Anti-human IgG1 Fc tag single-domain antibody and application thereof

Abstract

The invention relates to a single-domain antibody of an anti-human IgG1 Fc tag. The single-domain antibody has the following complementarity determining regions: CDR1, CDR2 and CDR3; wherein the amino acid sequence of the CDR1 is as shown in any one of SEQ ID NO.44-86, the amino acid sequence of the CDR2 is as shown in any one of SEQ ID NO.87-129, and the amino acid sequence of the CDR3 is as shown in any one of SEQ ID NO.130-172. The single-domain antibody of the anti-human IgG1 Fc tag is short in research and development cycle, high in antibody quality and low in development and production cost, has outstanding advantages in the aspects of antibody affinity, targeting specificity, small molecular size, stability, modifiability, production cost and the like, has very high tissue penetrability and stability, and can be used for preparing the anti-human IgG1 Fc tag. According to the present invention, with the application of the polypeptide, the polypeptide can easily penetrate through the tissue and the cell barrier, penetrate into the lesion tissue to provide effects, and can tolerate the temperature, the pH value and the chemical environment change in a wide range so as to provide advantages in various application scenarios.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to single-domain antibodies against human IgG1 Fc tags and their applications. Background Art

[0002] Human IgG1 is a subtype of human immunoglobulin G (IgG). IgG is an antibody secreted by plasma cells, and its basic structure is a Y-shaped molecule formed by two identical heavy chains (γ chains) and two identical light chains (κ or λ chains) connected by interchain disulfide bonds. The molecular weight of the heavy chain is about 50 - 70 kDa, and the molecular weight of the light chain is about 25 kDa. This structure endows the IgG1 molecule with sufficient stability and flexibility to meet different immune function requirements. The heavy chain of IgG1 belongs to the γ1 subclass, and its structure determines its various functions. IgG1 can specifically recognize and bind to antigens on the surfaces of foreign pathogens (such as bacteria, viruses, etc.). For example, when a virus invades the human body, the variable region of IgG1 can precisely recognize specific protein antigens on the virus surface. This specific binding is like a key fitting a lock, providing a target for subsequent immune responses. Through antigen-antibody binding, IgG1 can neutralize the activity of the virus and prevent the virus from entering host cells. For some bacterial toxins, IgG1 can also bind to them, rendering them non-toxic, thereby protecting the body from damage. IgG1 is also an activator of the classical pathway of complement activation. IgG1 shows potential application values in the fields of disease diagnosis, immunotherapy, and vaccine research and development, contributing to the further development and innovation of related fields.

[0003] The Fc fragment (fragment crystallizable) of human IgG1 is composed of the C-terminal parts (CH2 and CH3) of two heavy chains. The Fc fragment plays a core role in various functions of IgG1. It contains binding sites for complement. When IgG1 binds to an antigen, the conformation of the Fc fragment changes, enabling complement C1q to bind to it. C1q is the starting molecule of the classical activation pathway of the complement system, and its binding triggers a series of cascade reactions, thereby initiating the classical pathway of complement activation. In addition, the Fc fragment also contains binding sites for Fc receptors on the surfaces of immune cells (such as FcγRIII), and can bind to Fc receptors (FcγRIII) on the surfaces of immune cells such as natural killer (NK) cells and macrophages. This enables IgG1 to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and opsonophagocytosis. When IgG1 binds to an antigen on the surface of a target cell (such as a virus-infected cell or a tumor cell), immune cells can recognize the target cell through the binding of the Fc receptor to IgG1. Through the interaction between the Fc receptor and immune cells, IgG1 can direct immune cells to kill or phagocytose the target cell.

[0004] The human IgG1 Fc fragment has been widely used as a tag for recombinant protein antigens in animal immunization experiments or in vivo studies. In animal immunization, it can promote the dimerization of recombinant proteins, thereby enhancing immunogenicity (while the tag itself does not have strong immunogenicity), facilitating the easy expression, purification, good activity, long half-life, and good stability of recombinant proteins, and helping to obtain a better antigen-specific immune response. In experimental applications, the IgG1 Fc-tag antibody can be used for affinity chromatography. During the expression of recombinant proteins, the target protein is fused with the human IgG1 Fc tag for expression. Since the Fc-tag antibody can specifically recognize the Fc tag, the Fc-tag antibody can be immobilized on a chromatography medium (such as agarose gel) to obtain highly pure target proteins. At the same time, the human IgG1 Fc-tag antibody can also be used in experiments such as immunoblotting, ELISA, and immunoprecipitation. In cell biology research, the human IgG1 Fc-tag antibody can be used to study the intracellular localization of proteins by binding to proteins with Fc tags. For example, through immunofluorescence technology, the Fc-tag antibody is fluorescently labeled and then bound to the Fc-tag fusion protein in cells, and then the distribution of the fluorescence signal is observed under a fluorescence microscope to determine the specific location of the fusion protein in cells (such as in the nucleus, cytoplasm, or cell membrane, etc.). Moreover, in cell function research, the human IgG1 Fc-tag antibody can be used to interfere with or regulate the function of proteins with Fc tags. For example, by introducing the human IgG1 Fc-tag antibody into cells, the antibody binds to the human IgG1 Fc-tag protein, which may block the normal binding of the Fc-tag protein to other ligands or receptors, and thus observe the impact of this interference on cell functions (such as cell proliferation, differentiation, or signal transduction, etc.), helping to deeply understand the functional mechanism of proteins. Therefore, developing antibodies against human IgG1 Fc has many important uses. In terms of detection and quantitative analysis, it can be used as a primary antibody in experiments such as Western Blot and ELISA or for capturing recombinant proteins containing this tag, determining the expression of recombinant proteins and quantifying their concentrations, and assisting in optimizing expression conditions and quality control. For purifying recombinant proteins, it can be immobilized on the matrix of a chromatography column, and specifically bind to efficiently separate recombinant proteins from the mixture, improving protein purity to meet the requirements of subsequent research. In studying the mechanism of immune response, it helps to analyze the internal principle of this tag enhancing the immunogenicity of recombinant proteins, explore the interaction between antibodies and tags and recombinant proteins, and provide a basis for the development of immunotherapy strategies. In the process of developing nanobodies, it can evaluate the binding of nanobodies to the IgG1 Fc tag, optimize the design and screening, and also monitor the stability and distribution of the nanobody-recombinant protein complex in vivo, providing key references for the in vivo application of nanobodies and promoting the in-depth development of related research and applications.

[0005] Since Hamers et al. discovered heavy chain antibodies lacking light chains in camel blood in 1993, single domain antibodies (sdAbs) have gradually replaced other small antibodies and become a hot topic in the research and development of new antibody drugs. Single domain antibodies, also known as Nanobodies, usually weigh about 15KDa, approximately one-tenth the size of traditional antibodies. They have disulfide bonds inside and a large number of hydrophilic residues on the surface, showing strong resistance to heat and pH. The lack of the Fc segment and light chain in sdAbs enables them to recognize cryptic epitopes or small epitopes that traditional antibodies cannot recognize, and avoid complement reactions. In addition, single domain antibodies also have many advantages such as high stability, low toxicity, strong solubility, easy target screening, easy direct expression in prokaryotic microorganisms, and good economy. Sequence homology analysis shows that the germline gene sequence of camel sdAb VHH is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than those in humans, and CDR3 protrudes outwards in the tertiary structure, so it is speculated that they have higher antigen-binding specificity and affinity. Developing a single domain antibody against the human IgG1 Fc tag based on the excellent characteristics of the human IgG1 Fc tag will better exert its important functions and be even more perfect.

[0006] The production of antibodies against the human IgG1 Fc tag involves multiple steps, including antigen preparation, animal immunization, screening, purification, etc. Each step has problems of cost control and time cycle. With the continuous development of the biopharmaceutical industry, the demand for new and high-performance antibodies is increasing day by day. How to reduce production costs, shorten the experimental cycle, and ensure antibody quality at the same time is an important challenge faced by antibody production enterprises. Therefore, screening high-affinity single domain antibodies against the human IgG1 Fc tag efficiently for the biopharmaceutical diagnosis and treatment field has broad and profound significance. Summary of the Invention

[0007] In view of this, a single domain antibody against the human IgG1 Fc tag and its application are provided. It has a short R & D cycle, high antibody quality, low development and production costs, and has outstanding advantages in aspects such as antibody affinity and targeting specificity, small molecule size, stability and modifiability, production cost, etc. It also has strong tissue penetration and stability, can more easily penetrate tissue and cell barriers, penetrate deep into the diseased tissue to play a role, and can also tolerate a wide range of temperature, pH value, and chemical environment changes.

[0008] A single domain antibody against the human IgG1 Fc tag, the single domain antibody has the following complementarity determining regions: CDR1, CDR2, and CDR3; Among them, the amino acid sequence of CDR1 is shown as any one of SEQ ID NOs. 44 - 86, the amino acid sequence of CDR2 is shown as any one of SEQ ID NOs. 87 - 129, and the amino acid sequence of CDR3 is shown as any one of SEQ ID NOs. 130 - 172.

