TNF (Tumor Necrosis Factor) alpha targeting humanized single-domain antibody and application thereof

Abstract

The invention discloses a TNF (Tumor Necrosis Factor) alpha targeting humanized single-domain antibody and application thereof, and belongs to the field of biological medicines. The amino acid sequence of the single-domain antibody comprises three complementary determining regions CDR1-CDR3 and four skeleton regions FR1-FR4. The invention also provides a recombinant protein of the single-domain antibody, a nucleic acid molecule for coding the single-domain antibody or the recombinant protein, a carrier containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the carrier, and an application of the single-domain antibody or the recombinant protein in preparation of drugs for treating diseases by taking TNF alpha as a target spot.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a humanized single-domain antibody targeting TNFα and applications thereof. Background Art

[0002] Tumor necrosis factor α (TNFα) is an important cytokine produced by activated macrophages, monocytes and T lymphocytes. It plays a key role in normal inflammatory and immune responses. By binding to its receptors (TNF-r1 and TNF-r2), it activates signaling pathways such as MAPK, NF-κB and JNK, thereby regulating cell survival, proliferation, apoptosis, and inflammatory and necrotic processes. However, abnormally high levels of TNFα expression are closely related to a variety of autoimmune and inflammatory diseases, such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, sepsis, inflammatory bowel disease, multiple sclerosis, psoriasis, psoriatic arthritis and autoimmune uveitis. Therefore, TNFα has become a key cytokine for clinical detection of these diseases and an important drug target for the treatment of these diseases.

[0003] At present, there are many TNFα antagonists used in the clinic to treat related diseases, such as etanercept, infliximab, adalimumab, golimumab and certolizumab. However, these traditional TNFα antagonists have limitations such as large molecular weight and weak penetration. Therefore, the development of new antibody forms to improve these limitations has become a hot topic in current research.

[0004] As a new type of antibody, single-domain antibodies (VHH or nanobodies) have attracted much attention due to their small molecular weight, strong penetration, high specificity and high affinity. These antibodies are easy to express and modify, and have high stability, showing broad application prospects in the fields of disease treatment, diagnosis and material detection. In recent years, single-domain antibody drugs have developed rapidly, and many single-domain antibody drugs have been successfully approved for marketing. For example, Caplacizumab, the world's first single-domain antibody drug, has been approved by the European Union for the treatment of thrombotic thrombocytopenic purpura. Alphamab's PD-L1 single-domain antibody Fc fusion protein KN-035 has also been approved for marketing in China, becoming the world's first subcutaneously injected single-domain antibody drug. Ozoralizumab developed by Ablynx, as a bispecific single-domain antibody, has been approved in Japan for the treatment of rheumatoid arthritis, proving the huge potential of single-domain antibodies in drug development.

[0005] Based on the above background, the present invention is committed to developing a single-domain antibody targeting TNFα, providing a new option for the development of TNFα blocking drugs and the diagnosis and treatment of related diseases. Summary of the invention

[0006] The purpose of the present invention is to provide a humanized single-domain antibody targeting TNFα and its application, so as to solve the problems existing in the above-mentioned prior art. By taking humanized TNF-α as the target, bio-panning and screening the humanized single-domain antibody phage display library, the gene sequence of the single-domain antibody targeting TNFα is obtained; then these gene sequences are used to construct a prokaryotic expression system to prepare a recombinant protein of the single-domain antibody targeting TNFα, and experiments have proved that the recombinant protein of the single-domain antibody targeting TNFα can be used to treat rheumatoid arthritis.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The first aspect of the present invention provides a humanized single domain antibody targeting human TNF-α, wherein the amino acid sequence of the single domain antibody includes three complementary determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are any one of 1) to 16):

[0009] 1) CDR1: VTFNAKDMS, CDR2: TITNPS, CDR3: SSLDKSSSRCAREADVPY;

[0010] 2) CDR1: VRLIYEVMS, CDR2: TILNND, CDR3: SSDMESEYELLPQTLKS;

[0011] 3) CDR1: VRVITESMA, CDR2: GILITN, CDR3: TMYYFKRREVETTMRF;

[0012] 4) CDR1: VMFSYEDMA, CDR2: AIRNHD, CDR3: TCSDKPKEDAIRY;

[0013] 5) CDR1: DTITNETMA, CDR2: SIISNS, CDR3: SWLLECDIHTWVAKDLEY;

[0014] 6) CDR1: DTVNYKAMS, CDR2: SISNPD, CDR3: TDCMARASRPPALHY;

[0015] 7) CDR1: DMVSHEDMG, CDR2: TINSQD, CDR3: TVSSDQTAYEPVAF;

[0016] 8) CDR1: YSFTSEYMG, CDR2: TINTRN, CDR3: AGRVAEKNMGY;

