or scaffolds. Each VH typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The dimensions of the framework regions and CDRs can be precisely identified using methodologies known in the art, for example, the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition.

[0018] A library may contain one or more sub-libraries. For example, since the lengths of CDRs 1, 2, and 3 within a library may vary, multiple sub-libraries within the library may be created to account for this.

[0019] More specifically, the present invention provides a single-domain antibody having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a library thereof,

[0020] Here, CDR1 is of the formula X1-X2-X3-M-X4 (SEQ ID NO: 53), where X1 is D, G, N, or S, X2 is D or Y, X3 is A or D, and X4 is D or H.

[0021] FR2 is any one selected from the group consisting of sequence numbers 33 to 36,

[0022] CDR2 is of the formula X5-I-X6-X7-X8-X9-X10-X11-X12-YYADSVKG (SEQ ID NO: 54), where X5 is A, G, L, S, V, or W, X6 is S or Y, X7 is H, P, S, or Y, X8 is D, G, N, or S, X9 is D, G, N, or S, X10 is G or S, X11 is N or S, and X12 is I, K, or T.

[0023] CDR3 is of the formula ARXXXXFDY (SEQ ID NO: 55); of the formula ARXXXXXXXXXFDY (SEQ ID NO: 56); or of the formula ARXXXXXXXXXXXXXXMDV (SEQ ID NO: 57), where X is any amino acid.

[0024] Also provided herein are (synthetic) single-domain antibody libraries comprising any of the single-domain antibodies mentioned above.

[0025] A library is provided comprising a single-domain antibody having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR2 is any one selected from the group consisting of SEQ ID NOs: 33 to 36. The framework or scaffold structure according to the present invention is designed based on the VH3-23 region mutation.

[0026] The term "antibody library" of the present invention refers to a collection of various antibody genes having different sequences. In order to isolate specific antibodies against any antigen from an antibody library, a very high diversity is required, and generally, 10 9 ~ 10 11 A library consisting of different antibody clones is constructed and used. The antibody genes constituting this library are cloned into a phagemid vector and transformed into Escherichia coli.

[0027] The size of a library is one of the important factors that determines the quality of antibodies selected from it. The antigen-binding sites of an antibody library theoretically have a random diversity that is not specific to any specific antigen, and antibodies that selectively bind to a specific antigen appear from this random diversity purely by chance. Therefore, as the library size increases, i.e., as the number of different antibodies in the library increases, the probability of discovering an antibody with high selectivity and affinity by chance increases. Generally, at least 10 8 It is recognized that larger sizes are required, and many antibody libraries are 10 9 ~ 10 11 It has the size of a certain degree.

[0028] The functional diversity of a library refers to the proportion of clones within the library that can actually express antibodies. Even if the library size is large, if functional diversity is low, the actual library size will be small. This low functional diversity is largely due to errors in DNA synthesis and amplification during the library construction process. Antibody libraries are constructed through multiple steps of the polymerase chain reaction (PCR), and due to the nature of the enzymes and reactions, low-frequency errors inevitably occur. Accumulating these errors reduces the functional diversity of the final library. Furthermore, synthetic libraries, in particular, are prone to introducing errors due to inefficiencies in base synthesis reactions. Therefore, functional diversity issues are particularly prominent in synthetic libraries, and most synthetic libraries must incorporate design considerations to avoid these issues.

[0029] The quality of the individual clones that make up the library, including their expression, stability, and immunogenicity, is also a factor that determines the performance of the antibody library. To select clones with superior antibody engineering, these factors must be considered from the design stage of the synthetic antibody library. In particular, when introducing artificial diversity into existing antibody genes, the resulting diversity must be designed to be compatible with the antibody framework.

[0030] In some embodiments, the single-domain antibody library comprises at least 10 8 , 10 9 , 10 10 or 10 11 The synthetic single-domain antibody libraries disclosed herein contain unique antibody sequences. That is, the synthetic single-domain antibody libraries disclosed herein can provide a variety of antibodies by introducing high CDR diversity into the selected scaffold sequence. In some embodiments, the positions of each amino acid sequence of the synthetic CDR1 and synthetic CDR2 are rationally designed to mimic the natural diversity of CDRs found in the human repertoire. The length of the CDR3 sequence can affect the binding potential of an antibody to different epitope shapes, particularly to epitopes in protein cavities. Therefore, the disclosed single-domain monoclonal antibody libraries have CDR3 sequences of varying lengths.

[0031] In a single-domain antibody having the formula of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1 may be EVQLLESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 50), FR3 may be RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 51), and FR4 may be WGQGTLVTVSS (SEQ ID NO: 52), but is not limited thereto. Such FR1, FR3, and / or FR4 may also be applied with a mutant sequence suitable for use as a scaffold for an antibody library and may be used in the present invention.

[0032] In some embodiments of single-domain antibodies, CDR1 can be five amino acids. Specifically, X1-X2-X3-M-X4, where X1 is D, G, N, or S, X2 is D or Y, X3 is A or D, and X4 is D or H.

[0033] In some embodiments of the single-domain antibody, the CDR2 can be 17 amino acids. Specifically, the CDR2 can be X5-I-X6-X7-X8-X9-X10-X11-X12-YYADSVKG, wherein X5 is A, G, L, S, V, or W, X6 is S or Y, X7 is H, P, S, or Y, X8 is D, G, N, or S, X9 is D, G, N, or S, X10 is G or S, X11 is N or S, and X12 is I, K, or T.

[0034] In some embodiments of the single-domain antibody, CDR3 may be, for example, 9, 14, or 20 amino acids. Specifically, it is any one selected from the group consisting of ARXXXXFDY; ARXXXXXXXXXFDY; ARXXXXXXXXXXXXXMDV, wherein X is any amino acid. Additionally, any of the above amino acids may form a disulfide bond, if desired. Preferably, a disulfide bond may also be formed in ARXXXXXXXXXXXXXXMDV.

