Anti-tnf alpha nanobodies and uses thereof

Abstract

The application provides an anti-TNF alpha nanobody and application thereof, and relates to a nanobody, a polypeptide containing the nanobody and application thereof, and the like, a variable region in an amino acid sequence of the nanobody comprises three complementarity determining regions CDR and a framework region FR, the complementarity determining regions CDR comprise a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein the most important site participating in antigen recognition and combination is RXXXXXE on the CDR1 and ATYSDSPWNXXSFYXLSGVGA on the CDR3, the nanobody and the polypeptide thereof have high affinity and activity, can specifically recognize and combine TNF alpha, and can be used for adsorption, removal and detection of TNF alpha and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to an anti-TNFα nanobody, a polypeptide comprising the nanobody, and preparation and application thereof. BACKGROUND

[0002] Tumor necrosis factor-α (TNF-α) is an important cytokine, mainly secreted by macrophages, and is a homotrimeric protein composed of 157 amino acids. It plays a key role in inflammatory response, immune regulation, and the development of various diseases.

[0003] When the human body encounters infection, immune response, chronic inflammation, tumor, and genetic factors, these complex biological processes often lead to overexpression of TNF-α. As a key cytokine, TNF-α plays a crucial role in regulating immune response and inflammatory processes. When this balance is broken, and the level of TNF-α exceeds the normal range, it can become a trigger for a series of inflammatory reactions.

[0004] Excessive TNF-α not only exacerbates the existing inflammatory state, but also can trigger a series of chain reactions, leading to the overactivation of other inflammatory factors. These inflammatory factors, including but not limited to interleukins (ILs), interferons (IFNs), and chemokines, further amplify the inflammatory signal under the drive of TNF-α, forming a self-enhancing vicious cycle. This excessive TNF-α and the resulting inflammatory factor storm can also cause tissue damage, organ dysfunction, and even multiple organ failure in extreme cases.

[0005] Therefore, controlling the level of TNF-α is crucial for maintaining the health and stability of the body. Some drugs targeting TNF-α have been developed, such as Etanercept, Infliximab, Adalimumab, and Certolizumab, which bind to TNF-α and prevent its binding to receptors, thereby reducing inflammatory responses and immune cell activity, and are widely used in the treatment of rheumatoid arthritis, Crohn's disease, and other diseases. However, for excessive TNF-α, traditional drug therapy may not achieve the desired control effect. In this case, blood purification technology provides an effective solution. The existing removal methods mainly include non-specific adsorption methods, including: 1. Based on the adsorption of polystyrene divinylbenzene copolymer, a variety of inflammatory mediators with molecular weight in the range of 10,000-60,000, including TNF-α, are non-specifically adsorbed by hydrophobic force. Membrane: The surface is modified with a multilayer linear polyethyleneimine cationic polymer to carry positive charges, so as to effectively adsorb endotoxins and other inflammatory factors with negative charges.

[0006] The above methods are all non-specific adsorption, which removes harmful substances while also removing other beneficial molecules, affecting the physiological balance of the patient and increasing the physical burden of the patient. In contrast, specific adsorption has many incomparable advantages: high selectivity, high efficiency, fast response, and good safety. Antibodies can achieve specific adsorption of TNFα, but the volume of traditional antibodies is large and the preparation cost is high, so it limits its practical application in capture, detection and other fields, especially in the field of blood purification. SUMMARY

[0007] The purpose of the present application is to solve the above-mentioned problems of TNFα capture, adsorption or detection. In the first aspect, according to the anti-TNFα nanobody in some embodiments of the present application, the complementarity determining region CDR of the nanobody includes CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of the CDR1 is as shown in SEQ ID NO. 50, (2) the amino acid sequence of the CDR2 is as shown in SEQ ID NO. 66; and (3) the amino acid sequence of the CDR3 is as shown in SEQ ID NO. 33; or (II): an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequences of (1), (2) and (3) of (I), and an amino acid sequence that is functionally equivalent to the amino acid sequence of (I).

[0008] According to the anti-TNFa Nanobody in some embodiments of the present application, wherein: the amino acid sequence of (1), (2), (3) of (I) is substituted by one or more amino acids to obtain an amino acid sequence, including (II-1): the amino acid sequence of CDR1 is shown as SEQ ID NO. 51, the amino acid sequence of CDR2 is shown as SEQ ID NO. 67, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 34; or (II-2): the amino acid sequence of CDR1 is shown as SEQ ID NO. 52, the amino acid sequence of CDR2 is shown as SEQ ID NO. 66, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 33; or (II-3): the amino acid sequence of CDR1 is shown as SEQ ID NO. 53, the amino acid sequence of CDR2 is shown as SEQ ID NO. 68, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 35; or (II-4): the amino acid sequence of CDR1 is shown as SEQ ID NO. 54, the amino acid sequence of CDR2 is shown as SEQ ID NO. 69, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 36; or (II-5): the amino acid sequence of CDR1 is shown as SEQ ID NO. 54, the amino acid sequence of CDR2 is shown as SEQ ID NO. 70, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 37; or (II-6): the amino acid sequence of CDR1 is shown as SEQ ID NO. 55, the amino acid sequence of CDR2 is shown as SEQ ID NO. 71, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 38; or (II-7): the amino acid sequence of CDR1 is shown as SEQ ID NO. 55, the amino acid sequence of CDR2 is shown as SEQ ID NO. 72, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 39; or (II-8): the amino acid sequence of CDR1 is shown as SEQ ID NO. 56, the amino acid sequence of CDR2 is shown as SEQ ID NO. 73, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 40; or (II-9): the amino acid sequence of CDR1 is shown as SEQ ID NO. 57, the amino acid sequence of CDR2 is shown as SEQ ID NO. 74, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 40; or (II-10): the amino acid sequence of CDR1 is shown as SEQ ID NO. 58, the amino acid sequence of CDR2 is shown as SEQ ID NO. 74, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 41; or (II-11): the amino acid sequence of CDR1 is shown as SEQ ID NO. 59, the amino acid sequence of CDR2 is shown as SEQ ID NO.72, the amino acid sequence of CDR3 is shown in SEQ ID NO. 42; or (II-12): the amino acid sequence of CDR1 is shown in SEQ ID NO. 60, the amino acid sequence of CDR2 is shown in SEQ ID NO. 75, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 43; or (II-13): the amino acid sequence of CDR1 is shown in SEQ ID NO. 61, the amino acid sequence of CDR2 is shown in SEQ ID NO. 76, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 44; or (II-14): the amino acid sequence of CDR1 is shown in SEQ ID NO. 62, the amino acid sequence of CDR2 is shown in SEQ ID NO. 77, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 45; or (II-15): the amino acid sequence of CDR1 is shown in SEQ ID NO. 63, the amino acid sequence of CDR2 is shown in SEQ ID NO. 78, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 43; or (II-16): the amino acid sequence of CDR1 is shown in SEQ ID NO. 63, the amino acid sequence of CDR2 is shown in SEQ ID NO. 79, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 46; or (II-17): the amino acid sequence of CDR1 is shown in SEQ ID NO. 64, the amino acid sequence of CDR2 is shown in SEQ ID NO. 77, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 47; or (II-18): the amino acid sequence of CDR1 is shown in SEQ ID NO. 64, the amino acid sequence of CDR2 is shown in SEQ ID NO. 77, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 48; or (II-19): the amino acid sequence of CDR1 is shown in SEQ ID NO. 65, the amino acid sequence of CDR2 is shown in SEQ ID NO. 80, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 49.

[0009] According to the anti-TNFa Nanobody in some embodiments of the application, wherein: (III) the framework region FR of the Nanobody comprises FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is shown in SEQ ID NO. 81, (2) the amino acid sequence of FR2 is shown in SEQ ID NO. 82, (3) the amino acid sequence of FR3 is shown in SEQ ID NO. 84, (4) the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV) an amino acid sequence that is more than 50% homologous to the amino acid sequences shown in (III) (1), (2), (3), (4).