[0017] 9) CDR1: FIISNEVMA, CDR2: AIEMPN, CDR3: GVASDNVDSKQISS;

[0018] 10) CDR1: DSVSAEVMT, CDR2: AILNTN, CDR3: GSMQSIVPRGQKAVYY;

[0019] 11) CDR1: FTFNDDSMG, CDR2: GIPEHN, CDR3: SRKPAMTQPMKF;

[0020] 12) CDR1: DSLSSENMS, CDR2: GIEEPD, CDR3: SVGEDLEGDTTFGY;

[0021] 13) CDR1: DTVINEAMG, CDR2: SIVVEG, CDR3: ACVAMGQESFSY;

[0022] 14)CDR1: YIFSYENMS, CDR2: AIGDHD, CDR3: TYAWLMSVNYLTVAHEMDY;

[0023] 15)CDR1: FMVSDEDMA, CDR2: GINTTN, CDR3: RDYGSRVDQHLES;

[0024] 16) CDR1: YRLTAENMG, CDR2: AIYRQD, CDR3: SRFLSIFVGSYPKLEF.

[0025] Preferably, the above-mentioned single domain antibody can specifically bind to TNFα with high affinity, especially the single domain antibody having the complementary determining regions shown in items 1), 3), 5), 6), 7), 8), 9), 10) and 15) above.

[0026] Furthermore, single domain antibodies having the complementarity determining regions shown in the above items 1), 7), 9) and 10) are preferred.

[0027] Furthermore, the above-mentioned single domain antibody also includes four framework regions FR1, FR2, FR3 and FR4, and the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 are respectively as follows:

[0028] FR1: QVQLLESGGGLVQPGGSLRLSCAASG,

[0029] FR2: WVRQAPGKGLEWVS,

[0030] FR3:GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA,

[0031] FR4:WGQGTLVTVSS.

[0032] Furthermore, the structure of the above single domain antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0033] The second aspect of the present invention provides a recombinant protein, which comprises the above-mentioned single domain antibody.

[0034] Furthermore, the above-mentioned single-domain antibodies can be fused with other polypeptides, proteins or functional substances to achieve diversified application goals. For example, fusion with markers such as protein tags (such as 6xHis, albumin signal peptide, GFP, RFP, etc.), enzymes (such as horseradish peroxidase, alkaline phosphatase, etc.) or radioactive isotopes can significantly improve the convenience and sensitivity of TNFα target detection or facilitate protein purification; for example, through tandem fusion technology, multiple identical single-domain antibody fragments are connected into a bivalent or multivalent antibody form, which can not only effectively improve the binding affinity of antibodies to TNFα, but also prolong their half-life in the body, thereby enhancing the therapeutic effect and reducing the frequency of administration; for example, single-domain antibodies can be connected to small molecule drugs with biological activity through specific linkers (such as cleavable linkers, non-cleavable linkers, etc.) to form antibody-coupled drugs to more efficiently exert their efficacy.

[0035] The third aspect of the present invention also provides a nucleic acid molecule, which encodes the above-mentioned single domain antibody or recombinant protein.

[0036] The fourth aspect of the present invention provides a vector, which contains the above-mentioned nucleic acid molecule. The recombinant vector can be a plasmid or a virus, etc. These vectors can be replicated in host cells, thereby achieving efficient expression of nucleic acid molecules. In particular, the plasmid can be a prokaryotic expression vector or a eukaryotic expression vector, and the virus can be a lentivirus, an adenovirus, an adeno-associated virus, a phage or a retrovirus or a combination thereof to meet the needs of different host cells.

[0037] The fifth aspect of the present invention provides a host cell, wherein the host cell comprises the above-mentioned nucleic acid molecule or vector. The host cell can be a prokaryotic cell, a eukaryotic cell, a bacteriophage or a combination thereof, and the specific selection depends on the desired expression system and production scale.

[0038] The sixth aspect of the present invention provides a method for preparing the above-mentioned single domain antibody or recombinant protein, which comprises the steps of culturing the above-mentioned host cell and isolating and purifying the single domain antibody or recombinant protein from the culture.

[0039] The seventh aspect of the present invention provides a pharmaceutical composition, comprising the single domain antibody or the nucleic acid molecule or the vector or the host cell.

[0040] The eighth aspect of the present invention provides the use of the above-mentioned single domain antibodies and recombinant proteins in the preparation of drugs targeting TNFα for treating diseases, wherein the diseases include autoimmune and inflammatory diseases caused by abnormal TNFα, such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, sepsis, inflammatory bowel disease, multiple sclerosis, psoriasis, psoriatic arthritis, neovascular eye disease, autoimmune uveitis and tumors.