[0035] The present invention provides a nucleic acid molecule encoding the single-domain monoclonal antibody. This nucleic acid molecule may be operably linked to a promoter. Furthermore, a vector comprising the nucleic acid molecule is provided. Furthermore, a host cell comprising the nucleic acid molecule or vector is provided. The host cell may be a mammalian cell or a bacterial cell. Preferably, it is Escherichia coli. More specifically, an E. coli strain having a SupE44 mutation and an F plasmid, including TG1, ER2537, and XL1-Blue, may be used. Preferably, it is TG1 electrocompetent E. coli.

[0036] The term "framework" of the present invention refers to a portion of a variable domain sequence other than a complementarity-determining region, and refers to a portion that has lower sequence variability and diversity than the complementarity-determining region and generally does not participate in an antigen-antibody reaction.

[0037] The framework areas are FR1, FR2, FR3, and FR4.

[0038] The present invention is characterized by using a modified FR2, which may be any one selected from the group consisting of SEQ ID NOs: 33 to 36. In one embodiment of the present invention, camelid V is used to generate an optimized scaffold for constructing a human sdAb library. H We engineered the FR2 sequence of human VH3-23 (DP47) with high homology to H. Specifically, four FR2 residues (Val37, Gly44, Leu45, Trp47), which form part of the VH-VL interface through hydrophobic interactions in normal antibodies but contribute to the poor biophysical properties of many unengineered non-camelid VH sdAbs, were randomized to present a VH library with diverse FR2s on the phage surface. Inclusion of these modified backbone structures can achieve thermostability and high water solubility.

[0039] M13 bacteriophage retained full infectivity even after heat treatment at 80°C for 1 h, allowing the FR2-diversified VH phage display library to be briefly heated up to 80°C before binding to immobilized protein A, which has been shown to have affinity for correctly folded human VH3. FR2 variants that were resistant to heat denaturation or capable of rapid renaturation were preferentially enriched by protein A binding, and after eight rounds of panning, the output sequences showed a pattern of convergence.

[0040] Notably, the consensus FR2 sequence from the 8th round was similar to the corresponding region of human Vκ. The VL domain has been reported to exhibit better biophysical properties than the VH domain as sdAbs, suggesting that the observed convergence toward VL-like FR2 sequences facilitated the enrichment of sdAb sequences that were thermally stable during panning, and that the reported stability of VL sdAbs could be partly explained by the FR2 sequence.

[0041] CDR diversity was introduced into the scaffold according to the present invention, and as a result, the sdAb library successfully generated target-specific binders with desirable properties. These results indicate that the scaffold according to the present invention is monomeric and stable, yet flexible enough to accommodate a variety of CDR loop conformations.

[0042] Finally, it was confirmed that the scaffold protein composed of the amino acid sequence of SEQ ID NOs: 33 to 36 according to the present invention exhibits structural and functional compatibility with various CDR sequences and is an effective scaffold for constructing a high-function sdAbs library.

[0043] Optionally, one or more framework regions may have one or more mutations that provide improvements in protein expression, protein folding, protein purification, binding affinity, downstream target signal transduction, and / or signal transduction inhibition. In one embodiment of the present invention, FR1, FR3, or FR4 may be SEQ ID NO: 50, 51, or 52, respectively.

[0044] By utilizing these characteristics, the present invention can be utilized as a library scaffold for selecting ligands targeting antigens, i.e. VH domain antibodies.

[0045] Specifically, by maintaining the selected framework and inserting random amino acids into CDR1, CDR2, and CDR3, a library can be constructed, and antibodies with binding ability to the antigen can be selected using a conventional method called panning.

[0046] The construction of the above CDR1, CDR2, and CDR3 by inserting random amino acids can be constructed as mentioned above.

[0047] In one embodiment of the present invention, the antibody comprises a recombinant single chain Fv fragment (scFv), including without limitation bivalent or bispecific molecules, diabodies, triabodies and tetrabodies.

[0048] When selecting antibodies for a specific antigen using a manufactured antibody library, the panning method is usually used for selection. This process involves coating a tube with a ligand antigen, flowing the library phage to induce binding, and washing away unbound library phage to obtain bound phage. After performing the panning process in this way, the degree of binding can be indirectly confirmed using ELISA. By coating an ELISA plate with a ligand antigen, inducing binding with the selected monophage, and then detecting with an antibody that recognizes the bacteriophage, the binding strength to the antigen can be indirectly measured.

[0049] Specifically, the above panning refers to the process of selectively amplifying only clones that bind to a specific molecule from a library of proteins, such as antibodies, displayed on the surface of phage. The phage library is added to the target molecule immobilized on the surface to induce binding, unbound phage clones are washed away, and only the bound phage clones are eluted and re-infected with an E. coli host, and the target-binding phage clones are amplified using a helper phage. In most cases, this process is repeated 3 to 4 times or more to maximize the proportion of binding clones.

[0050] One aspect of the present invention provides a method for producing an antibody phage surface-display library, comprising the step of producing a nucleic acid sequence of a single domain antibody (sdAb) in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are linked in a row, comprising a nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36, which are FR2 variant polypeptides.

[0051] The heavy chain CDR1, CDR2 and / or CDR3 populations of the antibody library may be derived from naturally occurring human antibodies.

[0052] Preferably, in the above FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4,

[0053] CDR1 is a formula X1-X2-X3-M-X4, where X1 is D, G, N, or S, X2 is D or Y, X3 is A or D, and X4 is D or H.

[0054] FR2 is any one selected from the group consisting of sequence numbers 33 to 36,

[0055] CDR2 is of the formula X5-I-X6-X7-X8-X9-X10-X11-X12-YYADSVKG, where X5 is A, G, L, S, V, or W, X6 is S or Y, X7 is H, P, S, or Y, X8 is D, G, N, or S, X9 is D, G, N, or S, X10 is G or S, X11 is N or S, and X12 is I, K, or T.

