A nano-antibody against TNF-alpha and its preparation method and application

Abstract

The application belongs to the technical field of immunology, and particularly relates to an anti-TNF-alpha nanobody as well as a preparation method and application thereof. The application obtains an anti-TNF-alpha nanobody sequence with high affinity through four rounds of optimized phage screening. Further, the application uses a pET26b (+) -BL21 expression system in combination with a codon optimization technology to improve the soluble expression amount of the nanobody. The nanobody can be used for preparing a therapeutic or diagnostic detection reagent for TNF-alpha related diseases, and has extremely high practical application value.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, and particularly relates to an anti-TNF-α nanobody, its preparation method, and its application. Background Technology

[0002] Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine secreted by immune cells such as macrophages and monocytes, playing a crucial role in the regulation of the body's immune response, inflammatory response, and apoptosis. TNF-α binds to TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) on the cell surface, initiating downstream signaling pathways, thereby inducing the release of inflammatory factors, apoptosis, and tissue damage. In autoimmune diseases such as rheumatoid arthritis, ulcerative colitis, and Crohn's disease, abnormally high expression of TNF-α leads to persistent inflammatory responses, causing pathological changes such as articular cartilage destruction and intestinal mucosal damage. TNF antagonists can block the interaction between TNF-α and TNFR1 and TNFR2, thereby neutralizing the function of TNF-α.

[0003] Currently, the main TNF antagonists used clinically to treat TNF-α-related diseases are infliximab and adalimumab. However, these antibodies have large molecular weights (150 kDa) and complex structures, making it difficult for them to efficiently penetrate tissue barriers. Furthermore, their production processes are complex and require expression in mammalian cells, resulting in high production costs and limiting their widespread application.

[0004] Nanobodies (VHHs), as single-domain antibodies derived from camel-derived heavy-chain antibodies, have a molecular weight of only about 15 kDa and possess significant advantages such as high stability, strong affinity, good tissue penetration, and efficient expression in prokaryotic cells. However, natural camel-derived nanobodies may trigger an immune response in the human body, and the affinity and specificity of existing TNF-α nanobodies still need to be improved.

[0005] Therefore, developing a high-efficiency, low-cost, and high-affinity TNF-α nanobody has significant clinical application value and broad market prospects. Summary of the Invention

[0006] To address the aforementioned problems, the purpose of this invention is to provide an anti-TNF-α nanobody, its preparation method, and its application, thereby overcoming the shortcomings of the prior art.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides an anti-TNF-α nanobody, wherein the VHH chain of the nanobody includes a complementarity-determining region and a framework region;

[0009] The complementarity determination region includes:

[0010] (a1) CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 14; or,

[0011] (a2) CDR1 shown in SEQ ID NO.9, CDR2 shown in SEQ ID NO.12, and CDR3 shown in SEQ ID NO.15; or,

[0012] (a3) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.13, and CDR3 shown in SEQ ID NO.16.

[0013] In a second aspect, the invention provides isolated nucleic acid molecules that encode the nanobodies described in the first aspect of the invention.

[0014] A third aspect of the invention provides a carrier comprising the nucleic acid molecule described in the second aspect of the invention.

[0015] In a fourth aspect, the invention provides a host cell comprising the nucleic acid molecule described in the second aspect or the vector described in the third aspect.

[0016] A fifth aspect of the present invention provides a method for preparing the nanobody described in the first aspect of the present invention, comprising: culturing the host cell described in the fourth aspect of the present invention under conditions that allow protein expression, and recovering the nanobody from the cultured host cell culture.

[0017] A sixth aspect of the invention provides a conjugate comprising the nanobody described in the first aspect of the invention and the conjugation portion.

[0018] A seventh aspect of the present invention provides a pharmaceutical composition comprising a nanobody as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a carrier as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, or a conjugate as described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0019] An eighth aspect of the present invention provides the use of the nanobody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the conjugate described in the sixth aspect of the present invention in any of the following:

[0020] (c1) Use in the preparation of medicaments for the prevention and / or treatment of diseases related to TNF-α;

[0021] (c2) Application in the preparation of diagnostic reagents for detecting the presence or level of TNF-α in a sample or for diagnosing whether a subject has a disease related to TNF-α.

[0022] Compared with existing technologies, the above technical solutions have the following advantages:

[0023] (1) High screening efficiency: This invention 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%.