[0041] The ninth aspect of the present invention provides a kit for detecting TNFα, which comprises the above-mentioned single domain antibody or recombinant protein. These antibodies combined with other reagents can be used to detect the level of TNFα in vitro or in vivo, providing a powerful tool for disease diagnosis and evaluation of treatment effects.

[0042] The present invention discloses the following technical effects:

[0043] The present invention uses humanized TNFα as the target, bio-panning and screening of humanized single-domain antibody phage display libraries to obtain gene sequences of single-domain antibodies targeting TNFα. The present invention uses these gene sequences to construct a prokaryotic expression system to prepare recombinant proteins of single-domain antibodies targeting TNFα; these antibodies or recombinant proteins have unique CDR sequences and can specifically bind to TNFα, providing new options for the development of TNFα blocking drugs and the diagnosis and treatment of related diseases. Finally, at multiple levels such as in vitro, cells, and rheumatoid arthritis model mice, it is verified that single-domain antibodies targeting TNFα can treat rheumatoid arthritis. The present invention provides new candidate drugs for rheumatoid arthritis and provides new means for the diagnosis and detection of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 The degree of phage-specific enrichment was analyzed by polyclonal phage ELISA (*P<0.05, ##P<0.01);

[0046] Figure 2 The amino acid sequence alignment results of single-domain antibodies targeting TNFα (sdTNFs);

[0047] Figure 3 The results of prokaryotic expression, purification and identification of sdTNFs; (A) SDS-PAGE Coomassie Brilliant Blue staining results, (B) Western blot analysis results, (C) sdTNFs HiPrep 26 / 10Desalting representative images, (D) ImageJ software analysis of sdTNFs protein purity obtained in Figure A;

[0048] Figure 4 The affinity of sdTNFs binding to hTNFα was analyzed by ELISA;

[0049] Figure 5 sdTNFs inhibited the killing effect of hTNFα on L929 cells; (A) statistical graph, (B) microscopic observation results; Control, negative control; Model, cell model group;

[0050] Figure 6 EC for sdTNFs 50 Measurement results;

[0051] Figure 7 The results of the temperature stability of sdTNFs determined by MTT method;

[0052] Figure 8 Hoechst / PI double staining inverted fluorescence microscope was used to observe the inhibition of hTNF-α-induced apoptosis of L929 cells by sdTNF07; (A) fluorescence image, (B) statistical graph;

[0053] Fig. 9 Hoechst / PI double staining inverted fluorescence microscope was used to observe the inhibition of hTNF-α-induced apoptosis of L929 cells by sdTNF08; (A) fluorescence image, (B) data statistics graph;

[0054] Fig.10 Hoechst / PI double staining inverted fluorescence microscopy was used to observe the inhibition of hTNF-α-induced apoptosis of L929 cells by sdTNF09; (A) fluorescence image, (B) data statistics graph;

[0055] Fig.11 Annexin V-FITC / PI flow cytometric analysis of sdTNF07 inhibiting hTNF-α-induced apoptosis in L929 cells; (A) flow cytometric detection diagram, (B) data statistics diagram;

[0056] Fig.12 Hoechst / PI double staining inverted fluorescence microscope was used to observe the inhibition of hTNF-α-induced apoptosis of L929 cells by sdTNF08; (A) Flow cytometry detection diagram, (B) data statistics diagram;

[0057] Fig.13 Hoechst / PI double staining inverted fluorescence microscope was used to observe the inhibition of hTNF-α-induced apoptosis of L929 cells by sdTNF09; (A) flow cytometry detection diagram, (B) data statistics diagram;

[0058] Fig.14 Representative images showing that sdTNFs improves arthritis symptoms and immune function in the hind paws of CIA mice;

[0059] Fig.15 Effects of sdTNFs on arthritis symptoms and immune function of hind paws of CIA mice; (A) Arthritis score index-time curve of hind paws of mice, in which the table shows the statistical results of the corresponding line graph; (B) Spleen index of mice in each group (*P<0.05, **P<0.01, ****P<0.0001); (C) Body weight-time curve of mice in each group;

[0060] Fig.16 Effects of sdTNFs on arthritis tissue pathology in CIA mice; (A) H&E histological images of the ankle joints of the hind limbs of mice on day 48 after treatment; (B) Histological scores of synovial inflammation; (C) Pannus scores;

[0061] Fig.17 ELISA was used to detect and analyze the effects of sdTNFs on serum cytokines in CIA mice. DETAILED DESCRIPTION

[0062] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0063] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0064] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0065] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0066] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0067] Example 1 Biopanning of single domain antibodies targeting TNF-α (sdTNFs)

[0068] 1. First round of screening

[0069] (1) The target antigen TNF-α was coated on the ELISA plate at a final concentration of 10 μg / mL, 100 μL per well, and PBS buffer was set as a blank control.

[0070] (2) After incubation at 4°C overnight (8-12 h), wash the plate 5 times with 0.1% PBST and once with PBS.