[0010] According to the anti-TNFa Nanobody in some embodiments of the present application, the amino acid sequence of (IV) as described in (III) (1), (2), (3), (4) has an amino acid sequence with 50% or more homology, including (IV-1): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 84, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-2): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 85, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-3): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 86, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-4): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 87, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-5): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 88, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-6): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 84, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-7): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 87, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-8): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 89, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-9): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO.82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 89, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-10): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 87, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-11): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 87, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-12): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 86, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-13): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 84, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-14): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 89, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-15): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 86, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-16): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 84, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-17): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 82, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 89, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 90; or (IV-18): the amino acid sequence of FR1 is as set forth in SEQ ID NO. 81, the amino acid sequence of FR2 is as set forth in SEQ ID NO. 83, the amino acid sequence of FR3 is as set forth in SEQ ID NO.89, the amino acid sequence of FR4 is shown as SEQ ID NO. 90; or (IV-19): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 89, and the amino acid sequence of FR4 is shown as SEQ ID NO. 90.

[0011] According to the anti-TNFa nanobody in some embodiments of the present application, the nanobody has (V) the amino acid sequence shown as SEQ ID NO. 1; or (VI) the amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids from the amino acid sequence shown as (V), and the amino acid sequence which is functionally identical to the amino acid sequence shown as (I). According to the anti-TNFa nanobody in some embodiments of the present application, the amino acid sequence of the nanobody of (VI) is shown as any one of SEQ ID NO. 2 to SEQ ID NO. 20.

[0012] In the second aspect, according to the humanized nanobody in some embodiments of the present application, (VII): the amino acid sequence of the humanized nanobody is shown as any one of SEQ ID NO. 21 to SEQ ID NO. 32.

[0013] In the third aspect, according to the polypeptide in some embodiments of the present application, it comprises any one of the nanobodies.

[0014] In the fourth aspect, according to the nucleic acid molecule encoding any one of the nanobodies in some embodiments of the present application.

[0015] In the fifth aspect, according to the expression vector in some embodiments of the present application, it comprises the nucleic acid molecule.

[0016] In the sixth aspect, according to the host cell transformed or transfected with the expression vector in some embodiments of the present application.

[0017] In the seventh aspect, according to the conjugate or complex in some embodiments of the present application, it comprises any one of the nanobodies chemically or biologically labeled.

[0018] In the eighth aspect, according to the adsorbent in some embodiments of the present application, it comprises any one of the nanobodies; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the complex, and a carrier.

[0019] In a ninth aspect, a kit according to some embodiments of the present application comprises any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the adsorbents; or the kits; or the devices.

[0020] In a tenth aspect, an apparatus according to some embodiments of the present application for capturing, adsorbing and / or detecting TNFα comprises any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the adsorbents; or the kits; or the devices.

[0021] In an eleventh aspect, the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the adsorbents; or the kits; or the devices according to some embodiments of the present application are used in the preparation of a preparation for specifically capturing, adsorbing and / or detecting TNFα; in the preparation of a preparation for specifically capturing, adsorbing and / or detecting TNFα in an infection, immune response, chronic inflammation, tumor detection; in the preparation of a preparation for specifically capturing, adsorbing and / or detecting TNFα in enrichment and / or purification of cells; in the preparation of a reagent for immunofluorescence analysis or immunohistochemical analysis of TNFα.

[0022] Beneficial effects: The nanobodies of the present application are nanobodies with new amino acid sequences for screening and finding anti-TNFα, which have high affinity and activity, can specifically recognize and bind TNFα, and can be used for TNFα detection and purification, as well as capture and detection of TNFα positive cells, and can be applied to immunofluorescence analysis or immunohistochemical analysis through appropriate antibody labeling technology. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the overall effect of the T00 and TNFα docking diagram in the embodiments of the present application.

[0024] Figure 2 is a partial enlarged view of the first angle of the molecular docking diagram, and b. is a partial enlarged view of the second angle of the molecular docking diagram.

[0025] Figure 3 is a schematic diagram of the interaction between T00 and TNFα in the embodiments of the present application.

[0026] Figure 4Figure 1 is a schematic diagram of the intra-molecular interaction of T00 molecule in embodiments of the present application, wherein a. partial enlargement of the first angle of the three-dimensional structure diagram, b. partial enlargement of the second angle of the three-dimensional structure diagram.

[0027] Figure 5 Figure 2 is a kinetic curve of the nanobody in embodiments of the present application.

[0028] Figure 6 Figure 3 is a static capture adsorption curve in embodiments of the present application.

[0029] Figure 7 Figure 4 is a standard curve of sandwich ELISA detection in embodiments of the present application. DETAILED DESCRIPTION

[0030] The above and other aspects of the present application will become more apparent by describing in further detail embodiments thereof, among which:

[0031] (1) Unless otherwise indicated, the term "sequence" as used herein (e.g., in the terms "antibody sequence", "variable region sequence", "V HH sequence", or "protein sequence") shall be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the same, unless the context requires a more narrow interpretation.

[0032] (2) Unless otherwise indicated, all methods, procedures, techniques and operations not specifically described are known and as familiar to those skilled in the art. For example, reference is still made to the comprehensive background art cited above and to other references cited therein.

[0033] (3) The term "specificity" refers to the ability of a particular antigen-binding molecule (e.g., a nanobody or polypeptide of the present application) to bind to different types of antigens or antigenic determinants. The specificity of an antigen-binding molecule can be determined according to its affinity and / or activity. Affinity is expressed as the dissociation equilibrium constant (K D ) between an antigen and an antigen-binding molecule, the smaller the K D value, the stronger the binding strength between the antigen and the antigen-binding molecule, and vice versa, the larger the K D value, the weaker the binding strength between the antigen and the antigen-binding molecule. K a represents the association constant, the larger the K a value, the faster the binding, the smaller the K a value, the slower the binding; K d represents the dissociation constant, the larger the K d value, the faster the dissociation, the smaller the K d value, the slower the dissociation; and K D = K d / K a .

[0034] (4) The term "family" refers to a family of Nanobody sequences binding to the same antigen, having the same number of amino acids and having an amino acid sequence identity of more than 75%.

[0035] (5) Amino acid substitution can generally be described as follows, wherein an amino acid residue can be substituted by an amino acid having a similar chemical structure, but also by an amino acid having a different chemical structure, as long as there is little or essentially no effect on the function, activity or other biological property of the polypeptide. Preferably, an amino acid residue can be substituted by an amino acid having a similar chemical structure.

[0036] For the above-mentioned substitution, the cases disclosed in the documents WO 04 / 037999, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300 can be cited, but are not limiting, and in addition, the (preferred) type and / or combination of the substitutions can be selected based on the information in WO 04 / 037999 and WO 06 / 122786 referring to other references.

[0037] The amino acid substitution according to the present application can be cited, but is not limited to, the following substitution, wherein one amino acid of the following groups (a) to (e) is substituted by another amino acid of the same group: (a) Ala, Ser, Thr, Pro and Gly; (b) Asp, Asn, Glu and Gin; (c) His, Lys and Arg; (d) Met, Leu, lie, Val and Cys; (e) Phe, Tyr and Trp.

[0038] The preferred amino acid substitution can be cited, but is not limited to, the following: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gin or His; Asp is substituted by Glu; Cys is substituted by Ser or Thr; Gin is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gin; lie is substituted by Leu or Val; Leu is substituted by lie or Val; Lys is substituted by Arg, Glu or Gin; Met is substituted by Leu, Tyr or lie; Phe is substituted by Met, Tyr or Leu; Ser is substituted by Thr; Thr is substituted by Ser; Tyr is substituted by Trp; Trp is substituted by Tyr.

[0039] The framework regions are more conserved than the complementarity determining regions. The sequence and structure of the framework regions can be reasonably selected by the person skilled in the art according to the actual use and function of the nanobody. As the amino acid sequence of the framework region, an amino acid sequence having a homology of 50% or more is preferred, an amino acid sequence having a homology of 70% or more is further preferred, and an amino acid sequence having a homology of 95% or more is further preferred. The following table is the framework region sequence information of each antibody in Example 3:

[0040]

[0041]

[0042] The framework region contributes less to the affinity, so the amino acid substitution of the framework region generally does not affect the affinity of the nanobody, and the amino acid of the framework region is also suitable for the above-mentioned amino acid substitution mode. Among them, humanization is a typical example of amino acid substitution of the framework region, and in the present application, SEQ ID No: 21-SEQ ID No: 32 are four humanized forms of SEQ ID No: 1, SEQ ID No: 10, SEQ ID No: 13, which do not affect the affinity of the original sequence.

[0043] In addition, the total number of residues of the nanobody can be in the range of 110-120. However, the parts, fragments or analogs of the nanobody are not particularly limited to their length and / or size, as long as such parts, fragments or analogs meet the further requirements listed below and are also suitable for the purposes described herein.

