Highly purifiable single domain antibody

Abstract

The present application belongs to the field of biotechnology and antibody engineering pharmacy, and particularly relates to a single-domain antibody and a preparation method thereof. The present application is based on the key amino acid sites involved in the combination with Protein A, and the amino acids at the key sites of other single-domain antibodies not combined with Protein A are mutated, so that the combination ability of the mutated single-domain antibody with Protein A is significantly improved, and the mutated single-domain antibody can be purified through Protein A filler, and meanwhile the characteristics of the single-domain antibody are not changed.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and antibody engineering pharmaceuticals, and specifically relates to a single-domain antibody and its preparation method. Background Technology

[0002] Antibody drugs are widely used and have advantages such as good targeting, high specificity, and low toxicity (TRIKHA M, CORRINGHAM R, KLEIN B, et al. Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence [J]. Clinical Cancer Research, 2003, 9(13): 4653-4665.). They occupy a pivotal position in biopharmaceuticals, one of the main reasons being the mature production and purification processes for antibody drugs. Among these, affinity chromatography is the most widely used technique in the purification process. According to statistics, about 70-80% of marketed antibody drugs use Protein A or Protein G affinity chromatography for initial purification. Protein A is a cell wall surface protein from Staphylococcus aureus, containing five highly homologous immunoglobulin-binding domains. It can specifically bind to the constant region (Fc) of the heavy chain of IgG, IgM, and IgA antibodies, exhibiting high selectivity for the Fc region. Protein A affinity chromatography platform technology is suitable for most antibody production processes. Due to its high selectivity, high flow rate, and ability to remove HCP, DNA, and cell culture medium components, protein A affinity chromatography effectively removes most impurities, achieving antibody purity of over 95%. Therefore, when coupled to the matrix as an affinity ligand, it specifically binds to antibody molecules in the sample while allowing other impurities, such as nucleic acids, extraneous proteins, and potentially present viruses, to flow through. Furthermore, compared to Protein G, Protein A requires milder elution conditions, making it more widely used in the antibody capture stage and considered the "gold method" for downstream antibody purification processes.

[0003] In 1993, a natural, small, functional antibody, namely heavy chain antibody (HCAb), was first reported in the serum of camelids (HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMANS S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature, 1993, 363(6428):446-448.). Unlike traditional antibodies with heterotetrameric structures, camelid-derived HCAb does not contain light chains, nor does it contain the first fixed structural region (CH1) of the heavy chain. Its molecular weight is approximately 90 kDa. In contrast, the antigen-binding fragment in HCAbs contains only a single variable domain, called the antigen-binding variable domain of the H-chain of heavy-chain antibodies (VHH), also known as single-domain antibodies (sdAb) or nanobodies (YU X, XU Q, WU Y, et al. Nanobodies derived from camelids represent versatile). biomolecules for biomedical applications[J]. Biomaterials Science, 2020, 8(13):3559-3573.), while these single-domain antibodies have the ability to independently recognize and bind antigens after in vitro expression (MUYLDERMANS S. Applications of nanobodies[J]. Annual Review of AnimalBiosciences, 2021, 9: 401-421.).

[0004] Because single-domain antibodies only have the heavy chain variable region and lack the Fc fragment, they cannot be captured using Protein A affinity chromatography. Currently, most nanobodies are purified by adding a tag, such as histidine, MBP, or SUMO, to their N-terminus or C-terminus, using specific immobilized metal affinity chromatography (IMAC). Taking nickel affinity chromatography as an example, a histidine tag is generally added to the C-terminus of the nanobody to bind with Ni on the medium, thereby achieving specific purification of the nanobody. However, nickel affinity chromatography has poor specificity, and it is usually difficult to obtain high-purity antibodies in one step. Most importantly, the tags on nanobodies may cause various side effects, such as inducing immune responses in the body and altering the original pharmacokinetics of the drug. Therefore, the Chinese Pharmacopoeia clearly stipulates that "antibody drugs cannot contain exogenous tags." Currently, in production, exogenous tags must be removed after purification, which is not only cumbersome, with low recovery rates and difficulty in removing impurities, but also greatly increases production costs and makes it difficult to guarantee product quality. Fully human single-domain antibodies also lack the Fc region and cannot be purified by Protein A, which greatly limits the application of single-domain antibodies. Summary of the Invention

[0005] Nanobodies, also known as single-domain antibodies (sdAbs), are a special type of antibody fragment. They consist of only a single monomeric immunoglobulin domain, with a molecular weight of approximately 15 kDa, only one-tenth the size of a traditional intact antibody (approximately 150 kDa), hence the name "nano" antibody. Nanobodies typically consist of about 120-130 amino acids, and their structure follows the classic folding pattern of immunoglobulin variable regions, consisting of four framework regions (FR1-FR4) and three complementarity-determining regions (CDR1-CDR3). The CDR regions are the sites of direct contact between the antibody and the antigen, determining the antibody's specificity and affinity. The framework regions (FRs) primarily maintain the overall three-dimensional structure of the antibody (β-sandwich folding).

[0006] This invention engineered single-domain antibodies based on key amino acid sites involved in Protein A binding, significantly improving their binding affinity to Protein A. Furthermore, the engineered antibodies could all be purified using Protein A-containing packing materials. Based on this, the invention was completed.