[0024] (2) Strong antibody affinity: The three nanobodies EC obtained in this invention 50 All are below 100 nM, with C12 having an EC 50 It reaches 3.25 nM and has a higher affinity than most existing anti-TNF-α antibodies.

[0025] (3) High efficiency of expression system: The present invention uses the pET26b(+)-BL21 expression system, combined with codon optimization technology, which increases the soluble expression level of nanobody by 3.4 times and the yield is 30 mg / L, significantly reducing production cost.

[0026] (4) Broad application prospects: This nanobody can be used to prepare treatment or diagnostic reagents for TNF-α related diseases, and has extremely high practical application value. Attached Figure Description

[0027] Figure 1 This is a curve illustrating the screening effect of TNF-α nanobody in an embodiment of the present invention.

[0028] Figure 2 This is a gel electrophoresis image of the linearized pET26b(+) plasmid in an embodiment of the present invention;

[0029] Figure 3 This is a gel electrophoresis image of PCR amplification of antibody genes in an embodiment of the present invention;

[0030] Figure 4 This is a protein gel electrophoresis image showing the entire process of expression and purification of the TNF-α nanobody (A10) in this embodiment of the invention;

[0031] Figure 5 These are gel electrophoresis images of all TNF-α nanobody proteins in the embodiments of this invention;

[0032] Figure 6 This is an affinity curve of three TNF-α nanobodies in the embodiments of the present invention;

[0033] Figure 7This is a schematic diagram comparing the expression yield of C12 nanoantibody before and after codon optimization in an embodiment of the present invention. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods described in the following examples, including molecular biology experimental methods and immunoassays used in this invention, are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will understand that the examples illustrate the invention by way of example and are not intended to limit the scope of the invention.

[0036] Traditional three-round screening often leads to the omission of high-affinity clones, while overly stringent screening strategies (such as high-concentration competitive elution) reduce clonal diversity. Furthermore, *E. coli*, a commonly used expression host, exhibits codon usage preferences different from those of camel genes. For example, the presence of rare codons (such as AGA and AGG) in the camel VHH gene can cause translational arrest and protein degradation. Therefore, this invention provides a TNF-α nanobody that combines high affinity and low cost through a rationally designed four-round screening strategy and codon optimization of the antibody sequence.

[0037] In one specific embodiment of the present invention, an anti-TNF-α nanobody is provided, wherein the VHH chain of the nanobody includes a complementarity-determining region and a framework region; the complementarity-determining region includes:

[0038] (a1) CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 14; or,

[0039] (a2) CDR1 shown in SEQ ID NO.9, CDR2 shown in SEQ ID NO.12, and CDR3 shown in SEQ ID NO.15; or,

[0040] (a3) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.13, and CDR3 shown in SEQ ID NO.16.

[0041] The nanobodies described herein typically comprise 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 contains at least a portion of the nanobody, sufficient to confer the fragment the ability to specifically bind TNF-α. In some embodiments, the nanobodies of this invention may be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0042] In some embodiments, the nanobody comprises a heavy chain framework region derived from human immunoglobulins (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), the heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to camel residues.

[0043] In some embodiments, the nanobody comprises a framework region derived from a camel-derived heavy chain antibody.

[0044] In some implementations, the frame region includes:

[0045] (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,

[0046] (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,

[0047] (b3) FR1 shown in SEQ ID NO.19, FR2 shown in SEQ ID NO.22, FR3 shown in SEQ ID NO.25, and FR4 shown in SEQ ID NO.26.

[0048] In some embodiments, the nanobody comprises the sequence shown in any one of SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5. The sequences shown herein do not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at their N-terminus. Those skilled in the art will understand that during the preparation of proteins through genetic engineering, the first digit of the resulting polypeptide chain is often an amino acid (such as Met) encoded by the start codon due to the effect of the start codon. The nanobody or antigen-binding fragment of the present invention encompasses not only amino acid sequences that do not contain an amino acid (such as Met) encoded by a start codon at their N-terminus, but also amino acid sequences that do contain an amino acid (such as Met) encoded by a start codon at their N-terminus. Therefore, sequences that further contain an amino acid (such as Met) encoded by a start codon at the N-terminus of the aforementioned amino acid sequences are also within the scope of protection of the present invention.

[0049] In this invention, the nanobodies or antigen-binding fragments thereof may include variants that differ from their derived nanobodies or antigen-binding fragments only in one or more (e.g., up to 20, 15, 10, or 5 amino acid substitutions) of conserved amino acid residues, or have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with their derived antibodies or antigen-binding fragments, and substantially retain the biological function of their derived nanobodies or antigen-binding fragments (e.g., specific binding to TNF-α, neutralizing the biological activity of TNF-α).