[0071] (3) Block with 3% PBSM blocking buffer at 37°C for 2 h.

[0072] (4) Add the humanized single-domain antibody phage library HuSdL-2 (HuSdL-2 phage display humans single-domain antibody library, titer 10^12 pfu / mL) and incubate at 37°C for 2 h.

[0073] (5) After washing, add elution buffer for elution and immediately add neutralization buffer.

[0074] (6) The obtained phages are used for a new round of phage library amplification and titer determination.

[0075] 2. Phage library titer determination

[0076] (1) Gradient dilution of phage solution and mixed infection with JM101 Escherichia coli.

[0077] (2) Apply an appropriate amount of the mixture onto a 2×YT agar plate containing ampicillin and culture at 37°C overnight.

[0078] (3) Count the colonies on the plate and calculate the titer.

[0079] 3. Phage library amplification

[0080] (1) Infect JM101 Escherichia coli and add helper phage M13KO7.

[0081] (2) After expansion, centrifuge and collect the supernatant.

[0082] (3) Purify the phages by PEG precipitation to obtain the amplified phage library.

[0083] 4. Second, third and fourth rounds of screening

[0084] (1) Repeat the steps of the first round of screening, but reduce the antigen coating concentration in each round (5 μg / mL, 2.5 μg / mL and 1 μg / mL).

[0085] (2) Increase the dilution multiple when coating the plates in successive rounds to enrich for high-affinity phage clones.

[0086] 5. Results

[0087] Through four rounds of bio-panning, it was observed that the Output / Input ratio of phage, i.e., the recovery rate, increased with the increase in the number of screening rounds, indicating that the positive phages were effectively enriched (Table 1). This example successfully screened out high-affinity phage clones targeting TNFα from the humanized single-domain antibody phage library.

[0088] Table 1 Enrichment effect of biopanning phage targeting hTNFα single domain antibody

[0089]

[0090] Note: pfu: colony forming unit.

[0091] Example 2 Polyclonal ELISA to detect the specific enrichment of phage library after 4 rounds of screening

[0092] 1. Coating

[0093] (1) Dilute TNFα with coating buffer to a final concentration of 5 μg / mL, add 100 μL to each well for coating, and incubate at 4°C overnight.

[0094] (2) Use PBS with the same volume as the original TNFα solution as a control, and add coating buffer to 100 μL / well. Set up 3 replicate wells for each group.

[0095] 2. Closed

[0096] The plate was washed twice with 0.1% PBST, and 300 μL of 2% BSA was added to each well for blocking at room temperature for 2 hours.

[0097] 3. Incubate with primary antibody

[0098] (1) Wash the plate twice with 0.1% PBST, take 50 μL of the amplified phage solution after each round of screening and mix it with 50 μL of 2% BSA, and react at room temperature for 15 minutes.

[0099] (2) Add the mixed solution to the corresponding wells and incubate at room temperature for 2 hours.

[0100] 4. Incubate with secondary antibody

[0101] (1) Wash the plate 5 times with 0.1% PBST, add 100 μL of HRP-M13 antibody diluted 5000-fold with 2% BSA to each well, and incubate at room temperature for 1 hour.

[0102] (2) Wash the plate again 4 times with 0.1% PBST.

[0103] 5. Color reaction

[0104] Add 100 μl of TMB colorimetric substrate to each well and incubate at room temperature for 15 minutes (protected from light).

[0105] 6. Termination of the reaction

[0106] (1) Add 50 μL 1M H2SO4 to each well to terminate the color development reaction.

[0107] (2) Use an ELISA reader to measure the OD of each well. 450nm The absorbance value at .

[0108] The specific enrichment of the phage library for the target antigen TNFα after four rounds of bio-panning was detected by polyclonal ELISA. Figure 1As shown, the results show that with the increase of the screening rounds, the absorbance value of the phage binding to TNFα is on the rise. The phage after the third round of screening showed the highest binding ability, and although the absorbance value decreased slightly after the fourth round of screening, it still remained at a high level. It is speculated that the amount of positive phages has reached a plateau in the fourth round of screening. This shows that through four rounds of bio-panning, the specific phages for TNFα in the phage library have been effectively enriched. According to this result, the present invention selects the phage library after the fourth round of screening for subsequent plating operations, and picks monoclonal clones therefrom for DNA sequencing analysis.

[0109] Example 3 PCR-based DNA sequencing analysis to obtain the effectiveness of single-domain antibodies targeting TNFα (sdTNFs)

[0110] 1. Preparation of monoclonal phage

[0111] (1) The phages after the fourth round of screening were used to infect JM101 Escherichia coli and plated at an appropriate density on 2×YT medium (2×YT) plates containing 100 μg / mL ampicillin and 1% (W / V) glucose (about 100-300 clones / plate).