[0007] In a first aspect, the present invention provides a single-domain antibody, which is obtained by mutating amino acids at key sites in its frame region, and the mutated single-domain antibody has a significantly improved binding ability to Protein A; the frame region is selected from the FR1 region and / or the FR3 region, and the specific key sites are selected from one or more of positions 16, 18, 20, 65, 66, 67, 72, 74, 75, 76, 77, 79, 80, 81, 88, 90, 92, and 95 (IMGT numbering rule).

[0008] Furthermore, the single-domain antibody exhibits enhanced binding ability to the D domain of Protein A.

[0009] Furthermore, the single-domain antibody exhibits enhanced binding ability to helical II and helical III of the D domain of Protein A.

[0010] Furthermore, the amino acid mutations include one or more of the following: aspartic acid (Asp, D) scan mutation, glutamate (Glu, E) scan mutation, leucine (Leu, L) scan mutation, asparagine (Asn, N) scan mutation, glutamine (Gln, Q) scan mutation, glycine (Gly, G) scan mutation, serine (S, Ser, S) scan mutation, phenylalanine (Phe, F) scan mutation, isoleucine (Ile, I) scan mutation, arginine (Arg, R) scan mutation, tyrosine (Tyr, Y) scan mutation, alanine (Ala, A) scan mutation, and / or valine (Val, V) scan mutation.

[0011] Furthermore, the amino acid mutations include one or more of the following: glutamine scanning mutation, glycine scanning mutation, serine scanning mutation, phenylalanine scanning mutation, isoleucine scanning mutation, arginine scanning mutation, tyrosine scanning mutation, alanine scanning mutation, and / or valine scanning mutation.

[0012] Furthermore, the specific mutations at the key sites are selected from one or more of G16, S18 and / or R20 in the FR1 region, or one or more of T65, Y66, Y67, K72, G74, R75, F76, T77, S79, R80, D81, Y88, Q90, N92 and / or R95 in the FR3 region (IMGT numbering rule); The specific mutations at the aforementioned key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0013] Furthermore, the specific mutations at the key sites are selected from one or more of the following in the FR3 region: T65, Y66, Y67, K72, G74, R75, F76, T77, S79, R80, D81, Y88, Q90, N92 and / or R95 (IMGT numbering rules). The specific mutations at the aforementioned key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0014] Preferably, the specific mutation at the key site is selected from one or more of K72, G74, R75, F76, R80, Y88, Q90 and / or R95 in the FR3 region; The specific mutations at the aforementioned key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0015] Furthermore, the amino acid sequence of the FR1 region of the single-domain antibody has 80%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 76%-100% homology with SEQ ID NO:27.

[0016] Furthermore, the amino acid sequence of the FR1 region of the single-domain antibody has 85%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 80%-100% homology with SEQ ID NO:27.

[0017] Preferably, the amino acid sequence of the FR1 region of the single-domain antibody has 90%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 84%-100% homology with SEQ ID NO:27.

[0018] More preferably, the amino acid sequence of the FR1 region of the single-domain antibody has 95%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 86%-100% homology with SEQ ID NO:27.

[0019] Furthermore, the single-domain antibody includes humanized single-domain antibodies or camel-derived single-domain antibodies.

[0020] Furthermore, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 80%-100% homology with SEQ ID NO.26.

[0021] Preferably, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 85%-100% homology with SEQ ID NO.26.

[0022] More preferably, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 90%-100% homology with SEQ ID NO.26.

[0023] Furthermore, the amino acid sequence of the FR3 region of the humanized single-domain antibody has 84%-100% homology with SEQ ID NO.27.

[0024] Furthermore, the humanized single-domain antibody is obtained by mutating amino acid residues at positions 72, 74, 75, 76, 80, 81, 88, 90 and / or 95 of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4.

[0025] Furthermore, the humanized single-domain antibody is obtained by mutating amino acid residues 72, 74, 75, 76, 80, 81, 88, 90 and / or 95 of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4 with Q, R, L, G, Y, N, E, D, F, A, V, S and / or I.

[0026] Furthermore, the recommended mutations for the corresponding sites of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4 are K72, G74, R75, F76, R80, D81, Y88, L90 and / or R95 sites (IMGT numbering rules). The recommended mutations for the above key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0027] Preferably, the amino acid sequences of the humanized single-domain antibody are shown in SEQ ID NO.6-SEQ ID NO.20.

[0028] More preferably, the amino acid sequences of the humanized single-domain antibody are as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9-SEQ ID NO.10, SEQ ID NO.12-SEQ ID NO.15, and SEQ ID NO.18-SEQ ID NO.20.

[0029] Furthermore, the amino acid sequence of the camel-derived single-domain antibody in the FR1 region has 80%-100% homology with SEQ ID NO.26, and the amino acid sequence of the FR3 region has 76%-100% homology with SEQ ID NO.27.

[0030] Furthermore, the camel-derived single-domain antibody is obtained by mutating amino acid residues at positions 66, 72, 80, 81, 88, 90 and / or 95 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.5.

[0031] Furthermore, the camel-derived single-domain antibody is obtained by mutating amino acid residues at positions 66, 72, 80, 81, 88, 90 and / or 95 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.5 with Q, R, L, G, Y, N and / or E mutations.

[0032] Furthermore, the recommended mutations for the corresponding sites of the single-domain antibody shown in SEQ ID NO.5 are Y66, K72, R80, D81, Y88, Q90 and / or R95 (IMGT numbering rules). The specific mutations at the aforementioned key sites are Y66, K72, and R80, for example. Y66 refers to the mutation of amino acid 66 of the single-domain antibody to Y; K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; R80 refers to the mutation of amino acid 80 of the single-domain antibody to R, and so on.