[0009] Preferably, the complementary determining regions of the single - domain antibody against the human IgG1 Fc tag are shown as any one of the following (1) - (43): (1) CDR1 is shown as SEQ ID NO.44, CDR2 is shown as SEQ ID NO.87, and CDR3 is shown as SEQ ID NO.130; (2) CDR1 is shown as SEQ ID NO.45, CDR2 is shown as SEQ ID NO.88, and CDR3 is shown as SEQ ID NO.131; (3) CDR1 is shown as SEQ ID NO.46, CDR2 is shown as SEQ ID NO.89, and CDR3 is shown as SEQ ID NO.132; (4) CDR1 is shown as SEQ ID NO.47, CDR2 is shown as SEQ ID NO.90, and CDR3 is shown as SEQ ID NO.133; (5) CDR1 is shown as SEQ ID NO.48, CDR2 is shown as SEQ ID NO.91, and CDR3 is shown as SEQ ID NO.134; (6) CDR1 is shown as SEQ ID NO.49, CDR2 is shown as SEQ ID NO.92, and CDR3 is shown as SEQ ID NO.135; (7) CDR1 is shown as SEQ ID NO.50, CDR2 is shown as SEQ ID NO.93, and CDR3 is shown as SEQ ID NO.136; (8) CDR1 is shown as SEQ ID NO.51, CDR2 is shown as SEQ ID NO.94, and CDR3 is shown as SEQ ID NO.137; (9) CDR1 is shown as SEQ ID NO.52, CDR2 is shown as SEQ ID NO.95, and CDR3 is shown as SEQ ID NO.138; (10) CDR1 is shown as SEQ ID NO.53, CDR2 is shown as SEQ ID NO.96, and CDR3 is shown as SEQ ID NO.139; (11) CDR1 is as shown in SEQ ID NO.54, CDR2 is as shown in SEQ ID NO.97, and CDR3 is as shown in SEQ ID NO.140; (12) CDR1 is as shown in SEQ ID NO.55, CDR2 is as shown in SEQ ID NO.98, and CDR3 is as shown in SEQ ID NO.141; (13) CDR1 is as shown in SEQ ID NO.56, CDR2 is as shown in SEQ ID NO.99, and CDR3 is as shown in SEQ ID NO.142; (14) CDR1 is as shown in SEQ ID NO.57, CDR2 is as shown in SEQ ID NO.100, and CDR3 is as shown in SEQ ID NO.143; (15) CDR1 is as shown in SEQ ID NO.58, CDR2 is as shown in SEQ ID NO.101, and CDR3 is as shown in SEQ ID NO.144; (16) CDR1 is as shown in SEQ ID NO.59, CDR2 is as shown in SEQ ID NO.102, and CDR3 is as shown in SEQ ID NO.145; (17) CDR1 is as shown in SEQ ID NO.60, CDR2 is as shown in SEQ ID NO.103, and CDR3 is as shown in SEQ ID NO.146; (18) CDR1 is as shown in SEQ ID NO.61, CDR2 is as shown in SEQ ID NO.104, and CDR3 is as shown in SEQ ID NO.147; (19) CDR1 is as shown in SEQ ID NO.62, CDR2 is as shown in SEQ ID NO.105, and CDR3 is as shown in SEQ ID NO.148; (20) CDR1 is as shown in SEQ ID NO.63, CDR2 is as shown in SEQ ID NO.106, and CDR3 is as shown in SEQ ID NO.149; (21) CDR1 is as shown in SEQ ID NO.64, CDR2 is as shown in SEQ ID NO.107, and CDR3 is as shown in SEQ ID NO.150; (22) CDR1 is as shown in SEQ ID NO.65, CDR2 is as shown in SEQ ID NO.108, and CDR3 is as shown in SEQ ID NO.151; (23) CDR1 is as shown in SEQ ID NO.66, CDR2 is as shown in SEQ ID NO.109, and CDR3 is as shown in SEQ ID NO.152; (24) CDR1 is as shown in SEQ ID NO.67, CDR2 is as shown in SEQ ID NO.110, and CDR3 is as shown in SEQ ID NO.153; (25) CDR1 is as shown in SEQ ID NO.68, CDR2 is as shown in SEQ ID NO.111, and CDR3 is as shown in SEQ ID NO.154; (26) CDR1 is as shown in SEQ ID NO.69, CDR2 is as shown in SEQ ID NO.112, and CDR3 is as shown in SEQ ID NO.155; (27) CDR1 is as shown in SEQ ID NO.70, CDR2 is as shown in SEQ ID NO.113, and CDR3 is as shown in SEQ ID NO.156; (28) CDR1 is as shown in SEQ ID NO.71, CDR2 is as shown in SEQ ID NO.114, and CDR3 is as shown in SEQ ID NO.157; (29) CDR1 is as shown in SEQ ID NO.72, CDR2 is as shown in SEQ ID NO.115, and CDR3 is as shown in SEQ ID NO.158; (30) CDR1 is as shown in SEQ ID NO.73, CDR2 is as shown in SEQ ID NO.116, and CDR3 is as shown in SEQ ID NO.159; (31) CDR1 is as shown in SEQ ID NO.74, CDR2 is as shown in SEQ ID NO.117, and CDR3 is as shown in SEQ ID NO.160; (32) CDR1 is as shown in SEQ ID NO.75, CDR2 is as shown in SEQ ID NO.118, and CDR3 is as shown in SEQ ID NO.161; (33) CDR1 is as shown in SEQ ID NO.76, CDR2 is as shown in SEQ ID NO.119, and CDR3 is as shown in SEQ ID NO.162; (34) CDR1 is as shown in SEQ ID NO.77, CDR2 is as shown in SEQ ID NO.120, and CDR3 is as shown in SEQ ID NO.163; (35) CDR1 is as shown in SEQ ID NO.78, CDR2 is as shown in SEQ ID NO.121, and CDR3 is as shown in SEQ ID NO.164; (36) CDR1 is as shown in SEQ ID NO.79, CDR2 is as shown in SEQ ID NO.122, and CDR3 is as shown in SEQ ID NO.165; (37) CDR1 is as shown in SEQ ID NO.80, CDR2 is as shown in SEQ ID NO.123, and CDR3 is as shown in SEQ ID NO.166; (38) CDR1 is as shown in SEQ ID NO.81, CDR2 is as shown in SEQ ID NO.124, and CDR3 is as shown in SEQ ID NO.167; (39) CDR1 is as shown in SEQ ID NO.82, CDR2 is as shown in SEQ ID NO.125, and CDR3 is as shown in SEQ ID NO.168; (40) CDR1 is as shown in SEQ ID NO.83, CDR2 is as shown in SEQ ID NO.126, and CDR3 is as shown in SEQ ID NO.169; (41) CDR1 is as shown in SEQ ID NO.84, CDR2 is as shown in SEQ ID NO.127, and CDR3 is as shown in SEQ ID NO.170; (42) CDR1 is as shown in SEQ ID NO.85, CDR2 is as shown in SEQ ID NO.128, and CDR3 is as shown in SEQ ID NO.171; (43) CDR1 is as shown in SEQ ID NO.86, CDR2 is as shown in SEQ ID NO.129, and CDR3 is as shown in SEQ ID NO.172.

[0010] Preferably, the single-domain antibody against the human IgG1 Fc tag has the following framework regions: FR1, FR2, FR3, FR4; wherein, the amino acid sequence of FR1 is shown as any one of SEQ ID NO.173 - 215; the amino acid sequence of FR2 is shown as any one of SEQ ID NO.216 - 258; the amino acid sequence of FR2 is shown as any one of SEQ ID NO.259 - 301; the amino acid sequence of FR4 is shown as any one of SEQ ID NO.302 - 344.

