Synthetic single-domain library

Abstract

The present invention relates to the identification of a fully humanized single domain antibody scaffold and its use in the generation of synthetic single domain antibodies. The invention further relates to antigen binding proteins comprising this single domain antibody scaffold and their use in therapy.

Description

[Technical Field]

[0001] This invention relates to the identification of highly stable synthetic single-domain antibody scaffolds and their use in the production of synthetic single-domain antibody libraries. Furthermore, this invention relates to antigen-binding proteins containing these stable single-domain antibody scaffolds, and their use, in particular, for cancer treatment, especially as therapeutic agents. [Background technology]

[0002] Over the past decade, antibodies have become one of the most promising therapeutic approaches, particularly in the field of oncology, and have also become an important source of research and diagnostic tools.

[0003] Immunoglobulin G (IgG) is the basic structure of typical antibodies and contains two heterodimers, a heavy chain and a light chain, linked by disulfide crosslinks. However, naturally occurring single-chain antibodies have been found in at least two groups of animals: camelids (Hamers-Casterman et al., 1993, Nature, pp. 363, 446-448) and sharks (Greenberg et al., Nature, 1995, Mar 9; 374(6518): pp. 168-73). Such single-chain antibodies constitute a further class of IgG lacking a light chain. The recognition region of such naturally occurring single-chain antibodies contains only the variable domain of the heavy chain, called VHH. VHH includes four frameworks (FRs) that form the scaffold of the IgG domain and three complementarity-determining regions (CDRs) involved in antigen binding.

[0004] Many advantages of the VHH scaffold have been reported, including the absence of interchain disulfide crosslinks and generally greater solubility and stability in reducing environments (Wesolowski et al., 2009 Med Microbiol Immunol. Aug;198(3): pp. 157-74). Furthermore, VHH has been reported to have high solubility, expression yield, and thermal stability due to its small size (15 kDa) (Jobling SA et al., Nat Biotechnol. 2003 Jan; 21 (1): pp. 77-80). Moreover, the VHH framework shows high sequence and structural homology to the human VH domain of the III family (Muyldermans et al., 2001. J Biotechnol. Jun; 74 (4): pp. 277-302), and VHH possesses immunogenicity comparable to human VH, making it a very interesting drug for therapeutic application.

[0005] The properties of VHH scaffolds offer many advantages for therapeutic use, including better tissue penetration, faster clearance in the kidneys, high specificity, and reduced immunogenicity.

[0006] A camelid antibody library is described, for example, in U.S. Patent Application Publication No. 2006 / 0246058 (National Research Council of Canada). The described phage display library contains fragments of llama antibodies, in particular single-domain fragments of variable heavy chains (VHH and VH). This library was generated using lymphocyte genomes of non-immunized animals (naive library). The resulting phage display library also contains contaminants of conventional VH antibody fragments.

[0007] Furthermore, U.S. Patent No. 7,371,849 (Institute For Antibodies Co., Ltd.) reports a method for generating a VHH library from camel VHH genes. The diversity of such libraries was obtained by improving conventional methods for isolating VHH variable regions from a naive repertoire. However, such prior art does not address the immunogenicity problem of non-human antibodies. Even if some of these are identified to bind to a specific target of interest, they cannot be administered to patients for use as therapeutic agents that do not carry the risk of activating the human immune system.

[0008] A method for humanizing single-domain antibodies from camels is described by Vincke et al., 2008, JBC Vol 284(5) pp. 3273-3284.

[0009] U.S. Patent No. 8,367,586 discloses a collection of synthetic antibodies or fragments thereof. Such antibodies include pairs of variable heavy and variable light chains, and these framework regions contain a portion of an optimal germline gene sequence. This incorporation of human sequences allows for a reduction in the risk of immunogenicity for therapeutic use.

[0010] Monegal et al. (2012, Dev Comp Immunol. 36(1): pp. 150-156) reported that single-domain antibodies with VH hallmarks are repeatedly identified during biopanning of naive libraries. In fact, VH hallmarks are identified more frequently than VHH hallmarks on binders selected from VHH naive libraries. For example, Monegal et al. showed that while 5% of VH hallmarks are found in naive libraries, 20% of these VH hallmarks are found in antibodies selected following biopanning against antigens.

[0011] Recently, in Moutel et al. (eLife 2016;5:e16228) and WO 2015 / 063331, synthetic libraries of humanized nanobodies and intrabodies that result in functional high-affinity antibodies have been disclosed.

[0012] However, despite such knowledge, there remains a need for further single-domain antibody libraries that have improved humanization and that retain the specificity and advantages of single domains, in particular their high solubility and expression yields.

Prior Art Documents

Patent Documents

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Patent Document 1

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Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0014]

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Non-Patent Document 4

[0015] Accordingly, one aspect of the present invention is to provide a fully humanized recombinant single-domain antibody library capable of generating highly stable single-domain antibodies with high diversity and high affinity for specific antigens. Another aspect is to provide a library rich in single-domain antibodies active in the intracellular environment. Yet another aspect is to provide a library rich in single-domain antibodies with high thermal stability. Typically, the single-domain antibodies obtained by this disclosure also have high expression yields. Furthermore, typically, these single-domain antibodies can overcome the classic technical problems of mAbs, such as delayed blood clearance, limited penetration into solid tumors, nonspecific uptake by healthy tissues, and inability to contact recessed epitopes. [Means for solving the problem]

[0016] Among 10 amino acids distinct from human amino acids, four VHH hallmark amino acids have been identified in two regions of the VHH framework. The inventors have surprisingly discovered that these four camelid hallmarks can be replaced with four typical human hallmarks while preserving the VHH properties. The results provided herein demonstrate that the synthetic single-domain antibody library disclosed herein makes it possible to obtain synthetic humanized sdAbs with nano / picomolecular affinity to these targets with high specificity. We obtained sdAbs directed to various cellular targets that can be used as intrabodies for intracellular labeling in living cells. These sdAbs can be used to stain target cells. Furthermore, they can inhibit the downstream activation of these targets (i.e., the FGFR4 pathway). These sdAbs can also be used using CAR-T cell techniques to deliver loaders to target cells, arm T cells, and destroy targeted cells. Thus, these results provide evidence that the synthetic single-domain antibody library of the present invention provides single-domain antibodies highly suitable for cell labeling, diagnostic, and therapeutic applications.

[0017] The objective of the present invention is, i) A process of introducing diverse nucleic acids encoding CDR1, CDR2, and CDR3 between the respective framework coding regions of a humanized synthetic single-domain antibody (hereinafter referred to as "hs2dAb") to generate diverse nucleic acids encoding synthetic single-domain antibodies having the same synthetic single-domain scaffold amino acid sequence. A method for generating a synthetic single-domain antibody library, wherein the synthetic single-domain antibody scaffold amino acid sequence comprises at least the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, and FRW2-W14.

[0018] In some embodiments, the single-domain antibody scaffold is derived from a llama species.

[0019] In some embodiments, the synthetic single-domain antibody (hs2dAb) disclosed herein is, - FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2 and FRW4-L7, and / or - The group consisting of FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23 and FRW3-S27, in particular the following combinations: FRW1-P14, FRW2-S16, FRW3-S17, FRW3-R29 and FRW3-A30 It further comprises at least one amino acid residue selected from.

[0020] In some embodiments, the synthetic single-domain scaffold amino acid sequence comprises at least one of the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7, and optionally further comprises one or more of the following residues: FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, FRW3-S27.

[0021] In some embodiments, the synthetic single-domain scaffold amino acid sequence includes the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7, FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, and FRW3-S27.

[0022] In some of the embodiments described above, the synthetic single-domain antibody further comprises at least one amino acid residue selected from the group consisting of FRW2-V5, FRW3-V21, and FRW4-R2.