[0033] Preferably, the amino acid sequence of the camel-derived single-domain antibody is shown in SEQ ID NO.21-SEQ ID NO.25.

[0034] More preferably, the amino acid sequence of the camel-derived single-domain antibody is shown in SEQ ID NO.21 or SEQ ID NO.23.

[0035] In one embodiment of the present invention, the humanized single-domain antibodies shown in SEQ ID NO.6-SEQ ID NO.10 are obtained by mutating the 72nd, 74th and / or 76th amino acid residues (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.2 with Q, G, S, F, D and / or I mutations.

[0036] In one embodiment of the present invention, the humanized single-domain antibodies shown in SEQ ID NO.11-SEQ ID NO.15 are obtained by mutating the amino acid residues at positions 72, 74, 80 and / or 76 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.3 by R, G, A and / or V mutations.

[0037] In one embodiment of the present invention, the humanized single-domain antibodies shown in SEQ ID NO.16-SEQ ID NO.20 are obtained by D, R, F, E, L and / or G mutations in amino acid residues at positions 74, 75, 76 and / or 72 (IMGT numbering rules) of the single-domain antibody shown in SEQ ID NO.4.

[0038] In one embodiment of the present invention, the camel-derived single-domain antibodies shown in SEQ ID NO.21-SEQ ID NO.25 are obtained by mutating amino acid residues at positions 90, 95, 72, 80, 81, 88 and / or 66 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.5 with Q, R, L, G, Y, N and / or E mutations.

[0039] In a second aspect, the present invention provides a nucleic acid molecule that encodes the single-domain antibody described in the first aspect.

[0040] Furthermore, the amino acid sequence of the FR1 region of the single-domain antibody has 80%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 76%-100% homology with SEQ ID NO:27.

[0041] Furthermore, the amino acid sequence of the FR1 region of the single-domain antibody has 85%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 80%-100% homology with SEQ ID NO:27.

[0042] Preferably, the amino acid sequence of the FR1 region of the single-domain antibody has 90%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 84%-100% homology with SEQ ID NO:27.

[0043] More preferably, the amino acid sequence of the FR1 region of the single-domain antibody has 95%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 86%-100% homology with SEQ ID NO:27.

[0044] Furthermore, the single-domain antibody includes humanized single-domain antibodies or camel-derived single-domain antibodies.

[0045] Furthermore, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 80%-100% homology with SEQ ID NO.26.

[0046] Preferably, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 85%-100% homology with SEQ ID NO.26.

[0047] More preferably, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 90%-100% homology with SEQ ID NO.26.

[0048] Furthermore, the amino acid sequence of the FR3 region of the humanized single-domain antibody has 84%-100% homology with SEQ ID NO.27.

[0049] Furthermore, the humanized single-domain antibody is obtained by mutating amino acid residues at positions 72, 74, 75, 76, 80, 81, 88, 90 and / or 95 of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4.

[0050] Furthermore, the humanized single-domain antibody is obtained by mutating amino acid residues 72, 74, 75, 76, 80, 81, 88, 90 and / or 95 of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4 with Q, R, L, G, Y, N, E, D, F, A, V, S and / or I.

[0051] Furthermore, the recommended mutations for the corresponding sites of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4 are K72, G74, R75, F76, R80, D81, Y88, L90 and / or R95 sites (IMGT numbering rules); The specific mutations at the aforementioned key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0052] Preferably, the amino acid sequences of the humanized single-domain antibody are shown in SEQ ID NO.6-SEQ ID NO.20.

[0053] More preferably, the amino acid sequences of the humanized single-domain antibody are as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9-SEQ ID NO.10, SEQ ID NO.12-SEQ ID NO.15, and SEQ ID NO.18-SEQ ID NO.20.

[0054] Furthermore, the amino acid sequence of the camel-derived single-domain antibody in the FR1 region has 80%-100% homology with SEQ ID NO.26, and the amino acid sequence in the FR3 region has 76%-100% homology with SEQ ID NO.27.

[0055] Furthermore, the camel-derived single-domain antibody is obtained by mutating amino acid residues at positions 66, 72, 80, 81, 88, 90 and / or 95 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.5.

[0056] Furthermore, the camel-derived single-domain antibody is obtained by mutating amino acid residues at positions 66, 72, 80, 81, 88, 90 and / or 95 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.5 with Q, R, L, G, Y, N and / or E mutations.

[0057] Furthermore, the recommended mutations for the corresponding sites of the single-domain antibody shown in SEQ ID NO.5 are Y66, K72, R80, D81, Y88, Q90 and / or R95 (IMGT numbering rules). The specific mutations at the aforementioned key sites are Y66, K72, and R80, for example. Y66 refers to the mutation of amino acid 66 of the single-domain antibody to Y; K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; R80 refers to the mutation of amino acid 80 of the single-domain antibody to R, and so on.

[0058] Preferably, the amino acid sequence of the camel-derived single-domain antibody is shown in SEQ ID NO.21-SEQ ID NO.25.

[0059] More preferably, the amino acid sequence of the camel-derived single-domain antibody is shown in SEQ ID NO.21 or SEQ ID NO.23.

[0060] Thirdly, an expression vector comprising a nucleic acid molecule as described in the second aspect of the invention.

[0061] Furthermore, the expression vector includes, but is not limited to, plasmids, bacteriophages, granules, or viral vectors.

[0062] Fourthly, a host cell comprising the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, wherein the host cell is transformed or transfected by the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect.

[0063] Furthermore, the host cells include bacteria, fungi, and / or animal cells.

