Anti-TNF-alpha nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of immunology, and particularly relates to an anti-TNF-alpha nano antibody as well as a preparation method and application thereof. The anti-TNF-alpha nano antibody sequence with high affinity is obtained through four rounds of optimized phage screening, and further, the soluble expression quantity of the nano antibody is improved by utilizing a pET26a (+)-BL21 expression system and combining a codon optimization technology. The nano antibody can be used for preparing a reagent for treating or diagnosing TNF-alpha related diseases, and has extremely high practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunology, and particularly relates to an anti-TNF-alpha nanobody and a preparation method and application thereof. BACKGROUND

[0002] Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine secreted by immune cells such as macrophages and monocytes, which plays a crucial role in immune response, inflammatory response and apoptosis regulation. TNF-alpha binds to TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) on the cell surface, initiates downstream signaling pathways, and then induces the release of inflammatory factors, apoptosis and tissue damage. In autoimmune diseases such as rheumatoid arthritis, ulcerative colitis and Crohn's disease, abnormal high expression of TNF-alpha can lead to persistent inflammatory response, causing pathological changes such as joint cartilage destruction and intestinal mucosa damage. TNF antagonists can block the interaction between TNF-alpha and TNFR1 and TNFR2, thereby neutralizing the function of TNF-alpha.

[0003] At present, the main TNF antagonists for treating TNF-alpha related diseases in clinical are infliximab and adalimumab. However, these antibody molecules have large molecular weight (150 kDa) and complex structure, which are difficult to efficiently penetrate tissue barriers, and the production process is complex, which needs to rely on mammalian cell expression, resulting in high production cost, which limits its wide application.

[0004] Nanobody (VHH) as a single domain antibody is derived from single domain heavy chain antibody of Camelidae, with a molecular weight of only about 15 kDa, and has the advantages of high stability, strong affinity, good tissue penetration and efficient expression in prokaryotic cells. However, natural camel-derived nanobody may trigger an immune response in the human body, and the affinity and specificity of existing TNF-alpha nanobody still need to be improved.

[0005] Therefore, it is of important clinical application value and broad market prospect to develop a TNF-alpha nanobody with high efficiency, low cost and high affinity. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to provide an anti-TNF-alpha nanobody and a preparation method and application thereof, so as to solve the problems of the prior art.

[0007] The purpose of the present application can be achieved by the following technical solutions: In a first aspect of the present application, an anti-TNF-alpha nanobody is provided, wherein the VHH chain of the nanobody comprises a complementarity determining region and a framework region. The complementarity determining region comprises: (a1) CDR1 as shown in SEQ ID NO. 8, CDR2 as shown in SEQ ID NO. 11, and CDR3 as shown in SEQ ID NO. 14; or, (a2) CDR1 as shown in SEQ ID NO. 9, CDR2 as shown in SEQ ID NO. 12, and CDR3 as shown in SEQ ID NO. 15; or, (a3) CDR1 as shown in SEQ ID NO. 10, CDR2 as shown in SEQ ID NO. 13, and CDR3 as shown in SEQ ID NO. 16.

[0008] In a second aspect of the present application, there is provided an isolated nucleic acid molecule encoding the Nanobody according to the first aspect of the present application.

[0009] In a third aspect of the present application, there is provided a vector comprising the nucleic acid molecule according to the second aspect of the present application.

[0010] In a fourth aspect of the present application, there is provided a host cell comprising the nucleic acid molecule according to the second aspect of the present application or the vector according to the third aspect of the present application.

[0011] In a fifth aspect of the present application, there is provided a method for producing the Nanobody according to the first aspect of the present application, comprising culturing the host cell according to the fourth aspect of the present application under conditions permitting protein expression, and recovering the Nanobody from the culture of the host cell.

[0012] In a sixth aspect of the present application, there is provided a conjugate comprising the Nanobody according to the first aspect of the present application and a coupling moiety.

[0013] In a seventh aspect of the present application, there is provided a pharmaceutical composition comprising the Nanobody according to the first aspect of the present application or the nucleic acid molecule according to the second aspect of the present application or the vector according to the third aspect of the present application or the host cell according to the fourth aspect of the present application or the conjugate according to the sixth aspect of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0014] In an eighth aspect of the present application, there is provided the Nanobody according to the first aspect of the present application or the nucleic acid molecule according to the second aspect of the present application or the vector according to the third aspect of the present application or the host cell according to the fourth aspect of the present application or the conjugate according to the sixth aspect of the present application for use in any one of: (c1) the manufacture of a medicament for the prevention and / or treatment of a disease associated with TNF-alpha; (c2) the manufacture of a detection reagent for detecting the presence or level of TNF-alpha in a sample or for diagnosing whether a subject has a disease associated with TNF-alpha.

[0015] The above technical solution has the following beneficial effects relative to the prior art: (1) High screening efficiency: The present application significantly improves the enrichment efficiency of high-affinity antibodies through four rounds of optimized phage screening, with a high-affinity positive clone rate of 12.9%.