[0056] CDR3 is of the formula ARXXXXFDY; formula ARXXXXXXXXXFDY; or formula ARXXXXXXXXXXXXXXMDV, where X is any amino acid.

[0057] The above CDR1 and CDR2 diversity may be provided by designing and synthesizing using degenerate oligonucleotides to mimic the amino acid usage of human germline CDR-H1 and -H2 sequences.

[0058] More specifically, but not limited to, with respect to CDR3, any of the above Xs may be included at each desired position in the amino acid ratios below.

[0059] [Table 1] Proportion (%) of each amino acid in the randomized sequence of ARXXXXFDY (XXXX corresponds to residue positions 95-98 based on Kabat numbering)

[0060]

[0061] [Table 2] The ratio (%) of each amino acid in the randomized sequence of ARXXXXXXXXXXFDY (XXXXXXXXX corresponds to residue positions 95-100, 100A, 100B, and 100C based on Kabat numbering)

[0062]

[0063] [Table 3] Proportion (%) of each amino acid in the randomized sequence of ARXXXXXXXXXXXXXXMDV (XXXXXXXXXXXXXXX corresponds to residue positions 95-100, 100A to 100I based on Kabat numbering) (excluding disulfide bonds)

[0064]

[0065] [Table 4] Proportion (%) of each amino acid in the randomized sequence of ARXXXXXXXXXXXXXXMDV (XXXXXXXXXXXXXXX corresponds to residue positions 95-100, 100A to 100I based on Kabat numbering) (if including disulfide bond)

[0066]

[0067] Each number mentioned in Tables 1 to 4 above represents a percentage of the probability that the corresponding amino acid may exist. Accordingly, the description of each amino acid and its probability of existence mentioned in Tables 1 to 4 above may specifically refer to the existence of each X in the present specification. The amino acid ratios in Tables 1 to 4 above are approximations that mimic the amino acid ratios at each position in the sequences of known human antibody CDR-H3s having the corresponding lengths.

[0068] That is, it may be designed as a codon frequency system for the synthesis of CDR3 diversity through trinucleotide phosphoramidite chemistry based on the amino acid usage of human CDR-H3 as specifically described in FIG. 2 and Tables 1 to 4 above.

[0069] Additionally, the above FR2 may be sequence number 33 or 36.

[0070] Additionally, the FR1 may be sequence number 50, FR3 may be sequence number 51, and FR4 may be sequence number 52.

[0071] Specifically, a) a step of preparing a nucleic acid sequence of a single domain antibody (sdAb) in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are linked in a row from the N-terminus to the C-terminus by overlapping and polymerizing a nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36, which is an FR2 variant polypeptide, and nucleic acid fragments of FR1, CDR2, FR3, CDR3 and FR4;

[0072] b) a step of cleaving the single domain antibody (sdAb) and phagemid vector obtained in step a) with a restriction enzyme and assembling them through a ligation reaction; and

[0073] c) A method for producing an antibody phage surface-displayed library, comprising: a step of transforming the phagemid vector assembled in step b) into a host strain and then culturing the same;

[0074] The term "phage display" of the present invention refers to a technique in which the genes of M13 bacteriophage are manipulated to fuse the gene of a foreign protein to one of the genes of its surface proteins, and the foreign protein is fused to the surface protein of the produced phage and displayed on the surface of the phage. When displaying proteins on the surface, the foreign gene is often fused to the 5' side of the gIII gene.

[0075] The term "phagemid" vector of the present invention refers to a plasmid DNA having a phage origin of replication. Typically, it has an antibiotic resistance gene as a selection marker. In the case of a phage midi vector used for phage surface display, the gIII gene of the M13 phage or a part thereof is included, and the library gene is ligated to the 5' end of the gIII gene and expressed as a fusion protein in E. coli. The pComb3X vector used in the present invention is an example of a phagemid vector.

[0076] Phage display is a technique that genetically manipulates bacteriophages, which are bacterial hosts, to link the genotype (genes) and phenotype (proteins) within a single phage particle. In this case, the genotype gene is inserted as part of the phage genome, and the phenotype protein is displayed on the surface of the phage particle containing the protein's gene. This physical linkage of genotype and phenotype is a crucial concept in protein engineering, enabling the genes of protein clones selected for their phenotypic characteristics to be easily obtained, cloned, amplified, analyzed, and manipulated.

[0077] Antibodies are a particularly useful application of phage surface display technology. By presenting a highly diverse antibody library on the surface of phage and then binding it to surface-adsorbed antigens, the genes of antibody clones that selectively bind to the antigen can be obtained. This is a highly effective method for obtaining antibodies without using experimental animals, and because it allows the generation of human antibodies against any antigen, it has significant potential for the development of therapeutic antibody drugs with low immune responses in the human body. To obtain high-quality antibodies with high binding affinity, library quality is crucial, particularly library size and functional diversity, as well as clone quality.

[0078] A method for manufacturing a library according to one embodiment of the present invention is described in detail as follows.

[0079] <Step a): A step of producing a single-chain fragment domain antibody (scFvsdAb) in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are linked in a row from the N-terminus to the C-terminus by overlapping and polymerizing a nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36, which is an FR2 variant polypeptide, and a nucleic acid fragment of FR1, CDR2, FR3, CDR3 and FR4>

[0080] This step is a step of producing a nucleic acid sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are linked in a row to produce a single chain fragment domain antibody of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and a nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36, which are FR2 variant polypeptides.

[0081] The term “CDR (complementarity determining region)” of the present invention refers to a region in which three CDRs exist in each of the light chain and heavy chain of the variable domain, and is a region in which the variability of the amino acid sequence is particularly high among the variable domains, and due to this high variability, specific antibodies can be found for various antigens. Among these, the CDR according to the present invention can use the heavy chain CDR, and the three complementarity determining regions of the heavy chain are sequentially referred to as CDR-H1, CDR-H2, and CDR-H3 from the N-terminus to the C-terminus. Several methods for defining the amino acid numbering and the positions of CDRs on the antibody variable region sequence are known, and the Kabat definition is followed in the present invention.