[0023] In some embodiments, the synthetic single-domain antibody includes a framework region consisting of FRWl of SEQ ID NO: 1, FRW2 of SEQ ID NO: 2, FRW3 of SEQ ID NO: 3, and FRW4 of SEQ ID NO: 4, or, for example, a functional variant framework region having one, two, or three or fewer conserved amino acid substitutions within each framework region. In some such embodiments, the synthetic single-domain antibody scaffold includes at least amino acid residues consisting of FRW2-V4, FRW2-G11, FRW2-L12, and FRW2-W14. In even more specific embodiments, the scaffold includes at least one amino acid residue from the group consisting of FRW2-V5, FRW3-V21, and FRW4-R2.

[0024] In one preferred embodiment, the amino acid residues of synthetic CDR1 and CDR2 follow the following rules: First letter of CDR1: Y, R, S, T, F, G, A, or D Second CDR1: Y, S, F, G or T, Third item on CDR1: Y, S, F, or W, The fourth letter of CDR1: Y, R, S, T, F, G, A, W, D, E, K or N, Fifth letter of CDR1: S, T, F, G, A, W, D, E, N, I, H, R, Q or L, The sixth letter of CDR1: S, T, Y, D, or E The 7th letter of CDR1: S, T, G, A, D, E, N, I or V, First of CDR2: R, S, F, G, A, W, D, E or Y, CDR2 second: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y, Third in CDR2: S, T, F, G, A, W, D, E, N, H, Q, P, The fourth letter of CDR2: G, S, T, N, or D Fifth in CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M, The sixth letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W or K, The seventh letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V It is determined by [the following].

[0025] In one related embodiment that can be combined with the embodiments described above, this CDR3 amino acid sequence contains 9 to 18 amino acids. In one related embodiment that can be combined with the embodiments described above, this CDR3 amino acid sequence contains amino acid residues selected from one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

[0026] Furthermore, the present invention is available by the above method, and at least 3.10 9 This relates to a synthetic single-domain antibody library containing distinct single-domain antibody coding sequences.

[0027] The present invention further relates to a screening method for identifying synthetic single-domain antibodies that bind to a target of interest, such as human proteins, and to the use of this synthetic single-domain antibody library in, for example, phage display.

[0028] Ultimately, the present invention provides an antigen-binding protein comprising a synthetic single-domain antibody of the following formula: FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, wherein the framework regions FRW1, FRW2, FRW3 and FRW4 consist of at least the following amino acid residues FRW2-V4 / , FRW2-G11, FRW2-L12 and FRW2-W14, and optionally the following amino acid residues - FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7 and / or - FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, FRW3-S27 This involves handling antigen-binding proteins, including one or more of the following:

[0029] In some embodiments, the antigen-binding protein includes at least one of the following amino acid residues: FRW2-V4 / , FRW2-G11, FRW2-L12, and FRW2-W14, and optionally one or more of the following amino acid residues: FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7, FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, and FRW3-S27.

[0030] In some embodiments, the antigen-binding protein amino acid sequence includes the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7, FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, and FRW3-S27.

[0031] In some of these embodiments, the antigen-binding protein comprises at least amino acid residues consisting of FRW2-V4, FRW2-G11, FRW2-L12, and FRW2-W14. In more specific embodiments, the scaffold comprises at least one amino acid residue from the group consisting of FRW2-V5, FRW3-V21, and FRW4-R2.

[0032] In a particular embodiment that can be combined with the embodiments described above, the antigen-binding protein has the following functional properties: a) It can be expressed as a soluble single-domain antibody in the periplasm of Escherichia coli (E. coli). b) It can be expressed as a soluble intrabody in the cytosol of Escherichia coli, yeast, or other eukaryotic organisms. c) When expressed in mammalian cells, it does not aggregate and contains fusion proteins (e.g., fluorescent protein fusions). It includes a synthetic single-domain antibody having one or more of the following:

[0033] In some embodiments, the antigen-binding protein framework region is derived from the llama species VHH framework regions FRW1, FRW2, FRW3, and FRW4.

[0034] In some embodiments, the antigen-binding protein defined above has a framework region consisting of FRWl of SEQ ID NO: 1, FRW2 of SEQ ID NO: 2, FRW3 of SEQ ID NO: 3, and FR4 of SEQ ID NO: 4.

[0035] In a preferred embodiment that can be combined with the above-described embodiment, the amino acid residues of synthetic CDR1 and CDR2 are arranged as follows: First letter of CDR1: Y, R, S, T, F, G, A, or D Second CDR1: Y, S, F, G or T, Third of CDR1: Y, S, S, S, F or W, The fourth letter of CDR1: Y, R, S, T, F, G, A, W, D, E, K or N, Fifth letter of CDR1: S, T, F, G, A, W, D, E, N, I, H, R, Q or L, The sixth letter of CDR1: S, T, Y, D, or E The 7th letter of CDR1: S, T, G, A, D, E, N, I or V, First of CDR2: R, S, F, G, A, W, D, E or Y, CDR2 second: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y, Third in CDR2: S, T, F, G, A, W, D, E, N, H, Q, P, The fourth letter of CDR2: G, S, T, N, or D Fifth in CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M, The sixth letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W or K, The seventh letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P or V, The CDR3 amino acid sequence contains 9 to 18 amino acids selected from one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, and M. [Brief explanation of the drawing]

[0036] [Figure 1] (A) This figure shows the affinity determination of nanobodies to recombinant proteins by surface plasmon resonance spectroscopy. A single-cycle dynamic analysis was performed on FGFR4 immobilized by covalent amine bonds on a dextran-based sensor chip. After injecting the analytes F8 and mCh at five different concentrations, the process moved to the dissociation phase. The final dissociation step was added after the last injection step to determine the Koff rate and calculate the KD. The black curve represents the measured data, and the red curve shows the compatibility analysis (heteroligand model) performed by BIA evaluation software. [Modes for carrying out the invention]

[0037] In this specification, the positions of amino acid residues in synthetic single-domain antibodies or fragments thereof are indicated according to their positions (from left to right) in each individual sequence shown in Table 1 below.

[0038] [Table 1]

[0039] In this invention, i. A process of introducing diverse synthetic nucleic acids encoding CDR1, CDR2, and CDR3 between the respective framework coding regions of a synthetic single-domain antibody to generate nucleic acids encoding diverse synthetic single-domain antibodies having the same synthetic single-domain antibody scaffold amino acid sequence. The present invention provides a method for generating a synthetic single-domain antibody library, wherein the synthetic single-domain scaffold amino acid sequence comprises at least the following amino acid residues FRW2-V4, FRW2-G11, FRW2-L12, and FRW2-W14, and optionally further comprises one or more of the following residues FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, and FRW4-L7.

[0040] In some embodiments, the synthetic single-domain scaffold amino acid sequence includes at least one of the following amino acid residues: FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, and FRW2-S16.

[0041] In some embodiments, the synthetic single-domain scaffold amino acid sequence includes at least one of the following amino acid residues: FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, and FRW3-S27.

[0042] In some embodiments, the synthetic single-domain scaffold amino acid sequence comprises at least one of the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7, and optionally further comprises one or more of the following residues: FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, FRW3-S27.

[0043] Synthetic single-domain antibody scaffold of the present invention This disclosure relates to the acquisition of highly stable single-domain antibody scaffolds and the identification of unique features in the framework region of single-domain antibodies for use in the production of synthetic single-domain antibody libraries, such as synthetic single-domain antibody phage display libraries. The resulting hs2dAb with this unique scaffold is highly stable and has a very low risk of immunogenicity. Furthermore, the resulting hs2dAb exhibits high solubility and high expression yield, supporting the promotion of therapeutic use.

[0044] We can provide nucleic acids encoding single-domain antibodies as starting materials for library generation.

[0045] As used herein, the terms “single-domain antibody” or “Nanobody®” (a trademark of Ablynx) refer to antibody fragments having a molecular weight of only 12–15 kDa, consisting of a single monomeric variable antibody domain derived from a heavy chain. Such single-domain antibodies (designated VHH) can be found in camelid mammals and naturally lack a light chain. For an overview of single-domain antibodies, see also the prior art cited above, as well as European Patent No. 0368684, Ward et al. (Nature 1989 Oct 12; 341 (6242): pp. 544–546), Holt et al., Trends Biotechnol, 2003, 21(1 l): pp. 484–490, and International Publication Nos. 06 / 030220 and 06 / 003388.