[0011] Preferably, the framework region of the single-domain antibody is shown as any one of the following (44)-(86): (44) FR1 is as shown in SEQ ID NO.173, FR2 is as shown in SEQ ID NO.216, FR3 is as shown in SEQ ID NO.259, FR4 is as shown in SEQ ID NO.302; (45) FR1 is as shown in SEQ ID NO.174, FR2 is as shown in SEQ ID NO.217, FR3 is as shown in SEQ ID NO.260, FR4 is as shown in SEQ ID NO.303; (46) FR1 is as shown in SEQ ID NO.175, FR2 is as shown in SEQ ID NO.218, FR3 is as shown in SEQ ID NO.261, FR4 is as shown in SEQ ID NO.304; (47) FR1 is as shown in SEQ ID NO.176, FR2 is as shown in SEQ ID NO.219, FR3 is as shown in SEQ ID NO.262, FR4 is as shown in SEQ ID NO.305; (48) FR1 is as shown in SEQ ID NO.177, FR2 is as shown in SEQ ID NO.220, FR3 is as shown in SEQ ID NO.263, FR4 is as shown in SEQ ID NO.306; (49) FR1 is as shown in SEQ ID NO.178, FR2 is as shown in SEQ ID NO.221, FR3 is as shown in SEQ ID NO.264, FR4 is as shown in SEQ ID NO.307; (50) FR1 is as shown in SEQ ID NO.179, FR2 is as shown in SEQ ID NO.222, FR3 is as shown in SEQ ID NO.265, FR4 is as shown in SEQ ID NO.308; (51) FR1 is as shown in SEQ ID NO.180, FR2 is as shown in SEQ ID NO.223, FR3 is as shown in SEQ ID NO.266, FR4 is as shown in SEQ ID NO.309; (52) FR1 is as shown in SEQ ID NO.181, FR2 is as shown in SEQ ID NO.224, FR3 is as shown in SEQ ID NO.267, FR4 is as shown in SEQ ID NO.310; (53) FR1 is as shown in SEQ ID NO.182, FR2 is as shown in SEQ ID NO.225, FR3 is as shown in SEQ ID NO.268, FR4 is as shown in SEQ ID NO.311; (54) FR1 is as shown in SEQ ID NO.183, FR2 is as shown in SEQ ID NO.226, FR3 is as shown in SEQ ID NO.269, and FR4 is as shown in SEQ ID NO.312; (55) FR1 is as shown in SEQ ID NO.184, FR2 is as shown in SEQ ID NO.227, FR3 is as shown in SEQ ID NO.270, and FR4 is as shown in SEQ ID NO.313; (56) FR1 is as shown in SEQ ID NO.185, FR2 is as shown in SEQ ID NO.228, FR3 is as shown in SEQ ID NO.271, and FR4 is as shown in SEQ ID NO.314; (57) FR1 is as shown in SEQ ID NO.186, FR2 is as shown in SEQ ID NO.229, FR3 is as shown in SEQ ID NO.272, and FR4 is as shown in SEQ ID NO.315; (58) FR1 is as shown in SEQ ID NO.187, FR2 is as shown in SEQ ID NO.230, FR3 is as shown in SEQ ID NO.273, and FR4 is as shown in SEQ ID NO.316; (59) FR1 is as shown in SEQ ID NO.188, FR2 is as shown in SEQ ID NO.231, FR3 is as shown in SEQ ID NO.274, and FR4 is as shown in SEQ ID NO.317; (60) FR1 is as shown in SEQ ID NO.189, FR2 is as shown in SEQ ID NO.232, FR3 is as shown in SEQ ID NO.275, and FR4 is as shown in SEQ ID NO.318; (61) FR1 is as shown in SEQ ID NO.190, FR2 is as shown in SEQ ID NO.233, FR3 is as shown in SEQ ID NO.276, and FR4 is as shown in SEQ ID NO.319; (62) FR1 is as shown in SEQ ID NO.191, FR2 is as shown in SEQ ID NO.234, FR3 is as shown in SEQ ID NO.277, and FR4 is as shown in SEQ ID NO.320; (63) FR1 is as shown in SEQ ID NO.192, FR2 is as shown in SEQ ID NO.235, FR3 is as shown in SEQ ID NO.278, and FR4 is as shown in SEQ ID NO.321; (64) FR1 is as shown in SEQ ID NO.193, FR2 is as shown in SEQ ID NO.236, FR3 is as shown in SEQ ID NO.279, FR4 is as shown in SEQ ID NO.322; (65) FR1 is as shown in SEQ ID NO.194, FR2 is as shown in SEQ ID NO.237, FR3 is as shown in SEQ ID NO.280, FR4 is as shown in SEQ ID NO.323; (66) FR1 is as shown in SEQ ID NO.195, FR2 is as shown in SEQ ID NO.238, FR3 is as shown in SEQ ID NO.281, FR4 is as shown in SEQ ID NO.324; (67) FR1 is as shown in SEQ ID NO.196, FR2 is as shown in SEQ ID NO.239, FR3 is as shown in SEQ ID NO.282, FR4 is as shown in SEQ ID NO.325; (68) FR1 is as shown in SEQ ID NO.197, FR2 is as shown in SEQ ID NO.240, FR3 is as shown in SEQ ID NO.283, FR4 is as shown in SEQ ID NO.326; (69) FR1 is as shown in SEQ ID NO.198, FR2 is as shown in SEQ ID NO.241, FR3 is as shown in SEQ ID NO.284, FR4 is as shown in SEQ ID NO.327; (70) FR1 is as shown in SEQ ID NO.199, FR2 is as shown in SEQ ID NO.242, FR3 is as shown in SEQ ID NO.285, FR4 is as shown in SEQ ID NO.328; (71) FR1 is as shown in SEQ ID NO.200, FR2 is as shown in SEQ ID NO.243, FR3 is as shown in SEQ ID NO.286, FR4 is as shown in SEQ ID NO.329; (72) FR1 is as shown in SEQ ID NO.201, FR2 is as shown in SEQ ID NO.244, FR3 is as shown in SEQ ID NO.287, FR4 is as shown in SEQ ID NO.330; (73) FR1 is as shown in SEQ ID NO.202, FR2 is as shown in SEQ ID NO.245, FR3 is as shown in SEQ ID NO.288, FR4 is as shown in SEQ ID NO.331; (74) FR1 is as shown in SEQ ID NO.203, FR2 is as shown in SEQ ID NO.246, FR3 is as shown in SEQ ID NO.289, and FR4 is as shown in SEQ ID NO.332; (75) FR1 is as shown in SEQ ID NO.204, FR2 is as shown in SEQ ID NO.247, FR3 is as shown in SEQ ID NO.290, and FR4 is as shown in SEQ ID NO.333; (76) FR1 is as shown in SEQ ID NO.205, FR2 is as shown in SEQ ID NO.248, FR3 is as shown in SEQ ID NO.291, and FR4 is as shown in SEQ ID NO.334; (77) FR1 is as shown in SEQ ID NO.206, FR2 is as shown in SEQ ID NO.249, FR3 is as shown in SEQ ID NO.292, and FR4 is as shown in SEQ ID NO.335; (78) FR1 is as shown in SEQ ID NO.207, FR2 is as shown in SEQ ID NO.250, FR3 is as shown in SEQ ID NO.293, and FR4 is as shown in SEQ ID NO.336; (79) FR1 is as shown in SEQ ID NO.208, FR2 is as shown in SEQ ID NO.251, FR3 is as shown in SEQ ID NO.294, and FR4 is as shown in SEQ ID NO.337; (80) FR1 is as shown in SEQ ID NO.209, FR2 is as shown in SEQ ID NO.252, FR3 is as shown in SEQ ID NO.295, and FR4 is as shown in SEQ ID NO.338; (81) FR1 is as shown in SEQ ID NO.210, FR2 is as shown in SEQ ID NO.253, FR3 is as shown in SEQ ID NO.296, and FR4 is as shown in SEQ ID NO.339; (82) FR1 is as shown in SEQ ID NO.211, FR2 is as shown in SEQ ID NO.254, FR3 is as shown in SEQ ID NO.297, and FR4 is as shown in SEQ ID NO.340; (83) FR1 is as shown in SEQ ID NO.212, FR2 is as shown in SEQ ID NO.255, FR3 is as shown in SEQ ID NO.298, and FR4 is as shown in SEQ ID NO.341; (84) FR1 is as shown in SEQ ID NO.213, FR2 is as shown in SEQ ID NO.256, FR3 is as shown in SEQ ID NO.299, and FR4 is as shown in SEQ ID NO.342; (85) FR1 is as shown in SEQ ID NO.214, FR2 is as shown in SEQ ID NO.257, FR3 is as shown in SEQ ID NO.300, and FR4 is as shown in SEQ ID NO.343; (86) FR1 is as shown in SEQ ID NO.215, FR2 is as shown in SEQ ID NO.258, FR3 is as shown in SEQ ID NO.301, and FR4 is as shown in SEQ ID NO.344.

[0012] Preferably, the amino acid sequence of the single-domain antibody is as shown in any one of SEQ ID NOs. 1-43.

[0013] Preferably, the framework region is a heavy-chain framework region, and at least a part of the heavy-chain framework region independently comes from an alpaca-derived antibody.

[0014] Preferably, the single-domain antibody against the human IgG1 Fc tag is obtained by immunizing a camel with a recombinant human IgG1 Fc protein antigen, collecting the peripheral blood cells of the immunized camel, isolating the affinity lymphocytes for human IgG1 Fc therefrom, extracting total RNA and reverse-transcribing it into cDNA, then cloning the V region of the camel heavy-chain antibody by Nest-PCR technology, inserting it into the phagemid pMES4 to construct a phage expression library, then performing multiple rounds of screening on the human IgG1 Fc antigen by phage display technology, and verifying the binding ability of the obtained single-domain antibody by enzyme-linked immunosorbent assay, so as to screen out a single-domain antibody against the human IgG1 Fc tag with high affinity activity.

[0015] Preferably, the DNA sequence of the single-domain antibody against the human IgG1 Fc tag is as shown in any one of SEQ ID NOs. 345-387.

[0016] On the other hand, the present application provides a fusion protein containing the single-domain antibody against the human IgG1 Fc tag as described above.