[0082] The CDR according to the present invention has polymorphism, and the corresponding region is a human-derived random heavy chain complementarity determining region.

[0083] According to the present invention, a nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36 and FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 nucleic acids are overlapped and polymerized to provide a nucleic acid sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are linked in a row. Specifically, the sequences therebetween can be linked through an overlap extension polymerase chain reaction to provide a single sequence. Since OE-PCR is used to assemble several small double-stranded DNA fragments into a larger DNA sequence, the desired nucleic acid sequence can be provided by designing OE-PCR by a method commonly known to connect the nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36 and the nucleic acids FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 individually or in series of two or more.

[0084] The fragment according to the present invention is produced through a polymerization reaction using a specific primer set using VH3-23 scFv as a template. Preferred examples of the fragment produced in this step and the primer set for its production are as follows.

[0085] FR1-CDR1 fragment - primer set having nucleic acid sequences of SEQ ID NOs: 3 and 37

[0086] FR2 fragment - primer set having nucleic acid sequences of SEQ ID NOs: 38 and 39

[0087] CDR2-FR3 fragment (B) - Primer set having nucleic acid sequences of SEQ ID NOs: 40 and 41

[0088] FR4-CDR3 9, 14 length fragment (C) - Primer set having nucleic acid sequences of SEQ ID NOs: 45 and 4

[0089] FR4-CDR3 20, SS length fragment (D) - Primer set having nucleic acid sequences of SEQ ID NOs: 46 and 4

[0090] At this time, the SS length is 20 aa CDR with a disulfide bond within the loop.

[0091] When designing the CDR-H3 sequence of the above method, CDR-H3 sequences having different lengths can be separated by each length (specifically, a CDRH3 sequence composed of 9 to 16 amino acids) and applied to OE-PCR, respectively.

[0092] OE-PCR can provide a nucleic acid sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are linked in a row through overlapping fragment assembly through a combination of the above-mentioned nucleic acid sequences.

[0093] Finally, nucleic acid sequences for four types of single domain antibodies (sdAb) (CDR3 9 length sub-library, CDR3 14 length sub-library, CDR3 20 length sub-library, and CDR3 SS length sub-library) can be produced.

[0094] <Step b): A step of cutting the single domain antibody (sdAb) and phagemid vector obtained in the above step a) with a restriction enzyme and assembling them through a ligation reaction>

[0095] This step involves cleaving the nucleic acid sequence for the single-domain antibody (sdAb) and the pComb3X phagemid vector with restriction enzymes and ligating them. Any restriction enzyme present in the phagemid can be cleaved and ligated. Specifically, a combination of recombination and restriction enzyme cloning methods can be used for library construction.

[0096] In one embodiment of the present invention, the restriction enzyme is SfiI, and the phagemid vector is a pComb3X phagemid vector.

[0097] <Step c): A step of transforming the phagemid vector assembled in step b) into a host strain and then culturing it>

[0098]

[0099] This step is a process of transforming the phagemid vector assembled in step b) above into a host strain and then culturing it in a medium containing antibiotics.

[0100] At this time, the host strain may be an E. coli strain, for example, TG1, ER2537, XL1-Blue. SupE44 mutant E. coli strains may be used. Preferably, TG1 electrocompetent E. coli is used.

[0101] The phagemid vector according to the present invention has an antibiotic resistance gene as a selection marker, so that when a host strain is cultured in the antibiotic, only a strain that has been well transformed survives, and thus a host strain having a single domain antibody (sdAb) according to the present invention can be easily selected.

[0102] In addition, another aspect of the present invention provides an antibody phage surface display library manufactured by the above-described antibody phage surface display library manufacturing method.

[0103] In another aspect of the present invention, a screening method for identifying synthetic single-domain antibodies that bind to a target of interest is provided herein.

[0104] Also provided herein is a screening method for identifying synthetic single-domain monoclonal antibodies that bind to a target of interest, comprising using an antibody phage surface-displayed library.

[0105] In some embodiments, the screening method comprises the use of a synthetic single-domain antibody library disclosed herein. In some embodiments, the target of interest is a microbial antigen, such as a viral antigen, bacterial antigen, fungal antigen, or parasitic antigen. The screening method is a phage display method.

[0106] Single-domain antibodies are used in screening methods, such as those for identifying single-domain antibodies that specifically bind to a target of interest. Any known screening method for identifying binders with specific affinity for a target of interest can be used in conjunction with the synthetic single-domain antibody libraries disclosed herein. Such methods include, but are not limited to, phage display technology, bacterial cell display, yeast cell display, mammalian cell display, or ribosome display. In a specific embodiment, the screening method is phage display.

[0107] In some embodiments, the target of interest is a therapeutic target, and a synthetic single-domain monoclonal antibody library is used to identify synthetic single-domain antibodies that specifically bind to the therapeutic target. In certain embodiments, the target of interest is an antigen or comprises at least an antigenic determinant. For example, the target may be a saccharide or polysaccharide, a protein or glycoprotein, or a lipid. In one specific embodiment, the target of interest is of plant, yeast, fungal, insect, mammalian, or other eukaryotic cell origin. In another specific embodiment, the target of interest is of bacterial, protozoan, or viral origin. In certain embodiments, the target is an antigen, such as a viral, bacterial, fungal, or protozoan antigen.

[0108] The present disclosure provides single-domain antibodies selected from the single-domain antibody library disclosed above. Given the high diversity of synthetic single-domain antibodies in the disclosed library, one skilled in the art can obtain single-domain antibodies with high affinity and specificity for a target (antigen) of interest through conventional screening methods such as phage display / panning. The selected single-domain antibodies can optionally be further modified to produce appropriate antigen-binding properties. In particular, CDR residues can be modified to increase antibody affinity for the target of interest, improve folding or production, or reduce viscosity, for example, using techniques known in the art (e.g., mutagenesis, affinity maturation).