[0016] (2) Strong antibody affinity: The three nanobodies EC 50 obtained by the present application are all lower than 100 nM, among which the EC 50 of C12 is 3.25 nM, with an affinity superior to most anti-TNF-α antibodies in the prior art.

[0017] (3) Efficient expression system: The present application uses the pET26b (+) -BL21 expression system combined with codon optimization technology, which improves the soluble expression of nanobodies by 3.4 times, with a yield of 30 mg / L, significantly reducing production costs.

[0018] (4) Wide application prospect: The nanobodies can be used to prepare therapeutic or diagnostic reagents for TNF-α related diseases, with high practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a TNF-α nanobody screening effect verification curve in the embodiments of the present application; Figure 2 is a linear pET26b (+) plasmid gel electrophoresis graph in the embodiments of the present application; Figure 3 is a PCR amplified antibody gene gel electrophoresis graph in the embodiments of the present application; Figure 4 is a TNF-α nanobody (A10) expression and purification whole process protein gel electrophoresis graph in the embodiments of the present application; Figure 5 is a protein gel electrophoresis graph of all TNF-α nanobodies in the embodiments of the present application; Figure 6 is an affinity curve graph of three TNF-α nanobodies in the embodiments of the present application; Figure 7 is a schematic diagram of the expression yield of C12 nanobody before and after codon optimization in the embodiments of the present application. DETAILED DESCRIPTION

[0020] The present application will be further described in detail below in conjunction with specific embodiments, and the embodiments given are only to illustrate the present application, not to limit the scope of the present application. The embodiments provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0021] The experimental procedures in the following examples were performed essentially as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc, 1995, unless otherwise specified. Those skilled in the art will appreciate that the examples describe the application by way of example only, and are not intended to limit the scope of the application as claimed.

[0022] Traditional three-round screening often results in missing high-affinity clones, while overly stringent screening strategies (e.g. high concentration of competitive elution) reduce the diversity of clones. Moreover, as a commonly used expression host, E. coli has codon usage bias different from that of camelids, for example, the presence of rare codons (e.g. AGA, AGG) in camelid VHH genes can cause translational stalling and protein degradation. In view of this, the present application provides a TNF-α nanobody with high affinity and low cost advantage by reasonably designing a four-round screening strategy and codon optimizing the antibody sequence.

[0023] In one embodiment of the present application, a nanobody against TNF-α is provided, the VHH chain of the nanobody comprises a complementarity determining region and a framework region; the complementarity determining region comprises: (a1) CDR1 as shown in SEQ ID NO. 8, CDR2 as shown in SEQ ID NO. 11, and CDR3 as shown in SEQ ID NO. 14; or, (a2) CDR1 as shown in SEQ ID NO. 9, CDR2 as shown in SEQ ID NO. 12, and CDR3 as shown in SEQ ID NO. 15; or, (a3) CDR1 as shown in SEQ ID NO. 10, CDR2 as shown in SEQ ID NO. 13, and CDR3 as shown in SEQ ID NO. 16.

[0024] The nanobody described herein generally comprises a VHH consisting of four framework regions (FRs) and three complementarity determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, the antigen-binding fragment comprising at least a portion of the nanobody sufficient to impart the ability to the fragment to specifically bind TNF-α. In some embodiments, the nanobody described in the present application can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, so long as the antigen binding and specificity are substantially maintained.

[0025] In certain embodiments, the nanobody comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene), which optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from a human-derived residue to a camelid-derived residue.

[0026] In certain embodiments, the nanobody comprises a framework region derived from a camelid heavy chain antibody.

[0027] In certain embodiments, the framework region comprises: (b1) FR1 as shown in SEQ ID NO. 17, FR2 as shown in SEQ ID NO. 20, FR3 as shown in SEQ ID NO. 23, and FR4 as shown in SEQ ID NO. 26; or, (b2) FR1 as shown in SEQ ID NO. 18, FR2 as shown in SEQ ID NO. 21, FR3 as shown in SEQ ID NO. 24, and FR4 as shown in SEQ ID NO. 26; or, (b3) FR1 as shown in SEQ ID NO. 19, FR2 as shown in SEQ ID NO. 22, FR3 as shown in SEQ ID NO. 25, and FR4 as shown in SEQ ID NO. 26.

[0028] In certain embodiments, the nanobody comprises a sequence as shown in any one of SEQ ID NO. 1, SEQ ID NO. 3, and SEQ ID NO. 5. The sequence shown herein does not comprise an amino acid (e.g., methionine (Met)) encoded by a start codon (e.g., ATG) at its N-terminus. Those skilled in the art understand that, in the process of producing a protein by genetic engineering, the first amino acid of the polypeptide chain produced is often an amino acid (e.g., Met) encoded by a start codon. The nanobody or antigen-binding fragment thereof of the present application encompasses not only an amino acid sequence that does not comprise an amino acid (e.g., Met) encoded by a start codon (e.g., ATG) at its N-terminus, but also an amino acid sequence that comprises an amino acid (e.g., Met) encoded by a start codon (e.g., ATG) at its N-terminus. Therefore, a sequence further comprising an amino acid (e.g., Met) encoded by a start codon (e.g., ATG) at the N-terminus of the above-mentioned amino acid sequence is also within the scope of the present application.