[0017] In addition, the present application also provides a product containing the single-domain antibody against the human IgG1 Fc tag as described above or the fusion protein as described above, and the product is a diagnostic reagent, an in vitro diagnostic reagent, an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to a site expressing human IgG1 Fc.

[0018] This application combines the use of phage display technology, which can intuitively obtain antibody affinity information and obtain high-affinity human IgG1 Fc single-domain antibody genes in a relatively short time. In addition, this application provides a preparation scheme for the supernatant of the above-mentioned human IgG1 Fc single-domain antibody, which performs a small-scale expression of the human IgG1 Fc single-domain antibody, effectively reducing the development and production costs of human IgG1 Fc antibodies. The single-domain antibody has been verified by the ELISA system to have high specificity and high affinity for targeting human IgG1 Fc, indicating that the human IgG1 Fc single-domain antibody obtained by the present invention has the value for further development. In addition, the single-domain antibody against the human IgG1 Fc tag and its application of this application have a short R & D cycle and high antibody quality, and have outstanding advantages in terms of antibody affinity and targeting specificity, small molecular size, stability and modifiability, production cost, etc. In addition, the human IgG1 Fc single-domain antibody also has strong tissue penetrability and stability. Due to its small molecular weight, the human IgG1 Fc tag single-domain antibody has good tissue penetrability and can more easily penetrate tissue and cell barriers and play a role deep inside the diseased tissue. Moreover, the single-domain antibody can also tolerate a wide range of temperature, pH value, and chemical environment changes and still maintain its antigen-binding activity in complex in vivo and in vitro environments, which makes it have advantages in a variety of application scenarios (such as in vivo treatment, in vitro diagnostic reagent development, etc.). It has more advantages in the early diagnosis of diseases (especially in cases where rapid detection and high-sensitivity detection are required), tumor-targeted imaging and treatment (due to its good tissue penetrability), and some research and application scenarios that require precise recognition of subtle differences in antigens, such as the development of highly specific diagnostic reagents and new targeted therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the detection result of the titer of the camel immune serum of the single-domain antibody against the human IgG1 Fc tag.

[0020] Figure 2 It is a diagram of the panning result of the phage library of the affinity human IgG1 Fc single-domain antibody.

[0021] Figure 3 It is a diagram of the ELISA detection result for evaluating the enrichment degree of specific antibodies.

[0022] Figure 4 It is a diagram of the ELISA verification result of the screening result of the positive clone of the single-domain antibody for identifying the specificity of human IgG1 Fc, showing the results of ELISA 1-65.

[0023] Figure 5 It is a diagram of the ELISA verification result of the screening result of the positive clone of the single-domain antibody for identifying the specificity of human IgG1 Fc, showing the results of ELISA 66-130.

[0024] Figure 6 Show the analysis results of the unique sequences of positive clones.

[0025] Figure 7 Show the ELISA verification results based on the phage supernatant.

[0026] Figure 8 Show the ELISA verification results based on the periplasmic expression product (VHH nanobody). Detailed implementation manners

[0027] The present invention will be described in detail below in conjunction with specific embodiments and the accompanying drawings.

[0028] An embodiment of the present invention provides a single-domain antibody against the human IgG1 Fc tag, which is characterized in that the single-domain antibody has the following complementarity-determining regions: CDR1, CDR2, and CDR3; wherein, the amino acid sequence of CDR1 is shown as any one of SEQ ID NO.44-86, the amino acid sequence of CDR2 is shown as any one of SEQ ID NO.87-129, and the amino acid sequence of CDR3 is shown as any one of SEQ ID NO.130-172. Preferably, in the complementarity-determining regions of the single-domain antibody against the human IgG1 Fc tag, CDR1, CDR2, and CDR3 are matched in groups as shown in the sequence listing. Preferably, the single-domain antibody against the human IgG1 Fc tag has the following heavy-chain framework regions, namely FR1, FR2, FR3, and FR4; wherein, the amino acid sequence of FR1 is shown as any one of SEQ ID NO.173-215; the amino acid sequence of FR2 is shown as any one of SEQ ID NO.216-258; the amino acid sequence of FR2 is shown as any one of SEQ ID NO.259-301; the amino acid sequence of FR4 is shown as any one of SEQ ID NO.302-344. Preferably, the amino acid sequence of the single-domain antibody is shown as any one of SEQ ID NO.1-43. Preferably, the framework region is a heavy-chain framework region, and at least a part of the heavy-chain framework region independently comes from a llama-derived antibody. Preferably, the DNA sequence of the single-domain antibody against the human IgG1 Fc tag is shown as any one of SEQ ID NO.345-387.

[0029] Compared with traditional human IgG1 Fc-tagged antibodies, the functional characteristics of human IgG1 Fc-tagged single-domain antibodies are mainly manifested in the following aspects. First, it has outstanding advantages in antigen recognition specificity. The VHH domain of human IgG1 Fc-tagged single-domain antibodies has a highly specific antigen recognition ability. It can recognize and bind to specific epitopes on the antigen, and this binding is based on the amino acid sequence complementarity and spatial structure complementarity of the VHH domain, forming a tight non-covalent bond with the antigen. Due to the simpler structure of single-domain antibodies, the epitopes they bind to may be inaccessible to traditional antibodies, such as some epitopes hidden inside antigen molecules, which provides the possibility for the discovery and application of new antigen targets. Second, it can bind to antigens with high affinity. Although there is only one antigen-binding site, the VHH domain of single-domain antibodies can bind to antigens with relatively high affinity. Its affinity constant (Kd) can usually reach 10 -7 - 10 -9 M or even lower levels, enabling effective antigen binding at relatively low antibody concentrations, which is of great significance in disease diagnosis and treatment. For example, it can be used to develop highly sensitive diagnostic reagents or highly effective therapeutic antibodies. Although the affinity of a single binding site may be relatively low, it has advantages in recognizing some hidden antigen epitopes that are difficult for traditional antibodies to access. Moreover, its binding specificity is strong, enabling more precise recognition of subtle differences on antigens, such as distinguishing different isomers or mutants of antigens. Third, it has strong tissue penetration and stability. Due to its small molecular weight, human IgG1 Fc-tagged single-domain antibodies have good tissue penetration and can more easily penetrate tissue and cell barriers to play a role deep inside diseased tissues. Fourth, it also has high stability and can tolerate a wide range of temperature, pH value, and chemical environment changes, and still maintain its antigen-binding activity in complex in vivo and in vitro environments, which makes it have advantages in various application scenarios (such as in vivo treatment, in vitro diagnostic reagent development, etc.). It has more advantages in the early diagnosis of diseases (especially in cases where rapid and highly sensitive detection is required), tumor targeted imaging and treatment (due to its good tissue penetration), and some research and application scenarios that require precise recognition of subtle antigen differences, such as developing highly specific diagnostic reagents and new targeted therapeutic drugs.

[0030] Preferably, the anti-human IgG1 Fc-tag single-domain antibody is obtained by immunizing a camel with human IgG1 Fc recombinant protein antigen, collecting the peripheral blood cells of the immunized camel, isolating the affinity lymphocytes against human IgG1 Fc therefrom, extracting total RNA and reverse-transcribing it into cDNA, cloning the V region of the camel heavy-chain antibody by Nest-PCR technology, inserting it into the phage plasmid pMES4 to construct a phage expression library, then performing multiple rounds of screening on the human IgG1 Fc antigen by phage display technology, and verifying the binding ability of the obtained single-domain antibody by enzyme-linked immunosorbent assay, so as to screen out the anti-human IgG1 Fc-tag single-domain antibody with high affinity activity.

[0031] Therefore, the screening of the anti-human IgG1 Fc-tag single-domain antibody in this application is divided into the following stages: ① Camel immunization and serum titer determination; ② Construction of a camel single-domain antibody phage display library; ③ Amplification and rescue of the camel single-domain antibody phage display library; ④ Panning for single-domain antibodies specifically binding to human IgG1 Fc by phage display technology; ⑤ Identification of positive clones of single-domain antibodies specific for human IgG1 Fc; ⑥ Sequence analysis of positive clones; ⑦ Verification of Unique clones specifically binding to human IgG1 Fc.

[0032] The above stages will be illustrated by specific examples below.

[0033] ① Camel immunization and serum titer determination (1) Immunization of a camel with human IgG1 Fc Mix 1 mg of human IgG1 Fc with an equal volume of Freund's adjuvant and inject it subcutaneously at 3 - 5 points on the camel's neck. Before immunization, collect blood from the marginal ear vein of the camel. Immunize once every two weeks for a total of 4 immunizations; each time immunize, collect 5 mL of peripheral blood from the camel. When collecting blood, fix the camel's head to one side, shave the skin at the blood collection site of the animal, disinfect it with 75% alcohol, and wait for it to dry before collecting blood. Press the jugular vein groove with fingers. After the blood vessels dilate, disinfect the blood collection site and insert a needle to collect blood. After collecting 5 mL of blood and allowing it to stand, use it to prepare serum for titer evaluation. Seven days after the last immunization, collect 50 mL of blood into an EDTA anticoagulant tube, immediately shake it continuously and slowly, mix it well, place it on ice, and transport it back to the laboratory.