[0050] In another specific embodiment of the invention, isolated nucleic acid molecules are provided, said nucleic acid molecules encoding the nanobody described in the invention.

[0051] In some embodiments, the nucleic acid molecule encoding the VHH chain of the nanobody described in this invention is selected from the sequences shown in any one of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 7.

[0052] In some embodiments, the nucleic acid molecule encoding CDR1 shown in SEQ ID NO. 8 is selected from the nucleotide sequence shown in SEQ ID NO. 34.

[0053] In some embodiments, the nucleic acid molecule encoding CDR2 shown in SEQ ID NO.11 is selected from the nucleotide sequence shown in SEQ ID NO.38.

[0054] In some embodiments, the nucleic acid molecule encoding CDR3 shown in SEQ ID NO.14 is selected from the nucleotide sequence shown in SEQ ID NO.42.

[0055] In some embodiments, the nucleic acid molecule encoding CDR1 shown in SEQ ID NO. 9 is selected from the nucleotide sequence shown in SEQ ID NO. 35.

[0056] In some embodiments, the nucleic acid molecule encoding CDR2 shown in SEQ ID NO.12 is selected from the nucleotide sequence shown in SEQ ID NO.39.

[0057] In some embodiments, the nucleic acid molecule encoding CDR3 shown in SEQ ID NO.15 is selected from the nucleotide sequence shown in SEQ ID NO.43.

[0058] In some embodiments, the nucleic acid molecule encoding CDR1 shown in SEQ ID NO. 10 is selected from the nucleotide sequence shown in SEQ ID NO. 36 or 37.

[0059] In some embodiments, the nucleic acid molecule encoding CDR2 shown in SEQ ID NO. 13 is selected from the nucleotide sequence shown in SEQ ID NO. 40 or 41.

[0060] In some embodiments, the nucleic acid molecule encoding CDR3 shown in SEQ ID NO. 16 is selected from the nucleotide sequence shown in SEQ ID NO. 44 or 45.

[0061] In some embodiments, the nucleic acid molecule encoding FR1 shown in SEQ ID NO.17 is selected from the nucleotide sequence shown in SEQ ID NO.46.

[0062] In some embodiments, the nucleic acid molecule encoding FR2 shown in SEQ ID NO.20 is selected from the nucleotide sequence shown in SEQ ID NO.50.

[0063] In some embodiments, the nucleic acid molecule encoding FR3 as shown in SEQ ID NO.23 is selected from the nucleotide sequence shown in SEQ ID NO.54.

[0064] In some embodiments, the nucleic acid molecule encoding FR4 as shown in SEQ ID NO.26 is selected from the nucleotide sequence shown in SEQ ID NO.58, 59, 60, or 61.

[0065] In some embodiments, the nucleic acid molecule encoding FR1 shown in SEQ ID NO.18 is selected from the nucleotide sequence shown in SEQ ID NO.47.

[0066] In some embodiments, the nucleic acid molecule encoding FR2 as shown in SEQ ID NO.21 is selected from the nucleotide sequence shown in SEQ ID NO.51.

[0067] In some embodiments, the nucleic acid molecule encoding FR3 as shown in SEQ ID NO.24 is selected from the nucleotide sequence shown in SEQ ID NO.55.

[0068] In some embodiments, the nucleic acid molecule encoding FR1 shown in SEQ ID NO.19 is selected from the nucleotide sequence shown in SEQ ID NO.48 or 49.

[0069] In some embodiments, the nucleic acid molecule encoding FR2 as shown in SEQ ID NO.22 is selected from the nucleotide sequence shown in SEQ ID NO.52 or 53.

[0070] In some embodiments, the nucleic acid molecule encoding FR3 as shown in SEQ ID NO.25 is selected from the nucleotide sequence shown in SEQ ID NO.56 or 57.

[0071] In another specific embodiment of the present invention, a carrier is provided which comprises the nucleic acid molecule described in the present invention.

[0072] In some embodiments, the vector of the present invention may be a plasmid, a granule, a bacteriophage, etc.

[0073] In another specific embodiment of the invention, a host cell is provided, which comprises the nucleic acid molecule or the vector described in the invention. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0074] In another specific embodiment of the present invention, the method for preparing the nanobody according to the first aspect of the present invention includes: culturing the host cell according to the present invention under conditions that allow protein expression, and recovering the nanobody from the cultured host cell culture.