[0029] In the present application, the Nanobodies or antigen-binding fragments thereof of the application can include variants that differ from the Nanobodies or antigen-binding fragments thereof from which they are derived only in the conservative substitution of one or more (e.g., up to 20, up to 15, up to 10, or up to 5 amino acids) amino acid residues, or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the antibody or antigen-binding fragment thereof from which they are derived, and substantially retain the biological function (e.g., specifically binding to TNF-a, neutralizing the biological activity of TNF-a) of the Nanobodies or antigen-binding fragments thereof from which they are derived.

[0030] In yet another embodiment of the application, there is provided an isolated nucleic acid molecule encoding a Nanobody according to the application.

[0031] In certain embodiments, the nucleic acid molecule encoding the VHH chain of a Nanobody according to the application is selected from the sequence set forth in any one of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7.

[0032] In certain embodiments, the nucleic acid molecule encoding CDR1 set forth in SEQ ID NO. 8 is selected from the nucleotide sequence set forth in SEQ ID NO. 34.

[0033] In certain embodiments, the nucleic acid molecule encoding CDR2 set forth in SEQ ID NO. 11 is selected from the nucleotide sequence set forth in SEQ ID NO. 38.

[0034] In certain embodiments, the nucleic acid molecule encoding CDR3 set forth in SEQ ID NO. 14 is selected from the nucleotide sequence set forth in SEQ ID NO. 42.

[0035] In certain embodiments, the nucleic acid molecule encoding CDR1 set forth in SEQ ID NO. 9 is selected from the nucleotide sequence set forth in SEQ ID NO. 35.

[0036] In certain embodiments, the nucleic acid molecule encoding CDR2 set forth in SEQ ID NO. 12 is selected from the nucleotide sequence set forth in SEQ ID NO. 39.

[0037] In certain embodiments, the nucleic acid molecule encoding CDR3 set forth in SEQ ID NO. 15 is selected from the nucleotide sequence set forth in SEQ ID NO. 43.

[0038] In certain embodiments, the nucleic acid molecule encoding CDR1 set forth in SEQ ID NO. 10 is selected from the nucleotide sequence set forth in SEQ ID NO. 36 or 37.

[0039] In certain embodiments, the nucleic acid molecule encoding CDR2 set forth in SEQ ID NO. 13 is selected from the nucleotide sequence set forth in SEQ ID NO. 40 or 41.

[0040] In certain embodiments, the nucleic acid molecule encoding CDR3 set forth in SEQ ID NO. 16 is selected from the nucleotide sequence set forth in SEQ ID NO. 44 or 45.

[0041] In certain embodiments, the nucleic acid molecule encoding FR1 set forth in SEQ ID NO. 17 is selected from the nucleotide sequence set forth in SEQ ID NO. 46.

[0042] In certain embodiments, the nucleic acid molecule encoding FR2 set forth in SEQ ID NO. 20 is selected from the nucleotide sequence set forth in SEQ ID NO. 50.

[0043] In certain embodiments, the nucleic acid molecule encoding FR3 set forth in SEQ ID NO. 23 is selected from the nucleotide sequence set forth in SEQ ID NO. 54.

[0044] In certain embodiments, the nucleic acid molecule encoding FR4 set forth in SEQ ID NO. 26 is selected from the nucleotide sequence set forth in SEQ ID NO. 58 or 59 or 60 or 61.

[0045] In certain embodiments, the nucleic acid molecule encoding FR1 set forth in SEQ ID NO. 18 is selected from the nucleotide sequence set forth in SEQ ID NO. 47.

[0046] In certain embodiments, the nucleic acid molecule encoding FR2 set forth in SEQ ID NO. 21 is selected from the nucleotide sequence set forth in SEQ ID NO. 51.

[0047] In certain embodiments, the nucleic acid molecule encoding FR3 set forth in SEQ ID NO. 24 is selected from the nucleotide sequence set forth in SEQ ID NO. 55.

[0048] In certain embodiments, the nucleic acid molecule encoding FR1 set forth in SEQ ID NO. 19 is selected from the nucleotide sequence set forth in SEQ ID NO. 48 or 49.

[0049] In certain embodiments, the nucleic acid molecule encoding FR2 set forth in SEQ ID NO. 22 is selected from the nucleotide sequence set forth in SEQ ID NO. 52 or 53.

[0050] In certain embodiments, the nucleic acid molecule encoding the FR3 of SEQ ID NO. 25 is selected from the nucleotide sequence of SEQ ID NO. 56 or 57.

[0051] In yet another specific embodiment of the present application, a vector comprising the nucleic acid molecule of the present application is provided.

[0052] In certain embodiments, the vector of the present application can be a plasmid, a cosmid, a phage, etc.

[0053] In yet another specific embodiment of the present application, a host cell comprising the nucleic acid molecule of the present application or the vector of the present application is provided. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (e.g., mammalian cells such as mouse cells, human cells, etc.).