[0044] The nanobody in the present application belongs to the same family of nanobodies, which have the same total length of amino acid sequence, the same length of each framework region FR and antigen binding region CDR and high sequence identity, similar structure, and substantially equivalent antigen binding capacity.

[0045] As a method for preparing "nanobodies", it is not limited to specific biological resources or specific preparation methods in its broadest sense. For example, the nanobody of the present application can be obtained by: (1) isolating the V HH domain from a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding a naturally occurring V HH domain; (3) "humanizing" a naturally occurring V HH domain (as described below) or by expressing a nucleic acid encoding the humanized V HH domain; (4) preparing a protein, polypeptide or other amino acid sequence using synthetic or semi-synthetic techniques; (5) preparing a nucleic acid encoding a nanobody by using nucleic acid synthesis techniques, and then expressing the nucleic acid thus obtained; and / or (6) by any combination of the foregoing.

[0046] Furthermore, a variant based on a Nanobody according to the application also comprises a Nanobody having an amino acid sequence which corresponds to the naturally occurring V HH domains of a conventional 4-chain antibody from a human, but which has been humanized, i.e. in which one or more amino acid residues from the corresponding position in the naturally occurring V H domains have been replaced by one or more amino acid residues from the corresponding position in the naturally occurring V HH domains of a conventional 4-chain antibody from a human, but which has been humanized, i.e. in which one or more amino acid residues from the corresponding position in the naturally occurring V

[0047] According to a non-limiting embodiment of the application, the above polypeptide consists essentially of a Nanobody. "Consists essentially of" means that the amino acid sequence of the polypeptide according to the application is identical to or corresponds to the amino acid sequence of a Nanobody, wherein a limited number of amino acid residues, such as 1 to 10 amino acid residues, and preferably 1 to 6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, are added to the amino-terminal (N-terminal) and / or carboxy-terminal (C-terminal) end of said Nanobody or polypeptide.

[0048] The above amino acid residues can not alter the biological properties of the Nanobody, and can add other functionalities to the Nanobody. For example, the amino acid residues can:

[0049] a is a purification tag, i.e. an amino acid sequence or residue which facilitates purification of the Nanobody, e.g. using affinity techniques directed to said sequence or residue. Some preferred, but non-limiting, examples of such residues are poly-His tags (His6or His8), GST-tag, MBP-tag, Myc-tag, Strep-tag, Flag-tag, HA-tag, V5-tag, S-tag, E-tag;

[0050] b is a solubility tag, i.e. a tag which facilitates increased solubility of the Nanobody, e.g. SUMO;

[0051] c is an N-terminal amino acid residue, e.g. Met, Ala, Gin or MetAlaGin, AlaGin, whereby expression in a heterologous host cell or host organism can be facilitated;

[0052] d is a C-terminal Cys residue, e.g. whereby reaction with a -SH on a ligand or reaction with an Au surface can be facilitated;

[0053] e is a hinge, to provide linking or spacing of the Nanobody to other groups, e.g. a combination of GlySer, an IgG hinge, an IgA hinge, or other artificially synthesized hinge;

[0054] f is one or more amino acid residues that can be provided with a functional group and / or have been functionalized in a known manner, for example, as is known in the art, amino acid residues such as lysine or cysteine allow PEG groups to be attached.

[0055] The polypeptides of the application can also comprise two or more of said Nanobodies, also referred to as multivalent polypeptides.

[0056] A bivalent polypeptide comprises two Nanobodies, optionally linked by a hinge sequence, a trivalent polypeptide comprises three Nanobodies, optionally linked by two hinge sequences, a tetravalent polypeptide comprises four Nanobodies, optionally linked by three hinge sequences. The multivalent polypeptides can bind to the same antigenic epitope or to different antigenic epitopes, the latter also referred to as multispecific polypeptides.

[0057] With respect to multivalent and multispecific polypeptides comprising one or more V HH Domains and their preparation, reference is made to the description in EP 0822985.

[0058] Hinges for use in multivalent and multispecific polypeptides are well known to the person skilled in the art, for example comprising Gly-Ser, such as (Gly4Ser)3 or (Gly3Ser2)3 as described in WO 99 / 42077; or naturally occurring heavy chain antibody hinge regions or partial regions thereof. For other suitable hinges, reference is also made to the general background art cited above.

[0059] In addition, the polypeptides of the application can comprise functional groups, moieties or residues in addition to said one or more Nanobodies, such as therapeutically active substances, and / or labels, such as fluorescein labels, isotopic labels, biotin labels and enzymatic catalytic labels.

[0060] In addition, the dissociation equilibrium constant (K D ) of the Nanobodies or polypeptides of the application for binding to TNFα is between 10 -8 and 10 -10 molar (M). The dissociation equilibrium constant of the application is measured using the technology of plasmon resonance.

[0061] The specific binding between the above-mentioned antigens and antigen binding molecules can be determined using any suitable method known in the art, including Scatchard analysis and / or competition binding assays such as radioimmunoassays (RIA) and enzyme-linked immunoassays (ELISA), and other new methods known in the art, such as the technology of plasmon resonance (SPR) and / or the technology of bio-layer interferometry (BLI). And the person skilled in the art will know that the affinity parameters determined using different methods can differ considerably, even by a factor of 2 to 3.

[0062] The nanobody structure of the present application can be determined by any suitable method known in the art, including Alohafold2 multimer v3 (a protein structure prediction tool used to predict the three-dimensional structure of protein complexes, which can help researchers understand how different proteins interact with each other to form complexes), Amber (a widely used molecular dynamics simulation software package that can be used to simulate the behavior of proteins and other biological molecules. In structural biology, Amber is commonly used to perform energy minimization, side-chain optimization, and molecular dynamics simulations), Relax Process (in molecular dynamics simulations, the Relax Process generally refers to the process of energy minimization of a protein structure to optimize the geometry of side chains, reduce internal conflicts and unfavorable geometry of molecules), ADT (AutoDock Tools, a software toolset for molecular docking and molecular dynamics simulations, which can help researchers predict the binding mode and affinity between ligands and receptors), and / or Ligplot+ (a tool for analyzing protein-ligand complex interfaces, which can identify and visualize hydrogen bonds, hydrophobic interactions, and other non-covalent interactions).

[0063] The nanobodies, polypeptides, and nucleic acids encoding the same of the present application can be prepared in a known manner, which will be apparent to those skilled in the art from the further description herein. A particularly useful method for preparing the nanobodies, polypeptides, and nucleic acids generally comprises the following steps:

[0064] (1) expressing the nucleic acid encoding the nanobody or polypeptide of the present application in a suitable host cell or host organism or in another suitable expression system, optionally followed by;

[0065] (2) isolating and / or purifying the nanobody or polypeptide of the present application thus obtained.

[0066] Alternatively, other methods comprising the following steps can be employed:

[0067] (3) culturing and / or maintaining the host of the present application under conditions such that the host of the present application expresses and / or produces a nanobody and / or polypeptide of the present application; optionally followed by;

[0068] (4) isolating and / or purifying the nanobody or polypeptide of the present application thus obtained.

[0069] The nucleic acids of the present application can be in the form of single- or double-stranded DNA or RNA, and preferably in the form of double-stranded DNA. For example, the nucleic acid sequences of the present application can be genomic DNA, cDNA, or synthetic DNA (e.g., DNA with codon usage specifically adapted for expression in the host cell or host organism to be used, i.e., codon-optimized).

[0070] The nucleic acids of the application can be prepared or obtained in essentially known methods, based on the information given herein for the amino acid sequences of the Nanobodies or polypeptides of the application, and / or can be isolated from appropriate natural sources. For example, the naturally occurring V HH The nucleic acid sequences of the domains are subjected to site-directed mutagenesis to provide the nucleic acids of the application encoding said analogs.

[0071] The nucleic acids of the application can also be in a form, present and / or be part of a genetic construct, as is well known to the person skilled in the art. Such genetic constructs typically comprise at least one nucleic acid of the application, can be in the form of a vector, such as a plasmid, a YAC, a viral vector or a transposon. In particular, the vector can be an expression vector, i.e. a vector that can provide for expression in vitro and in vivo (e.g. in a suitable host cell, host organism and / or expression system).