[0075] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification, the resulting DNA molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells can then be cultured under specific conditions to express the antibodies of the present invention.

[0076] In another specific embodiment of the present invention, a conjugate is provided, comprising the nanobody described in the present invention and the conjugation portion.

[0077] In some embodiments, the nanobodies of the present invention are optionally conjugated to the coupling portion via a linker.

[0078] In some embodiments, the coupling portion is selected from protein tags. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification, detection, or tracing).

[0079] In some embodiments, the coupling portion is selected from detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-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 cyanine dye derivatives (e.g., Cy7, Alexa 750), luminescent substances (e.g., chemiluminescent substances, such as acridinium esters), magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastics (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.).

[0080] In some embodiments, the detectable markers described above can be linked to the nanobodies or antigen-binding fragments of the present invention via linkers of different lengths to reduce potential steric hindrance.

[0081] In some embodiments, the coupling portion is selected from therapeutic agents, such as anti-inflammatory drugs or immunosuppressants.

[0082] In some embodiments, the coupling portion is selected from other bioactive peptides.

[0083] In another specific embodiment of the invention, a pharmaceutical composition is provided comprising the nanobody, isolated nucleic acid molecule, carrier, host cell, or conjugate described herein, and pharmaceutically acceptable carrier and / or excipient.

[0084] In some embodiments, the pharmaceutical composition may also contain additional pharmaceutically active agents.

[0085] In some embodiments, the nanobodies, isolated nucleic acid molecules, carriers, host cells, or conjugates of the present invention, along with the additional pharmaceutically active agents, can be provided as separate components or as mixed components in the pharmaceutical composition. Therefore, the isolated nucleic acid molecules, carriers, host cells, or conjugates of the nanobodies of the present invention, along with the additional pharmaceutically active agents, can be administered simultaneously, separately, or sequentially.

[0086] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0087] The pharmaceutical compositions of the present invention may include, in a “therapeutic effective amount” or “preventative effective amount”, nanobodies, isolated nucleic acid molecules, carriers, host cells, or conjugates described herein. A “preventative effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Therapeutic effective amounts may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0088] In another specific embodiment of the present invention, the use of the nanobody, isolated nucleic acid molecule, carrier, host cell, or conjugate described herein is provided in any of the following:

[0089] (c1) Use in the preparation of medicaments for the prevention and / or treatment of diseases related to TNF-α;

[0090] (c2) Application in the preparation of diagnostic reagents for detecting the presence or level of TNF-α in a sample or for diagnosing whether a subject has a disease related to TNF-α.

[0091] In some embodiments, the TNF-α-related disease is characterized by elevated TNF-α expression and / or excessive TNF-α activity.

[0092] In some implementations, the TNF-α-related disease is an inflammatory disease or an autoimmune disease.

[0093] In some embodiments, the TNF-α-related diseases are 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, and psoriasis.

[0094] In some implementations, the subject is a mammal, such as a human.

[0095] In some embodiments, the nanobody, isolated nucleic acid molecule, carrier, host cell, or pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents (e.g., anti-inflammatory drugs or immunosuppressants).

[0096] The nanobodies, isolated nucleic acid molecules, carriers, host cells, or conjugates of the present invention, or the pharmaceutical compositions of the present invention, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use.

[0097] A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0098] The nanobodies, isolated nucleic acid molecules, carriers, host cells, or conjugates of the present invention, or the pharmaceutical compositions of the present invention, may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intrabladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the nanobodies, isolated nucleic acid molecules, carriers, host cells, conjugates, or pharmaceutical compositions of the present invention are administered by intravenous injection or bolus.

[0099] In some embodiments, the present invention provides a method for detecting the presence or amount of TNF-α in a sample, comprising using the nanobody or antigen-binding fragment or conjugate of the present invention.

[0100] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0101] In some embodiments, the conjugate used in the method comprises the nanobody of the present invention and a detectable label.

[0102] In some embodiments, the nanobodies used in the method are labeled with detectable tags.

[0103] In some embodiments, the nanobodies or antigen-binding fragments thereof used in the method do not carry a detectable label. Therefore, the method may also include using other reagents (such as a second antibody) carrying a detectable label to detect the nanobodies or antigen-binding fragments thereof of the present invention.