[0046] In some embodiments, this single-domain antibody is - Fragments of naturally occurring antibodies lacking a light chain, for example, so-called VHH antibodies derived from camel antibodies, or so-called VNAR fragments derived from shark antibodies, or - Human antibodies It may also be derived from the amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, and optionally, - FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7 and / or - The group consisting of FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, and FRW3-S27, in particular the combination of FRW1-P14, FRW2-S16, FRW3-S17, FRW3-R29, and FRW3-A30. It has at least one amino acid residue selected from.

[0047] Therefore, a single-domain antibody contains at least four framework regions with three high-frequency variable CDR regions in between, resulting in the following typical antibody variable domain structure: FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4. This single domain does not need to interact with the light chain antibody variable region to form a conventional heterodimer heavy and light chain antigen-binding antibody structure for activation.

[0048] As used herein, the term “synthetic” means that such antibodies are not obtained from naturally occurring antibody fragments but are produced from recombinant nucleic acids containing artificial coding sequences.

[0049] In particular, the synthetic single-domain antibody library of the present invention is produced by the synthesis of an artificial framework and a CDR coding sequence. In contrast to libraries obtained by amplification of a naive repertoire derived from non-immune llama animals, the synthetic single-domain antibody library of the present invention does not contain a mixture of frameworks, in particular a mixture of VHH and conventional VH antibodies.

[0050] Advantageously, in a preferred embodiment of the synthetic single-domain antibody library of the present invention, all single-domain antibody clones include the same framework region, thereby providing a unique synthetic single-domain antibody scaffold.

[0051] As used herein, the term “scaffold” refers to the four framework regions of the synthetic single-domain antibodies in the library of the present invention. Typically, all single-domain antibodies in the library of the present invention have the same scaffold amino acid sequence, but their CDRs may differ (i.e., the diversity of each library is limited to the CDR region).

[0052] The synthetic single-domain antibody scaffold according to the present invention comprises amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, and optionally, - FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7 and / or - FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, FRW3-S27, in particular FRW1-P14, FRW2-S16, FRW3-S17, FRW3-R29 and FRW3-A30 It contains at least one amino acid residue selected from the group consisting of the following.

[0053] In some embodiments, the synthetic single-domain scaffold amino acid sequence includes the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7, FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, and FRW3-S27.

[0054] These unique characteristics result in highly stable synthetic single-domain antibodies with a low risk of immunogenicity.

[0055] In certain embodiments, the synthetic single-domain antibody scaffold includes a framework region consisting of FRW1 of SEQ ID NO: 1, FRW2 of SEQ ID NO: 2, FRW3 of SEQ ID NO: 3, and FRW4 of SEQ ID NO: 4, or a functional variant framework region having, for example, one, two, or three or fewer conservative amino acid substitutions within each framework region, more preferably within a single framework region.

[0056] In some of these embodiments, the synthetic single-domain antibody scaffold comprises at least amino acid residues consisting of FRW2-V4, FRW2-G11, FRW2-L12, and FRW2-W14. In more specific embodiments, the scaffold comprises at least one amino acid residue from the group consisting of FRW2-V5, FRW3-V21, and FRW4-R2. As previously mentioned, such amino acid residues at the designation site make it possible to obtain a single-domain antibody with reduced immunogenicity (particularly due to the FR2V5 residue) and improved thermal stability, solubility, and biological productivity (particularly due to the FRW4-E2 residue).

[0057] Conservative amino acid substitution involves substituting an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Such families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0058] In another embodiment, the synthetic single-domain antibody scaffold comprises functional variants of the FRW1, FRW2, FRW3, and FRW4 framework regions having at least 90%, preferably 95% or 99%, identity with SEQ ID NOs: 1-4, respectively. Typically, amino acid residues FRW2-V4, FRW2-G11, FRW2-L12, and FRW2-W14 are conserved.

[0059] As used herein, the percentage identity between two arrays is a function of the number of identical positions shared by the arrays (i.e., % identity = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap. These must be introduced for optimal alignment of the two arrays. The comparison of arrays and the determination of their percentage identity can be achieved using mathematical algorithms as described below.

[0060] The percentage identity between two amino acid sequences can be determined using the algorithm by E. Myers and W. Miller (Comput. Appl. Biosci. 4: 1 pp. 1-17, 1988), which is incorporated into the ALIGN program. In addition, the percentage identity between two amino acid sequences can be determined using the algorithm by Needleman and Wunsch (J. Mol. Biol. 48: pp. 443-453, 1970), which is incorporated into the GAP program of the GCG software package. Another program for determining percentage identity is CLUSTAL (first described by M. Larkin et al., Bioinformatics 23: pp. 2947-2948, 2007; D. Higgins and P. Sharp, Gene 73: pp. 237-244, 1988), which is available as a standalone program or via a web server (see http: / / www.clustal.org / ).

[0061] Functional variants can be examined for their ability to retain the advantageous properties of the synthetic single-domain scaffold of the present invention. In particular, they possess the following properties: i. It can be expressed as a soluble single-domain antibody in the periplasm of E. coli. ii. It can be expressed as a soluble intrabody in the cytosol of Escherichia coli, yeast, or other eukaryotic organisms. iii. When expressed in mammalian cells, it does not aggregate and contains fusion proteins (e.g., fluorescent protein fusions). The ability to hold at least one or more of these may be examined.

[0062] The assay for testing the above characteristics is described in the examples.

[0063] For example, a reference synthetic single-domain antibody coding sequence can be constructed by transplanting a reference CDR coding sequence (e.g., the CDR of clone F8 of sequence number 9) onto a variant scaffold coding sequence to be tested (which has homologous sequences to sequence numbers 1-4). This reference synthetic single-domain antibody coding sequence makes it possible to generate a reference synthetic single-domain antibody that can quantify the above-mentioned properties.

[0064] Introducing CDR diversity to selected single-domain antibody scaffolds In particular, methods for generating CDR diversity in antibody libraries by random or specific synthesis of CDR coding sequences, and for cloning them to corresponding framework sequences, have been widely described in the art.

[0065] The synthetic single-domain antibody library of the present invention can also be generated by introducing CDR high diversity into selected unique scaffold sequences, for example, as described in Lindner, T., H. Kolmar, U. Haberkorn, and W. Mier. 2011. Molecules. 16: pp. 1625-1641.

[0066] In a preferred embodiment of the present invention, the positions of the amino acid sequences of synthetic CDR1 and CDR2 are rationally designed to mimic the natural diversity of CDRs in the human repatrophyll.

[0067] Cysteine ​​is optionally avoided because these thiol groups can interfere with intracellular expression and functionality. Furthermore, arginine and hydrophobic residues can also be avoided due to the increased risk of antibody aggregation. A low proline ratio is also preferable as it provides greater mobility in the CDR. Preferably, serine, threonine, and tyrosine are the most frequent residues in all three CDRs because they are involved in epitope binding. Aspartate and glutamate can also be abundant at certain positions to enhance solubility. In the CDR3 sequence, length can affect different epitope shapes, particularly the ability to bind to cavities. Therefore, CDR3 sequences of various lengths can be introduced into the library.

[0068] In a particular embodiment, those skilled in the art will know the following rule: First letter of CDR1: Y, R, S, T, F, G, A, or D Second CDR1: Y, S, F, G or T, Third item on CDR1: Y, S, F, or W, The fourth letter of CDR1: Y, R, S, T, F, G, A, W, D, E, K or N, Fifth letter of CDR1: S, T, F, G, A, W, D, E, N, I, H, R, Q or L, The sixth letter of CDR1: S, T, Y, D, or E The 7th letter of CDR1: S, T, G, A, D, E, N, I or V, First of CDR2: R, S, F, G, A, W, D, E or Y, CDR2 second: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y, Third in CDR2: S, T, F, G, A, W, D, E, N, H, Q, P, The fourth letter of CDR2: G, S, T, N, or D Fifth in CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M, The sixth letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W or K, The seventh letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V The amino acid residues of synthetic CDR1 and CDR2 can be selected accordingly.