[0109] The present invention also provides a nucleic acid molecule encoding a single-domain antibody selected from the single-domain antibody library disclosed herein. In some embodiments, the nucleic acid molecule is operably linked to a promoter, such as a heterologous promoter. The nucleic acid may be present in whole cells, a cell lysate, or may be in a partially purified or substantially pure form. In some embodiments, the nucleic acid molecule is DNA or RNA and may or may not contain intronic sequences.

[0110] Nucleic acid molecules encoding single-domain antibodies can be further engineered using standard recombinant DNA techniques to include, for example, a signal sequence for appropriate secretion in an expression system, a purification tag, and / or a cleavable tag for further purification steps. In such manipulations, the nucleic acid molecule (such as a DNA molecule) is operably linked to another DNA molecule or to a fragment encoding another protein, such as a purification / secretion tag or a flexible linker.

[0111] Additionally provided are isolated host cells suitable for the production of single-domain antibodies. In some embodiments, the host cell may be a mammalian cell or Escherichia coli.

[0112] Also provided is a method for producing a single-domain monoclonal antibody, comprising the steps of culturing a host cell under suitable conditions for producing a single-domain monoclonal antibody, and isolating the single-domain monoclonal antibody.

[0113] The scaffold according to the present invention is monomeric, stable, maintains proper folding, and is flexible enough to withstand various CDR loop conformations, thereby being able to accommodate various CDR sequences and conformations, making it highly suitable for use as a scaffold for antibody libraries.

[0114] Figure 1 shows the size exclusion chromatography results for YSPW, YESPW, YTPW, and YETPW variants.

[0115] Figure 2 shows the results of confirming CDR3 diversity for application to an antibody library.

[0116] Figure 3 shows the size exclusion chromatography results for VH scaffolds (QS, QT, KS, and KT).

[0117] Figure 4 shows the size exclusion chromatography results for VH scaffolds (SQT, FQT, SKS, and FKS).

[0118] Figure 5 shows the results of confirming the monomer content of the separated sdAb.

[0119] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0120] Experimental method

[0121] 1. Panning 1~8 times

[0122] Panning was performed against a VH3-23-based phage surface-displayed library in which four FR2 residues (Val37, Gly44, Leu45, and Trp47) were randomized. One to three rounds of panning were performed using a standard panning method. Specifically, protein A antigen (2 μg / mL in 1 mL of PBS) was immobilized on an immunotube (Nunc 470319, Thermo Scientific). After immobilization, the antigen-coated tube was blocked with a 3% skim milk solution diluted in PBS containing 0.05% Tween 20 (mPBS) for 1 h at room temperature. The FR2 random library phage stock was blocked with mPBS-0.05% Tween 20 (mPBST, total volume 1 mL) for 1 h and then transferred to the protein A-coated immunotube. After incubation at 37°C for 2 h, unbound phage was removed, and the tube was washed three times with PBST. Bound phage was eluted with 1 ml of 100 mM triethylamine solution and neutralized with 0.5 ml of 1 M Tris-HCl (pH 7.0).

[0123] Afterwards, 8.5 ml of mid-log phase TG1E. coli was added to the eluted phage, and the mixture was gently stirred (120 rpm) at 37°C for 1 h, centrifuged, and plated on LB-ampicillin agarose plates supplemented with 2% (w / v) glucose, followed by titration. The next day, the amplified cells were collected, and phage was obtained for the next round.

[0124] Phage harvesting was performed by inoculating 50 μl of the collected E. coli into 20 ml of SB-ampicillin. VSCM13 helper phage (10) was added to the mid-log phase culture for infection by gentle shaking (120 rpm) at 37°C for 1 h. 11pfu) and kanamycin (70 μg / ml) were added, and the bacteria were cultured overnight at 30°C. The next day, the phages secreted from the culture supernatant were precipitated with 5X PEG solution, resuspended in PBS, and used in the next panning round.

[0125] In rounds 4 to 6, the precipitated phage pool was heated at 70°C for 2 minutes before binding to the antigen and cooled on ice for 5 minutes.

[0126] In the 7th and 8th rounds, the sample was heated at 80°C for 10 minutes and cooled on ice for 10 minutes. After heat treatment, the panning process was performed in the same manner as described above.

[0127] 2. sdAb purification

[0128] For sdAb purification, E. coli cells were cultured in 50 ml of SB-ampicillin medium at OD 600 The cells were cultured at 37°C until the confluency reached 0.7. Protein expression was induced with 1 mM IPTG, and the cultures were incubated overnight at 30°C with shaking at 220 rpm. The cell pellet obtained by centrifugation the next day was resuspended in 4 ml of cold 1X TES buffer and incubated on ice for 30 min. Cold 0.2X TES (6 ml) and PMSF (final concentration 1 mM) were added, and the mixture was incubated on ice for an additional 30 min.

[0129] After centrifugation at 14,000 × g for 15 min, 5 mM MgCl2 was added to the supernatant, followed by 100 μl of Ni-NTA agarose bead suspension (Cat. #70666-4, Merck Millipore, Darmstadt, Germany). The suspension was incubated at room temperature with gentle agitation for 1 h and then transferred to an empty gravity column (Cat. #7311550, Bio-Rad, Hercules, CA, USA) to allow the liquid to flow through. The beads were washed twice with 5 ml of wash buffer (PBS containing 5 mM imidazole, pH 7.4), and 400 μl of elution buffer (PBS containing 250 mM imidazole, pH 7.4) was added to elute the purified sdAb.

[0130] 3. Size exclusion chromatography (SEC)

[0131] Size exclusion chromatography was performed on a Superdex AKTA PURE system (Cytiva, Marlborough, MA, USA). TM The assay was performed using a 75 increase 10 / 300 GL column (Cat. #17-5174-01, Cytiva, Marlborough, MA, USA). 400 μL of purified sdAb was injected at a concentration of 3–5 mg / mL, and the run was performed using 1X PBS buffer at a flow rate of 0.75 mL / min.