[0034] (2) Serum titer detection Coat 100 ng of human IgG1 Fc antigen overnight at 4 °C on a 96-well high-binding ELISA plate. After washing the coated antigen 3 times with PBST, block the antigen with 200 μL of 2% BSA at room temperature for 2 hours. Dilute the sera collected before immunization and after each immunization to different concentration gradients. Aspirate the blocking solution, wash the ELISA plate 3 - 5 times with PBST, add 100 μL of serum samples at different dilution gradients to each well, incubate at room temperature for 2 hours, then aspirate the sera, wash 5 times with PBST. Then dilute Camelid IgG-HRP with 1% BSA, add 100 μL to each well, incubate at room temperature for 1.5 hours in the dark, then wash the plate 5 times again. Add 100 μL of TMB chromogenic solution to each well, incubate at room temperature for 10 - 30 min, and read the OD450 after adding the stop solution. The results of serum titer detection are shown in the appendix Figure 1 。

[0035] (3)Separation of blood lymphocyte samples Separate lymphocytes from the blood samples collected after the last immunization. The separation method is as follows: i. Add 7 mL of lymphocyte separation medium Ficoll to each 15 mL centrifuge tube; ii. Add an equal volume of PBS (1×) or normal saline to the fresh whole blood with anticoagulant (EDTA) added, and mix well; iii. Take a 15 mL centrifuge tube containing lymphocyte separation medium, carefully and slowly transfer it to another 15 mL centrifuge tube already containing lymphocyte separation medium, and make the above mixture above the liquid level of the lymphocyte separation medium (i.e., the two liquids do not mix and a clear interface is retained), centrifuge at 3,000 g for 20 min; iv. Carefully transfer the supernatant to a 1.5 mL cell cryopreservation tube with a 1 mL pipette, write the animal number and plasma on it, put it in a small cloth bag with a rope, and store it in a liquid nitrogen tank. Carefully separate the white blood cell layer to a 15 mL centrifuge tube with a 1 mL pipette; fill it up to 15 mL with PBS (1×); wash the white blood cells with PBS (1×), centrifuge (3,000 g, 20 min), carefully pour out the supernatant without disturbing the cell pellet at the bottom of the tube, and recover the white blood cells in the remaining 0.1 - 0.2 mL of PBS.

[0036] vi. Add 5 volumes of RNA later, gently mix the cell pellet, divide it into 2 parts and transfer to 1.5 mL cell cryopreservation tubes, and store in a liquid nitrogen tank.

[0037] ② Construction of camel single-domain antibody phage display library (1)Total RNA extraction Take an aliquot of cryopreserved lymphocytes, add 1 mL of Trizol, let it stand at room temperature for 10 min, then add 0.2 mL of chloroform, shake vigorously, let it stand at room temperature until the solution separates (about 10 min), centrifuge at 12,000 rpm, collect the upper aqueous phase, add an equal volume of isopropanol, mix well, let it stand at room temperature for 15 min until nucleic acid precipitates, centrifuge at high speed to remove the supernatant, wash the RNA precipitate with 1 mL of 75% ethanol (prepared with DEPC water), centrifuge at high speed to remove the supernatant, air-dry, dissolve the RNA in nuclease-free water, and take 1 μL respectively for concentration and purity determination.

[0038] (2)cDNA synthesis Take 1 μg of RNA, and perform cDNA synthesis using a cDNA first-strand synthesis kit (Super Script TMIII First - Strand Synthesis SuperMix (Invitrogen)). Use Oligo dT as the reverse transcription primer, and store the synthesized cDNA at -20 °C. (3)Construction of phage display library PCR amplification Using the synthesized cDNA above as a template, amplify the V region (VHH) of camel heavy-chain antibody by Nest-PCR. The following table shows the names and sequences of Nest-PCR primers:

[0039] a. The reaction system for the first round of PCR: 1 μL of cDNA; 12.5 μL of Mix; 0.5 μL of CALL001; 0.5 μL of CALL002; make up to 25 μL with water. The reaction conditions for the first round of PCR: 95 °C for 5 min; 94 °C for 1 min, 57 °C for 1 min, 72 °C for 1 min, 35 cycles; 72 °C for 5 min. b. The reaction system for the second round of PCR: 40 ng of the product from the first round of PCR; 25 μL of Mix; 1 μL of VHH-Back; 1 μL of VHH -For; make up to 50 μL with water. The reaction conditions for the second round of PCR: 95 °C for 5 min; 94 °C for 45 s, 60 °C for 45 s, 72 °C for 45 s, 15 cycles; 72 °C for 5 min. c. After the PCR reaction, detect the PCR products by 1.5% agarose gel electrophoresis. The target gene fragment of the first round of PCR is at 700 bp. Cut the gel, and use the QIAEX II Gel Extraction kit to recover the target band, and perform the second round of PCR. The target gene fragment is at 500 bp. Cut the gel and recover the target band, namely the VHH fragment.

[0040] (4)Restriction enzyme digestion of phagemid vector and ligation of target fragment The VHH fragment and the pMES4 vector were digested with the restriction enzymes Eco91I and PstI respectively. The reaction systems were as follows: a. Vector digestion system: 20 μg of pMES4 vector; 10 μL of PstI; 20 μL of Eco91I; 50 μL of Cutsmart buffer; add H2O to 500 μL. b. Fragment digestion system: 5 μg of VHH fragment; 7 μL of PstI; 14 μL of Eco91I; 50 μL of Cutsmart buffer; add H2O to 500 μL. Incubate at 37 °C overnight for digestion. After agarose gel electrophoresis, cut and recover the gel. Mix the digestion products of the vector and the VHH fragment, and ligate overnight at 16 °C using T4 DNA Ligase.

[0041] ③ Amplification and rescue of camel single-domain antibody phage display library (1)Construction of phage display library After purification of the ligation product with PCR Purification Kit, 1 μL was taken to transform Escherichia coli TG1 competent cells. The cells were resuscitated at 37 °C for 2 h, serially diluted to 101, 102, 103, and 300 μL of each dilution was taken and spread on plates. Incubate at 37 °C overnight, count the number of colonies, approximately 105 colonies / plate. Using the same transformation method as above, perform large-scale transformation until the number of clones in the library reaches more than 108. Elute all the clones with LB, centrifuge at 5,000g for 5 min, resuspend the pellet in 2 mL of LB, add an equal volume of 30% glycerol, and store at -80 °C.

[0042] (2)Library diversity detection Randomly pick 80 clones from (1) for sequencing to detect the recombination rate and evaluate the quality of the library. The recombination rate of the constructed human IgG1Fc single-domain antibody library was 92.5%. Analyze the diversity of the human IgG1Fc single-domain antibody library. The sequencing results showed that 54 amino acid sequences were found among 64 monoclonal clones, indicating that the constructed library had good diversity.

[0043] (3)Phage amplification and rescue The phage library of human IgG1 Fc single-domain antibody was amplified and rescued using helper phage. The stored monoclonal library was inoculated into 100 mL of medium and cultured until the logarithmic growth phase. Helper phage with an MOI of 20 was added, and the mixture was left standing at room temperature for 30 min. After low-speed centrifugation, the precipitate was resuspended in medium and inoculated into 300 mL of medium and cultured overnight. The next day, it was centrifuged at 3,000 g for 30 min, and the supernatant was collected. PEG was added to precipitate the phage, and it was left standing on ice for 30 min and then centrifuged at 3,000 for 30 min. The precipitate was the phage library of human IgG1 Fc single-domain antibody. After suspending the precipitate with PBS, its titer was determined to be 2 x 10^13 pfu / mL.

[0044] ④ Screening for specific binding human IgG1 Fc single-domain antibody using phage display technology (1)Affinity panning of human IgG1 Fc single-domain antibody phage library A total of 3 rounds of panning were performed. An ELISA plate was coated with human IgG1 Fc antigen and incubated overnight at 4 °C. The next day, the rescued human IgG1 Fc single-domain antibody phage was added and incubated at room temperature for 2 h; the wells were washed 10 times with PBST, 100 μL of triethylamine was added, and it was incubated at room temperature for 30 min. The collected phage was the human IgG1 Fc single-domain antibody phage library obtained by affinity panning; 10 μL was taken to infect TG1 cells and spread on a plate for determining the number of clones after screening, and the remaining phage after screening was used for amplification. The results of each round of panning are shown in the appendix Figure 2 。

[0045] (2)Amplification and rescue of phage after screening The amplification and rescue methods were the same as above. The obtained PBS suspension was the phage after the first round of screening amplification, stored at 4 °C, and used for the next round of screening; according to the same screening steps as above, the antigen amount was gradually decreased successively, and 3 - 4 rounds of screening were performed.