[0069] Furthermore, in another specific embodiment, the CDR3 amino acid sequence includes 9 to 18 amino acids selected from one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

[0070] The above appearance rules are used as guidance for generating preferred libraries of the present invention, but other libraries with different appearance rules are also part of the present invention, as long as they contain the advantageous synthetic single-domain antibody scaffolds of the present invention.

[0071] In certain embodiments, only a significant clonal population of the library may strictly adhere to the occurrence rules described above. For example, statistically, at least 50%, 60%, 70%, 80%, or at least 90% of the clones in the library may adhere to the occurrence rules described above for amino acid residues at the CDR1, CDR2, and CDR3 positions.

[0072] In accordance with such occurrences of amino acid positions, advanced gene synthesis techniques are preferably used to avoid in-frame termination or cysteine ​​occurrence, or to reduce frameshifts. Such methods include, but are not limited to, double-stranded DNA triple-block, trinucleotide synthesis, or other codon-controlled, more generally, position-controlled degenerate synthesis techniques described by Van den Brulle et al., 2008, Biotechniques 45(3): pp. 340-33.

[0073] In certain embodiments, the codon bias can be further optimized, for example, for host cell type, such as mammalian host cell expression, using well-known methods.

[0074] In a particular embodiment, the coding sequence is designed so as not to include unwanted restriction sites, such as those used for cloning the coding sequence into a suitable cloning or expression vector.

[0075] The resulting diverse coding sequences are introduced into an expression or cloning vector suitable for an antibody library. In certain embodiments, the expression vector is a plasmid. In another preferred embodiment, the expression vector is suitable for the production of a phage display library. Two different types of vectors, phagemid vectors and phage vectors, can be used for the production of a phage display library.

[0076] Phagemids are derived from filamentous phage (Ff phage-derived) vectors containing plasmid origins of replication. The basic components of a phagemid primarily include the plasmid origin of replication, selection markers, intergenetic regions (IG regions, typically including minus and plus strand packing sequences and the origin of replication), the gene for the phage coat protein, restriction enzyme recognition sites, promoters, and DNA segments encoding signal peptides. In addition, they may include molecular tags to facilitate library screening based on phagemids. Phagemids can be converted into filamentous phage particles identical in morphology to Ff phages by co-infection with helper phages, such as R408, M13K07, and VCSM13 (Stratagene). An example of a phage vector is fd-tet (Zacher et al., gene, 1980, 9, pp. 127-140), which consists of an fd phage genome and a TnlO segment inserted near the phage genome origin of replication. Examples of promoters for use in phagemid vectors include, but are not limited to, PlacZ or PT7, and examples of signal peptides include, but are not limited to, pelB reader, gill, CAT reader, SRP, or OmpA signal peptide.

[0077] Other phage display methods use soluble phages such as T4 or T7. In addition, non-phage vectors, including vectors for bacterial cell display (Daugherty et al., 1999 Protein Eng. Jul;12(7):pp. 613-621; Georgiou et al., 1997 Nat Biotechnol. 1997 Jan;15(l):pp. 29-34), yeast cell display (Boder and Wittrup, Nat Biotechnol. 1997 Jun;15(6):pp. 553-557), or ribosome display (Zahnd C, Amstutz P, Pluckthun A. Nat Methods. 2007 Mar;4(3):pp. 269-79), can be used to generate display libraries. DNA display (Eldridge et al., Protein Engineering, Design & Selection, Vol. 22, No. 11, pp. 691-698, 2009) and surface display on mammalian cells (Rode HJ et al., Biotechniques. 1996 Oct;21(4):650, 652-653, 655-6, pp. 658) have also been reported. Non-display methods, such as yeast two-hybrids, can also be used for selecting appropriate binders from libraries (Visintin et al., 1999 Proc Natl Acad Sci USA 96, pp. 1723-1728).

[0078] In one preferred embodiment, to avoid the generation of empty vectors, positive selection of the recombinant coding sequence in the cloning vector containing the suicide gene is applied (see, for example, Philippe Bernard, 1996, BioTechniques, Vol. 21, No. 2, "Positive Selection of Recombinant DNA by CcdB").

[0079] Preferably, the logical diversity calculated from all possible combinations of CDR amino acid residues designed for antibody library generation is at least 10 11 or at least 10 12 , in particular, 10 23It is a unique array.

[0080] The present invention provides synthetic single-domain antibody libraries and their uses Ultimately, according to another embodiment, the present invention relates to a synthetic single-domain antibody library that is available or obtained by conventional methods.

[0081] Therefore, as used herein, the term “synthetic single-domain antibody library” encompasses a nucleic acid library that is highly diverse and optionally contains the synthetic single-domain antibody coding sequence in a cloning vector or expression vector. The term “synthetic single-domain antibody library” further includes any transformed host cell or organism having this nucleic acid library, more specifically, bacteria, yeast or filamentous fungi or mammalian cells transformed with this nucleic acid library, or bacteriophages or viruses containing this nucleic acid library. The term “synthetic single-domain antibody library” further includes a corresponding mixture of diverse antibodies encoded by this nucleic acid library. As used herein, the term “clone” refers to each unique individual in the antibody library, whether a nucleic acid, host cell, or single-domain antibody.

[0082] In a particular embodiment of the present invention, the synthetic single-domain antibody library of the present invention comprises at least 1 × 10⁶ 8 In particular, 1.6 × 10 9 Includes diverse clones.

[0083] This library can be used in screening methods for identifying synthetic single-domain antibodies that specifically bind to a target of interest. Any known screening method for identifying binders with specific affinity to a target of interest can be used in conjunction with the synthetic single-domain antibody library of the present invention. Such methods include, but are not limited to, phage display techniques, bacterial cell displays, yeast cell displays, mammalian cell displays, or ribosome displays.

[0084] Preferably, the screening method is phage display.

[0085] Preferably, the target of interest is a therapeutic target, and the synthetic single-domain antibody library is used for the identification of synthetic single-domain antibodies that specifically bind to this therapeutic target. In certain embodiments, the target of interest comprises at least an antigenic determinant. In certain embodiments, the target is a saccharide or polysaccharide, a protein or glycoprotein, a lipid. In one particular embodiment, this target of interest is of plant, yeast, fungal, insect, mammalian, or other eukaryotic cell origin. In another particular embodiment, this target of interest is of bacterial, protozoan, or viral origin.

[0086] In one particular embodiment, a "single-domain antibody that specifically binds to the target of interest" is intended to refer to a single-domain antibody that binds to the target of interest with a K D of 1 mM or less, 100 μM or less, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM or less. This does not exclude the possibility that this single-domain antibody also binds to other antigens.

[0087] The term "K D ", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K d to K a (i.e., K d / K a ) and is expressed as molar concentration -1 (M -1 ). The K D value of an antibody can be determined using methods well established in the art. Methods for determining the K D of an antibody are by use of surface plasmon resonance or by use of a biosensor system, e.g., a Biacore® system or a Proteon®.

[0088] The antigen-binding protein of the present invention Given the high diversity of the synthetic single-domain antibody library of the present invention, those skilled in the art can obtain synthetic single-domain antibodies having high affinity and high specificity for a target of interest using conventional screening methods, such as phage display.

[0089] The resulting synthetic single-domain antibody can then be further modified to produce an appropriate antigen-binding protein. In particular, CDR residues can be modified using techniques known in the art (mutaogenesis, affinity maturation) to enhance antibody affinity to the target of interest and improve its folding or production.