[0054] In yet another specific embodiment of the present application, the method for preparing the Nanobody of the first aspect of the present application comprises culturing the host cell of the present application under conditions permitting protein expression, and recovering the Nanobody from the culture of the host cell.

[0055] The antibody of the present application can be prepared by various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding the antibody of the present application is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then the host cell is transfected. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present application is expressed.

[0056] In yet another specific embodiment of the present application, a conjugate comprising the Nanobody of the present application and a coupling moiety is provided.

[0057] In certain embodiments, the Nanobody of the present application is optionally conjugated to the coupling moiety via a linker.

[0058] In certain embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP or biotin, and how to select a suitable protein tag according to the desired purpose (e.g., purification, detection or tracing) is known to those skilled in the art.

[0059] In certain embodiments, the conjugation moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. The detectable label of the present application can be any substance that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or a cyanine dye derivative (e.g., Cy7, Alexa 750), luminescent substances (e.g., chemiluminescent substances such as acridinium esters), magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.), and biotin for use with avidin modified to bind the above labels. In certain embodiments, such labels can be suitable for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.).

[0060] In certain embodiments, the detectable label as described above can be linked to the Nanobodies or antigen binding fragments thereof of the present application by linkers of varying lengths to reduce potential steric hindrance.

[0061] In certain embodiments, the conjugation moiety is selected from a therapeutic agent, such as an anti-inflammatory drug or an immunosuppressant.

[0062] In certain embodiments, the conjugation moiety is selected from another biologically active polypeptide.

[0063] In yet another specific embodiment of the present application, a pharmaceutical composition is provided comprising a Nanobody, isolated nucleic acid molecule, vector, host cell, or conjugate of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0064] In certain embodiments, the pharmaceutical composition can further comprise an additional pharmaceutically active agent.

[0065] In certain embodiments, the Nanobody, isolated nucleic acid molecule, vector, host cell, or conjugate of the present application and the additional pharmaceutically active agent in the pharmaceutical composition can be provided as separate components or as a combined component. Thus, the Nanobody, isolated nucleic acid molecule, vector, host cell, or conjugate of the present application and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0066] In certain embodiments, the pharmaceutically acceptable carrier and / or excipient can comprise a sterile injectable liquid, such as an aqueous or non-aqueous suspension or solution. In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof.

[0067] The pharmaceutical compositions of the present application can include a "therapeutically effective amount" or a "prophylactically effective amount" of a Nanobody, an isolated nucleic acid molecule, a vector, a host cell, or a conjugate according to the present application. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to prevent, retard, or delay the development of a disease. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to cure or at least partially arrest the symptoms of a disease and its complications in a patient already suffering from the disease. The therapeutically effective amount can vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of the drug, and other therapies being administered to the patient, etc.

[0068] In yet another specific embodiment of the present application, there is provided the use of a Nanobody, an isolated nucleic acid molecule, a vector, a host cell, or a conjugate according to the present application for any of the following: (c1 ) the manufacture of a medicament for the prevention and / or treatment of a TNF-α related disease; (c2) the manufacture of a diagnostic test for detecting the presence or level of TNF-α in a sample or for diagnosing whether a subject is suffering from a TNF-α related disease.

[0069] In certain embodiments, the TNF-α related disease is characterized by elevated TNF-α expression and / or excessive TNF-α activity.

[0070] In certain embodiments, the TNF-α related disease is an inflammatory disease or an autoimmune disease.

[0071] In certain embodiments, the TNF-α related disease is inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, psoriasis (e.g., plaque psoriasis), systemic lupus erythematosus, ankylosing spondylitis, graft versus host disease, hidradenitis suppurativa, psoriatic arthritis, polyarticular juvenile idiopathic arthritis, Behcet's syndrome, uveitis, psoriasis.

[0072] In certain embodiments, the subject is a mammal, e.g., a human.

[0073] In certain embodiments, the Nanobody, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent, e.g., an anti-inflammatory drug or an immunosuppressant.

[0074] The Nanobody, isolated nucleic acid molecule, vector, host cell, or conjugate of the application, or the pharmaceutical composition of the application, can be formulated into any dosage form known in the medical art, e.g., tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, pastilles, suppositories, injections (including sterile solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, and the like. The preferred dosage form will depend on the intended mode of administration and therapeutic use.

[0075] A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the antibody of the application, or antigen-binding fragment thereof, in the required amount in an appropriate solvent with one or more of the other ingredients enumerated above, as required, followed by filtered sterilization. Furthermore, sterile solutions can be prepared by incorporating the antibody of the application, or antigen-binding fragment thereof, in the required amount in the appropriate solvent with one or more of the other ingredients, as required, followed by filtered sterilization. In addition, sterile solutions can be prepared as sterile lyophilized powders for reconstitution with a suitable solvent (e.g., water, WFI, BWFI, sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof) prior to use.