[0046] (3)ELISA evaluation of the enrichment degree of specific antibodies An ELISA plate was coated with 100 ng of human IgG1 Fc antigen and incubated overnight at 4 °C; the next day, 2% BSA was added and blocked at room temperature for 1 h; in the experimental groups, the phage amplified after each round of panning was added respectively, and in the control group, an equal amount of wild-type phage was added, and it was incubated at room temperature for 2 h; the wells were washed 10 times with PBST to remove unbound phage; HRP-labeled anti-M13 antibody was added and incubated at room temperature for 1 h; chromogenic solution was added, and the reaction was carried out in the dark for 60 min, and the absorbance value was measured. The absorbance value gradually increased with the number of panning times, indicating that specific antibodies were enriched. The ELISA results are shown in the appendix Figure 3 。

[0047] ⑤ Identification of positive clones of human IgG1 Fc-specific single-domain antibody The experimental group ELISA plates were coated with 100 ng of human IgG1 Fc antigen, and the control group ELISA plates were coated with 100 μL of PBS, and incubated overnight at 4 °C; The plates coated with phages obtained from the third and final rounds of screening were taken, and 1056 monoclonal colonies were randomly picked into 1 mL of medium, cultured at 37 °C until the logarithmic phase, added with M13 and infected at room temperature for 30 min, then centrifuged to replace with fresh medium; The next day, the supernatant was collected by centrifugation; At the same time, ELISA plates were taken, added with 2% BSA and blocked at room temperature for 2 h; The supernatant was added to each well in both the experimental group and the control group, and incubated at room temperature for 2 h; Washed 5 times with PBST, added with M13 antibody, and incubated at room temperature for 1 h; Washed 3 - 5 times with PBST; Added with TMB substrate and reacted for 10 min, and the absorbance value was read on an enzyme - linked immunosorbent assay (ELISA) reader; When the ratio of the absorbance value to the control well was greater than 2, it was determined as a positive clone; The ELISA verification results showed that 130 positive clones were obtained. The screening results of positive clones are shown in the appendix Figure 4 and Figure 5 。

[0048] ⑥ Analysis of positive clone sequences The 130 obtained positive clones were sequenced. The sequencing results showed that 43 nucleotide sequences were obtained. The amino acid sequences were analyzed, and among them, all 43 sequences had the typical structure of single - domain antibodies, that is, composed of framework regions (FR1, FR2, FR3, and FR4) and complementarity - determining regions (CDR1, CDR2, and CDR3). The analysis of the unique sequences of positive clones is shown in the appendix Figure 6 。

[0049] ⑦ Unique clones for verifying the specificity of human IgG1 Fc The experimental group coated 100 ng of human IgG1 Fc antigen on the ELISA plate, and the control group coated 100 uL of PBS on the ELISA plate, and incubated overnight at 4 °C; 2 aliquots of 43 unique monoclonal antibodies to be verified were picked into 1 mL of medium and cultured at 37 °C until the logarithmic phase. One aliquot was added with M13 and infected at room temperature for 30 min, then centrifuged to replace with fresh medium; the next day, the supernatant was collected by centrifugation; at the same time, an ELISA plate was taken, and 2% BSA was added and blocked at room temperature for 2 h; the supernatant was added to each well of the experimental group and the control group, and incubated at room temperature for 2 h; washed 5 times with PBST, added M13-HRP secondary antibody, and incubated at room temperature for 1 h; the other aliquot was added with 1 mM IPTG and induced overnight; the next day, the bacterial pellet was collected by centrifugation, lysed, and centrifuged at 5,000 g for 15 min to collect the supernatant; at the same time, an ELISA plate was taken, and 2% BSA was added and blocked at room temperature for 2 h; the lysed supernatant of the monoclonal antibody was added to each well of the experimental group and the control group, and incubated at room temperature for 2 h; washed 5 times with PBST, added VHH-HRP secondary antibody, and incubated at room temperature for 1 h; after the incubation of the secondary antibody, washed 3 - 5 times with PBST; added TMB substrate and reacted for 10 min, and the absorbance value was read on an enzyme-linked immunosorbent assay (ELISA) reader; when the ratio of the absorbance value to the control well was greater than 2, it was determined as a positive clone; the ELISA verification results showed that the secondary verification results of all 43 unique clones were positive. It was indicated that all 43 positive sequences could specifically bind to human IgG1 Fc antigen. The verification results of the unique clones are shown in the appendix Figure 7 and appendix Figure 8 .

[0050] In the present invention, a single-domain antibody sequence specifically binding to human IgG1 Fc was successfully screened by phage display technology, and the binding ability of the single-domain antibody of human IgG1 Fc was analyzed by ELISA method.

[0051] Example 1: Detection of the enrichment degree of human IgG1 Fc-specific antibodies by polyclonal phage ELISA The enrichment degree of human IgG1 Fc antibodies in the rescued library after enrichment was detected by polyclonal phage ELISA. In the experimental group, the ELISA plate was coated with 100 ng of human IgG1 Fc, and in the blank group, the ELISA plate was coated with PBS without coating antigen, and incubated overnight at 4 °C; the next day, 2% BSA was added and blocked at room temperature for 1 h; the phage supernatant collected from each round of rescue library and the original library during the panning of human IgG1 Fc was used as the primary antibody for incubation. The antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 h; washed 10 times with PBST, added the secondary antibody, and incubated at room temperature for 1 h; added TMB substrate and reacted for 10 - 20 min, and the OD 450 absorbance value was read on an ELISA reader. The results showed that after 3 rounds of panning, the antibodies specifically binding to human IgG1 Fc were effectively enriched (appendix Figure 3 ).

[0052] Example 2: Analysis of the binding ability of human IgG1 Fc single-domain antibody by ELISA In the experimental group, the ELISA plate was coated with 100 ng of human IgG1 Fc protein, and the blank group was coated with PBS, and incubated overnight at 4 °C; the next day, 2% BSA was added for blocking at room temperature for 1 h; the M13 monoclonal supernatant of human IgG1 Fc was taken as the primary antibody for incubation, and the antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 h; washed 10 times with PBST, added the secondary antibody, and incubated at room temperature for 1 h; added the substrate, reacted for 10 - 20 min, and read the absorbance value on the microplate reader. The ELISA test results showed that the human IgG1 Fc single-domain antibody had good specific binding to the human IgG1 Fc antigen, and the signal values of the experimental group were far higher than those of the blank group (attached Figure 7 ).

[0053] Example 3: Analysis of the binding ability of human IgG1 Fc single-domain antibody by analyzing the periplasmic expression product by ELISA In the experimental group, the ELISA plate was coated with 100 ng of human IgG1 Fc protein, and the blank group was coated with PBS, and incubated overnight at 4 °C; the next day, 2% BSA was added for blocking at room temperature for 1 h; the periplasmic expression product of human IgG1 Fc induced by IPTG was taken as the primary antibody for incubation, and the antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 h; washed 10 times with PBST, added the secondary antibody, and incubated at room temperature for 1 h; added the substrate, reacted for 10 - 20 min, and read the absorbance value on the microplate reader. The ELISA test results showed that the human IgG1 Fc single-domain antibody had good specific binding to the human IgG1 Fc antigen, and the signal values of the experimental group were far higher than those of the blank group (attached Figure 8 ).

[0054] On the other hand, the present application provides a fusion protein containing the single-domain antibody against the human IgG1 Fc tag as described above.

[0055] In addition, the present application also provides a product containing the single-domain antibody against the human IgG1 Fc tag as described above or the fusion protein as described above, and the product is an anti-tumor drug, an immune cell for adoptive cell therapy, or a carrier for delivering a drug to a site expressing human IgG1 Fc.

[0056] In this application, camels were immunized with a recombinant protein antigen of human IgG1 Fc (human IgG1 Fc), and a human IgG1 Fc single-domain antibody with high affinity activity was successfully screened out. The heavy-chain variable region of the single-domain antibody has three CDR regions. Among them, CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure. Therefore, the single-domain antibody has higher antigen-binding specificity and affinity than traditional antibodies. In addition, in response to the problem that "the prior art for the development of human IgG1 Fc (human IgG1 Fc) antibodies focuses on monoclonal traditional antibodies, the traditional monoclonal antibody screening method is time-consuming and laborious, traditional antibodies cannot be expressed in prokaryotic systems, have a large molecular weight, complex structure, poor tissue permeability, long R & D cycle, high production cost, and large batch-to-batch differences, etc., which seriously limits the development of human IgG1 Fc antibody drugs in China and cannot meet the diagnostic and treatment needs of Chinese patients", this application provides a single-domain antibody based on an anti-human IgG1 Fc tag and its application, which has a short R & D cycle, high antibody quality, and outstanding advantages in terms of antibody affinity and targeting specificity, small molecular size, stability and modifiability, production cost, etc.

[0057] It should be noted that the present invention is not limited to the above embodiments. According to the creative spirit of the present invention, those skilled in the art can also make other changes, and these changes made based on the creative spirit of the present invention should be included within the scope of protection required by the present invention.