[0064] Fifthly, the present invention provides an antibody conjugate comprising an antibody portion and a conjugation portion conjugated to the antibody portion; the antibody portion comprising the single-domain antibody described in the first aspect.

[0065] Furthermore, the coupling portion is selected from drugs or other markers.

[0066] Furthermore, the drug is a small molecule drug; the label is selected from fluorescent labels, chemiluminescent labels, chromogenic labels, or enzymes.

[0067] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the domain antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or the antibody-drug conjugate described in the fifth aspect.

[0068] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0069] Furthermore, the dosage form of the pharmaceutical composition includes, but is not limited to, one or more of aerosols, solutions, suspensions, and / or emulsions.

[0070] In a seventh aspect, the present invention provides a method for improving the binding ability of a single-domain antibody to Protein A, the method comprising mutating amino acids at key sites in the frame region of the single-domain antibody to significantly improve its binding ability to Protein A; the frame region is selected from the FR1 region and / or the FR3 region, and the specific key sites are selected from one or more of positions 16, 18, 20, 65, 66, 67, 72, 74, 75, 76, 77, 79, 80, 81, 88, 90, 92, and 95 (IMGT numbering rule).

[0071] Furthermore, the single-domain antibody exhibits enhanced binding ability to the D domain of Protein A.

[0072] Furthermore, the single-domain antibody exhibits enhanced binding ability to helical II and helical III of the D domain of Protein A.

[0073] Furthermore, the amino acid mutations include one or more of the following: aspartic acid (Asp, D) scan mutations, glutamate (Glu, E) scan mutations, leucine (Leu, L) scan mutations, asparagine (Asn, N) scan mutations, glutamine (Gln, Q) scan mutations, glycine (Gly, G) scan mutations, serine (S), phenylalanine (Phe, F) scan mutations, isoleucine (Ile, I) scan mutations, arginine (Arg, R) scan mutations, tyrosine (Tyr, Y) scan mutations, alanine (Ala, A) scan mutations, and / or valine (Val, V) scan mutations.

[0074] Furthermore, the amino acid mutations include one or more of the following: glutamine scanning mutation, glycine scanning mutation, serine scanning mutation, phenylalanine scanning mutation, isoleucine scanning mutation, arginine scanning mutation, tyrosine scanning mutation, alanine scanning mutation, and / or valine scanning mutation.

[0075] Furthermore, the specific mutations at the key sites are selected from one or more of G16, S18 and / or R20 in the FR1 region, or one or more of T65, Y66, Y67, K72, G74, R75, F76, T77, S79, R80, D81, Y88, Q90, N92 and / or R95 in the FR3 region (IMGT numbering rule); The specific mutations at the aforementioned key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0076] Furthermore, the specific mutations at the key sites are selected from one or more of the following in the FR3 region: T65, Y66, Y67, K72, G74, R75, F76, T77, S79, R80, D81, Y88, Q90, N92 and / or R95 (IMGT numbering rules). The specific mutations at the aforementioned key sites, taking K72, G74, and R75 as examples, refer to the mutation of amino acid position 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid position 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid position 75 of the single-domain antibody to R, and so on. Preferably, the specific mutation at the key site is selected from one or more of K72, G74, R75, F76, R80, Y88, Q90 and / or R95 in the FR3 region; The specific mutations at the aforementioned key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0077] Furthermore, the amino acid sequence of the FR1 region of the single-domain antibody has 80%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 76%-100% homology with SEQ ID NO:27.

[0078] Furthermore, the amino acid sequence of the FR1 region of the single-domain antibody has 85%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 80%-100% homology with SEQ ID NO:27.

[0079] Preferably, the amino acid sequence of the FR1 region of the single-domain antibody has 90%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 84%-100% homology with SEQ ID NO:27.

[0080] More preferably, the amino acid sequence of the FR1 region of the single-domain antibody has 95%-100% homology with SEQ ID NO:26, and the amino acid sequence of the FR3 region has 86%-100% homology with SEQ ID NO:27.

[0081] Furthermore, the single-domain antibody includes humanized single-domain antibodies or camel-derived single-domain antibodies.

[0082] Furthermore, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 80%-100% homology with SEQ ID NO.26.

[0083] Preferably, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 85%-100% homology with SEQ ID NO.26.

[0084] More preferably, the amino acid sequence of the FR1 region of the humanized single-domain antibody has 90%-100% homology with SEQ ID NO.26.

[0085] Furthermore, the amino acid sequence of the FR3 region of the humanized single-domain antibody has 84%-100% homology with SEQ ID NO.27.

[0086] Furthermore, the humanized single-domain antibody is obtained by mutating amino acid residues at positions 72, 74, 75, 76, 80, 81, 88, 90 and / or 95 of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4.

[0087] Furthermore, the humanized single-domain antibody is obtained by mutating amino acid residues 72, 74, 75, 76, 80, 81, 88, 90 and / or 95 of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4 with Q, R, L, G, Y, N, E, D, F, A, V, S and / or I.

[0088] Furthermore, the recommended mutations for the corresponding sites of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4 are K72, G74, R75, F76, R80, D81, Y88, L90 and / or R95 sites (IMGT numbering rules). The specific mutations at the aforementioned key sites are K72, G74, and R75, for example. K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; G74 refers to the mutation of amino acid 74 of the single-domain antibody to G; R75 refers to the mutation of amino acid 75 of the single-domain antibody to R, and so on.