[0132] 4. Protein Thermal Shift (PTS) Analysis

[0133] The Tm (°C) of purified VH sdAb was measured by Protein Thermal Shift™ (PTS) assay using a StepOnePlus™ Real-Time PCR System (Cat. # 4379216, Applied Biosystems). Measurements were performed by mixing sdAb with the assay reagents of the Protein Thermal Shift™ Dye Kit (Cat. # 4462263, Applied Biosystems) in 1X PBS (pH 7.4) according to the manufacturer's instructions. The real-time PCR instrument was set up as described for the 7500 Fast Real-Time PCR system in the Protein Thermal Shift™ Starter Kit User Instructions from Applied Biosystems. Tm values ​​were calculated using Protein Thermal Shift™ Software v1.4 (Cat. # 4466038, Applied Biosystems).

[0134] 5. Surface plasmon resonance (SPR) analysis

[0135] SPR analysis was performed on a BIAcore 3000 (GE Healthcare, Piscataway, NJ, USA) instrument. Antigen (2–5 μg / mL in 10 mM acetate buffer [pH 4, 4.5, or 5]) was immobilized to 1,000 response units (RUs) on the flow cell of a CM5 sensor chip (GE Healthcare) using the amine coupling method according to the manufacturer's protocol. Purified sdAb was diluted in filtered and degassed PBS and run on the antigen-immobilized sensor chip. Binding kinetics were analyzed using BIAevaluation software using a 1:1 Langmuir binding model.

[0136] Example 1. Preparation of human VH3 FR2 variant scaffolds

[0137] 1. Isolation of stable human VH3 FR2 variants

[0138] To develop a human scaffold for a VH sdAb library, we first selected the VH domain of a scFv antibody (VH3-23-scFv) with high expression levels and excellent physicochemical properties isolated through preliminary experiments. To engineer stable variants of this VH domain (based on the human VH3-23 germline gene) into an sdAb format, the “signature” residues of FR2 (residues 37, 44, 45, and 47 according to Kabat numbering) were randomized to NNK degenerate codons (N = A, T, G, or C, and K = G or T).

[0139] The FR2 randomized VH library was assembled through overlap extension PCR using NNK randomized oligonucleotide primers as shown in Table 5 below, and the PCR conditions performed at this time were as follows:

[0140] Initial melting at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 minutes, for a total of 25 cycles; final extension at 72°C for 7 minutes.

[0141] [Table 5] NNK randomized oligonucleotide primers

[0142]

[0143] The randomized VH repertoire was ligated into the pComb3x phagemid vector and transformed into TG1 Escherichia coli cells. Finally, 1.1 X 10 7 We obtained a FR2 random library of size 10, which is the theoretical maximum diversity (NNK)4(~1 X 10 6 ) was enough to cover.

[0144] From the FR2 random library obtained above, stable human VH single domains were enriched through eight rounds of panning against protein A superantigens known to bind to antibody VH domains including human VH3 and the Fc portion of immunoglobulins of various species.

[0145] After three rounds of panning, the amplified phage pool was heated for 10 min (70°C for rounds 4–6, 80°C for rounds 7–8) prior to the protein A binding step. Inclusion of the heating step was expected to preferentially enrich for VH variants that were resistant to heat denaturation or that renatured rapidly after denaturation. After the final panning round, clones that showed binding to protein A (i.e., clones expressing correctly folded VH sdAbs) were identified by ELISA, at which point E. coli plasma extracts containing individual VH variants were heat treated prior to binding to surface-adsorbed protein A.

[0146] As shown in Table 6, the sequences of the ELISA-positive clones from the final panning round showed considerable sequence convergence. In particular, the consensus FR2 sequence of the ELISA-positive clones showed significant similarity to the FR2 sequence of human Vκ. Based on Kabat numbering, residues 37 and 44 were preferentially occupied by Tyr / Phe and Pro, respectively, and residue 45 was mostly occupied by Pro, whereas the consensus amino acids at the corresponding positions in human Vκ were Tyr, Ala / Ser, and Pro for Vκ residues 36, 43, and 44, respectively.

[0147]

[0148] Two of the isolated FR2 variants (8H1 (hereafter, YTPW) and 8G5 (hereafter, YPPG)) expressed well in Escherichia coli, confirming their potential as human VH sdAbs. YTPW, with its superior expression compared to YPPG, was selected as a prime candidate for further optimization and library construction. This clone also harbored a spontaneous F27S mutation in FR1 (located in CDR1 according to the Kabat definition and Chothia CDR definition), which was retained in subsequent sdAb library construction.

[0149] 2. Identification of optimized sdAb library scaffolds

[0150] VH Gln39 forms a hydrogen bond with VL Gln38 in the native VH-VL pairing and faces each other in the VH-VH homodimer at a distance of 6 Å. Therefore, we reasoned that introducing the Q39E mutation, which induces charge repulsion, could reduce the formation of VH single-domain homodimers. Accordingly, introducing the mutation into YTPW resulted in YETPW ( 36 WYREAPGKTPEWVS sequence number 20) was generated. A clone with a T44S mutation (YESPW (()) was generated during the Q39E mutation process. 36 WYREAPGKSPEWVS sequence number 21)) ) was identified. This clone was well expressed and was tested as a library scaffold because residue 43 of human Vκ2 and Vκ6 (corresponding to residue 44 of human VH, according to Kabat numbering) is mainly Ser.

[0151] For this purpose, YETPW, YESPW and their Gln39 forms (YTPW and YSPW(( 36 WYRQAPGKSPEWVS sequence number 22))) was expressed in Escherichia coli. The results of size exclusion chromatography (SEC) analysis of the purified VH sdAbs are shown in Table 7 below.

[0152]

[0153] As shown in Table 7 and Fig. 1 above, YETPW and YESPW were selected to evaluate their performance as library scaffolds because they had high monomer contents.