Claims

1. A single domain antibody against human IgG1 Fc tag, characterized in that: The single domain antibody has the following complementarity determining regions: CDR1, CDR2 and CDR3; Among them, the amino acid sequence of CDR1 is shown in any one of SEQ ID NOs.44-86, the amino acid sequence of CDR2 is shown in any one of SEQ ID NOs.87-129, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs.130-172.

2. The single domain antibody against human IgG1 Fc tag according to claim 1, characterized in that The complementarity determining region of the single domain antibody against human IgG1 Fc tag is shown in any one of the following (1)-(43): (1) CDR1 is shown in SEQ ID NO.44, CDR2 is shown in SEQ ID NO.87, and CDR3 is shown in SEQ ID NO.130; (2) CDR1 is shown in SEQ ID NO.45, CDR2 is shown in SEQ ID NO.88, and CDR3 is shown in SEQ ID NO.131; (3) CDR1 is shown in SEQ ID NO.46, CDR2 is shown in SEQ ID NO.89, and CDR3 is shown in SEQ ID NO.132; (4) CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.90, and CDR3 is shown in SEQ ID NO.133; (5) CDR1 is shown in SEQ ID NO.48, CDR2 is shown in SEQ ID NO.91, and CDR3 is shown in SEQ ID NO.134; (6) CDR1 is shown in SEQ ID NO.49, CDR2 is shown in SEQ ID NO.92, and CDR3 is shown in SEQ ID NO.135; (7) CDR1 is shown in SEQ ID NO.50, CDR2 is shown in SEQ ID NO.93, and CDR3 is shown in SEQ ID NO.136; (8) CDR1 is shown in SEQ ID NO.51, CDR2 is shown in SEQ ID NO.94, and CDR3 is shown in SEQ ID NO.137; (9) CDR1 is shown in SEQ ID NO.52, CDR2 is shown in SEQ ID NO.95, and CDR3 is shown in SEQ ID NO.138; (10) CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.96, and CDR3 is shown in SEQ ID NO.139; (11) CDR1 is shown in SEQ ID NO.54, CDR2 is shown in SEQ ID NO.97, and CDR3 is shown in SEQ ID NO.140; (12) CDR1 is shown in SEQ ID NO.55, CDR2 is shown in SEQ ID NO.98, and CDR3 is shown in SEQ ID NO.141; (13) CDR1 is shown in SEQ ID NO.56, CDR2 is shown in SEQ ID NO.99, and CDR3 is shown in SEQ ID NO.142; (14) CDR1 is shown in SEQ ID NO.57, CDR2 is shown in SEQ ID NO.100, and CDR3 is shown in SEQ ID NO.143; (15) CDR1 is shown in SEQ ID NO.58, CDR2 is shown in SEQ ID NO.101, and CDR3 is shown in SEQ ID NO.144; (16) CDR1 is shown in SEQ ID NO.59, CDR2 is shown in SEQ ID NO.102, and CDR3 is shown in SEQ ID NO.145; (17) CDR1 is shown in SEQ ID NO.60, CDR2 is shown in SEQ ID NO.103, and CDR3 is shown in SEQ ID NO.146; (18) CDR1 is shown in SEQ ID NO.61, CDR2 is shown in SEQ ID NO.104, and CDR3 is shown in SEQ ID NO.147; (19) CDR1 is shown in SEQ ID NO.62, CDR2 is shown in SEQ ID NO.105, and CDR3 is shown in SEQ ID NO.148; (20) CDR1 is shown in SEQ ID NO.63, CDR2 is shown in SEQ ID NO.106, and CDR3 is shown in SEQ ID NO.149; (21) CDR1 is shown in SEQ ID NO.64, CDR2 is shown in SEQ ID NO.107, and CDR3 is shown in SEQ ID NO.150; (22) CDR1 is shown in SEQ ID NO.65, CDR2 is shown in SEQ ID NO.108, and CDR3 is shown in SEQ ID NO.151; (23) CDR1 is shown in SEQ ID NO.66, CDR2 is shown in SEQ ID NO.109, and CDR3 is shown in SEQ ID NO.152; (24) CDR1 is shown in SEQ ID NO.67, CDR2 is shown in SEQ ID NO.110, and CDR3 is shown in SEQ ID NO.153; (25) CDR1 is shown in SEQ ID NO.68, CDR2 is shown in SEQ ID NO.111, and CDR3 is shown in SEQ ID NO.154; (26) CDR1 is shown in SEQ ID NO.69, CDR2 is shown in SEQ ID NO.112, and CDR3 is shown in SEQ ID NO.155; (27) CDR1 is shown in SEQ ID NO.70, CDR2 is shown in SEQ ID NO.113, and CDR3 is shown in SEQ ID NO.156; (28) CDR1 is shown in SEQ ID NO.71, CDR2 is shown in SEQ ID NO.114, and CDR3 is shown in SEQ ID NO.157; (29) CDR1 is shown in SEQ ID NO.72, CDR2 is shown in SEQ ID NO.115, and CDR3 is shown in SEQ ID NO.158; (30) CDR1 is shown in SEQ ID NO.73, CDR2 is shown in SEQ ID NO.116, and CDR3 is shown in SEQ ID NO.159; (31) CDR1 is shown in SEQ ID NO.74, CDR2 is shown in SEQ ID NO.117, and CDR3 is shown in SEQ ID NO.160; (32) CDR1 is shown in SEQ ID NO.75, CDR2 is shown in SEQ ID NO.118, and CDR3 is shown in SEQ ID NO.161; (33) CDR1 is shown in SEQ ID NO.76, CDR2 is shown in SEQ ID NO.119, and CDR3 is shown in SEQ ID NO.162; (34) CDR1 is shown in SEQ ID NO.77, CDR2 is shown in SEQ ID NO.120, and CDR3 is shown in SEQ ID NO.163; (35) CDR1 is shown in SEQ ID NO.78, CDR2 is shown in SEQ ID NO.121, and CDR3 is shown in SEQ ID NO.164; (36) CDR1 is shown in SEQ ID NO.79, CDR2 is shown in SEQ ID NO.122, and CDR3 is shown in SEQ ID NO.165; (37) CDR1 is shown in SEQ ID NO.80, CDR2 is shown in SEQ ID NO.123, and CDR3 is shown in SEQ ID NO.166; (38) CDR1 is shown in SEQ ID NO.81, CDR2 is shown in SEQ ID NO.124, and CDR3 is shown in SEQ ID NO.167; (39) CDR1 is shown in SEQ ID NO.82, CDR2 is shown in SEQ ID NO.125, and CDR3 is shown in SEQ ID NO.168; (40) CDR1 is shown in SEQ ID NO.83, CDR2 is shown in SEQ ID NO.126, and CDR3 is shown in SEQ ID NO.169; (41) CDR1 is shown in SEQ ID NO.84, CDR2 is shown in SEQ ID NO.127, and CDR3 is shown in SEQ ID NO.170; (42) CDR1 is shown in SEQ ID NO.85, CDR2 is shown in SEQ ID NO.128, and CDR3 is shown in SEQ ID NO.171; (43) CDR1 is shown in SEQ ID NO.86, CDR2 is shown in SEQ ID NO.129, and CDR3 is shown in SEQ ID NO.

172.

3. The single domain antibody against human IgG1 Fc tag according to claim 1, characterized in that: The single domain antibody against human IgG1 Fc tag has the following framework regions: FR1, FR2, FR3, FR4; in, The amino acid sequence of FR1 is shown in any one of SEQ ID NOs. 173-215; The amino acid sequence of FR2 is shown in any one of SEQ ID NOs. 216-258; The amino acid sequence of FR2 is shown in any one of SEQ ID NOs. 259-301; The amino acid sequence of FR4 is shown in any one of SEQ ID NOs. 302-344.