[0089] Furthermore, the amino acid sequence of the camel-derived single-domain antibody in the FR1 region has 80%-100% homology with SEQ ID NO.26, and the amino acid sequence of the FR3 region has 76%-100% homology with SEQ ID NO.27.

[0090] Furthermore, the camel-derived single-domain antibody is obtained by mutating amino acid residues at positions 66, 72, 80, 81, 88, 90 and / or 95 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.5.

[0091] Furthermore, the camel-derived single-domain antibody is obtained by mutating amino acid residues at positions 66, 72, 80, 81, 88, 90 and / or 95 (IMGT numbering rule) of the single-domain antibody shown in SEQ ID NO.5 with Q, R, L, G, Y, N and / or E mutations.

[0092] Furthermore, the recommended mutations for the corresponding sites of the single-domain antibody shown in SEQ ID NO.5 are Y66, K72, R80, D81, Y88, Q90 and / or R95 (IMGT numbering rules). The specific mutations at the aforementioned key sites are Y66, K72, and R80, for example. Y66 refers to the mutation of amino acid 66 of the single-domain antibody to Y; K72 refers to the mutation of amino acid 72 of the single-domain antibody to K; R80 refers to the mutation of amino acid 80 of the single-domain antibody to R, and so on.

[0093] Beneficial effects This invention analyzes the complex structure of fully human single-domain antibodies with nanomolar affinity for Protein A, and discovers that the FR1 and FR3 regions of the single-domain antibodies are key amino acid sites involved in binding to Protein A. Based on these sites, other fully human single-domain antibodies and camel-derived single-domain antibodies that do not bind to Protein A are engineered to significantly improve their binding ability to Protein A. Furthermore, the modified antibodies can be purified using commercially available Protein A packing material without altering their inherent properties. Attached Figure Description

[0094] Figure 1 This is the crystal structure of a complex of a fully human single-domain antibody and a Protein A domain protein. Note: A. Structure of the complex of the single-domain antibody and the Protein A domain; green represents the single-domain antibody, orange represents CDR1 / 2, and red represents CDR3; B. Interaction between the single-domain antibody and the Protein A domain; all interacting amino acids are indicated by stick, hydrogen bonds are indicated by black dashed lines, and salt bridges are indicated by red dashed lines.

[0095] Figure 2 This study aimed to identify key binding sites of fully human single-domain antibodies to the Protein A domain. Note: A. SDS-PAGE identification of wild-type n501 and its alanine mutant; B. ELISA detection of binding of wild-type n501 and its alanine mutant to the Protein A domain; C. BLI detection of binding of wild-type n501 and its alanine mutant to the Protein A domain.

[0096] Figure 3 The amino acid sequences of four single-domain antibodies to be modified are compared with those of n501. Note: The amino acid sequence comparison results of the antibodies to be modified n622, n67, n118 and VHH3 with the FR1 / FR3 amino acid sequence of n501 are shown.

[0097] Figure 4 To enhance the ability of single-domain antibodies to bind to Protein A through key site modification. Note: BLI assay was used to detect the affinity of the key site-modified mutants of antibodies n622(A), n67(B), n118(C), and VHH3 for the Protein A domain.

[0098] Figure 5 Sequence alignment of four mutant strains of single-domain antibodies.

[0099] Figure 6 This is the crystal structure of the complex of the camel-derived single-domain antibody mutant M1 and the Protein A domain protein. Note: A. Structure of the complex of the camel-derived single-domain antibody and the Protein A domain; pink indicates the camel-derived single-domain antibody, orange indicates CDR1 / 2, and red indicates CDR3; B. Interaction between the camel-derived single-domain antibody and the Protein A domain; all interacting amino acids are indicated by stick, hydrogen bonds are indicated by black dashed lines, and salt bridges are indicated by red dashed lines.

[0100] Figure 7 The results of SDS-PAGE analysis of the modified single-domain antibodies after purification with Protein A and Ni-NTA are shown. Note: Wild-type and mutant antibodies n622(A), n118(B), VHH3(C), and n67(D) were purified using Ni-NTA and Protein A packing materials, respectively. After elution with the same volume of elution buffer, the results were analyzed by SDS-PAGE.

[0101] Figure 8 This represents the change in the antigen-binding ability of the modified single-domain antibody. Note: ELISA was used to detect the antigen-binding ability of the wild-type and modified mutants of the antibodies n622(A), n67(B), n118(C), and VHH3(D). Detailed Implementation

[0102] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0103] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0104] This invention uses the IMGT numbering system to number single-domain antibody amino acid sites. The IMGT (International Immunogenetic Information System) protein numbering system is a standardized numbering system based on its own ontology (IMGT-ONTOLOGY). It is the "gold standard" in immunoinformatics, aiming to provide unified, accurate, and three-dimensional structure-related numbers for molecules such as immunoglobulins (antibodies, IG), T-cell receptors (TR), and major histocompatibility complex (MH). The core of this system is IMGT numbering, which achieves precise sequence description through a fixed set of "anchor points" and a standardized amino acid sequence.

[0105] The scanning mutation described in this invention is an amino acid scanning mutation, which refers to a mutation method in which each amino acid residue is replaced one by one with other amino acids in a specific region (structural domain, functional segment, protein-protein interaction interface, etc.) of a target protein in sequence (most commonly alanine, i.e., alanine scanning). By detecting the functional, binding activity, enzyme kinetics, stability or phenotypic changes of the mutant, the mutation site that makes a key contribution to the structure or function of the protein is located.