[0072] The nucleic acids of the application and / or the genetic constructs of the application can be used to transform a host cell or host organism, i.e. for expression and / or production of the Nanobodies or polypeptides of the application. Suitable hosts or host cells are well known to the person skilled in the art, e.g. can be any appropriate fungal, prokaryotic or eukaryotic cell or organelle or organism, and all other hosts or host cells essentially known for expression and production of antibodies and antibody fragments, including but not limited to single domain antibodies and ScFv fragments, are well known to the person skilled in the art.

[0073] For production, the Nanobodies and polypeptides of the application can be produced in the milk of transgenic mammals, e.g. in the milk of rabbits, cows, goats or sheep, but also in plants or parts of plants, including but not limited to their leaves, flowers, fruits, roots or seeds.

[0074] As mentioned above, one advantage of applying Nanobodies is that the polypeptides based thereon can be expressed and produced in prokaryotic systems, and suitable prokaryotic expression systems, vectors, host cells, etc. are well known to the person skilled in the art, as referenced above. It should be noted, however, that the application is not limited in its broadest sense to expression in bacterial systems.

[0075] Preferably, in the present application, the Nanobodies or polypeptides are produced in bacterial cells, in particular in bacterial cells suitable for large scale pharmaceutical production, as described above.

[0076] When expressed in a cell for production of a Nanobody or polypeptide of the application, the Nanobody or polypeptide of the application can be produced intracellularly (e.g. in the cytoplasm or periplasmic space), then isolated from the host cell, and optionally further purified; or can be produced extracellularly (i.e. secreted), then isolated from the culture medium, and optionally further purified.

[0077] Some preferred, but non-limiting, vectors for use with these host cells include vectors for expression in mammalian cells - pMANneo (Clonetech), pUCTtag (ATCC 37460) and pMCIneo (Stratagene); vectors for expression in bacterial cells - pET vectors (Novagen) and pQE vectors (Qiagen); vectors for expression in yeast or other fungal cells - pYES2 (Invitrogen) and Picha expression vector (Invitrogen); vectors for expression in insect cells - pBlueBac II (Invitrogen) and other baculovirus vectors; and the like.

[0078] The corresponding techniques for transforming a host or host cell of the application are well known to the person skilled in the art.

[0079] After transformation, those hosts which have been successfully transformed with the nucleotide sequence / genetic construct of the application can be detected and selected. The transformed host cell (which can be in the form of a stable cell line) or host organism (which can be in the form of a stable mutant line or strain) forms a further aspect of the application.

[0080] The amino acid sequence of the application can then be isolated from the host cell / host organism and / or from the culture medium in which the host cell or host organism is cultured, can be isolated by protein isolation and / or purification techniques known per se, such as (preparative) chromatographic and / or electrophoretic techniques, differential precipitation techniques, affinity techniques (e.g. using a specific / cleavable amino acid sequence fused to the amino acid sequence of the application) and / or preparative immunological techniques (i.e. using antibodies against the amino acid sequence to be isolated).

[0081] The adsorbent of the application can be used to specifically recognize TNFα.

[0082] The Nanobody or polypeptide or adsorbent of the application can be used for enriching, purifying, removing and detecting TNFα.

[0083] The application provides a nanobody with a specific structure of an amino acid sequence, a polypeptide comprising the nanobody and application thereof, to solve the problems of complex antibody preparation process, high cost, poor antibody affinity and stability in the aspects of TNFα enrichment, purification, detection and removal. Specifically, in a first aspect, the application provides a nanobody binding to TNFα, a variable region in the amino acid sequence of the nanobody comprising a complementarity determining region CDR and a framework region FR, the complementarity determining region CDR comprising a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein the most important site participating in antigen recognition and binding is RXXXXXE on CDR1 and ATYSDSPWNXXSFYXLSGVGA on CDR3.

[0084] Preferably, the amino acid sequence of the complementarity determining region CDR3 comprises SEQ ID No: 33-SEQ ID No: 49, the amino acid sequence of the complementarity determining region CDR1 comprises SEQ ID No: 50-SEQ ID No: 65, and a sequence with more than 75% homology thereto.

[0085] Preferably, the amino acid sequence of the nanobody comprises SEQ ID No: 1-SEQ ID No: 20.

[0086] Preferably, the nanobody is a humanized nanobody, and preferably, the humanized nanobody comprises SEQ ID No: 21-SEQ ID No: 32.

[0087] In a second aspect, the application provides a polypeptide obtained by N-terminal and / or C-terminal amino acid modification of the nanobody.

[0088] Preferably, the N-terminal and / or C-terminal amino acid modification of the nanobody comprises:

[0089] In a first mode, a tag is added to the N-terminal and / or C-terminal amino acid of the nanobody.

[0090] In a second mode, after a tag is added to the N-terminal and / or C-terminal amino acid of the nanobody, the tag is further protected by a hinge-attached amino acid.

[0091] Preferably, the tag comprises at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag and Flag-tag, the hinge comprises at least one of GS hinge, IgG hinge, IgA hinge and PEG, and the protected amino acid comprises Ala, Gln, Glu, Met or any two or more combinations of the foregoing amino acids.

[0092] Thirdly, the present invention provides a polypeptide obtained by multivalent synthesis of the aforementioned nanobody.

[0093] Fourthly, the present invention provides a nucleic acid that encodes the aforementioned nanobody or the aforementioned polypeptide.

[0094] Fifthly, the present invention provides an expression vector comprising the expression frame of the nucleic acid described above.

[0095] In a sixth aspect, the present invention provides a host cell containing the expression vector described above.

[0096] In a seventh aspect, the present invention provides the application of the nanobodies and / or the peptides described herein in immunoassay, enrichment and / or purification.

[0097] Preferably, the nanobody and / or the polypeptide are used in the preparation of TNFα adsorbents, TNFα purification kits, and TNFα detection kits.

[0098] Preferably, the nanobody and / or the polypeptide are used in the capture and detection of TNFα-positive cells.

[0099] Examples: The following examples illustrate specific implementations of the present invention. However, the implementation of the present invention is not limited to these examples, and any selections and modifications can be made within the scope of the technical effects to be achieved by the present invention.

[0100] Example 1: Construction of an anti-TNFα nanobody library.

[0101] The phage display library used in this invention is an immune library based on T7 phage, and the establishment steps are as follows:

[0102] (1) Alpacas (numbered 2208-1 and 2208-2) were immunized with TNFα. After four immunizations, jugular vein blood was collected from both alpacas, peripheral blood lymphocytes were isolated, and total RNA (PuerLink) was extracted. TM RNAMini Kit, Life Technologies: 12183018A);

[0103] (3) Total RNA was reverse transcribed into cDNA, and V was amplified using two rounds of nested PCR. HH Gene;

[0104] UP primer1: CTTGGTGGTCCTGGCTGCTCT, DOWN primer1: GGTACGTGCTGTTGAACTGTTCC, UP primer2: TATCTAGTC CGCCCAGGTGCAGCTC, DOWN primer2: AGCGACTAAGCTTTGAGGAGACGGTGAC

[0105]

[0106]

[0107] GAATTC

[0108]

[0109] (3) The PCR product was double digested with EcoRI and Hind III, and agarose electrophoresis was performed to recover the gene band of 350-500 bp, which was V HH gene fragment;

[0110] (4) The T7 vector (10-3 Cloning Kit, Merck Millipore 70550-3) and V HH gene fragment were connected by T4 ligase;

[0111] (5) The ligation product was mixed with packaging protein to form a complete T7 phage, and the mixture was amplified to obtain a phage original library;

[0112] (6) The titer of the original library was 9.56 x 10 9 pfu / mL, and the diversity was 6.6 x 10 6 .

[0113] Example 2: Screening of nanobodies.

[0114] ​​​​​​​Firstly, the antigen TNFα is diluted with TBS to 10 μg / mL, 100 μL is added to the 96-well plate, and incubated at 4°C for 12 h. The antigen diluent in the well is sucked out, the plate is washed with TBS for 3 times, dried, 1% protein-free blocking solution (purchased from Shengong Bioengineering Co., Ltd.) is added, 300 μL / well, and incubated at room temperature for 2 h (1% protein-free blocking solution and 1% BSA are used alternately during screening). The blocking agent in the well is sucked out, the plate is washed with TBST for 6 times, dried, and the amplified phage is added, 100 μL / well, and incubated at room temperature for 30 min. The plate is washed with TBST for 10 times, the phage is eluted with T7 elution buffer (1% SDS), incubated at room temperature for 30 min, and the eluent is amplified for the next round of screening.