[0154] CDR1 and CDR2 were randomized using degenerate oligonucleotides with limited success, and CDR3 was diversified using trinucleotide phosphoramidite chemistry as shown in Table 8 below. For the 20-aa CDR3, two separate repertoires were designed, with (SS AA) and without (20 AA) intra-loop disulfide bonds simulating the human immunoglobulin D2 segment.

[0155]

[0156] 10 9 A library of sdAbs estimated to have more than 100,000 diverse antibodies failed to produce binders when panned against several antigens, including hen egg lysozyme (HEL), human serum albumin (HSA), B-cell maturation antigen (BCMA), c-Met, and HER2.

[0157] Because scaffolds containing the Q39E mutation did not function well, the Glu at position 39 (based on Kabat numbering) was either reverted to its original Gln or converted to a positively charged Lys. Furthermore, position 44 was mutated between Ser and Thr to determine which residue would provide better scaffold properties, as shown in Table 9.

[0158]

[0159] As shown in Fig. 3, the four mutants (QT, QS, KT, KS) were expressed in E. coli and tested for aggregation propensity through SEC. As a result, QT, KT, and KS eluted as a single peak with a retention time similar to HEL (the molecular weights of HEL and VH sdAb were 14.3 and ~15 kDa, respectively), whereas the other mutant, QS, showed a retention time similar to scFv (~25 kDa), indicating that it exhibited the form of a VH homodimer.

[0160] For QT, KT, and KS, position 27, based on Kabat numbering, is Phe in human VH3-23, but as mentioned above, it spontaneously mutated to Ser during the selection process, resulting in a mutation between Ser and Phe. Panning selection of this mutation suggested a possible role for residue 27 in the physicochemical behavior of VH sdAbs.

[0161] Accordingly, as shown in Table 10 below, mutants SQT, SKS, FQT, and FKS were produced in E. coli and SEC and protein thermal shift (PTS) analyses were performed.

[0162]

[0163] As shown in Table 10 and Figure 4, all four variants exhibited monomeric behavior and Tm values ​​above 60°C, and among these clones, FKS eluted with a sharp monomer peak in SEC with minimal cleavage aggregation, and was selected for construction of the final sdAb library.

[0164] Example 2. Construction and validation of sdAb library

[0165] 1. Construction of sdAb library

[0166] Diversity in CDR1 and CDR2 was introduced into the FKS scaffold using partially denatured oligonucleotides. CDR3 was diversified using trinucleotide phosphoramidite-based randomization with length variations (9, 14, and 20 amino acids [aa]; Kabat CDR definition).

[0167] That is, to introduce CDR diversity into the selected VH scaffold (FKS scaffold) for constructing the final sdAb library, FR1-CDR1, FR2, CDR2-FR3, CDR3, and FR4 fragments were obtained by PCR and assembled by overlap extension PCR.

[0168] At this time, the primer information used to construct the library is shown in Table 11 below, and the PCR conditions are as follows:

[0169] Initial melting at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 minutes, for a total of 25 cycles; final extension at 72°C for 7 minutes.

[0170]

[0171] The assembled VH library was digested with SfiI restriction enzyme (New England Biolabs) and ligated into the SfiI-digested pComb3X phagemid vector, followed by transformation into TG1 electrocompetent E. coli (Lucigen, Meddleton, WI, USA). The transformed cells were plated on LB-agarose square plates (500 cm2) supplemented with 2% (w / v) glucose and 100 μg / mL ampicillin. The following day, the amplified cells were harvested and resuspended in 5 mL of SB medium (Super Broth; 3% [w / v] bactotryptone, 2% yeast extract, and 1% MOPS, pH 7.2) supplemented with 100 mg / mL ampicillin and 2% glucose. Half of the 50% glycerol solution was added (final glycerol 17%), 1 ml portions were rapidly frozen in liquid nitrogen and stored at -80°C.

[0172] As shown in Table 12 below, the total estimated size of the library inferred from the number of bacterial transformants is 1.6 X 10 9 It was confirmed as .

[0173]

[0174] 2. Verification

[0175] The sdAb library constructed above was validated by panning against a panel of test antigens (HEL, HSA, ovalbumin, cysteinyl tRNA synthetase 1 [CARS1]).

[0176] The library was panned four times against test antigens (HSA, HEL, CARS1, and ovalbumin) following the same protocol for protein A panning described above, except for the heat treatment step. To select antigen-specific VH binders, single colonies were picked from the final round and inoculated into 96-well microtiter plates containing 200 μl SB-ampicillin medium and incubated with shaking at 37°C until turbidity occurred (3–4 h). IPTG (final concentration 1 mM) was added to each well and incubated overnight at 30°C with shaking.

[0177] The next day, the plate was centrifuged, and the cell pellet was resuspended in 60 μl of cold 1X TES buffer (20% (w / v) sucrose, 1 mM EDTA, 50 mM Tris, pH 8.0) and incubated on ice for 30 min. Cold 0.2X TES (90 μl) was added, and the mixture was incubated on ice for 30 min. The plate was centrifuged, and the supernatant containing E. coli protoplast extract was transferred to an ELISA plate coated with antigen and blocked with 3% BSA. After binding for 1 h and washing three times with PBST, anti-HA-HRP antibody (Cat. #2999S, 1:3,000 dilution, Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated for 1 h at room temperature. After washing three times with PBST, binding activity was measured using chromogenic HRP-conjugated substrate TMB (tetramethylbenzidine), and the results are shown in Table 13.

[0178]

[0179] After performing four rounds of panning for each antigen, target-binding clones were identified through ELISA screening of the panning output clones. Multiple binders with unique sequences and minimal background binding signals were identified for HSA, ovalbumin, and CARS1, respectively. However, panning and screening for HEL did not produce any specific binders. This result is thought to be because the small, positively charged protein (14.6 kDa, pI = 10.7) may not have an epitope suitable for binding to the large convex paratope of the sdAb library.