[0106] Table 1. This invention relates to amino acid sequences. Example 1: Preparation of Protein A Resin

[0107] The pComb3X plasmid was transferred into HB2151 competent cells. Colonies were picked from solid agar plates and inoculated into liquid medium containing ampicillin (Amp) at a final concentration of 100 µg / mL. The culture was shaken until the medium became turbid. The bacterial culture was then transferred to SB liquid medium (Amp final concentration 100 µg / mL) and cultured until the OD value reached 0.6–0.8. IPTG (final concentration 1 mM) was added to induce protein expression, and the culture was continued. Protein A resin was used for purification. First, a gravity chromatography column was prepared. After rinsing, 0.5 mL of well-mixed Protein A resin was loaded into the column and washed with 5 times the resin volume of Binding / Wash Buffer to remove residual ethanol. The supernatant containing the target protein was added to the gravity chromatography column. After all the supernatant had flowed out, the column was washed with 30 times the resin volume of Binding / Wash Buffer to remove any contaminating proteins. The target protein was then eluted with 10-15 column volumes of Elution Buffer into Neutralization Buffer containing 1 / 10 of the Elution Buffer volume, maintaining the target protein at pH 7.4. Protein A resin was then washed again with Elution Buffer. Finally, the Protein A resin was recovered for reuse. Example 2: Crystal structure of the fully human single-domain antibody n501 complex with Protein A domain

[0108] A fully human single-domain antibody, n501, with nanomolar affinity for Protein A (Genscript, L00210) was selected. The sequence of n501 is shown in SEQ ID NO.1. The crystal structure of the complex of the fully human single-domain antibody n501 and the Protein A domain protein is shown in [image missing]. Figure 1 As shown, the fully human single-domain antibody interacts with helical II and helical III of the D domain of Protein A through its conserved backbone regions FR1 and FR3, wherein the three CDR regions of the antibody do not participate ( Figure 1 A) indicates that other single-domain antibodies can achieve Protein A binding performance by modifying their backbone region.

[0109] Further analysis revealed that the fully human single-domain antibody n501 involves a total of 12 amino acids in its interactions, including G16, S18, and R20 in the FR1 region and T65, Y67, K72, G74, T77, S79, Q90, N92, and R95 in the FR3 region. Six of these amino acids are located on the β chain of the FR region, while the other six are located between the chain and loop in the backbone region furthest from the CDR. Specifically, the amino acids involved in the interaction of the D domain of Protein A are A28, Q29, G32, F33, Q35, S36, and D39 in helix II, and N46, V47, E50, and K53 in helix III. D40, also consisting of 12 amino acids, is located in the loop between helix II and helix III. Both interacting surfaces are mainly composed of polar side chains. The fully human single-domain antibody has three positively charged amino acids (R20, K72, R95), which generate a strong electrostatic attraction with three negatively charged amino acids (D39, D40, E50) on the D domain. Two salt bridges are formed between R20 and D39. Figure 1 B). The fully human single-domain antibody formed 7 pairs of hydrogen bonds with the amino acids on the helical II of the D domain. Figure 1 B), where the K72 backbone, G74 and R95 and the four amino acids on the helical III of the D domain form hydrophobic interactions under the action of salt bridges. Example 3: Identification of key binding sites between fully human single-domain antibody n501 and Protein A domain protein

[0110] Analysis of the crystal structure of the fully human single-domain antibody complex with the Protein A domain revealed that 12 amino acid sites in the fully human single-domain antibody are involved in binding to Protein A. According to the IMGT numbering system, these sites are G16, S18, R20, T65, Y67, K72, G74, T77, S79, Q90, N92, and R95. After mutating these 12 sites to alanine using an alanine scan, changes in the binding affinity of these mutants to Protein A were detected using ELISA and BLI methods, respectively. Figure 2 As shown in Figure A, all 12 mutants could be expressed normally. In the ELISA experiment, the binding affinity of all mutants to Protein A was decreased, with changes at sites 20, 65, 67, 74, and 90 having the greatest impact on Protein A binding. Figure 2 B). In the BLI experiment, consistent with the ELISA results, the affinity of all mutants for Protein A decreased, indicating that these 12 sites are crucial for the binding of single-domain antibodies to Protein A. Figure 2 C). Example 4: The modified single-domain antibody acquired Protein A binding ability.

[0111] Three fully human single-domain antibodies that do not bind Protein A—n622 (sequence shown in SEQ ID NO.2), n67 (sequence shown in SEQ ID NO.3), and n118 (sequence shown in SEQ ID NO.4)—and one camel-derived single-domain antibody that does not bind Protein A—VHH3 (sequence shown in SEQ ID NO.5)—were selected. The amino acid sequences of the four single-domain antibodies to be modified were compared with those of n501 as follows: Figure 3 As shown. The FR1 amino acid sequence homology of the three fully human single-domain antibodies n622, n67, and n118 with that of n501 is 100%, and the homology of FR3 is 86%, 86%, and 84%, respectively. The amino acid sequence homology of the FR1 and FR3 of the camel-derived single-domain antibody VHH3 with that of n501 is 80% and 76%, respectively. For n622, n67, and n118, all but one of the 12 key amino acid sites that bind to protein A are identical to those of n501, except for amino acid position 74. Therefore, as... Figure 4 As shown in AC, different combinations of amino acid mutations were designed. Sequence alignment of the four single-domain antibody-modified mutants is as follows: Figure 5 As shown. Specifically: In n622, mutant M1 introduces mutations in K72Q and S74G, M2 introduces mutations in K72S, S74G, and V76F, M3 introduces mutations in K72D and S74G, M4 additionally introduces mutations in K72Q, S74G, and V76I, and M5 additionally introduces mutations in K72Q, S74G, and V76F. The sequences of the n622 mutants M1, M2, M3, M4, and M5 are shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10, respectively. The affinities of the five mutants to Protein A are 61.6 nM, 77.1 nM, 0 nM, 86.2 nM, and 316 nM, respectively. Figure 4 A).