[0115] Example 3: Construction of genetically engineered bacteria

[0116] (1) After four rounds of screening, the screening eluent is solidly amplified, and the phage plaques are picked to amplify the phage plaques. The amplified phage plaques are used as templates, UP primer 3 and DOWN primer 3 are used as upstream and downstream primers, and PCR amplification is performed;

[0117] UP primer 3: TTCCTTAA CATATG GCCCAGGTGCAGCTCGT,

[0118] DOWN primer 3: TTAAGGAA CTCGAG CACGGTGACCAGGGTC;

[0119] (2) A part of the PCR product is outsourced for sequencing, that is, the sequence information of the nanobody is obtained. According to the length and homology of the CDR region, 20 monoclonal sequences belonging to the same family are selected, and the nanobody naming and sequence number information are shown in Table 1.

[0120] (3) Another part of the PCR product is double-digested with NdeI and XhoI, and the digested product is recovered. At the same time, the same method is used for enzyme digestion and recovery of the vector, and the digested product and the vector are connected with T4 ligase. The ligation product is transformed into E. coli, and the genetically engineered bacteria expressing the TNFα-specific nanobody are obtained.

[0121] Table 1: Nanobody sequence number and CDR sequence information

[0122]

[0123] Example 4: Key amino acid calculation

[0124] Using sequences T00 and TNFα as model proteins, molecular docking is performed to predict the antigen binding epitope of VHH on TNFα and the key amino acids in the interaction.

[0125] First, the TNFα structure was obtained from the database, and after combining the TNFα sequence and the VHH antibody sequence, the complex structure prediction and search were performed using Alohafold2 multimer v3. After generating the complex structure, the Relax Process was performed on the complex structure using Amber to optimize the side chain structure, AutoDock Tools was used to check the hydrogen atoms and calculate the potential, Ligplot+ was used to analyze the hydrogen bonds and hydrophobic interaction network between the CDR regions of the VHH and the binding interface of TNFα, and the atomic contacts between the domains were checked, and the reasonable conformation meeting the above conditions was selected as the structure model of the VHH antibody and TNFα protein complex.

[0126] Through molecular docking, the binding epitopes of VHH on TNFα were calculated: RXXXXXE on CDR1 and ATYSDSPWNXXSFYXLSGVGA on CDR3 were involved in antigen recognition and binding. Among them, R25 and E31 on CDR1 of VHH collectively recognize and bind T72, E104, E107 and R138 on TNFα, and Y97, S98, D99, S100 and N103 on CDR3 of VHH collectively recognize and bind T72, H73, R138, K112 (chain B), L75 and N137 on TNFα.

[0127] The molecular docking results are shown in FIGS. 1, 2 and 3, wherein, Figure 1 and Figure 2 , wherein, Figure 1 FIGS. 1, 2 and 3 are molecular docking diagrams of the nanobody used for analysis in Example 6 and TNFα protein and partial enlargements thereof, in order to further more clearly show the respective sites and docking relationships, Figure 2 a of FIG. 1 and Figure 2 b of FIG. 2 show partial enlargements of the molecular docking diagrams from different angles. It can be seen that the main amino acid residues on VHH (blue) involved in antigen recognition and binding are R25, E31, Y97, S98, D99, S100 and N103 (yellow), which bind T72, E104, E107, H73, R138, K112 (chain B), L75 and N137 (pink) on TNFα (chain A is green and chain B is gray) through electrostatic interaction and hydrogen bond interaction. Figure 3 FIG. 3 is a schematic diagram of the antigen-antibody intermolecular interaction, wherein red represents electrostatic interaction and blue represents hydrogen bond interaction.

[0128] The interactions on the binding surface can be divided into four cores, which specifically include:

[0129] 1). R25 on T00, and E104 and E107 on TNFα;

[0130] 2) E31 and Y97 on T00, and T72 and R138 on TNFα;

[0131] 3) S98, D99, and S100 on T00, and K112 (chain B), H73, and R138 on TNFα;

[0132] 4) N103 on T00, and H73, L75 and N137 on TNFα.

[0133] At the same time, such as Figure 4 As shown in a and b of 4, the residues T96, P101, S106, G112, G114, and A115 (brown) on the CDR3 of T00 provide necessary support and directionality for CDR3 through hydrogen bond interactions with other residues within the framework region, thereby stabilizing the three-dimensional conformation of CDR3. These interactions not only ensure that the CDR3 region can maintain its functional morphology, but are also crucial for the formation of the large exposed convex rings in the CDR3 region. These convex rings, like "fingers," can penetrate deep into the gaps or cracks of the antigen, reaching epitopes that are difficult for traditional antibodies to access.

[0134] Furthermore, the W102 and F107 residues on CDR3 interact with the Y57 residue on the FR3 framework region via π-π stacking, further enhancing the stability of CDR3. This π-π interaction is a special type of non-covalent interaction that occurs between aromatic amino acid residues, enhancing molecular stability through the overlap of conjugated π-electron systems. This interaction is particularly important for the conformational stability of the CDR3 region, as it helps maintain the rigidity and correct spatial orientation of the CDR3 ring, enabling it to bind effectively to antigens.

[0135] These complex interaction networks not only play a crucial role in the formation of the large exposed convex ring in the CDR3 region, but are also essential for the stability of the entire nanobody conformation. Together, they enhance the affinity and stability of the nanobody for the antigen.

[0136] Sequence analysis revealed that in the homologous nanobody sequences described in Example 3, these two regions (RXXXXXE on CDR1 and ATYSDSPWNXXSFYXLSGVGA on CDR3) are strictly conserved. That is, the most important sites for antigen recognition and binding of the nanobodies T00-T19 of this invention are R25 and E31 on CDR1 of VHH, and Y97, S98, D99, S100, and N103 on CDR3 of VHH. Here, X is a general symbol in the art, representing any amino acid.

[0137] Example 5: Preparation of TNFα nanobody.

[0138] (1) The basic culture medium of the nanobody is TB culture medium, inoculated at 5% inoculation amount, cultured at 37°C for 3-5h, and then induced by adding inducer galactoside (IPTG) (final concentration 0.25mM, same below) for overnight;

[0139] (2) After the induction is completed, centrifuge the wet bacteria containing the nanobody at 4000rpm for 20min.

[0140] (3) Add lysis solution (10mM imidazole, 500mM NaCl, pH7.4 0.02M PB) to the obtained wet bacteria at a ratio of 1:10, and use a 700bar high-pressure homogenizer to break the cells;

[0141] (4) Centrifuge at 4°C, 10000rpm for 20min, and take the supernatant;

[0142] (5) Filter the supernatant through a 0.45μm filter, and then separate and purify the TNFα nanobody through an affinity chromatography column (GE Healthcare, US), wherein the filler of the affinity chromatography column is Ni Sepharose High Performance;

[0143] (6) The nanobody purified through the affinity chromatography is subjected to SDS-PAGE electrophoresis to determine the purity, and the protein solution with higher purity is selected to determine the protein concentration by BCA method.

[0144] Example 6: Affinity analysis.

[0145] SPR technology is used to analyze the binding capacity of the nanobody to human TNFα.

[0146] TNFα exists in two forms of trimer and monomer. The active form of TNFα is mainly trimer structure, which triggers a series of complex intracellular signal transduction pathways by binding to its specific receptors TNFR1 (tumor necrosis factor receptor 1) and TNFR2 (tumor necrosis factor receptor 2). This active form of TNFα not only plays a role in normal physiological processes, but also changes in expression and activity in various pathological states such as autoimmune diseases, sepsis, malignant tumors, heart failure and chronic inflammation, thereby having a profound impact on the progression and treatment of diseases.

[0147] In certain pathological conditions, monomeric form of TNFα can be detected in human body. For example, in autoimmune diseases such as rheumatoid arthritis, abnormal expression of TNFα can lead to inflammation and tissue damage of joints. In acute inflammatory responses such as sepsis, the level of TNFα rises sharply, and participates in the development of systemic inflammatory response syndrome (SIRS). In addition, TNFα monomer also plays a role in the development of malignant tumors, and it can promote tumor growth and metastasis by promoting inflammatory response in tumor microenvironment.

[0148] Therefore, both monomeric form and trimeric form of TNFα have important research value.