[0180] Accordingly, the affinity and physicochemical properties of the identified binding clones were analyzed in detail below.

[0181] Example 3. Characterization of Isolated Target-Specific sdAb

[0182] The sdAb of Example 2 (3 CARS1, 2 ovalbumin, 1 HSA) was expressed in E. coli and purified by immobilized metal ion affinity chromatography (IMAC). Approximately 1-3 mg of sdAb was purified from 50 ml of culture medium (purification yield 20-70 mg / L, Table 14).

[0183]

[0184] As shown in Figure 5, the SEC analysis results showed that the monomer content of the separated sdAb was at a normal to high level (66% to 86%) upon purification.

[0185] Meanwhile, the kinetic binding parameters of the purified monomeric sdAb were analyzed by surface plasmon resonance (SPR), and the thermal stability of the sdAb was analyzed by protein thermal shift (PTS), and the results are shown in Table 15 below.

[0186]

[0187] As shown in Table 15, the affinity (KD value) is 10 -9 ~ 10 -7 M range, kon is 10 3 - 10 5 M -1 s -1 , koff is 10 -3 s -1 It appeared in the range.

[0188] For comparison, the average kon, koff, and KD values ​​of a panel of 50 monoclonal antibodies raised against 19 different antigens were 1.9 X 10 5 M -1 s -1 , 2.4 X 10 -3 s -1 , 1.3 X 10 -8 It was shown as M, and the Tm value was observed in the range of 58~70℃, with an average of 67℃.

[0189] As shown in the above results, the affinity, monomer content, and Tm values ​​of the isolated sdAbs were comparable to those of typical nanobodies and murine monoclonal antibodies. These results suggest that the engineered VH scaffold FKS can accommodate diverse CDR sequences and conformations while maintaining proper folding.

Claims

1. A single-domain antibody having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR2 is a single-domain antibody selected from the group consisting of SEQ ID NOs: 33 to 36.

2. In paragraph 1, Here, CDR1 is the formula X1-X2-X3-M-X4, where X1 is D, G, N or S, X2 is D or Y, X3 is A or D, X4 is D or H, FR2 is any one selected from the group consisting of sequence numbers 33 to 36, CDR2 is of the formula X5-I-X6-X7-X8-X9-X10-X11-X12-YYADSVKG, where X5 is A, G, L, S, V, or W, X6 is S or Y, X7 is H, P, S, or Y, X8 is D, G, N, or S, X9 is D, G, N, or S, X10 is G or S, X11 is N or S, and X12 is I, K, or T. A single-domain antibody wherein CDR3 is of the formula ARXXXXFDY; formula ARXXXXXXXXXFDY; or formula ARXXXXXXXXXXXXXXMDV, wherein X is any amino acid.

3. A single-domain antibody according to claim 1, wherein FR2 has SEQ ID NO: 33 or 36.

4. A single-domain antibody according to claim 1, wherein FR1 is SEQ ID NO: 50, FR3 is SEQ ID NO: 51, and FR4 is SEQ ID NO:

52.

5. A single-domain antibody library comprising a single-domain antibody according to any one of claims 1 to 4.

6. In paragraph 5, the single-domain antibody library comprises at least 10 8 A single-domain antibody library comprising a unique antibody sequence of a dog.

7. A nucleic acid molecule encoding a single-domain antibody according to any one of claims 1 to 4.

8. A vector comprising a nucleic acid molecule according to paragraph 7.

9. A host cell comprising a vector according to paragraph 8. A method for producing an antibody phage surface-display library, comprising the step of producing a nucleic acid sequence of a single-domain antibody (sdAb) in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are linked in a row, comprising a nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36, which are FR2 variant polypeptides; 11. In paragraph 10, CDR1 is a formula X1-X2-X3-M-X4, where X1 is D, G, N, or S, X2 is D or Y, X3 is A or D, and X4 is D or H. FR2 is any one selected from the group consisting of sequence numbers 33 to 36, CDR2 is of the formula X5-I-X6-X7-X8-X9-X10-X11-X12-YYADSVKG, where X5 is A, G, L, S, V, or W, X6 is S or Y, X7 is H, P, S, or Y, X8 is D, G, N, or S, X9 is D, G, N, or S, X10 is G or S, X11 is N or S, and X12 is I, K, or T. A method for producing an antibody phage surface-displayed library, wherein CDR3 is of the formula ARXXXXFDY; formula ARXXXXXXXXXFDY; or formula ARXXXXXXXXXXXXXXMDV, wherein X is any amino acid.

12. A method for producing an antibody phage surface-displayed library in claim 10, wherein FR2 has sequence number 33 or 36.

13. A method for producing an antibody phage surface-display library in claim 10, wherein FR1 is sequence number 50, FR3 is sequence number 51, and FR4 is sequence number 52.

14. In the 10th paragraph, the method for producing an antibody phage surface-displayed library is as follows: a) A step of preparing a nucleic acid sequence of a single domain antibody (sdAb) in which FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are connected in a row from the N-terminus to the C-terminus by overlapping and polymerizing a nucleic acid encoding any one selected from the group consisting of SEQ ID NOs: 33 to 36, which is an FR2 variant polypeptide, and nucleic acid fragments of FR1, CDR2, FR3, CDR3 and FR4; b) a step of cleaving the single domain antibody (sdAb) and phagemid vector obtained in step a) with a restriction enzyme and assembling them through a ligation reaction; and c) A method for producing an antibody phage surface-displayed library, comprising: a step of transforming the phagemid vector assembled in step b) into a host strain and then culturing the same; 15. An antibody phage surface-display library manufactured by a method according to any one of claims 10 to 14.

16. A screening method for identifying a synthetic single-domain monoclonal antibody that binds to a target of interest, comprising using a single-domain antibody library according to paragraph 5.

17. A screening method for identifying a synthetic single-domain monoclonal antibody that binds to a target of interest, comprising using a single-domain antibody library according to claim 15.

Images