[0076] The Nanobody, isolated nucleic acid molecule, vector, host cell, or conjugate of the application, or the pharmaceutical composition of the application, can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracellular web, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the Nanobody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition of the application is administered by intravenous injection or bolus.

[0077] In certain embodiments, the application provides a method of detecting the presence of or the amount of TNF-alpha in a sample, comprising using a Nanobody or antigen-binding fragment or conjugate thereof of the application.

[0078] In certain embodiments, the method is an immunological detection, such as immunoblotting, enzyme immunoassay (e.g. ELISA), chemiluminescence immunoassay, fluorescence immunoassay or radioimmunoassay.

[0079] In certain embodiments, the conjugate used in the method comprises a Nanobody of the application and a detectable label.

[0080] In certain embodiments, the Nanobody used in the method is labeled with a detectable label.

[0081] In certain embodiments, the Nanobody or antigen-binding fragment thereof used in the method is not labeled with a detectable label. Thus, the method can further comprise using a further reagent (such as a secondary antibody) labeled with a detectable label to detect the Nanobody or antigen-binding fragment thereof of the application.

[0082] In certain embodiments, the method comprises the following steps: (1) contacting the sample with a Nanobody or antigen-binding fragment thereof or conjugate of the application; (2) detecting the formation of a complex between the Nanobody or antigen-binding fragment thereof or conjugate and TNF-alpha or detecting the amount of the complex.

[0083] The formation of the complex is indicative of the presence of TNF-alpha or of a cell expressing TNF-alpha.

[0084] The method can be used for non-diagnostic purposes (e.g. the sample is a cell sample, not a sample from a patient).

[0085] In certain embodiments, the method is used to diagnose whether a subject has a disease associated with TNF-alpha. In such embodiments, the method can further comprise the step of comparing the amount of TNF-alpha in the sample from the subject to a reference value. The reference value can be the level of TNF-alpha in a sample from a subject known not to have a disease associated with TNF-alpha (e.g. a healthy control) (also referred to as a "negative reference value"). For example, if the amount of TNF-alpha in the sample from the subject is elevated relative to the negative reference value, then this is indicative of the subject having a disease associated with TNF-alpha.

[0086] In certain embodiments, the disease associated with TNF-alpha is an inflammatory disease or an autoimmune disease.

[0087] In certain embodiments, the TNF-a related disease is inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, psoriasis (e.g., plaque psoriasis), systemic lupus erythematosus, ankylosing spondylitis, graft versus host disease, hidradenitis suppurativa, psoriatic arthritis, polyarticular juvenile idiopathic arthritis, Behcet's syndrome, uveitis, psoriasis.

[0088] In certain embodiments, the sample can be selected from the group consisting of urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, a biopsy or fine needle aspirate), histological preparations, and the like.

[0089] In certain embodiments, the TNF-a is human TNF-a.

[0090] In certain embodiments, the conjugate used to prepare the detection reagent comprises a Nanobody or antigen binding fragment thereof of the application and a detectable label.

[0091] In certain embodiments, the Nanobody or antigen binding fragment thereof used to prepare the detection reagent is labeled with a detectable label.

[0092] In certain embodiments, the Nanobody or antigen binding fragment thereof used to prepare the detection reagent is not labeled with a detectable label. In such embodiments, the detection reagent can further comprise an additional reagent (e.g., a secondary antibody) capable of detecting the Nanobody or antigen binding fragment thereof of the application.

[0093] As used herein, the term "Nanobody" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or shark antibody). Typically, a Nanobody is composed of four framework regions and three complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A Nanobody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or to lack one or both of those framework regions, so long as antigen binding and specificity are substantially maintained. Nanobodies are also known as single-domain antibodies (sdAbs), and the two terms are used interchangeably.

[0094] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in a Nanobody, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g. as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a skilled person will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between the different numbering systems is well known to the skilled person (see e.g. Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0095] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0096] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0097] The sequence information related to the present application is described in Table 1.

[0098] Table 1 Sequence information

[0099] The reagents used in the following examples are shown in Table 2.