[0149] In the present application, TNFα monomer and trimer are respectively amino-coupled to CM5 sensor chip with a density of 500-800 RU, and nanobodies are injected at 7 different concentrations in the range of 1-100 nM, and the flow rate is 45 μL / min in all experiments. The chip regeneration condition is glycine-HCl pH 1.5. The binding curves obtained at different nanobody concentrations are used to calculate the kinetic parameters K a , K d and K D . The kinetic parameters can be obtained by determining a single concentration of analyte, or multiple concentrations of analyte can be used. Figure 5 In the figure, from left to right, the first row shows the kinetic curves of antibodies T00, T01, T10, T11, the second row shows the kinetic curves of antibodies T02, T12, T14, T04, the third row shows the kinetic curves of antibodies T15, T03, T13, T05, the fourth row shows the kinetic curves of antibodies T06, T07, T18, T16, and the fifth row shows the kinetic curves of T17, T08, T09, T19. The black line is the theoretical calculation kinetic curve, and the colored line is the response curve, Figure 5 which shows that the theoretical calculation kinetic curve is fitted to the response curve, wherein the curves with poor fitting degree have been deleted. Figure 5 In the figure, the curves represent the response curves of antibodies at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM and 1.5625 nM, and the kinetic parameters shown in Table 2 are calculated by equation fitting. The experiment and calculation can be realized by using Biacore system.

[0150] As shown in Table 2, the nanobodies have high affinity to both monomeric form and trimeric form of TNFα, and the K D range is 10 -8 ~ 10 -10 (M).

[0151] Table 2 Affinity of nanobodies to TNFα

[0152]

[0153] SEQ ID: No. 1 >00: QLQESGGGLVQAGGSLSLSCAASG-RILDSYE-MGWFRQAPGKEREFVAA-INSDGSTT- YYADSVKGRFTISRDNAENTLYLQMFTLKPEDTAVYYC-ATYSDSPWNPHSFYALSGVGA- WGKGTQVTVSS

[0154] SEQ ID: No. 2 >01: QLQESGGGLVQAGGSLSLSCAASG-RIFDSFE-MGWFRQAPGKEREFVAA-INHGGSTT- YYADSVKGRFTISRDNAENTLYLQMFTLKPEDTAVYYC-ATYSDSPWNPHSFYVLSGVGA- WGKGTQVTVSS

[0155] SEQ ID: No. 3 >10: QLQESGGGLVQAGGSLSLSCAASG-RILDSFE-MGWFRQAPGKEREFVAA-INSDGSTT- YYANSVKGRFTISRDNAENTLYLQMFTLKPEDTAVYYC-ATYSDSPWNPHSFYALSGVGA- WGKGTQVTVSS

[0156] SEQ ID: No. 4 >11: QLQESGGGLVQAGGSLSLSCAASG-RIAGSFE-MGWFRQAPGKEREFVAA-INRGGSTT- YYADSVKGRFTISRDNAENTLYLQMFALKPEDTAVYYC-ATYSDSPWNGRSFYALSGVGA- WGKGTQVTVSS

[0157] SEQ ID: No. 5 >02: QLQESGGGLVQAGGSLSLSCAASG-RSLNSYE-MGWFRQAPGKEREFVAA-INTEGSNT- YYADSVKGRFTISRDNAKNTLYLQMFTLKPEDTAVYYC-ATYSDSPWNKHSFYELSGVGA- WGKGTQVTVSS

[0158] SEQ ID: No. 6 > T12: QLQESGGGLVQAGGSLSLSCAASG-RSLNSYE-MGWFRQAPGKEREFVAA- INSDGSNT-YYADSVKGRFTISRDNAKNTLYLQMFSLKPEDTAVYYC-ATYSDSPWNGHSFYALSGVGA- WGKGTQVTVSS

[0159] SEQ ID: No. 7 > T14: QLQESGGGLVQAGGSLSLSCAASG-RTFSSYE-MGWFRQAPGKEREFVAA- ITVSGSMT-YYADSVKGRFTISRDNAENTLYLQMFTLKPEDTAVYYC-ATYSDSPWNFDSFYSLSGVGA- WGKGTQVTVSS

[0160] SEQ ID: No. 8 > T04: QLQESGGGLVQAGGSLSLSCAASG-RTFSSYE-MGWFRQAPGKEREFVAA- ISVSGGLT-YYADSVKGRFTISRDNAKNTLYLQMFTLKPEDTAVYYC-ATYSDSPWNFNSFYSLSGVGA- WGKGTQVTVSS

[0161] SEQ ID: No. 9 > T15: QLQESGGGLVQAGGSLSLSCAASG-RTFSRYE-MGWFRQAPGKEREFVAA- ISMSGGLT-YYADSVKGRFTISRDNAKNTLYLQMFALKPEDTAVYYC-ATYSDSPWNSDSFYSLSGVGA- WGKGTQVTVSS

[0162] SEQ ID: No. 10 > T03: QLQESGGGLVQAGGSLSLSCAASG-RTLSMFE-MGWFRQAPGKEREFVAA- ISTSGGLT-YYADSVKGRFTISRDNAKNTLYLQMFALKPEDTAVYYC-ATYSDSPWNSDSFYSLSGVGA- WGKGTQVTVSS

[0163] SEQ ID: No. 11 >T13: QLQESGGGLVQAGGSLSLSCAASG-RTFSTYE-MGWFRQAPGKEREFVAA-ISTSGGLT- YYADSVKGRFTISRDNAKNTLYLQMFTLKPEDTAVYYC-ATYSDSPWNSNSFYSLSGVGA- WGKGTQVTVSS

[0164] SEQ ID: No. 12 >T05: QLQESGGGLVQAGGSLSLSCAASG-RSLSSYE-MGWFRQAPGKEREFVAA-ISVSGGLT- YYADSVKGRFTISRDNAKNTLYLQMFTLKPEDTAVYYC-ATYSDSPWNSDSFYELSGVGA- WGKGTQVTVSS

[0165] SEQ ID: No. 13 >T06: QLQESGGGLVQAGGSLSLSCAASG-RVLDMFE-MGWFRQAPGKEREFVAA-ITWSGSNT- YYADSVKGRFTISRDNAENTLYLQMFALKPEDTAVYYC-ATYSDSPWNYGSFYELSGVGA- WGKGTQVTVSS

[0166] SEQ ID: No. 14 >T07: QLQESGGGLVQAGGSLSLSCAASG-RVFDMFE-MGWFRQAPGKEREFVAA-INRSGSNT- YYADSVKGRFTISRDNAENTLYLQMFTLKPEDTAVYYC-ATYSDSPWNYGSFYALSGVGA- WGKGTQVTVSS

[0167] SEQ ID: No. 15 >T18: QLQESGGGLVQAGGSLSLSCAASG-RIFDMFE-MGWFRQAPGKEREFVAA-INWSGVNT- YYADSVKGRFTISRDNAKNTLYLQMFALKPEDTAVYYC-ATYSDSPWNYDSFYWLSGVGA- WGKGTQVTVSS

[0168] SEQ ID: No. 16 > T16: QLQESGGGLVQAGGSLSLSCAASG-RVAGSFE-MGWFRQAPGKEREFVAA-ITRGGSNT- YYADSVKGRFTISRDNAENTLYLQMFALKPEDTAVYYC-ATYSDSPWNYGSFYELSGVGA- WGKGTQVTVSS

[0169] SEQ ID: No. 17 > T17: QLQESGGGLVQAGGSLSLSCAASG-RVAGSFE-MGWFRQAPGKEREFVAA- INWSGSNT-YYADSVKGRFTISRDNAENTLYLQMFTLKPEDTAVYYC-ATYSDSPWNYESFYALSGVGA- WGKGTQVTVSS

[0170] SEQ ID: No. 18 > T08: QLQESGGGLVQAGGSLSLSCAASG-RAFSMFE-MGWFRQAPGKEREFVAA- INWSGVNT-YYADSVKGRFTISRDNAKNTLYLQMFALKPEDTAVYYC-ATYSDSPWNYDSFYYLSGVGA- WGKGTQVTVSS

[0171] SEQ ID: No. 19 > T09: QLQESGGGLVQAGGSLSLSCAASG-RAFSMFE-MGWFRQAPGQEREFVAA- INWSGVNT-YYADSVKGRFTISRDNAKNTLYLQMFALKPEDTAVYYC-ATYSDSPWNFGSFYDLSGVGA- WGKGTQVTVSS

[0172] SEQ ID: No. 20 > T19: QLQESGGGLVQAGGSLSLSCAASG-RISDMFE-MGWFRQAPGKEREFVAA- INSVAGNT-YYADSVKGRFTISRDNAKNTLYLQMFALKPEDTAVYYC-ATYSDSPWNFGSFYNLSGVGA- WGKGTQVTVSS

[0173] Note: 1. The “-” between FR and CDR is a connector, which is used to distinguish FR and CDR, and there is no gap site.