[0112] (2) Culture in a 37°C incubator overnight (8-12 hours).

[0113] (3) Use a sterile toothpick to pick up a single colony and transfer it to a 96-well cell culture plate containing 200 μL of 2×YT-AG medium.

[0114] (4) Incubate the culture medium at 37°C and 250 rpm with shaking until the medium becomes turbid, indicating that the phage has been amplified.

[0115] 2. Colony PCR amplification of phage-specific DNA fragments

[0116] (1) Using 2×PCR Master Mix reagent and specific forward and reverse primers (forward primer L1: 5′-TGGAATTGTGAGCGGATAACAATT-3′; and reverse primer S6: 5′-GTAAATGAATTTTCTGTATGAGG-3′), PCR amplification of a monoclonal phage-specific DNA fragment was performed.

[0117] (2) Prepare the PCR reaction system as shown in Table 2.

[0118] Table 2 PCR reaction system

[0119]

[0120] (3) Set the PCR program: pre-denaturation at 98°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 29 cycles; final extension at 72°C for 2 minutes; store at 4°C.

[0121] 3. Agarose gel electrophoresis and DNA sequencing analysis

[0122] (1) Perform 2% agarose gel electrophoresis on the PCR products, observe and take photos to record the results.

[0123] (2) Select PCR products with expected molecular weight (about 700 bp) for DNA sequencing.

[0124] After PCR amplification and agarose gel electrophoresis analysis, about 200 PCR products with expected molecular weight were screened from nearly 1,300 samples. After DNA sequencing and sequence analysis, 16 gene sequences of single-domain antibodies (sdTNF) targeting TNFα with complete open reading frames (ORFs) were obtained. These sequences were translated into corresponding polypeptide sequences and named sdTNF01 to sdTNF16. Amino acid sequence alignment was performed using CLC sequence Viewer 6.0 software to obtain the similarities and differences between these polypeptide sequences. The results are shown in Figure 2. Figure 2 shown.

[0125] Example 4 Preparation and identification of single domain antibodies targeting TNF-α (sdTNFs)

[0126] 1. Construction of sdTNFs genetically engineered bacteria

[0127] (1) Gene design and synthesis: Based on the codon preference of E. coli, 16 sdTNFs sequences (amino acid sequences such as SEQ ID NOs: 1-16, nucleotide sequences such as SEQ ID NOs: 17-32) were designed and synthesized. A 6XHis tag was added to the end of the gene to facilitate subsequent protein purification and identification.

[0128] (2) Cloning and transformation: The synthesized gene was cloned into the NdeI / XhoI restriction endonuclease sites of the pET-28a vector to construct the prokaryotic expression plasmid of sdTNFs. Then, these plasmids were transformed into competent cells E. coli BL21 (DE3).

[0129] (3) Screening of positive clones: Colony PCR and DNA sequencing analysis were used to screen positive clones from the transformed cells, i.e., genetically engineered strains expressing sdTNFs recombinant protein, named E. coli BL21 (DE3) / sdTNFs.

[0130] 2. Preparation of sdTNFs recombinant protein

[0131] (1) Bacterial culture and induction: The sdTNFs genetically engineered bacteria were revived and inoculated with LB medium containing kanamycin (20 μg / mL kanamycin) at a volume ratio of 1:100 and cultured (37°C / 225 rpm) until the OD 600nm =0.6; add 1 mM IPTG at a final concentration for 5 hours to induce the bacteria to express sdTNFs recombinant protein.

[0132] (2) Bacterial cell disruption and protein extraction: The induced bacterial cells were collected and disrupted by ultrasound to release the recombinant protein. Since the sdTNFs recombinant protein exists in the form of inclusion bodies, high-purity inclusion bodies were obtained through a series of centrifugation and washing steps.

[0133] (3) Protein purification and renaturation: 8 M urea was used to dissolve the inclusion bodies, and the supernatant was collected by centrifugation at 4°C / 10000 rpm / 10 min and filtered through a 0.22 μm pore micromembrane. The sdTNFs recombinant protein was purified using nickel ion magnetic beads and renatured by dialysis (the protein dialysis concentration was diluted to 0.1-1 mg / mL, the dialysis bag cutoff was 2 kDa, and the urea concentration in the dialysate 1L TGE Buffer decreased from 6 M, 4 M, 2 M and 1 M). After dialysis, the protein was concentrated using a 3 kDa ultrafiltration tube, purified using a Capto Q column to remove endotoxins, and desalted using a HiPrep 26 / 10 Desalting column. The concentration was determined by Nanon drop2000.

[0134] The amino acid sequences of sdTNF01-sdTNF16 correspond to SEQ ID NOs: 1-16, as follows:

[0135] sdTNF01:

[0136] QVQLLESGGGLVQPGGSLRLSCAASG VTFNAKDMS WVRQAPGKGLEWVS TITNPS GSTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA SSLDKSSSRCAREADVPY WGQGTLVTVSS.