[0104] In some implementations, the method includes the following steps:

[0105] (1) Contact the sample with the nanobody or its antigen-binding fragment or conjugate of the present invention;

[0106] (2) Detect the formation of a complex between the nanobody or its antigen-binding fragment or conjugate and TNF-α or detect the amount of the complex.

[0107] The formation of the complex indicates the presence of TNF-α or cells expressing TNF-α.

[0108] The method can be used for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0109] In some embodiments, the method is used to diagnose whether a subject has a TNF-α-related disease. In such embodiments, the method may further include the step of comparing the amount of TNF-α in a sample from the subject with a reference value. The reference value may be the level of TNF-α in a sample from a subject known not to have a TNF-α-related disease (e.g., a healthy control) (also referred to as a "negative reference value"). For example, if the amount of TNF-α in the sample from the subject is elevated relative to the negative reference value, it indicates that the subject has a TNF-α-related disease.

[0110] In some implementations, the TNF-α-related disease is an inflammatory disease or an autoimmune disease.

[0111] In some embodiments, the TNF-α-related diseases are 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, and psoriasis.

[0112] In some embodiments, the sample may be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (e.g., surgically removed tumor tissue, biopsy sections, or fine-needle aspiration tissue), histological preparations, etc.

[0113] In some embodiments, the TNF-α is human TNF-α.

[0114] In some embodiments, the conjugates used to prepare the detection reagents comprise the nanobodies of the present invention or antigen-binding fragments thereof, and a detectable label.

[0115] In some embodiments, the nanobodies or antigen-binding fragments thereof used to prepare the detection reagents are labeled with detectable tags.

[0116] In some embodiments, the nanobodies or antigen-binding fragments thereof used to prepare the detection reagents do not carry a detectable label. In such embodiments, the detection reagents may further comprise other reagents (such as a second antibody) capable of detecting the nanobodies or antigen-binding fragments thereof of the present invention.

[0117] As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting 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 camel or shark antibody). Typically, a nanobody consists of four framework regions and three complementarity-determining regions, having a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity. Nanobodies are also called single-domain antibodies (sdAbs), and the two terms are used interchangeably.

[0118] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. The nanobody contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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, those skilled in the art will readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefrance et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0119] As used herein, the term “frame region” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0120] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0121] The sequence information involved in this invention is described in Table 1.

[0122] Table 1 Sequence Information

[0123]

[0124] The sources of the reagents used in the following examples are shown in Table 2.

[0125] Table 2. Sources of Reagents

[0126]

[0127] Example 1

[0128] This embodiment provides a method for screening high-affinity anti-TNF-α nanobodies, including the following steps:

[0129] (a) Four rounds of phage screening

[0130] (1) Plate coating: 5 μg / mL of TNF-α antigen was coated onto an ELISA plate, and the blank group was coated with PBS buffer and incubated overnight at 4°C.

[0131] (2) Blocking the plate: Discard the coating solution and block the ELISA plate at room temperature for 2 hours using 1% BSA-PBS (changing the blocking solution each time can greatly reduce the enrichment of phages against a specific blocking solution);

[0132] (3) Blocking the library: 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;

[0133] (4) Binding: Discard the blocking solution, shake the microplate dry, add the phage library, shake at 220 rpm and incubate at 37°C for 2 h;

[0134] (5) Washing: Pour out the unbound phages from the ELISA plate, wash 5 times with PBST, and wash 3 times with PBS;

[0135] (6) Elution: Add 200 μL HCl (0.1 M, pH=2.2) to the enzyme-labeled wells, shake at 220 rpm and incubate at 37°C for 10 min to elute the bound phages;

[0136] (7) Neutralization: Neutralize using 1M Tris-HCl (pH=8.0);

[0137] (8) Amplification: 4 mL of TG1 Escherichia coli cells with OD 600 = 0.5 were infected with 400 μL of the neutralized solution and then cultured at 37°C with shaking for 1 h; then M13KO7 helper phage was added and cultured at 37°C with shaking for 1 h; finally, the cells were transferred to 50 mL of 2×YT / Carb / Kan medium and cultured overnight at 30°C.

[0138] (9) The titer determination was performed simultaneously with (8) the amplification step: the bacterial culture after infecting TG1 cells was serially diluted 10× with 2×YT (8 concentrations). 10 μL of each gradient was dropped into 2×YT / Carb solid medium and cultured overnight at 37°C. The bacterial culture medium in PBS wells was used as a negative control.