[0174] Example 7: Stability and humanization modification of nanobody and affinity determination.

[0175] In order to reduce the immunogenicity of nanobodies while maintaining or improving the stability and biological activity of nanobodies, the present application is based on the common humanization or high-stability nanobody scaffold to perform CDR region transplantation, complete the humanization and stability modification of nanobodies.

[0176] (1) Sequence design

[0177] The present application selects four nanobody scaffolds for modification, which are ah, YW-sdAb (hereinafter referred to as sdAb), hs2dAb (hereinafter referred to as hs) and com scaffolds.

[0178] The ah scaffold and the sdAb scaffold are universal fully humanized scaffolds, taken from the literature (Chi, X. J. et al. Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain. Nature Communications 11, doi:10.1038 / s41467-020-18387-8 (2020)).

[0179] hs is a high-stability, high-expression, partially humanized scaffold, from the literature (Moutel, S. et al. NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife 5, doi:10.7554 / eLife.16228 (2016)).

[0180] com is a high-stability, high-expression universal scaffold, from the literature (Ferrari, D., Garrapa, V., Locatelli, M. & Bolchi, A. A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries. Molecular Biotechnology 62, 43-55, doi:10.1007 / s12033-019-00224-z (2020)).

[0181] The sequence name and sequence number of the modified humanized sequence are shown in Table 3.

[0182] Table 3 Humanized sequence name and sequence number

[0183] Antibody name Sequence number Humanized antibody sequence number SEQ ID: No. 21 sdAb-00 SEQ ID: No. 21 SEQ ID: No. 22 ah-00 SEQ ID: No. 22 SEQ ID: No. 23 com-00 SEQ ID: No. 23 SEQ ID: No. 24 hs2-00 SEQ ID: No. 24 SEQ ID: No. 25 sdAb-03 SEQ ID: No. 25 SEQ ID: No. 26 ah-03 SEQ ID: No. 26 SEQ ID: No. 27 com-03 SEQ ID: No. 27 SEQ ID: No. 28 hs2-03 SEQ ID: No. 28 SEQ ID: No. 29 sdAb-06 SEQ ID: No. 29 SEQ ID: No. 30 ah-06 SEQ ID: No. 30 SEQ ID: No. 31 com-06 SEQ ID: No. 31 SEQ ID: No. 32 hs2-06 SEQ ID: No. 32

[0184] SEQ ID: No. 21 > sdAb-00: QLVESGGGLVQPGGSLRLSCAASG-RILDSYE-MGWFRQAPGKGLEAVAA- INSDGSTT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-ATYSDSPWNPHSFYALSGVGA- WGQGTQVTVSS

[0185] SEQ ID: No. 22 > ah-00: QLVESGGGLVQPGGSLRLSCAASG-RILDSYE-MGWFRQAPGKGLEAVAA- INSDGSTT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-ATYSDSPWNPHSFYALSGVGA- WGQGTLVTVSS

[0186] SEQ ID: No. 23 > com-00: QLQESGGGLVQAGGSLRLSCAASG-RILDSYE-MGWFRQAPGKEREFVAA- INSDGSTT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-ATYSDSPWNPHSFYALSGVGA- WGQGTQVTVSS

[0187] SEQ ID: No. 24 > hs2-00: QLQASGGGFVQPGGSLRLSCAASG-RILDSYE-MGWFRQAPGKEREFVSA- INSDGSTT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-ATYSDSPWNPHSFYALSGVGA- WGQGTQVTVSS

[0188] SEQ ID: No. 25 > sdAb-03: QLVESGGGLVQPGGSLRLSCAASG-RTLSMFE-MGWFRQAPGKGLEAVAA- ISTSGGLT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-ATYSDSPWNSDSFYSLSGVGA- WGQGTQVTVSS

[0189] SEQ ID: No. 26 > ah-03: QLVESGGGLVQPGGSLRLSCAASG-RTLSMFE-MGWFRQAPGKGLEAVAA-ISTSGGLT- YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-ATYSDSPWNSDSFYSLSGVGA- WGQGTLVTVSS

[0190] SEQ ID: No. 27 > com-03: QLQESGGGLVQAGGSLRLSCAASG-RTLSMFE-MGWFRQAPGKEREFVAA-ISTSGGLT- YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-ATYSDSPWNSDSFYSLSGVGA- WGQGTQVTVSS

[0191] SEQ ID: No. 28 > hs2-03: QLQASGGGFVQPGGSLRLSCAASG-RTLSMFE-MGWFRQAPGKEREFVSA-ISTSGGLT- YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-ATYSDSPWNSDSFYSLSGVGA- WGQGTQVTVSS

[0192] SEQ ID: No. 29 > sdAb-06: QLVESGGGLVQPGGSLRLSCAASG-RVLDMFE-MGWFRQAPGKGLEAVAA-ITWSGSNT- YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-ATYSDSPWNYGSFYELSGVGA- WGQGTQVTVSS

[0193] SEQ ID: No. 30 > ah-06: QLVESGGGLVQPGGSLRLSCAASG-RVLDMFE-MGWFRQAPGKGLEAVAA-ITWSGSNT- YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-ATYSDSPWNYGSFYELSGVGA- WGQGTLVTVSS

[0194] SEQ ID: No. 31 > com-06: QLQESGGGLVQAGGSLRLSCAASG-RVLDMFE-MGWFRQAPGKEREFVAA-ITWSGS NT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-ATYSDSPWNYGSFYELSGVGA- WGQGTQVTVSS

[0195] SEQ ID: No. 32 > hs2-06: QLQASGGGFVQPGGSLRLSCAASG-RVLDMFE-MGWFRQAPGKEREFVSA-ITWSGS NT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC-ATYSDSPWNYGSFYELSGVGA- WGQGTQVTVSS

[0196] (2) Nanobody preparation and affinity determination

[0197] The vector construction process is described in Example 3, the nanobody preparation process is described in Example 5, and the affinity characterization of the nanobody is described in Example 6. The binding curves obtained at different nanobody concentrations were used to calculate the kinetic parameters K a , K d and K D Table 4.

[0198] Table 4. Affinity results before and after humanization of nanobody

[0199]

[0200] Compared with the original sequence, the affinity of the humanized nanobody to the antigen TNFα did not decrease significantly, and the affinity to the related antigen was still maintained at the original level. For example, the affinity of nanobody T03 to TNFα monomer was 8.27 x 10 -8 M, and the affinity to TNFα trimer was 6.62 x 10 -8 M, and the affinities of the four humanized forms of the sequence sdAb-03, ah-03, com-03 and hs2-03 to TNFα monomer were still 2.99 x 10 -8 ~ 7.85 x 10 -8 M, and the affinities to TNFα trimer were still 2.74 x 10 -8 ~ 8.64 x 10 -8 M. This shows that the humanized nanobody of the application is successfully modified.

[0201] Example 8: Preparation of adsorbent and evaluation of effect

[0202] Activation of agarose gel. Take 2g of agarose microspheres, add 2mol / L NaOH and 0.8mL of 1,4-butanediol diglycidyl ether, mix according to this ratio, and react for at least 60min. After the reaction, wash the gel with plenty of deionized water and filter it into a wet cake.

[0203] Immobilization of nanobodies. The activated agarose gel carrier material was used, and the epoxy density on the gel was measured to be 15 μmol / g. Nanobody solutions T00 and T03 were added separately (PBS was used to prepare the nanobody solutions in this example; physiological saline or pure water can also be used). The coupling reaction was carried out at 37℃ and 250 rpm for 24 h. After the reaction, 3 volumes of ethanolamine (6% by volume, pH 9.0) were added for overnight blocking to obtain the TNFα adsorbent. The concentration of nanobody solution T00 was 10 mg / mL, the gel-to-water ratio was 1:3, and the final coupling amount on the gel was 12.91 mg / mL, denoted as adsorbent A; the concentration of nanobody solution T03 was 5 mg / mL, the gel-to-water ratio was 1:5, and the final coupling amount on the gel was 8.15 mg / mL, denoted as adsorbent B.