[0100] Table 2 Reagent sources

[0101] Example 1 This example provides a method for screening high-affinity anti-TNF-a nanobodies, comprising the following steps: (I) Four rounds of phage screening (1) Plate coating: 5 μg / mL of TNF-a antigen was coated on an enzyme-labeled plate, and PBS buffer was coated in the blank group, 4°C, overnight; (2) Blocking the well plate: Discard the coating solution, use 1% BSA-PBS (by changing the blocking solution every round, the enrichment of phage against specific blocking solution can be greatly reduced) to block the enzyme-labeled plate at room temperature for 2h; (3) Library blocking: Mix 90 μL of phage library, 180 μL of PBS, and 30 μL of 10% BSA-PBS solution, shake at 220 rpm and block at room temperature for 1 hour; (4) Binding: Discard the blocking solution, tap and dry the enzyme-labeled plate, add the phage library, shake at 220 rpm and incubate at 37°C for 2h; (5) Washing: Pour out the enzyme-labeled plate that does not bind phage, wash with PBST 5 times and PBS 3 times; (6) Elution: Add 200 μL of HCl (0.1 M, pH=2.2) to the enzyme-labeled well, shake at 220 rpm and incubate at 37°C for 10 min to elute the bound phage; (7) Neutralization: Neutralize with 1M Tris-HCl (pH=8.0); (8) Amplification: Use 400 μL of eluted neutralization solution to infect 4mL of TG1 E. coli cells with OD 600=0.5, then shake culture at 37°C for 1h; then add M13KO7 helper phage, shake at 37°C for 1h; finally, transfer into 50mL of 2×YT / Carb / Kan medium, and incubate at 30°C overnight; (9) Titer determination is performed simultaneously with step (8) amplification: Take the bacterial solution after infecting TG1 cells for gradient dilution with 2×YT (dilute 8 concentrations), take 10 μL of each gradient and drop on 2×YT / Carb solid medium, incubate at 37°C overnight, and use PBS well bacterial culture solution as negative control; (10) Purification of phage: For the amplified phage, PEG / NaCl precipitation method was used to purify the phage, and the OD 268nm absorption was measured by Nanodrop 2000c to calibrate the phage concentration, and then the next round of screening was carried out. According to this step, the key conditions of each round of screening were changed, and a total of four rounds of phage screening were carried out. The conditions and enrichment rates of the four rounds of screening are shown in Table 3.

[0102] Table 3 Conditions and enrichment rates of four rounds of screening

[0103] (II) ELISA verification of screening effect The phage library purified in each round was used for screening effect verification by ELISA, and the specific process was as follows: (1) Plate coating: 1 μg / mL of TNF-α antigen was coated in an enzyme-labeled plate at 4°C overnight; (2) Blocking: Discard the coating solution, and use 1% BSA-PBS to block the enzyme-labeled plate at room temperature for 2h; (3) Dilute the phage library: Dilute the phage library in each round to 1.0 OD, and use PBS to dilute it by 3 times (dilute 8 concentrations); (4) Binding: Discard the blocking solution, tap and dry the enzyme-labeled plate, add the gradient-diluted phage to the enzyme-labeled plate, and incubate at 37°C for 30 min; (5) Washing: Wash with PBST for 5 times; (6) Add secondary antibody: Add anti-M13 Bacteriophage-HRP secondary antibody (1:5000 dilution) to the enzyme-labeled plate, and incubate at 37°C for 1 h; (7) Wash again: Wash with PBST for 5 times; (8) Color development: Add 100 μL of TMB color developing solution, and develop color at 37°C for 15 min; (9) Termination: Add 2M sulfuric acid 50 μL to terminate the reaction; (10) Measurement: Measure the absorbance at 450 nm by an enzyme-labeled instrument.

[0104] The measured OD value was combined with the phage OD value to draw a fitting curve, and finally the point corresponding to half of the strongest signal was found, and the phage OD value was the EC 50 , EC 50 defined as the concentration producing 50% efficacy or binding. The four rounds of screening effect curve and EC50 results are shown in Figure 1 , wherein the first round EC 50 value is 1.2151, the second round EC 50 value is 0.2311, the third round EC 50 value is 0.0047, and the fourth round EC 50 value is 0.0033.

[0105] (III) ELISA screening and sequencing of positive clones After the fourth round of screening, 480 single colonies were randomly picked and inoculated in 96 deep well plates, each well of which had been added with 400 μL 2xYT / Carb and 4 μL M13K07 helper phage, and incubated at 37 °C overnight, and the phage supernatant was obtained by centrifugation at 3000 rpm.

[0106] The specific process of monoclonal ELISA screening is as follows: (1) Plate coating: 1 μg / mL of TNF-α antigen was coated in the enzyme-labeled plate, and PBS buffer was coated in the blank group, 4 °C, overnight; (2) Blocking: Discard the coating solution, use 1% BSA-PBS to block the enzyme-labeled plate at room temperature for 2 h; (3) Dilute the phage supernatant: Mix the phage supernatant with ELISA buffer 1:1 uniformly; (4) Combine: Discard the blocking solution, tap and dry the enzyme-labeled plate, add the diluted phage supernatant to the enzyme-labeled plate, and incubate at 37 °C for 1 h; (5) Wash: Wash 5 times with PBST; (6) Add secondary antibody: Add anti-M13 Bacteriophage-HRP secondary antibody (1:5000 dilution) to the enzyme-labeled plate, and incubate at 37 °C for 1 h; (7) Wash again: Wash 5 times with PBST; (8) Color development: Add 100 μL TMB color developing solution, and develop color at 37 °C for 15 min; (9) Termination: Add 50 μL 2M sulfuric acid to terminate the reaction; (10) Measurement: Measure the absorbance value at 450 nm with an enzyme-labeled instrument.

[0107] The clones with OD value bound to TNF-α / OD value bound to BSA≥2 were defined as positive clones, and the clones with OD value bound to TNF-α>0.5, OD value bound to BSA<0.25, and ratio≥5 were defined as high-affinity positive clones. The positive clone rate statistics of ELISA screening are shown in Table 4.