[0139] (10) Phage purification: The amplified phages were purified using the PEG / NaCl precipitation method. The phage concentration was determined by measuring the OD 268nm absorbance using a Nanodrop 2000c, and then the next round of screening was performed. The key conditions for each round of screening were changed according to this step, 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.

[0140] Table 3. Conditions and enrichment rates for the four rounds of screening

[0141]

[0142] (II) ELISA Validation of Screening Results

[0143] The purified phage libraries from each round were used to verify the screening effect via ELISA. The specific process is as follows:

[0144] (1) Coating: Coat the ELISA plate with 1 μg / mL TNF-α antigen and incubate overnight at 4℃; (2) Blocking: Discard the coating solution and block the ELISA plate with 1% BSA-PBS at room temperature for 2 h; (3) Diluting the phage library: Dilute each round of phage library to 1.0 OD and perform a 3× serial dilution with PBS (8 dilutions); (4) Binding: Discard the blocking solution, shake the ELISA plate dry, add the serially diluted phage to the ELISA plate, and incubate at 37℃ for 30 min; (5) Washing: Wash 5 times with PBST; (6) Adding secondary antibody: Add anti-M13 Bacteriophage-HRP secondary antibody (1:5000 dilution) to the ELISA plate and incubate at 37℃ for 1 h; (7) Washing again: Wash 5 times with PBST; (8) Color development: Add 100 μL TMB color development solution, place at 37℃, and develop for 15 minutes. min; (9) Termination: Add 50 μL of 2M sulfuric acid to terminate the reaction; (10) Measurement: Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0145] The measured OD values ​​are combined with phage OD values ​​to plot a fitting curve. Finally, the point where the strongest signal is halfway down is found, and the corresponding phage OD value is the EC value. 50 EC 50 Defined as the concentration that produces 50% efficacy or binding. The four rounds of screening effect curves and EC50 results are shown below. Figure 1 As shown, the first round of EC 50 The value is 1.2151, second round EC 50 The value is 0.2311, the third round of EC 50 The value is 0.0047, fourth round EC 50 The value is 0.0033.

[0146] (III) ELISA screening and sequencing of positive clones

[0147] After the fourth round of screening, 480 single colonies were randomly selected and inoculated into 96-well plates. Each well contained 400 μL of 2×YT / Carb and 4 μL of M13K07 helper phage. The plates were incubated overnight at 37°C and centrifuged at 3000 rpm to obtain the phage supernatant.

[0148] The specific process of monoclonal ELISA screening is as follows:

[0149] (1) Coating: Coat the ELISA plate with 1 μg / mL TNF-α antigen, and coat the blank group with PBS buffer. Incubate overnight at 4°C. (2) Blocking: Discard the coating solution and block the ELISA plate with 1% BSA-PBS at room temperature for 2 h. (3) Diluting the phage supernatant: Mix the phage supernatant with ELISA buffer at a 1:1 ratio. (4) Binding: Discard the blocking solution, shake the ELISA plate dry, add the diluted phage supernatant to the ELISA plate, and incubate at 37°C for 1 h. (5) Washing: Wash 5 times with PBST. (6) Adding secondary antibody: Add anti-M13 Bacteriophage-HRP secondary antibody (1:5000 dilution) to the ELISA plate and incubate at 37°C for 1 h. (7) Washing again: Wash 5 times with PBST. (8) Color development: Add 100 μL TMB color development solution, place at 37°C, and develop for 15 minutes. min; (9) Termination: Add 50 μL of 2M sulfuric acid to terminate the reaction; (10) Measurement: Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0150] Clones with an OD value ≥2 for binding to TNF-α and an OD value ≥2 for binding to BSA were defined as positive clones. Clones with an OD value >0.5 for binding to TNF-α and an OD value <0.25 for binding to BSA, and a ratio ≥5, were defined as high-affinity positive clones. The statistical results of the positive clone screening rate by ELISA are shown in Table 4.

[0151] Table 4. Statistics on the positive clone rate of ELISA screening

[0152]

[0153] Positive clones were selected for sequencing (sequencing primer sequences are shown in SEQ ID NO. 33). Through sequence alignment and cluster analysis, three high-affinity candidate sequences (A10, C2, and C12) were selected for further 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, and the nucleotide sequence of C12 is shown in SEQ ID NO. 6. Detailed antibody sequence information is shown in Table 1.

[0154] Example 2

[0155] This embodiment provides the expression and purification of nanobodies.