[0204] Adsorbent performance evaluation. Static adsorption experiments were conducted, with high-concentration (100 ng / mL) and low-concentration (50 ng / mL) groups to evaluate the adsorption performance of the TNFα adsorbent. The experimental method is briefly described as follows:

[0205] Human TNFα standard was diluted with bovine serum to 50 and 100 ng / mL, and mixed at a ratio of 1 mL adsorbent to 100 mL of serum containing TNFα. Static adsorption was performed by incubating on a horizontal shaker at room temperature. The remaining concentration of TNFα in serum was measured at t = 0 min, 15 min, and 30 min. Experimental conditions and removal rates are shown in Table 5. Figure 6 As shown.

[0206] Table 5 Static adsorption experimental conditions and removal rate

[0207]

[0208] The results showed that both adsorbents could effectively remove TNFα from serum, and the adsorption equilibrium was basically reached after 10 min of incubation. The final removal rate of TNFα by adsorbent A and adsorbent B prepared in this invention was between 77% and 89%.

[0209] Example 9: Preparation of TNFα detection kit.

[0210] Replace the T00 nanobody with PBS (pH 7.4) to a concentration of approximately 10 mg / mL, and use an HRP conjugation kit (Abcam, Lightning- (ab102890) was marked and thoroughly dialyzed and the solution changed.

[0211] Sandwich ELISA detection method:

[0212] Coat the capture ligand on the high hydrophobic 96-well plate, incubate overnight at 4℃ in the dark, wash the plate with PBS; block with 1%-3% skimmed milk powder, wash the plate with PBS; add TNFα standard or sample to be tested, incubate on a horizontal shaker for 2 hours, wash the plate with PBS; add HRP-conjugated nanobody T00, incubate on a horizontal shaker for 2 hours, wash the plate with PBS; add TMB working solution, incubate at room temperature in the dark for 30 min, add 2M sulfuric acid stop solution to terminate the reaction, and measure OD 450 .

[0213] The capture ligand can be nanobody T03 or other nanobodies in the application, or an anti-TNFα antibody, or other ligand molecules capable of binding TNFα. The capture antibody used in this embodiment is a recombinant rabbit anti-human TNFα monoclonal antibody (EPR19147, Abeam, ab183218).

[0214] The standard curve for measuring TNFα by the kit of the application is shown in Figure 7 The fitting curve used is a four-parameter Logistic curve fitting, and the fitting result is: y=(A-D) / [1+(x / C)^B]+D, where A=2.81219, B=-1.38233, C=85.60183, D=0.11842, R 2 =0.99955. R 2 can reach 0.99 or more, indicating that the ELISA detection method based on the nanobody of the application is highly reliable and sensitive.

[0215] Industrial applicability

[0216] The nanobody of the application is an anti-TNFα nanobody with a new amino acid sequence discovered by screening of a phage library. The nanobody and its polypeptide have high affinity and activity, can specifically recognize and bind TNFα, have strong adsorption capacity for TNFα, and the detection kit has high sensitivity, which can be applied to the fields of blood purification and TNFα detection, and is helpful for the diagnosis and treatment of TNFα-related diseases.

[0217] The above embodiments only express several embodiments of the application, which are described in detail and specifically, but should not be understood as limiting the scope of the patent of the application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are all within the protection scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. An anti-TNFa nanobody, characterized in that, The complementarity determining regions CDR of the nanobody include CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of the CDR1 is shown as SEQ ID NO. 50, (2) the amino acid sequence of the CDR2 is shown as SEQ ID NO. 66; and (3) the amino acid sequence of the CDR3 is shown as SEQ ID NO. 33; or (II-1): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 51, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 67, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 34; or (II-2): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 52, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 66, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 33; or (II-3): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 53, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 68, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 35; or (II-4): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 54, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 69, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 36; or (II-5): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 54, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 70, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 37; or (II-6): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 55, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 71, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 38; or (II-7): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 55, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 72, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 39; or (II-8): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 56, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 73, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 40; or (II-9): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 57, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 74, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 40; or (II-10): the amino acid sequence of the CDR1 is shown as SEQ ID NO. 58, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 74, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 41; or ​ (II-11): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 59, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 72, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 42; or (II-12): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 60, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 75, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 43; or (II-13): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 61, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 76, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 44; or (II-14): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 62, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 77, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 45; or (II-15): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 63, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 78, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 43; or (II-16): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 63, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 79, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 46; or (II-17): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 64, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 77, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 47; or (II-18): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 64, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 77, and the amino acid sequence of CDR3 is set forth in SEQ ID NO. 48; or (II-19): the amino acid sequence of CDR1 is set forth in SEQ ID NO. 65, the amino acid sequence of CDR2 is set forth in SEQ ID NO. 80, and the amino acid sequence of CDR3 is set forth in SEQ ID NO.

49.

2. The Nanobody according to claim 1, characterized in that, wherein: (III) the framework region FR of the Nanobody comprises FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of the FR1 is set forth in SEQ ID NO. 81, (2) the amino acid sequence of the FR2 is set forth in SEQ ID NO. 82, (3) the amino acid sequence of the FR3 is set forth in SEQ ID NO. 84, and (4) the amino acid sequence of the FR4 is set forth in SEQ ID NO. 90; or (IV-1): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 84, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-2): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 85, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-3): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 86, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-4): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 87, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-5): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 88, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-6): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 84, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-7): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 87, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-8): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 89, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-9): the amino acid sequence of FR1 is shown in SEQ ID NO. 81, the amino acid sequence of FR2 is shown in SEQ ID NO. 82, the amino acid sequence of FR3 is shown in SEQ ID NO. 89, and the amino acid sequence of FR4 is shown in SEQ ID NO. 90; or (IV-10): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 87, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-11): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 87, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-12): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 86, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-13): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 84, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-14): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 89, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-15): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 86, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-16): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 84, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-17): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 82, the amino acid sequence of FR3 is as represented in SEQ ID NO. 89, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-18): the amino acid sequence of FR1 is as represented in SEQ ID NO. 81, the amino acid sequence of FR2 is as represented in SEQ ID NO. 83, the amino acid sequence of FR3 is as represented in SEQ ID NO. 89, and the amino acid sequence of FR4 is as represented in SEQ ID NO. 90; or (IV-19): the amino acid sequence of FR1 is shown as SEQ ID NO. 81, the amino acid sequence of FR2 is shown as SEQ ID NO. 82, the amino acid sequence of FR3 is shown as SEQ ID NO. 89, and the amino acid sequence of FR4 is shown as SEQ ID NO.

90.

3. The Nanobody according to any one of claims 1-2, characterized in that, (V) the amino acid sequence of the Nanobody is shown as any one of SEQ ID NO. 1-20.

4. A humanized nanobody, characterized in that, (VII): the amino acid sequence of the humanized Nanobody is shown as any one of SEQ ID NO. 21-32.

5. A polypeptide, characterized in that, The Nanobody of any one of claims 1-4 is tagged with an amino acid at the N-terminus and / or C-terminus of the Nanobody.

6. The polypeptide of claim 5, wherein, The tag comprises at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, Flag-tag.

7. The polypeptide of claim 5, wherein, After the Nanobody is tagged with an amino acid at the N-terminus and / or C-terminus of the Nanobody, the tag protects the amino acid by a hinge.

8. The polypeptide of claim 7, wherein, The tag comprises at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, Flag-tag.

9. The polypeptide of claim 7, wherein, The hinge comprises at least one of GS hinge, IgG hinge, IgA hinge, PEG.

10. The polypeptide of claim 7, wherein, The protecting amino acid comprises Ala, Gin, Glu, Met, or any two or more combinations of the foregoing amino acids.

11. A nucleic acid molecule encoding the Nanobody of any one of claims 1-4.

12. An expression vector, characterized in that, The nucleic acid molecule of claim 11.

13. A host cell transformed or transfected with the expression vector of claim 12.

14. An adsorbent, characterized by, The Nanobody of any one of claims 1-4; or the polypeptide of any one of claims 5-10; and a carrier.

15. A kit comprising, The Nanobody of any one of claims 1-4; or the polypeptide of any one of claims 5-10; and a carrier.

16. Use of the Nanobody of any one of claims 1-4; or the polypeptide of any one of claims 5-10; for the manufacture of a preparation for specifically capturing, adsorbing and / or detecting TNFα; or for the manufacture of a preparation for specifically capturing, adsorbing and / or detecting TNFα-enriched and / or purified cells; or for the manufacture of an immunofluorescence assay or immunohistochemistry assay reagent for specifically capturing, adsorbing and / or detecting TNFα.

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