[0108] Table 4 Positive clone rate statistics of ELISA screening

[0109] The positive clones were picked for sequencing (the sequence of the sequencing primer is shown in SEQ ID NO. 33), and through sequence alignment and cluster analysis, 3 high-affinity candidate sequences (A10, C2, and C12, respectively) were selected for subsequent research. The amino acid sequence of A10 is shown in SEQ ID NO. 1, the nucleotide sequence of A10 is shown in SEQ ID NO. 2, the amino acid sequence of C2 is shown in SEQ ID NO. 3, the nucleotide sequence of C2 is shown in SEQ ID NO. 4, the amino acid sequence of C12 is shown in SEQ ID NO. 5, the nucleotide sequence of C12 is shown in SEQ ID NO. 6, and the detailed antibody sequence information is shown in Table 1.

[0110] Example 2 The present embodiment provides expression and purification of nanobodies.

[0111] (I) Construction of expression plasmid The pET26b(+) plasmid was linearized by enzyme digestion; then the positive clone plasmid was used as a template for PCR amplification of the nanobody gene fragment, and the pET26b(+) plasmid homologous arms were introduced at both ends of the antibody gene; finally, the linearized plasmid and the nanobody gene fragment were connected into a complete plasmid by homologous recombination. The specific construction steps are as follows.

[0112] (1) Linearization of plasmid The pET26b(+) plasmid was amplified in Top10 cells, extracted using a mini-prep kit, and the concentration was calibrated using a Nanodrop 2000c. The pET26(+) plasmid was digested with Nco I and Xho I restriction endonuclease, incubated at 37°C for 2 hours, and the enzyme digestion system is shown in Table 5.

[0113] Table 5 Enzyme digestion system

[0114] DNA agarose gel electrophoresis was performed, and the results are shown in Figure 2 , confirming that the target product is about 5000 bp. Finally, the gel was recovered to obtain the linearized plasmid, and the concentration was calibrated using a Nanodrop 2000c.

[0115] (2) PCR amplification of target antibody gene The PCR primers of the 3 candidate sequences are shown in Table 1, the PCR reaction system is shown in Table 6, and the PCR reaction program is shown in Table 7.

[0116] Table 6 PCR reaction system

[0117] Table 7 PCR reaction program

[0118] DNA agarose gel electrophoresis was performed, and the results are shown in Figure 3 , confirming that the target product is about 400 bp. Finally, the target product was recovered by PCR, and the concentration was calibrated using a Nanodrop 2000c.

[0119] (3) Homologous recombination The linearized pET26b(+) plasmid and the PCR-amplified target antibody gene were subjected to homologous recombination, and the reaction system is shown in Table 8.

[0120] Table 8 Homologous recombination reaction system

[0121] Incubate the ligation mixture at 50°C for 15 minutes for chemical transformation.

[0122] (4) Chemical transformation: Add 5 μL of the homologous recombination product to 50 μL of chemically competent BL21 cells, and incubate on ice for 30 minutes. After heat shock at 42°C for 30 seconds, quickly return to ice for 2 minutes. Add 950 μL of SOC medium, and incubate at 37°C, 220 rpm, for 1 hour. Take 100 μL of the culture and spread on LB / Kan solid medium, and incubate at 37°C overnight. The next day, colonies grow on the medium containing antibiotics, indicating successful homologous recombination of the plasmid.

[0123] (II) Nanobody expression and purification Induce the target nanobody to express in BL21 host, and purify the antibody using a Ni-NTA column, with the following specific steps.

[0124] (1) Antibody expression Pick BL21 cells containing the target antibody and inoculate in 5 mL of LB / Kan medium, and incubate at 37°C, 200 rpm, overnight. The next day, transfer 5 mL of the cell culture to 500 mL of LB / Kan medium, and incubate at 37°C, 200 rpm, until OD600=0.6. Add IPTG to 1 mM, and incubate at 25°C, 200 rpm, for about 16 hours to induce antibody expression. On the third day, centrifuge the induced culture at 5000 rpm, and harvest the cell pellet.

[0125] (2) Antibody purification Resuspend the cell pellet in 100 mL of lysis solution (0.5 M sucrose, 0.2 M Tris, pH 8, 0.5 mM EDTA), add 200 mL of deionized water, add a magnetic stirrer, and stir at 4°C for 45 minutes to release the nanobody. Then centrifuge at 12000 rpm for 20 minutes, and harvest the supernatant. Add the supernatant to a filter column assembled with 3 mL of Ni-NTA resin, and discard the filtrate. Wash the column with about 100 mL of high-salt washing solution (20 mM HEPES, 500 mM sodium chloride, 20 mM imidazole, pH 7.5), and then wash the column with about 100 mL of washing solution (20 mM HEPES, 100 mM sodium chloride, 20 mM imidazole, pH 7.5). Finally, elute the nanobody bound to the nickel column using 30 mL of elution solution (20 mM HEPES, 100 mM sodium chloride, 400 mM imidazole, pH 7.5).