[0156] (I) Construction of expression plasmids

[0157] The pET26b(+) plasmid was linearized by enzyme digestion; then, using the positive clone plasmid as a template, the gene fragment containing the nanobody was amplified by PCR, and homologous arms of the pET26b(+) plasmid were introduced at both ends of the antibody gene; finally, the linearized plasmid and the nanobody gene fragment were ligated into a complete plasmid through homologous recombination. The specific construction steps are as follows.

[0158] (1) Linearized plasmids

[0159] The pET26b(+) plasmid was amplified in Top10 cells, extracted using a mini-prep kit, and its concentration was determined using Nanodrop2000c. Nco I and Xho The pET26(+) plasmid was digested with restriction endonuclease I and incubated at 37°C for 2 hours. The digestion system is shown in Table 5.

[0160] Table 5 Enzyme digestion system

[0161]

[0162] DNA agarose gel electrophoresis was performed, and the results are as follows: Figure 2 As shown, the target product was confirmed to be approximately 5000 bp. Finally, linearized plasmids were obtained through gel recovery and their concentration was determined using a Nanodrop 2000c.

[0163] (2) PCR amplification of the target antibody gene

[0164] The PCR primers for the three candidate sequences are shown in Table 1, the PCR reaction system is shown in Table 6, and the PCR reaction procedure is shown in Table 7.

[0165] Table 6 PCR Reaction System

[0166]

[0167] Table 7 PCR reaction procedure

[0168]

[0169] DNA agarose gel electrophoresis was performed, and the results are as follows: Figure 3 As shown, the target product was confirmed to be approximately 400 bp. Finally, PCR product recovery was performed to obtain the target product, and its concentration was determined using a Nanodrop 2000c.

[0170] (3) Homologous recombination

[0171] The linearized pET26b(+) plasmid and the target antibody gene amplified by PCR were subjected to homologous recombination. The reaction system is shown in Table 8.

[0172] Table 8 Homologous recombination reaction system

[0173]

[0174] The ligation mixture was incubated at 50°C for 15 minutes for chemical transformation.

[0175] (4) Chemical transformation:

[0176] Add 5 μL of the homologous recombination product to 50 μL of chemocompetent BL21 cells and incubate on ice for 30 minutes. After a heat shock at 42°C for 30 seconds, immediately return to ice and incubate for 2 minutes. Add 950 μL of SOC medium and incubate at 37°C and 220 rpm for 1 hour. Spread 100 μL of the culture medium onto LB / Kan solid medium and incubate overnight at 37°C. The next day, clones grew on the antibiotic-containing medium, confirming successful plasmid homologous recombination.

[0177] (II) Expression and purification of nanobodies

[0178] The target nanobody was induced to express in the BL21 host and the antibody was purified using a Ni-NTA column. The specific steps are as follows.

[0179] (1) Antibody expression

[0180] BL21 cells containing the target antibody were seeded into 5 mL of LB / Kan medium and cultured overnight at 37°C and 200 rpm. The next day, 5 mL of cell culture medium was transferred to 500 mL of LB / Kan medium and cultured at 37°C and 200 rpm until OD600 = 0.6. IPTG was added to a final concentration of 1 mM, and the cells were cultured at 25°C and 200 rpm for approximately 16 hours to induce antibody expression. On the third day, the induced culture medium was centrifuged at 5000 rpm, and the cell pellet was harvested.

[0181] (2) Antibody purification

[0182] The cell pellet was resuspended in 100 mL of lysis buffer (0.5 M sucrose, 0.2 M Tris, pH 8, 0.5 mM EDTA), and 200 mL of deionized water was added. A magnetic stir bar was added, and the mixture was stirred at 4°C for 45 minutes to release the nanobodies. The mixture was then centrifuged at 12,000 rpm for 20 minutes, and the supernatant was collected. The supernatant was added to a 3 mL Ni-NTA resin-assembled filter column, and the filtrate was discarded. The column was washed with approximately 100 mL of high-salt washing buffer (20 mM HEPES, 500 mM sodium chloride, 20 mM imidazole, pH 7.5), followed by approximately 100 mL of washing buffer (20 mM HEPES, 100 mM sodium chloride, 20 mM imidazole, pH 7.5). Finally, the nanobodies bound to the nickel column were eluted with 30 mL of elution buffer (20 mM HEPES, 100 mM sodium chloride, 400 mM imidazole, pH 7.5).