[0137] sdTNF02:

[0138] QVQLLESGGGLVQPGGSLRLSCAASG VRLIYEVMS WVRQAPGKGLEWVS TILNND GSTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA SSDMESEYELLPQTLKSWGQGTLVTVSS。

[0139] sdTNF03:

[0140] QVQLLESGGGLVQPGGSLRLSCAASG VRVITESMA WVRQAPGKGLEWVS GILITN GSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA TMYYFKRREVETTMRF WGQGTLVTVSS。

[0141] sdTNF04:

[0142] QVQLLESGGGLVQPGGSLRLSCAASG VMFSYEDMA WVRQAPGKGLEWVS AIRNHD GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA TCSDKPKEDAIRY WGQGTLVTVSS。

[0143] sdTNF05:

[0144] QVQLLESGGGLVQPGGSLRLSCAASG DTITNETMA WVRQAPGKGLEWVS SIISNS GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA SWLLECDIHTWVAKDLEY WGQGTLVTVSS。

[0145] sdTNF06:

[0146] QVQLLESGGGLVQPGGSLRLSCAASG DTVNYKAMS WVRQAPGKGLEWVS SISNPD GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA TDCMARASRPPALHY WGQGTLVTVSS。

[0147] sdTNF07:

[0148] QVQLLESGGGLVQPGGSLRLSCAASG DMVSHEDMG WVRQAPGKGLEWVS TINSQ GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA TVSSDQTAYEPVAF WGQGTLVTVSS。

[0149] sdTNF08:

[0150] QVQLLESGGGLVQPGGSLRLSCAASG YSFTSEYMG WVRQAPGKGLEWVS TINTRN GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA AGRVAEKNMGY WGQGTLVTVSS。

[0151] sdTNF09:

[0152] QVQLLESGGGLVQPGGSLRLSCAASG FIISNEVMA WVRQAPGKGLEWVS AIEMPN GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA GVASDNVDSKQISS WGQGTLVTVSS。

[0153] sdTNF10:

[0154] QVQLLESGGGLVQPGGSLRLSCAASG DSVSAEVMT WVRQAPGKGLEWVS AILNTN GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA GSMQSIVPRGQKAVYY WGQGTLVTVSS。

[0155] sdTNF11:

[0156] QVQLLESGGGLVQPGGSLRLSCAASG FTFNDDSMG WVRQAPGKGLEWVS GIPEHN GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA SRKPAMTQPMKF WGQGTLVTVSS。

[0157] sdTNF12:

[0158] QVQLLESGGGLVQPGGSLRLSCAASG DSLSSENMS WVRQAPGKGLEWVS GIEEPD GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA SVGEDLEGDTTFGY WGQGTLVTVSS。

[0159] sdTNF13:

[0160] QVQLLESGGGLVQPGGSLRLSCAASG DTVINEAMG WVRQAPGKGLEWVS SIVVEG GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA ACVAMGQESFSY WGQGTLVTVSS.

[0161] sdTNF14:

[0162] QVQLLESGGGLVQPGGSLRLSCAASG YIFSYENMS WVRQAPGKGLEWVS AIGDHD GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA TYAWLMSVNYLTVAHEMDY WGQGTLVTVSS.

[0163] sdTNF15:

[0164] QVQLLESGGGLVQPGGSLRLSCAASG FMVSDEDMA WVRQAPGKGLEWVS GINTTN GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RDYGSRVDQHLES WGQGTLVTVSS.

[0165] sdTNF16:

[0166] QVQLLESGGGLVQPGGSLRLSCAASG YRLTAENMG WVRQAPGKGLEWVS QUR GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA SRFLSIFVGSYPKLEF WGQGTLVTVSS.

[0167] The three underlined sequences in each of the above amino acid sequences correspond to CDR1, CDR2 and CDR3 respectively. The sequences on both sides of the variable region (non-underlined parts) are the FR regions, and the structure is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, that is, each single-domain antibody includes four framework regions FR1-FR4.