[0090] Therefore, another aspect of the present invention is an antigen-binding protein comprising a synthetic single-domain antibody of the following formula: FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, wherein the framework regions FRW1, FRW2, FRW3 and FRW4 optionally consist of the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, - FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2 and FRW4-L7, and / or - FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, FRW3-S27, in particular FRW1-P14, FRW2-S16, FRW3-S17, FRW3-R29 and FRW3-A30 The present invention relates to antigen-binding proteins comprising at least one amino acid residue selected from the group consisting of the following.

[0091] In some embodiments, this synthetic single-domain scaffold of the present disclosure comprises amino acid residues FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2 and FRW4-L7 and / or FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, FRW3-S27, in particular FRW1-P14, FRW2-S16, FRW3-S17, FRW3-R29 and FRW3-A30.

[0092] In some embodiments, the synthetic single-domain scaffold amino acid sequence includes the following amino acid residues: FRW2-V4, FRW2-G11, FRW2-L12, FRW2-W14, FRW1-V5, FRW1-E6, FRW1-L11, FRW3-V35, FRW4-R2, FRW4-L7, FRW1-P14, FRW3-S17, FRW3-R29, FRW3-A30, FRW2-S16, FRW3-K18, FRW3-V21, FRW3-Y22, FRW3-L23, and FRW3-S27.

[0093] In some embodiments, the framework regions are derived from the llama species VHH framework regions FRW1, FRW2, FRW3, and FRW4.

[0094] In one preferred embodiment, the synthetic single-domain antibody has the following characteristics: (i) Framework areas FRW1 of sequence number 1, FRW2 of sequence number 2, FRW3 of sequence number 3, and FRW4 of sequence number 4, (ii) A functional variant framework region having one, two, or three or fewer conservative substitutions of amino acids and retaining advantageous synthetic single-domain properties, (iii) Functional variant framework regions FRW1, FRW2, FRW3, and FRW4 having at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity to SEQ ID NOs. 1-4, respectively, and retaining advantageous synthetic single-domain properties. Includes any of the following.

[0095] Typically, one or more amino acid residues within the framework region can be substituted with other amino acid residues from the same side-chain family, and novel polypeptide variants can be tested for retention of advantageous properties using the functional assays described herein.

[0096] Such advantageous characteristics are one or more of the following characteristics: i. It can be expressed as a soluble single-domain antibody in the periplasm of E. coli. When using simple centrifugation analysis, no aggregation is observed during expression, extraction, and purification from periplasm. Typically, yields exceeding 1 mg / L can be obtained with the pelB leader peptide, preferably in E. coli strains. ii. It can be expressed as a soluble intrabody in the cytosol of E. coli. When using simple centrifugation analysis, no agglutination is observed during expression, extraction, and purification from the periplasm. For example, the antibody can be expressed in E. coli strain BL21(DE3) with a yield exceeding 50 mg / liter using the T7 promoter. iii. When expressed as a fluorescent protein fusion in mammalian cell lines, aggregation does not occur.

[0097] Preferably, aggregation should not be detected when the antigen-binding protein containing a synthetic single-domain antibody is expressed as a fluorescent protein fusion. Analysis can be performed using simple fluorescence imaging.

[0098] Preferably, the amino acid residues of synthetic CDR1 and CDR2 are First letter of CDR1: Y, R, S, T, F, G, A, or D Second CDR1: Y, S, F, G or T, Third of CDR1: Y, S, S, S, F or W, The fourth letter of CDR1: Y, R, S, T, F, G, A, W, D, E, K or N, Fifth letter of CDR1: S, T, F, G, A, W, D, E, N, I, H, R, Q or L, The sixth letter of CDR1: S, T, Y, D, or E The 7th letter of CDR1: S, T, G, A, D, E, N, I or V, First of CDR2: R, S, F, G, A, W, D, E or Y, CDR2 second: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y, Third in CDR2: S, T, F, G, A, W, D, E, N, H, Q, P, The fourth letter of CDR2: G, S, T, N, or D Fifth in CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M, The sixth letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W or K, The seventh letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V The CDR3 amino acid sequence may also include 9 to 18 amino acids selected from one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

[0099] Therefore, in a preferred embodiment, the antigen-binding protein of the present invention essentially consists of a synthetic single-domain antibody of the general formula FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4.

[0100] In this embodiment, more preferably, FRW1 is SEQ ID NO: 1, or a functional variant of SEQ ID NO: 1 having one, two, or three amino acid substitutions; FRW2 is SEQ ID NO: 2, or a functional variant of SEQ ID NO: 2 having one, two, or three amino acid substitutions; FRW3 is SEQ ID NO: 3, or a functional variant of SEQ ID NO: 3 having one, two, or three amino acid substitutions; FRW4 is SEQ ID NO: 4, or a functional variant of SEQ ID NO: 4 having one, two, or three amino acid substitutions; and the amino acid sequences of CDR1 and CDR2 are the following amino acid residues: First letter of CDR1: Y, R, S, T, F, G, A, or D Second CDR1: Y, S, F, G or T, Third of CDR1: Y, S, S, S, F or W, The fourth letter of CDR1: Y, R, S, T, F, G, A, W, D, E, K or N, Fifth letter of CDR1: S, T, F, G, A, W, D, E, N, I, H, R, Q or L, The sixth letter of CDR1: S, T, Y, D, or E The 7th letter of CDR1: S, T, G, A, D, E, N, I or V, First of CDR2: R, S, F, G, A, W, D, E or Y, CDR2 second: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y, Third in CDR2: S, T, F, G, A, W, D, E, N, H, Q, P, The fourth letter of CDR2: G, S, T, N, or D Fifth in CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M, The sixth letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W or K, The seventh letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V The CDR3 amino acid sequence contains 9 to 18 amino acids selected from one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

[0101] Another aspect of the present invention relates to a nucleic acid molecule encoding the antigen-binding protein of the present invention. Accordingly, the present invention provides an isolated nucleic acid encoding at least this synthetic single-domain antibody portion of the antigen-binding protein.

[0102] Nucleic acids may be present in all cells in a cell lysate, or they may be partially purified or substantially pure forms of nucleic acids. When nucleic acids are purified from other cellular components or contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques including alkali / SDS treatment, CsCI band formation, column chromatography, agarose gel electrophoresis, and other techniques well known in the art, they shall be considered “isolated” or “substantially pure.” See F. Ausubel et al., ed., 1987 Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids of this invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In embodiments, the nucleic acid is a DNA molecule. The nucleic acid may be present in a vector, e.g., a phage display vector, or a recombinant plasmid vector. Accordingly, in a particular embodiment, the present invention provides an isolated nucleic acid or a cloning or expression vector comprising at least one or more variants that have at least 90% identity to these sequences, encoding the framework regions FRW1, FRW2, FRW3, and FRW4 of SEQ ID NOs. 1 to 4, respectively: SEQ ID NOs. 5, 6, 7, 8, or functional variants of FRW1, FRW2, FRW3, and FRW4 of SEQ ID NOs. 1 to 4.

[0103] The DNA fragments encoding the antigen-binding proteins described above and in the examples can be further manipulated by standard recombinant DNA techniques to include, for example, any signal sequence for appropriate secretion in an expression system, any purification tag, and a cleavage tag for further purification steps. In such manipulation, the DNA fragment is operably linked to another DNA molecule or to another protein, such as a fragment encoding a purification / secretion tag or a mobile linker. The term "operably linked," as used in this context, is intended to mean that the two DNA fragments are ligated functionally, for example, such that the amino acid sequence encoded by the two DNA fragments remains in frame, or that the protein is expressed under the control of a desired promoter.

[0104] The antigen-binding proteins of the present invention can be generated in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods, as is well known in the art. In the expression and generation of the recombinant antigen-binding proteins of the present invention in host cell transfectomas, those skilled in the art can advantageously utilize their general knowledge relating to the expression and recombinant generation of antibody molecules or single-domain antibody molecules.