[0183] After dialyzing the eluent in PBS, 12% SDS-PAGE protein gel electrophoresis was performed to confirm that the nanobody was approximately 15 kDa, and the nanobody concentration was determined using Nanodrop 2000c. Finally, 50% glycerol was added, and the nanobody was aliquoted and stored at -20°C.

[0184] Taking A10 as an example, the entire process of nanobody expression and purification, including protein gel electrophoresis, is as follows: Figure 4 As shown. The purified proteins from the A10, C2, and C12 nanobodies were subjected to gel electrophoresis as follows. Figure 5 As shown.

[0185] Example 3

[0186] This embodiment provides nanobody affinity ELISA verification, and the specific steps are as follows:

[0187] (1) Coating: Coat the ELISA plate with 1 μg / mL TNF-α antigen and incubate overnight at 4°C; (2) Blocking: Discard the coating solution and block the ELISA plate with 1% BSA-PBS at room temperature for 2 h; (3) Diluting the antibody: Dilute the A10, C2, and C12 nanobodies to 2 μM and perform a 3× serial dilution with PBS (8 concentrations); (4) Binding: Discard the blocking solution, shake the ELISA plate dry, add the diluted nanobodies to the ELISA plate, and incubate at 37°C for 1 h; (5) Washing: Wash 5 times with PBST; (6) Adding secondary antibody: Add anti-His-HRP secondary antibody (1:5000 dilution) to the ELISA plate and incubate at 37°C for 1 h; (7) Washing again: Wash 5 times with PBST; (8) Color development: Add 100 μL of TMB color development solution, place at 37°C, and develop for 15 minutes. min; (9) Termination: Add 50 μL of 2M sulfuric acid to terminate the reaction; (10) Measurement: Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0188] Record the obtained OD values, plot the fitting curve in conjunction with the antibody concentration, and calculate the EC50 of each antibody. 50 Values. Affinity results for the three nanobodies are as follows: Figure 6 As shown, EC of A10 50 The value is 67.56 nM, EC of C2 50 The value is 86.58 nM, EC of C12 50 The value is 3.25 nM.

[0189] Example 4

[0190] This embodiment provides nanobody codon optimization and verification.

[0191] Affinity testing revealed that C12 possesses high affinity, but its yield during expression production was low, only 8–9 mg / L. Sequence optimization was performed on the nucleotide sequence of C12. The optimized nanobody nucleotide sequence is shown in SEQ ID NO. 7.

[0192] The optimized gene sequence was obtained through chemical synthesis, cloned into the pET26b(+) vector, transformed into BL21 expression bacteria, and after induction and purification, the final yield of the C12 nanobody increased from 8.8 mg / L to 30 mg / L, an increase of 3.4 times. The expression yield before and after codon optimization is compared as follows: Figure 7 As shown, codon optimization has a significant effect on increasing the yield of nanobodies.

[0193] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention 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 substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anti-TNF-a Nanobody, characterized in that, The VHH chain of the Nanobody comprises complementarity determining regions and framework regions; The complementarity determining regions comprise: (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.

2. The Nanobody of claim 1, wherein The framework regions comprise: (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.

3. The nanobody of claim 1, wherein The sequence of the Nanobody is as shown in SEQ ID NO.

5.

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

5. Vector, characterized in that, comprising the nucleic acid molecule of claim 4.

6. A host cell characterized in that, comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A method of producing a Nanobody according to any one of claims 1-3, characterized in that, comprising: culturing the host cell of claim 6 under conditions that allow expression of the protein, and recovering the Nanobody from the cultured host cell culture.

8. A pharmaceutical composition, characterized by, comprising the Nanobody of any one of claims 1 to 3 or the nucleic acid molecule of claim 4 or the vector of claim 5 or the host cell of claim 6.

9. The Nanobody of any one of claims 1 to 3 or the nucleic acid molecule of claim 4 or the vector of claim 5 or the host cell of claim 6 or the pharmaceutical composition of claim 8 for use in: the manufacture of a medicament for the prevention and / or treatment of a TNF-α related disease; the TNF-α related disease is inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, ankylosing spondylitis, hidradenitis suppurativa, psoriatic arthritis, polyarticular juvenile idiopathic arthritis, uveitis.

10. The Nanobody of any one of claims 1 to 3 or the nucleic acid molecule of claim 4 or the vector of claim 5 or the host cell of claim 6 for use in: the manufacture of a detection reagent for detecting the presence or level of TNF-α in a sample.

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