[0112] In n67, mutant M1 introduces mutations in K72R, S74G, and R80A; M2 introduces mutations in S74G and R80A; M3 introduces mutations in S74G, V76A, and R80A; M4 introduces mutations in S74G and R80V; and M5 introduces a mutation in S74G. The sequences of n67 mutants M1, M2, M3, M4, and M5 are shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively. The affinities of the five mutants to Protein A are 0 nM, 181.1 nM, 719.4 nM, 186.3 nM, and 34.11 nM, respectively. Figure 4 B).

[0113] In n118, mutant M1 introduces mutations in S74D, L75R, and V76F; M2 introduces mutations in K72E, L75R, and V76L; M3 introduces mutations in S74G, L75R, and V76F; M4 introduces mutations in K72R, S74G, and L75R; and M5 introduces mutations in S74G and L75R. The sequences of the n118 mutants M1, M2, M3, M4, and M5 are shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, respectively. The affinities of the five mutants to Protein A are 0 nM, 0 nM, 62.7 nM, 127 nM, and 147.8 nM, respectively. Figure 4 C).

[0114] For VHH3, all but one of the 12 key amino acid sites that bind to protein A are identical to n501, except for amino acid position 90. For example... Figure 4 As shown in D, different combinations of amino acid mutations were designed. M1 introduced mutations of L90Q and K95R; M2 introduced mutations of K72R, R80L, and D81G; M3 introduced mutations of D88Y, L90Q, and K95R; M4 introduced mutations of Y66N and K72E; and M5 introduced a mutation of K72R. The sequences of the VHH3 mutants M1, M2, M3, M4, and M5 are shown in SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25, respectively. The affinities of the five mutants to Protein A were 504 nM, 0 nM, 451 nM, 0 nM, and 0 nM, respectively.

[0115] In summary, by simultaneously mutating different combinations of amino acids around key sites, the affinity of four single-domain antibodies for protein A was significantly improved. Example 5: Crystal structure of the complex of camel-derived single-domain antibody VHH3 mutant M1 and Protein A domain protein

[0116] The crystal structure of the complex of camel-derived single-domain antibody VHH3 mutant M1 and Protein A domain protein is shown below. Figure 6 As shown, camel-derived single-domain antibodies interact with helical II and helical III of the Protein AD domain through their conserved backbone regions FR1 and FR3, while the three CDR regions of the antibody do not participate ( Figure 6 A). Further analysis revealed that 13 amino acids in the camel-derived single-domain antibody VHH3 mutant M1 are involved in binding to Protein A, including G16, S18, and R20 in the FR1 region and T65, Y67, K72, G74, T77, S79, Q90, N92, S93, and R95 in the FR3 region. Six of these amino acids are located on the β chain of the FR domain, while the other seven are located between the chain and ring in the backbone region furthest from the CDR. The amino acids involved in the D domain interaction are specifically A28, Q29, G32, F33, Q35, S36, and D39 in helix II, and N46, V47, E50, and L54 in helix III. Both interacting surfaces are mainly composed of polar side chains. The camel-derived single-domain antibody has three positively charged amino acids (R20, K72, R95), which generate a strong electrostatic attraction with the three negatively charged amino acids (D39, D40, E50) on the D domain. Among them, two salt bridges are formed between R20 and D39. Figure 6 B). The camel-derived single-domain antibody forms 7 pairs of hydrogen bonds with the amino acids on the helix II of the D domain. The K72 backbone, G74, and R95 on the antibody form hydrophobic interactions with the 4 amino acids on the helix III of the D domain through the action of salt bridges. Example 6: The modified single-domain antibody can be captured by Protein A affinity chromatography.

[0117] The ability to purify the modified single-domain antibodies (mutants M1, M2, M4, and M5 of n622, M3, M4, and M5 of n118, M2, M3, M4, and M5 of n67, and M1 and M3 of VHH3) using Protein A affinity chromatography was verified. The supernatant obtained from the lysed bacterial cells after induction was simultaneously purified using commercially available Protein A (GenScript, L00210) packing material. Figure 7The results showed that these modified antibodies were better captured by Protein A filler than wild-type antibodies, and the protein yield was similar to that obtained by Ni-NTA purification. Example 7: Changes in the antigen-binding ability of the modified single-domain antibody

[0118] The ability of the modified single-domain antibodies (mutants M1, M2, M4, and M5 of n622, mutants M3, M4, and M5 of n118, mutants M2, M3, M4, and M5 of n67, and mutants M1 and M3 of VHH3) to bind to the antigen was validated. Figure 8 As shown, most of the successfully modified single-domain antibodies did not exhibit significant changes in antigen-binding ability compared to the wild type. Specifically: In n622, wild-type n622 (n622-WT) binds to its antigen CEACAM5 via EC2. 50 The EC50 of the four mutants M1, M2, M4, and M5 was 3.014 nM. 50 The values ​​were 2.248 nM, 0.952 nM, 1.392 nM, and 1.482 nM, respectively, showing no significant decrease.