[0105] Accordingly, the present invention provides recombinant host cells suitable for the production of this antigen-binding protein of the present invention, comprising nucleic acids and, optionally, secretory signals. In a preferred embodiment, the host cells of the present invention are mammalian cell lines. The present invention further provides the previously described method for the production of an antigen-binding protein, comprising the steps of culturing the host cells under conditions suitable for the production of the antigen-binding protein and isolating the protein.

[0106] Mammalian host cells for secreting the antigen-binding protein of the present invention may be CHO cells used in conjunction with a DHFR selection marker, e.g., as described in RJ Kaufman and PA Sharp, 1982 Mol. Biol. 159:601-621, e.g., dhfr-CHO cells (as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), NSO melanoma cells, or Invivogen's pFuse expression system, multi-Fc type system for recombinant antibody generation, BMC Biotechnol 9, 14, COS cells, and SP2 cells, or human cell lines (PER-C6 cell line, Crucell or HEK293 cells, Yves) This includes Durocher et al., 2002, Nucleic Acids Research, Vol. 30, No. 2, p. 9. When this nucleic acid encoding the antigen-binding protein of the present invention is introduced into mammalian host cells, the antigen-binding protein is produced by the expression of recombinant polypeptide in the host cells, or by the secretion of recombinant polypeptide into the culture medium for growing the host cells, and by culturing the host cells for a period of time sufficient to allow for appropriate refolding for the production of this antigen-binding protein.

[0107] The antigen-binding protein can then be recovered from the culture medium using standard protein purification methods.

[0108] In a particular embodiment, the present invention provides a multivalent antigen-binding protein, for example in the form of a complex, comprising at least two identical or different synthetic single-domain antibody amino acid sequences of the present invention. In one embodiment, the multivalent protein comprises at least two, three, or four synthetic single-domain antibody amino acid sequences. The synthetic single-domain amino acid sequences can be linked via protein fusion or covalent or non-covalent bonds.

[0109] In another embodiment, the present invention provides a composition, such as a pharmaceutical composition, comprising one or a combination of the antigen-binding proteins of the present invention, formulated with one or more pharmaceutically acceptable solvents or carriers.

[0110] The pharmaceutical formulations of the present invention can be prepared in the form of aqueous solutions, lyophilized or other dry formulations by mixing a protein of desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer (Remington: The Science and Practice of Pharmacy, 20th edition (2000)) for preservation.

[0111] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and organic esters for injection (e.g., ethyl oleate). Appropriate fluidity can be maintained, for example, by the use of a coating substance (e.g., lecithin), maintaining the particle size required in the case of a dispersion, and using a surfactant.

[0112] Furthermore, such compositions may contain adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by both the sterilization procedure described above and the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, sustained absorption of injectable pharmaceutical preparations can be achieved by the inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin.

[0113] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The use of such culture media and agents for pharmaceutically active substances is known in the art. Their use is considered in the pharmaceutical compositions of the present invention, except insofar as any conventional culture media or agent is incompatible with the active compound. Additional active compounds may also be incorporated into the compositions.

[0114] The therapeutic composition must be typically sterile and stable under manufacturing and storage conditions.

[0115] In the following, the present invention will be illustrated by the following embodiments and drawings. [Examples]

[0116] Verification of fully humanized s2dAb scaffolding Scaffold validation was performed using CDR transplantation. VHH antibody CDRs were inserted into the fully humanized single-domain scaffold described herein. Antibodies targeting various antigens (GFP, mCherry, alpha-tubulin, MUC18) were inserted, and the resulting sdAbs were used to investigate whether such fully human sdAbs functioned as their parental VHH counterparts in terms of antigen detection, presentation on phage surfaces, expression in bacterial periplasm, expression in bacterial cytosol, and expression in mammalian cell cytosol. Despite the absence of camel-specific amino acids considered essential for stability and robustness, this demonstrated that efficient generation and stability in reducing environments are possible with these fully human sdAbs.

[0117] Construction of a synthetic phage display library based on the design described herein Several methods exist for constructing synthetic and diverse libraries, but we used an oligonucleotide-based approach (provided by Twist Bioscience) here. Synthetic genes based on the design described herein were ordered and inserted into a modified pHEN2 plasmid with three myc tags. 1.6 109 I built the Gimli-1 library, which is a clone library.

[0118] Use of fully humanized sdAb Examples of fully humanized sdAbs (resulting from CDR transplantation or selection from the Gimli-1 library) were examined to validate the use of the designs disclosed herein for various antibody-based applications, including immunostaining, signaling inhibition, or cell targeting, including CAR-T cell generation. Fully human sdAbs were used as monomeric soluble forms, presented on phages, and fused to the Fc domain or to CAR-T scaffolds.

[0119] GFP-Specific Nanobody Phage Display Selection Screening was performed under natural conditions using the GFP protein as a target. Immunofluorescence detected GFP-Rab6 in 4 of the 80 non-redundant clones analyzed. Importantly, we observed that such antibodies can be used as intrabodies against recombinant GFP expressed in HeLa cells (see, e.g., anti-GFP_Gimli_D8, SEQ ID NO: 10).

[0120] Phage display options for tubulin nanobodies Screening was performed under natural conditions using biotinylated tubulin (Cytoskeleton) as the target (see Nizak, 2005, above). After three rounds of selection, 80 clones were randomly screened by immunofluorescence on methanol-fixed HeLa cells. Endogenous tubulin (34 unique sequences) was stained by 71 recombinant Abs (see, e.g., anti-tubulin Gimli B1, SEQ ID NO: 11).

[0121] Phage display selection of FGFR4-specific nanobody The identification of antibodies targeting the cell surface of cancer cells was illustrated by screening of FGFR4-targeted sdAbs. Using the fully humanized sdAb library Gimli-1, we screened for FGFR4-binding nanobodies. We performed phage display selection by three rounds of biopanning against recombinant FGFR4. To validate binding specificity to FGFR4, we used RMS cells (M. Bernasconi, University of Zurich), which are FGFR4 knockout cells, and examined 80 phage clones to screen for binding to Rh4 FGFR4 wild-type cells (Rh4-FR4wt) and Rh4 FGFR4 knockout cells (Rh4-FR4ko). Flow cytometry analysis revealed that 55 phage clones from the Gimli-1 library bound only to Rh4-FR4wt cells. Sanger sequencing of the 55 phage clones revealed 28 unique nanobodies derived from the Gimli-1 library. Next, phage clones from the Gimli library (i.e., Gimli-1: A4, F8, F11, H2) that showed the best binding to Rh4-FR4wt by flow cytometry were recombinantly expressed. As a negative control, we expressed anti-mCherry nanobodies (mCh). Recombinant nanobodies of approximately 17 kDa were modified to express a C-terminal Myc / 6xHis tag and additional cysteine ​​for maleimide coupling. High-purity protein was obtained by 6xHis tag purification and size exclusion chromatography, yielding yields ranging from 3 to 16 mg per liter of bacterial culture.

[0122] The selected nanobody binds to FGFR4-expressing cells. The binding of recombinant nanobodies to the surface of FGFR4-expressing cells was verified by flow cytometry using Rh4-FR4wt and Rh4-FR4ko cells. Surface-bound nanobodies were detected using a FITC-labeled anti-6XHis tag antibody. The three recombinant nanobodies examined did not show significant binding to Rh4-FR4wt cells (A4, F11, H2, data not shown), but recombinant nanobodies F8 (SEQ ID NO: 9) showed specific binding to Rh4-FR4wt cells but not to Rh4-FR4ko cells. As expected, the anti-mCherry negative control nanobodies did not bind to either Rh4-FR4wt or Rh4-FR4ko cells. The median fluorescence intensity (MFI) of the FGFR4 binder incubated with Rh4-FR4wt cells was in the range of 400, while anti-mCherry negative controls or anti-6xHis tag antibodies exhibited an MFI of only 200, similar to that of binding to Rh4-FR4ko cells.