[0126] The eluate was dialyzed against PBS and subjected to 12% SDS-PAGE protein gel electrophoresis to confirm that the nanobody was approximately 15 kDa. The nanobody concentration was calibrated using a Nanodrop 2000c. Finally, 50% glycerol was added, and the solution was aliquoted and stored at -20°C.

[0127] Taking A10 as an example, the protein gel electrophoresis of the whole process of nanobody expression and purification is as follows Figure 4 As shown. Protein gel electrophoresis of purified A10, C2, and C12 nanobodies is shown. Figure 5 shown.

[0128] Example 3 This example provides a nanobody affinity ELISA validation, the specific steps are as follows: (1) Coating: Coat 1 μg / mL TNF-α antigen on the ELISA plate at 4°C overnight; (2) Blocking: Discard the coating solution and block the ELISA plate with 1% BSA-PBS at room temperature for 2 h; (3) Dilute the antibody: Dilute the A10, C2, and C12 nanoantibodies to 2 μM and perform a 3× gradient dilution with PBS (8 dilutions); (4) Binding: Discard the blocking solution, pat the ELISA plate dry, add the diluted nanoantibodies to the ELISA plate, and incubate at 37°C for 1 h; (5) Washing: Wash 5 times with PBST; (6) Add secondary antibody: Add anti-His-HRP secondary antibody (1:5000 dilution) to the ELISA plate and incubate at 37°C for 1 h; (7) Wash again: Wash 5 times with PBST; (8) Color development: Add 100 μL TMB color development solution, incubate at 37°C for 15 min; (9) Termination: Add 50 μL of 2M sulfuric acid to terminate the reaction; (10) Measurement: Measure the absorbance at 450 nm using a microplate reader.

[0129] Record the obtained OD values, draw a fitting curve based on the antibody concentration, and calculate the EC of each antibody 50 The affinity results of the three nanoantibodies are as follows Figure 6 As shown, the EC of A10 50 The value was 67.56 nM, and the EC of C2 50 The value was 86.58 nM, and the EC of C12 50 The value is 3.25 nM.

[0130] Example 4 This example provides nanobody codon optimization and verification Affinity validation revealed that C12 exhibited high affinity, but its production yield was low, at only 8-9 mg / L. The nucleotide sequence of C12 was optimized. The optimized Nanobody nucleotide sequence is shown in SEQ ID NO. 7.

[0131] The optimized gene sequence is obtained by chemical synthesis, cloned into pET26b(+) vector, transformed into BL21 expression bacteria, and after induction and purification, the final yield of C12 nanobody is increased from 8.8 mg / L to 30 mg / L, increased by 3.4 times. The expression yield before and after codon optimization is compared as shown in the table. Figure 7 As shown in the table, it is proved that codon optimization has obvious effect on improving the yield of nanobody.

[0132] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some of them. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An anti-TNF-α nanobody, characterized in that The VHH chain of the Nanobody includes a complementarity determining region and a framework region; The complementarity determining regions include: (a1) CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 14; or (a2) CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 12, and CDR3 shown in SEQ ID NO. 15; or (a3) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 13, and CDR3 shown in SEQ ID NO.

16.

2. The Nanobody according to claim 1, wherein The framework region includes: (b1) FR1 shown in SEQ ID NO. 17, FR2 shown in SEQ ID NO. 20, FR3 shown in SEQ ID NO. 23, and FR4 shown in SEQ ID NO. 26; or (b2) FR1 shown in SEQ ID NO. 18, FR2 shown in SEQ ID NO. 21, FR3 shown in SEQ ID NO. 24, and FR4 shown in SEQ ID NO. 26; or (b3) FR1 represented by SEQ ID NO. 19, FR2 represented by SEQ ID NO. 22, FR3 represented by SEQ ID NO. 25, and FR4 represented by SEQ ID NO.

26.

3. The Nanobody according to claim 1, wherein The nanobody comprises the sequence shown in any one of SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.

5.

4. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Nanobody according to any one of claims 1 to 3.

5. A carrier, characterized in that Comprising the nucleic acid molecule of claim 4.

6. A host cell, characterized in that Comprising the nucleic acid molecule according to claim 4 or the vector according to claim 5.

7. The method for preparing the Nanobody according to any one of claims 1 to 3, characterized in that: include: The host cell according to claim 6 is cultured under conditions that allow protein expression, and the Nanobody is recovered from the cultured host cell culture.

8. A conjugate, characterized in that Comprising the Nanobody according to any one of claims 1 to 3 and a coupling portion.

9. A pharmaceutical composition, characterized in that Comprising the Nanobody of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, or the conjugate of claim 8, and a pharmaceutically acceptable carrier and / or excipient.

10. Use of the Nanobody according to any one of claims 1 to 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the host cell according to claim 6, or the conjugate according to claim 8, or the pharmaceutical composition according to claim 9 in any of the following: (c1) Use in the preparation of drugs for preventing and / or treating diseases related to TNF-α; (c2) Use in the preparation of a detection reagent for detecting the presence or level of TNF-α in a sample or for diagnosing whether a subject suffers from a disease associated with TNF-α.

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