4. The single domain antibody against human IgG1 Fc tag according to claim 3, characterized in that: The framework region of the single domain antibody is as shown in any one of the following (44)-(86): (44) FR1 is shown in SEQ ID NO.173, FR2 is shown in SEQ ID NO.216, FR3 is shown in SEQ ID NO.259, and FR4 is shown in SEQ ID NO.302; (45) FR1 is shown in SEQ ID NO.174, FR2 is shown in SEQ ID NO.217, FR3 is shown in SEQ ID NO.260, and FR4 is shown in SEQ ID NO.303; (46) FR1 is shown in SEQ ID NO.175, FR2 is shown in SEQ ID NO.218, FR3 is shown in SEQ ID NO.261, and FR4 is shown in SEQ ID NO.304; (47) FR1 is shown in SEQ ID NO.176, FR2 is shown in SEQ ID NO.219, FR3 is shown in SEQ ID NO.262, and FR4 is shown in SEQ ID NO.305; (48) FR1 is shown in SEQ ID NO.177, FR2 is shown in SEQ ID NO.220, FR3 is shown in SEQ ID NO.263, and FR4 is shown in SEQ ID NO.306; (49) FR1 is shown in SEQ ID NO.178, FR2 is shown in SEQ ID NO.221, FR3 is shown in SEQ ID NO.264, and FR4 is shown in SEQ ID NO.307; (50) FR1 is shown in SEQ ID NO.179, FR2 is shown in SEQ ID NO.222, FR3 is shown in SEQ ID NO.265, and FR4 is shown in SEQ ID NO.308; (51) FR1 is shown in SEQ ID NO.180, FR2 is shown in SEQ ID NO.223, FR3 is shown in SEQ ID NO.266, and FR4 is shown in SEQ ID NO.309; (52) FR1 is shown in SEQ ID NO.181, FR2 is shown in SEQ ID NO.224, FR3 is shown in SEQ ID NO.267, and FR4 is shown in SEQ ID NO.310; (53) FR1 is shown in SEQ ID NO.182, FR2 is shown in SEQ ID NO.225, FR3 is shown in SEQ ID NO.268, and FR4 is shown in SEQ ID NO.311; (54) FR1 is shown in SEQ ID NO.183, FR2 is shown in SEQ ID NO.226, FR3 is shown in SEQ ID NO.269, and FR4 is shown in SEQ ID NO.312; (55) FR1 is shown in SEQ ID NO.184, FR2 is shown in SEQ ID NO.227, FR3 is shown in SEQ ID NO.270, and FR4 is shown in SEQ ID NO.313; (56) FR1 is shown in SEQ ID NO.185, FR2 is shown in SEQ ID NO.228, FR3 is shown in SEQ ID NO.271, and FR4 is shown in SEQ ID NO.314; (57) FR1 is shown in SEQ ID NO.186, FR2 is shown in SEQ ID NO.229, FR3 is shown in SEQ ID NO.272, and FR4 is shown in SEQ ID NO.315; (58) FR1 is shown in SEQ ID NO.187, FR2 is shown in SEQ ID NO.230, FR3 is shown in SEQ ID NO.273, and FR4 is shown in SEQ ID NO.316; (59) FR1 is shown in SEQ ID NO.188, FR2 is shown in SEQ ID NO.231, FR3 is shown in SEQ ID NO.274, and FR4 is shown in SEQ ID NO.317; (60) FR1 is shown in SEQ ID NO.189, FR2 is shown in SEQ ID NO.232, FR3 is shown in SEQ ID NO.275, and FR4 is shown in SEQ ID NO.318; (61) FR1 is shown in SEQ ID NO.190, FR2 is shown in SEQ ID NO.233, FR3 is shown in SEQ ID NO.276, and FR4 is shown in SEQ ID NO.319; (62) FR1 is shown in SEQ ID NO.191, FR2 is shown in SEQ ID NO.234, FR3 is shown in SEQ ID NO.277, and FR4 is shown in SEQ ID NO.320; (63) FR1 is shown in SEQ ID NO.192, FR2 is shown in SEQ ID NO.235, FR3 is shown in SEQ ID NO.278, and FR4 is shown in SEQ ID NO.321; (64) FR1 is shown in SEQ ID NO.193, FR2 is shown in SEQ ID NO.236, FR3 is shown in SEQ ID NO.279, and FR4 is shown in SEQ ID NO.322; (65) FR1 is shown in SEQ ID NO.194, FR2 is shown in SEQ ID NO.237, FR3 is shown in SEQ ID NO.280, and FR4 is shown in SEQ ID NO.323; (66) FR1 is shown in SEQ ID NO.195, FR2 is shown in SEQ ID NO.238, FR3 is shown in SEQ ID NO.281, and FR4 is shown in SEQ ID NO.324; (67) FR1 is shown in SEQ ID NO.196, FR2 is shown in SEQ ID NO.239, FR3 is shown in SEQ ID NO.282, and FR4 is shown in SEQ ID NO.325; (68) FR1 is shown in SEQ ID NO.197, FR2 is shown in SEQ ID NO.240, FR3 is shown in SEQ ID NO.283, and FR4 is shown in SEQ ID NO.326; (69) FR1 is shown in SEQ ID NO.198, FR2 is shown in SEQ ID NO.241, FR3 is shown in SEQ ID NO.284, and FR4 is shown in SEQ ID NO.327; (70) FR1 is shown in SEQ ID NO.199, FR2 is shown in SEQ ID NO.242, FR3 is shown in SEQ ID NO.285, and FR4 is shown in SEQ ID NO.328; (71) FR1 is shown in SEQ ID NO.200, FR2 is shown in SEQ ID NO.243, FR3 is shown in SEQ ID NO.286, and FR4 is shown in SEQ ID NO.329; (72) FR1 is shown in SEQ ID NO.201, FR2 is shown in SEQ ID NO.244, FR3 is shown in SEQ ID NO.287, and FR4 is shown in SEQ ID NO.330; (73) FR1 is shown in SEQ ID NO.202, FR2 is shown in SEQ ID NO.245, FR3 is shown in SEQ ID NO.288, and FR4 is shown in SEQ ID NO.331; (74) FR1 is shown in SEQ ID NO.203, FR2 is shown in SEQ ID NO.246, FR3 is shown in SEQ ID NO.289, and FR4 is shown in SEQ ID NO.332; (75) FR1 is shown in SEQ ID NO.204, FR2 is shown in SEQ ID NO.247, FR3 is shown in SEQ ID NO.290, and FR4 is shown in SEQ ID NO.333; (76) FR1 is shown in SEQ ID NO.205, FR2 is shown in SEQ ID NO.248, FR3 is shown in SEQ ID NO.291, and FR4 is shown in SEQ ID NO.334; (77) FR1 is shown in SEQ ID NO.206, FR2 is shown in SEQ ID NO.249, FR3 is shown in SEQ ID NO.292, and FR4 is shown in SEQ ID NO.335; (78) FR1 is shown in SEQ ID NO.207, FR2 is shown in SEQ ID NO.250, FR3 is shown in SEQ ID NO.293, and FR4 is shown in SEQ ID NO.336; (79) FR1 is shown in SEQ ID NO.208, FR2 is shown in SEQ ID NO.251, FR3 is shown in SEQ ID NO.294, and FR4 is shown in SEQ ID NO.337; (80) FR1 is shown in SEQ ID NO.209, FR2 is shown in SEQ ID NO.252, FR3 is shown in SEQ ID NO.295, and FR4 is shown in SEQ ID NO.338; (81) FR1 is shown in SEQ ID NO.210, FR2 is shown in SEQ ID NO.253, FR3 is shown in SEQ ID NO.296, and FR4 is shown in SEQ ID NO.339; (82) FR1 is shown in SEQ ID NO.211, FR2 is shown in SEQ ID NO.254, FR3 is shown in SEQ ID NO.297, and FR4 is shown in SEQ ID NO.340; (83) FR1 is shown in SEQ ID NO.212, FR2 is shown in SEQ ID NO.255, FR3 is shown in SEQ ID NO.298, and FR4 is shown in SEQ ID NO.341; (84) FR1 is shown in SEQ ID NO.213, FR2 is shown in SEQ ID NO.256, FR3 is shown in SEQ ID NO.299, and FR4 is shown in SEQ ID NO.342; (85) FR1 is shown in SEQ ID NO.214, FR2 is shown in SEQ ID NO.257, FR3 is shown in SEQ ID NO.300, and FR4 is shown in SEQ ID NO.343; (86) FR1 is shown in SEQ ID NO.215, FR2 is shown in SEQ ID NO.258, FR3 is shown in SEQ ID NO.301, and FR4 is shown in SEQ ID NO.

344.

5. The single domain antibody against human IgG1 Fc tag according to claim 1, characterized in that: The amino acid sequence of the single domain antibody is shown in any one of SEQ ID NOs. 1-43.

6. The single domain antibody against human IgG1 Fc tag according to claim 4, characterized in that: The framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from an alpaca-derived antibody.

7. The single domain antibody against human IgG1 Fc tag according to claim 1, characterized in that: The single-domain antibody against human IgG1 Fc tag is prepared by immunizing camels with human IgG1 Fc recombinant protein antigen, collecting peripheral blood cells of the immunized camels, separating human IgG1 Fc affinity lymphocytes therefrom, extracting total RNA and reversely transcribing it into cDNA, cloning the V region of the camel heavy chain antibody by Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the human IgG1 Fc antigen by phage display technology, and verifying the binding force of the obtained single-domain antibody by enzyme-linked immunosorbent assay, thereby screening out the single-domain antibody against human IgG1 Fc tag with high affinity activity.

8. The single domain antibody against human IgG1 Fc tag according to claim 1, characterized in that: The DNA sequence of the single domain antibody against human IgG1 Fc tag is shown in any one of SEQ ID NOs. 345-387.

9. A fusion protein, characterized in that: It contains the single domain antibody with anti-human IgG1 Fc tag as described in any one of claims 1 to 8.

10. A product, characterized in that It contains the single-domain antibody with an anti-human IgG1 Fc tag as described in any one of claims 1 to 8 or the fusion protein as described in claim 9, and the product is an in vitro diagnostic reagent, an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to a site expressing human IgG1 Fc.

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