[0123] Nanobody that binds to FGFR4 with high affinity To determine the binding affinity of nanobodies to FGFR4, we performed surface plasmon resonance (SPR) spectroscopy using recombinant FGFR4. As already mentioned above, FGFR1 and FGFR2 were expressed on Rh4-FR4ko cells, and flow cytometry analysis did not show nanobodies binding to the cells. To further confirm FGFR4 specificity, we also set up affinity measurements using recombinant FGFR1, FGFR2, and FGFR3. Nanobodies F8 and mCh were injected onto FGFR-coated chips at five different concentrations (see Table 1). Excluding the negative control mCh, K for FGFR4 binding was calculated. D The values ​​were within the nano and picomolar ranges (Figure 1; Table 1). Affinity parameters could not be fitted to a 1:1 binding model; the best fit was obtained using the heterologous ligand model in the BIA evaluation software, with two K values ​​for each candidate. DValues ​​were generated. Measurement of affinity for receptor family isoforms FGFR1 and FGFR3 did not show the expected binding of the analyte. SPR data confirmed strong binding of F8 to FGFR4, further suggesting that F8 possesses strict specificity.

[0124] [Table 2]

[0125] Table 2: Surface plasmon resonance spectroscopy determination of nanobody binding affinity for FGFR4 The measured data is fitted to a heterologous ligand model to determine the association and dissociation constants (k on and k off The ) was clarified and used to calculate affinity with respect to the equilibrium dissociation constant KD (koff / kon). The maximum analytic binding signal Rmax was determined from the K D Both are indicated by RU. This is analogous to their proportions in the total amount of bonded nanobodies.

[0126] Materials and methods CDR porting In silico design was performed to transplant a CDR of VHH that binds to known targets, e.g., mCherry (as well as GFP, tubulin, or MUC18), onto the scaffold disclosed herein. Synthetic genes were ordered and cloned into pHEN2-derived plasmids for expression in E. coli, phage display, and fusion to fluorescent proteins for expression in mammalian cytosols.

[0127] Soluble expression in E. coli periplasm A single-domain antibody fragment can be subcloned into a pHEN2-derived bacterial periplasm expression vector and expressed downstream of the pelB secretion sequence. Newly transformed colonies can be grown in Terrific Broth medium supplemented with 1% glucose and 100 μg / ml ampicillin antibiotic until A600 = 0.6–0.8 is reached. The expression of the 6His-tagged antibody fragment can then be induced with 500 μM isopropyl PD-thiogalactopyranoside at 16°C for 16 hours or at 28°C for 4 hours, followed by centrifugation. After centrifugation, the cell precipitate can be incubated in Tris-EDTA-sucrose osmotic shock buffer and centrifuged again. The cell lysates can be clarified and mounted on an IMAC resin affinity column for polyhistidine tagging. The eluted fraction is dialyzed, and protein purity is typically analyzed by SDS-PAGE.

[0128] Soluble expression of intrabodies in E. coli cytosol Single-domain antibody fragments can be subcloned into bacterial expression vectors under the control of the T7 promoter. Plasmid constructs can be transformed into E. coli BL21(DE3) cells. Single colonies can be grown in LB medium supplemented with 1% glucose and 100 μg / ml ampicillin antibiotic until A600 = 0.6–0.8 is reached. Antibody fragment expression can then be induced with 500 μM isopropyl β-D-thiogalactopyranoside at 16°C for 16 hours, followed by centrifugation. After centrifugation, the cell precipitate is lysed and centrifuged again. The cell lysates are clarified and typically loaded onto an IMAC resin affinity column for polyhistidine tagging. The eluted fractions are dialyzed, and protein purity is typically analyzed by SDS-PAGE.

[0129] Aggregation assays in mammalian cell expression systems Functional expression as intracellular antibodies in eukaryotic cells Single-domain antibody fragments can be subcloned into mammalian expression vectors and expressed as fusions with fluorescent proteins, typically under the control of the CMV promoter. Mammalian cell lines were transfected, and intracellular fluorescence was observed 24 or 48 hours post-transfection.

[0130] cell line Cell lines Rh4 (donated by Peter Houghton of Research Institute at Nationwide Children's Hospital, Columbus, OH), Rh30, HEK293ft, and HEK293T (purchased from LGC Promochem, ATCC) were maintained at 37°C and 5% CO2 in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin (both Thermo Fisher Scientific). The RMS cell lines were tested and validated by cell line typing analysis (STR profiling) in 2014 / 2015 and were positively matched. 48 All cell lines tested negative for mycoplasma.

[0131] Phage display selection Screening for soluble proteins was performed under natural conditions using biotinylated targets or SBP-tagged targets (e.g., extracellular FGFR4- G&P Biosciences) (as described in Nizak, C., Moutel, S., Goud, B., and Perez, F. Methods Enzymol. 403, pp. 135-153 (2005)). The single-domain antibody library disclosed herein contains 1.6 × 10⁶ units. 9It consisted of fully humanized hs2dAb. Briefly, the biotinylated antigen or SBP antigen was diluted to 10-20 nM (ultimately 1.5 mL) and ensured that it was fully collected on 50 μL of streptavidin-coated magnetic beads (Dynal). For reference, a 10 nM solution of 100-kDa protein represents 1 μg / mL of protein (and therefore per selection round). The proportion of bound and unbound samples can then be compared by Western blotting using streptavidin HRP or anti-AviTag antibody. For screening, a sufficient amount of biotinylated antigen-coated beads was left for 2 hours in a phage library (PBS + 0.1% Tween 20 + 2% nonfat milk 1 mL diluted 10 13 Incubate with the phage. The phage has previously been adsorbed onto empty streptavidin-coated magnetic beads (to remove nonspecific binders). Collect the phage bound to the streptavidin-coated beads onto the magnet. Wash 10 times (1 round) or 20 times (2 and 3 rounds) on the magnet using PBS + Tween 0.1%. Elute the bound phage with triethylamine (TEA, 100 mM), and neutralize the eluted phage with 1 M Tris pH 7.4. Elution is performed twice on the beads. Then, infect E. coli (TG1) with the eluted phage. Typically, only 10 times are used in 2 and 3 rounds. 12 Please note that phages were used as input.

[0132] Protein expression and purification Periplasmic expression of nanobodies was performed in E. coli MC1061 containing a pSB_init vector capable of producing proteins with a C-terminal cysteine ​​and a 6xHis tag. 20 ml of overnight pre-culture grown in Terrific Broth medium (25 μg / ml chloramphenicol) was diluted in 2000 ml of fresh medium and grown at 37°C for 2 hours. The temperature was then reduced to 25°C, and protein expression was induced with 0.02% L-arabinose after 1 hour. The bacterial culture was grown overnight at 25°C, and cells were collected by centrifugation (12000 g, 15 minutes). Periplasmic protein extraction was performed by osmotic shock. Cells were resuspended in 50 ml of lysis buffer 1 (50 mM Tris / HCl, pH 8.0, 20% sucrose, 0.5 mM EDTA, 5 μg / ml lysozyme, 2 mM DTT) and incubated on ice for 30 minutes. After adding ice-cold lysis buffer 2 (PBS, pH 7.5, 1 mM MgCl2, 2 mM DTT), the cell debris was collected by centrifugation (3800 g, 15 min), and imidazole was added to the protein-containing supernatant to a final concentration of 10 mM. 2+ 10 ml of bead slurry (HisPur cobalt resin, Thermo Fisher Scientific) was washed with washing buffer (PBS, pH 7.5, 30 mM imidazole, 2 mM DTT), and the supernatant was added to the beads. After incubation at 4°C for 1 hour, the beads were washed with 20 ml of washing buffer, and the bound protein was eluted with 20 ml of elution buffer (PBS, pH 7.5, 300 mM imidazole, 2 mM DTT). Prior to size exclusion chromatography (SEC), the protein eluate was dialyzed overnight with PBS, pH 7.5, 2 mM DTT, and concentrated by spin-filter centrifugation (Amicon Ultra 15, 3 kDa, Merck Millipore).