[0119] In n118, wild-type n118 (n118-WT) binds to its antigen CD16A via EC. 50 The EC50 of the three mutants M3, M4, and M5 was 0.323 nM. 50 The values ​​were 0.658 nM, 0.994 nM, and 2.002 nM, respectively, showing no significant decrease. In VHH3, wild-type VHH3 (VHH3-WT) binds to its antigen TNFα via EC. 50 The EC50 of the two mutants, M1 and M3, was 1.868 nM. 50 The values ​​were 2.605 nM and 2.388 nM, respectively, showing no significant decrease.

[0120] However, in n67, wild-type n67 (n67-WT) binds to its antigen Zika E protein via EC. 50 The EC50 of the four mutants M2, M3, M4, and M5 is 155 nM. 50 The values ​​were 2675 nM, 4127 nM, 12555 nM, and 156.4 nM, respectively. Except for M5, the binding ability of the other three mutants was significantly reduced.

[0121] Table 2-1 n501 and its mutants, other modified single-domain antibodies (FR1 sequence) and their affinity for ProA Table 2-2 FR2 sequences of n501 and its mutants, other modified single-domain antibodies, and their affinity for ProA. Table 2-3 FR3 sequences of n501 and its mutants, other modified single-domain antibodies, and their affinity for ProA. Table 2-4 FR3 sequences of n501 and its mutants, other modified single-domain antibodies, and their affinity for ProA. Table 2-5 FR4 sequences of n501 and its mutants, other modified single-domain antibodies, and their affinity for ProA. Table 2-6 CDR1 and CDR2 sequences of n501 and its mutants, other modified single-domain antibodies, and their affinity for ProA. Note: In the table above, numbers 1-128 are used to number the amino acid sites of the single-domain antibody described in this invention according to the IMGT numbering system.

Claims

1. A single-domain antibody, wherein the single-domain antibody is obtained by mutating amino acids at key sites in its framework region, and the mutated single-domain antibody has a significantly improved binding ability to Protein A; The frame region is selected from the FR1 region and / or FR3 region, and the specific key sites are selected from one or more of 16, 18, 20, 65, 66, 67, 72, 74, 75, 76, 77, 79, 80, 81, 88, 90, 92, and 95. The amino acid mutations include one or more of the following: aspartic acid scanning mutation, glutamic acid scanning mutation, leucine scanning mutation, asparagine scanning mutation, glutamine scanning mutation, glycine scanning mutation, serine scanning mutation, phenylalanine scanning mutation, isoleucine scanning mutation, arginine scanning mutation, tyrosine scanning mutation, alanine scanning mutation, and / or valine scanning mutation.

2. The single-domain antibody as described in claim 1, wherein the single-domain antibody comprises a humanized single-domain antibody or a camel-derived single-domain antibody; The amino acid sequence of the humanized single-domain antibody in the FR1 region has 80%-100% homology with SEQ ID NO.26, and the amino acid sequence of the FR3 region has 84%-100% homology with SEQ ID NO.

27. The amino acid sequence of the camel-derived single-domain antibody in the FR1 region has 80%-100% homology with SEQ ID NO.26, and the amino acid sequence in the FR3 region has 76%-100% homology with SEQ ID NO.

27.

3. The single-domain antibody as claimed in claim 1, wherein the humanized single-domain antibody is obtained by mutating amino acid residues 72, 74, 75, 76, 80, 81, 88, 90 and / or 95 of the single-domain antibodies shown in SEQ ID NO.2-SEQ ID NO.4 by Q, R, L, G, Y, N, E, D, F, A, V, S and / or I mutations. The camel-derived single-domain antibody was obtained by mutating amino acid residues at positions 66, 72, 80, 81, 88, 90 and / or 95 (IMGT numbering rules) of the single-domain antibody shown in SEQ ID NO.5 with Q, R, L, G, Y, N and / or E mutations.

4. The single-domain antibody as described in claim 1, wherein the amino acid sequence of the humanized single-domain antibody is shown in SEQ ID NO.6-SEQ ID NO.20, and the amino acid sequence of the camel-derived single-domain antibody is shown in SEQ ID NO.21-SEQ ID NO.

25.

5. A nucleic acid molecule, said nucleic acid molecule encoding the single-domain antibody of claim 1.

6. An expression vector comprising the nucleic acid molecule as described in claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the expression vector of claim 6, wherein the host cell is transformed or transfected by the nucleic acid molecule of claim 5 and / or the expression vector of claim 6.

8. An antibody conjugate comprising an antibody portion and a conjugation portion conjugated to the antibody portion; wherein the antibody portion comprises the single-domain antibody of claim 1.

9. A pharmaceutical composition comprising the single-domain antibody of claim 1, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, or the antibody-drug conjugate of claim 8.

10. A method for improving the binding ability of a single-domain antibody to Protein A, the method comprising mutating amino acids at key sites in the frame region of the single-domain antibody to significantly improve its binding ability to Protein A; wherein the frame region is selected from the FR1 region and / or the FR3 region, and the specific key sites are selected from one or more of positions 16, 18, 20, 65, 66, 67, 72, 74, 75, 76, 77, 79, 80, 81, 88, 90, 92, and 95; wherein the amino acid mutation includes one or more of the following: aspartic acid scanning mutation, glutamic acid scanning mutation, leucine scanning mutation, asparagine scanning mutation, glutamine scanning mutation, glycine scanning mutation, serine scanning mutation, phenylalanine scanning mutation, isoleucine scanning mutation, arginine scanning mutation, tyrosine scanning mutation, alanine scanning mutation, and / or valine scanning mutation.

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