[0133] Flow cytometry The binding of selected phages and recombinant nanobodies was validated in Rh4-FR4wt and Rh4-FR4ko cells. The specificity of the selected phage clones that bind to FGFR4 was determined by flow cytometry using a 96-well plate (Becton Dickinson). Cell surface staining of Rh4-FR4wt or Rh4-FR4ko cells was performed in PBS supplemented with 1% FBS on ice. 80 μL of phage + 20 μL of PBS / 1% milk was placed on 1 × 10⁶ cells on ice. 5 The cells were incubated for 1 hour. After two washes with PBS, phage binding was detected on ice for 1 hour using a 1:250 dilution of anti-M13 antibody (27-9420-01, GE Healthcare), followed by detection for 45 minutes using a 1:400 dilution of A488 conjugate anti-mouse antibody (715-545-151, Jackson ImmunoResearch, Europe Ltd). After two washes by flow cytometry on a MACSQuant cytometer (Miltenyi), the samples were analyzed, and the results were analyzed using FlowJo software (BD Biosciences, France). Phage display was performed using anti-mCherry nanobodies as a negative control. 24 As a positive control, we used an anti-FGFR4 antibody (donated by J. Khan's lab, BT53, NCI, Bethesda, MD). For recombinant nanobody binding testing, cells were separated with acetylase (Stemcell Technologies) and washed with PBS. All subsequent steps were performed on ice: 4 × 10⁻⁶ 5 Cells were incubated with nanobodies at a concentration of 30 μg / ml for 1 hour, washed once with PBS, and incubated for a further 30 minutes with anti-His-tagged FITC-labeled antibody (LS-C57341, LSBioscience, 1:10 dilution). Cells were washed again with PBS and analyzed. Cells were washed twice with PBS and separated with acetylase. All flow cytometry measurements were performed using a Fortessa flow cytometer (BD Biosciences), and data were analyzed using FlowJo® 10.4.1 software.

[0134] Western blotting SDS-PAGE samples were divided onto 4-12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific) and blotted onto Trans-Blot Turbo Transfer Blot membranes (Biorad). After blocking the membranes with blocking buffer (5% milk / TBST) for 1 hour at room temperature, the primary antibody was added at a 1:1000 dilution and incubated overnight at 4°C. The secondary HRP conjugate antibody was diluted at 1:10,000 with blocking buffer and added to the membranes, which had been washed at room temperature for 1 hour. After incubation with Amersham® ECLTM detection reagent (GE Healthcare) or SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) using a ChemiDoc® Touch imaging system (BioRad), chemiluminescence was detected.

[0135] surface plasmon resonance spectroscopy Single-cycle kinetic analysis was performed on a CMD200M sensor chip (XanTec bioanalytics GmbH) activated with a mixture of 300 mM NHS (N-hydroxysuccinimide) and 50 mM EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) using a BIAcore T200 instrument (GE Healthcare). Recombinant FGFR1, FGFR2, FGFR3, and FGFR4 (G&P Biosciences) were immobilized onto the activated biosensor (800-12,000 RU, 1 RU = 1 pg / mm³). 2 A blocking step was performed using 1M ethanolamine. Background correction was performed using one channel per chip as a reference. After implanting nanobodies at five different concentrations, the process moved to the dissociation phase. The final dissociation step after the last implantation resulted in K offThe rate was determined. Measurements using FGFR4 were performed on each nanobody on newly immobilized proteins due to strong binding and incomplete dissociation from the surface. The immobilization flow rate was 5 μl / min, and the binding test was performed at 30 μl / min. Binding parameters were determined by heterologous ligand model fitting using BIAevaluation software. The black curve represents the measured data, and the red curve represents the fitted analysis performed.

[0136] [Table 3]

Claims

1. A process to generate nucleic acids encoding diverse synthetic single-domain antibodies having the same synthetic single-domain antibody scaffold by introducing diverse nucleic acids encoding CDR1, CDR2, and CDR3 between the respective framework coding regions of a synthetic single-domain antibody. A method for generating a synthetic single-domain antibody library, wherein the synthetic single-domain antibody scaffold includes a framework region comprising FRW1 of SEQ ID NO: 1, FRW2 of SEQ ID NO: 2, FRW3 of SEQ ID NO: 3, and FRW4 of SEQ ID NO:

4.

2. The amino acid residues of synthetic CDR1 and CDR2 follow the following rules: First letter of CDR1: Y, R, S, T, F, G, A, or D Second CDR1: Y, S, F, G or T, Third item on CDR1: Y, S, F, or W, The fourth letter of CDR1: Y, R, S, T, F, G, A, W, D, E, K or N, Fifth letter of CDR1: S, T, F, G, A, W, D, E, N, I, H, R, Q or L, The sixth letter of CDR1: S, T, Y, D, or E The 7th letter of CDR1: S, T, G, A, D, E, N, I or V, First of CDR2: R, S, F, G, A, W, D, E or Y, CDR2 second: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y, Third in CDR2: S, T, F, G, A, W, D, E, N, H, Q, P, The fourth letter of CDR2: G, S, T, N, or D Fifth in CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M, The sixth letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W or K, The seventh letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V The method according to claim 1, wherein the CDR3 amino acid sequence is determined by and comprises 9 to 18 amino acids randomly selected from one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

3. A synthetic single-domain antibody library obtained by the method described in claim 1 or 2.

4. at least 1 × 10 9 A synthetic single-domain antibody library according to claim 3, comprising several distinct antibody coding sequences.

5. Use of the synthetic single-domain antibody library according to claim 3 or 4 in a screening method for identifying synthetic single-domain antibodies that bind to a target of interest.

6. The use according to claim 5, wherein the screening method is a phage display.

7. The following formula: An antigen-binding protein containing a synthetic single-domain antibody of FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, An antigen-binding protein containing a framework region consisting of FRW1 (SEQ ID NO: 1), FRW2 (SEQ ID NO: 2), FRW3 (SEQ ID NO: 3), and FRW4 (SEQ ID NO: 4).

8. The aforementioned synthetic single-domain antibody has the following functional characteristics: i. It can be expressed as a soluble single-domain antibody in the periplasm of E. coli. ii. It can be expressed as a soluble intrabody in the E. coli cytosol. iii. When expressed as a fluorescent protein fusion in mammalian cell lines, it does not aggregate. The antigen-binding protein according to claim 7, having one or more of the above.

9. The amino acid residues of synthetic CDR1 and CDR2 are First letter of CDR1: Y, R, S, T, F, G, A, or D Second CDR1: Y, S, F, G or T, Third item on CDR1: Y, S, F, or W, The fourth letter of CDR1: Y, R, S, T, F, G, A, W, D, E, K or N, Fifth letter of CDR1: S, T, F, G, A, W, D, E, N, I, H, R, Q or L, The sixth letter of CDR1: S, T, Y, D, or E The 7th letter of CDR1: S, T, G, A, D, E, N, I or V, First of CDR2: R, S, F, G, A, W, D, E or Y, CDR2 second: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y, Third in CDR2: S, T, F, G, A, W, D, E, N, H, Q, P, The fourth letter of CDR2: G, S, T, N, or D Fifth in CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M, The sixth letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W or K, The seventh letter of CDR2: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V The antigen-binding protein according to claim 7 or 8, wherein the CDR3 amino acid sequence comprises 9 to 18 amino acids selected from one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

10. The antigen-binding protein according to any one of claims 7 to 9, further comprising an F-box domain for targeting the protein to the proteasome.

11. An isolated nucleic acid encoding an antigen-binding protein according to any one of claims 7 to 10, comprising the following nucleic acid sequences encoding framework regions FRW1, FRW2, FRW3, and FRW4 of SEQ ID NOs. 1 to 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs.

8.

12. Recombinant host cells for the production of antigen-binding proteins according to any one of claims 7 to 10, wherein the production is i. A step of culturing host cells under conditions suitable for the production of antigen-binding proteins, and ii. Step of isolating the antigen-binding protein. Recombinant host cells, including those containing the recombinant host cells.

13. Recombinant host cells according to claim 12, transformed with the isolated nucleic acid according to claim 11.

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