[0168] The nucleotide sequences of sdTNF01-sdTNF016 correspond to SEQ ID NOs: 17-32, as follows:

[0169] sdTNF01:

[0170] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGTTACCTTTAATGCCAAAGATATGTCATGGGTTCGTCAGGCACCGGGTAAAGGTTTAGAATGGGTGAGTACCATTACTAATCCGAGCGGCAGCACCTATTATGCGGATAGTGTTAAGGGGCGCTTTACCATTAGCCGTGATAATAGCAAAAATACCTTATATCTGCAGATGAATAGCCTGCGTGCCGAAGACACAGCAGTTTATTATTGTGCAAGCAGCTTAGATAAAAGTAGCAGTCGTTGCGCAAGAGAAGCAGATGTGCCGTACTGGGGCCAGGGTACCCTTGTGACCGTGAGCAGC。

[0171] sdTNF02:

[0172] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGTTCGTTTAATTTATGAAGTTATGAGCTGGGTTCGTCAGGCCCCGGGCAAGGGTCTGGAGTGGGTGAGTACAATTTTAAATAATGATGGTAGCACCTACTACGCAGATAGTGTTAAGGGTCGTTTTACCATTAGTCGTGACAATAGCAAAAATACCCTGTATCTGCAAATGAACAGCCTGCGGGCAGAAGACACGGCAGTGTATTATTGTGCCAGCAGCGATATGGAGAGTGAATATGAACTGCTGCCGCAGACCCTGAAAAGCTGGGGTCAGGGCACCTTAGTTACCGTGTCATCA。

[0173] sdTNF03:

[0174] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGTTCGTGTTATTACCGAGAGCATGGCATGGGTTCGTCAGGCACCGGGCAAGGGTCTGGAGTGGGTTAGTGGGATTTTAATTACGAATGGAAGCACATATTACGCGGATAGTGTGAAGGGTCGTTTTACAATAAGCAGAGACAATAGTAAAAACACACTGTATCTGCAGATGAATAGCCTGCGTGCAGAAGACACCGCAGTTTACTATTGTGCAACCATGTATTATTTCAAGCGTCGTGAAGTTGAAACCACCATGCGCTTCTGGGGTCAGGGGACCCTGGTTACCGTTTCTAGC。

[0175] sdTNF04:

[0176] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGTTATGTTTAGTTATGAAGATATGGCATGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAGTGGGTTAGTGCGATTCGTAATCATGATGGTAGCACCTATTATGCGGATAGTGTTAAGGGTCGGTTTACCATTAGCCGTGATAATAGCAAAAATACCTTATATCTGCAGATGAACAGCCTGCGTGCAGAAGACACCGCCGTCTATTATTGTGCAACCTGTAGTGATAAGCCGAAAGAAGATGCAATCCGTTATTGGGGGCAGGGAACCCTGGTTACGGTTAGCAGC。

[0177] sdTNF05:

[0178] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGATACCATTACAAATGAAACCATGGCATGGGTTCGTCAGGCACCGGGAAAAGGTCTGGAATGGGTGAGCAGTATTATTAGCAATAGTGGATCTACCTATTACGCGGATAGTGTTAAGGGGCGCTTTACCATCAGCCGCGACAATAGCAAAAATACCCTGTATCTGCAGATGAACAGCCTGCGGGCAGAAGATACGGCAGTGTATTATTGTGCAAGCTGGTTACTGGAGTGTGATATTCATACCTGGGTTGCAAAAGATCTGGAATATTGGGGCCAAGGAACCTTAGTTACAGTTAGTAGC。

[0179] sdTNF06:

[0180] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGATACCGTTAATTATAAAGCAATGAGCTGGGTTAGACAGGCACCGGGTAAAGGTCTGGAATGGGTTAGTTCGATTAGCAATCCGGATGGAAGCACCTATTATGCGGATAGTGTGAAGGGGCGCTTTACCATTAGCCGTGACAATAGCAAAAATACCTTATATCTGCAAATGAACAGCCTGCGTGCAGAAGACACAGCCGTCTATTATTGTGCAACCGACTGTATGGCGCGTGCAAGCCGTCCGCCCGCACTGCATTATTGGGGACAGGGCACACTGGTTACCGTTAGCAGC。

[0181] sdTNF07:

[0182] CAGGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTGATATGGTTTCTCATGAAGATATGGGTTGGGTTCGTCAGGCCCCGGGTAAAGGTCTGGAATGGGTTAGTACCATTAATAGTCAGGATGGTAGCACATATTATGCGGATAGTGTTAAAGGTCGTTTTACCATTAGCCGTGATAATAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGTGCAGAAGATACCGCCGTTTATTATTGTGCAACCGTTAGTAGCGATCAGACCGCATACGAACCAGTTGCGTTTTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGC。

[0183] sdTNF08:

[0184] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTTATAGCTTTACCAGTGAATATATGGGTTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAGTGGGTGAGTACGATTAATACCCGTAATGGAAGCACATACTACGCGGATAGTGTTAAAGGGAGATTTACGATTAGTCGTGACAATAGCAAGAATACACTGTATCTGCAAATGAATAGTCTGAGAGCAGAAGACACCGCAGTTTACTATTGTGCAGCCGGCCGTGTGGCGGAGAAAAATATGGGCTATTGGGGGCAAGGAACACTGGTTACAGTCAGCAGC。

[0185] sdTNF09:

[0186] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTTTTATTATTTCAAATGAAGTTATGGCCTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAGTGGGTTAGTGCAATTGAAATGCCGAATGGATCCACCTATTATGCGGATAGTGTTAAGGGTCGTTTTACCATCAGCCGGGACAATAGCAAAAATACCCTGTACCTGCAGATGAATTCACTGCGTGCAGAAGACACAGCCGTTTACTATTGTGCAGGTGTTGCAAGTGATAATGTTGATTCTAAGCAGATTAGCAGCTGGGGCCAGGGCACCCTGGTTACCGTGAGCAGC。

[0187] sdTNF10:

[0188] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGATAGCGTTAGCGCGGAAGTTATGACCTGGGTTAGACAGGCACCGGGAAAAGGTCTGGAATGGGTTAGTGCAATATTAAATACGAATGGATCTACCTACTACGCAGATAGTGTTAAGGGAAGATTTACAATCAGCAGAGACAATAGCAAAAATACCCTGTATCTGCAGATGAATAGCCTGCGTGCGGAAGACACAGCAGTTTATTATTGTGCAGGCAGCATGCAGTCGATTGTTCCGCGGGGTCAGAAAGCAGTTTATTACTGGGGCCAAGGAACCCTGGTTACCGTGAGCAGC。

[0189] sdTNF11:

[0190] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTTTTACCTTTAATGATGATTCAATGGGTTGGGTTCGTCAGGCCCCGGGCAAGGGTCTGGAGTGGGTTAGTGGGATTCCGGAACATAATGGGAGCACCTATTATGCGGATAGTGTTAAGGGCCGTTTTACCATTAGCCGTGATAATAGTAAGAATACACTTTACCTGCAAATGAATAGTCTGCGTGCAGAGGATACAGCAGTTTATTATTGTGCAAGCCGTAAGCCGGCGATGACGCAGCCTATGAAATTTTGGGGTCAGGGAACCCTGGTGACCGTTTCCTCA。

[0191] sdTNF12:

[0192] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGATAGCTTATCTAGTGAAAATATGAGCTGGGTTAGACAGGCACCGGGAAAAGGTCTGGAATGGGTTAGTGGTATTGAAGAACCGGATGGATCCACCTATTATGCTGATAGCGTTAAGGGGAGATTTACCATTAGCCGGGATAACAGCAAAAATACCCTGTATCTGCAAATGAATAGCCTGCGTGCAGAAGATACAGCAGTTTATTACTGCGCATCCGTTGGTGAGGATCTGGAAGGTGATACCACCTTTGGATATTGGGGCCAGGGAACCCTGGTTACCGTTAGCAGC。

[0193] sdTNF13:

[0194] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTGATACCGTTATTAATGAAGCAATGGGTTGGGTCAGACAGGCACCGGGTAAAGGTCTGGAATGGGTTAGTTCGATTGTTGTTGAGGGTGGATCCACCTATTATGCGGATAGTGTTAAGGGGCGCTTTACCATAAGTCGTGATAATTCTAAGAACACACTTTATCTGCAGATGAATTCACTGCGTGCCGAAGATACCGCGGTTTATTATTGTGCAGCCTGTGTTGCAATGGGTCAGGAGAGTTTTAGCTACTGGGGTCAGGGGACCCTGGTGACCGTTTCTTCT。

[0195] sdTNF14:

[0196] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTTATATTTTTAGCTATGAAAATATGAGCTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAGTGGGTGAGTGCAATTGGTGATCATGATGGGAGCACCTATTATGCGGATAGCGTTAAAGGGCGGTTTACAATTAGCCGGGATAATAGTAAAAACACACTGTATCTGCAGATGAATAGCCTGCGTGCGGAAGACACAGCAGTTTATTATTGTGCAACCTATGCATGGTTAATGAGCGTTAATTACCTGACCGTTGCACATGAAATGGACTACTGGGGTCAAGGCACCCTGGTGACGGTTAGCAGT。

[0197] sdTNF15:

[0198] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTTTTATGGTTTCAGATGAAGATATGGCATGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAGTGGGTGAGTGGTATTAATACCACAAATGGATCCACCTATTATGCGGATTCAGTTAAGGGTCGGTTTACCATTAGCCGTGATAATAGCAAGAATACCTTATATCTGCAGATGAATTCACTGCGTGCGGAAGATACCGCAGTTTATTATTGTGCACGTGATTATGGTTCCCGTGTTGATCAGCATCTGGAAAGCTGGGGTCAGGGGACGCTGGTTACCGTTTCTAGC。

[0199] sdTNF16:

[0200] CAGGTTCAATTACTTGAAAGCGGTGGTGGTTTAGTTCAGCCGGGTGGTAGCTTACGTTTATCATGTGCAGCAAGCGGTTATCGTTTAACCGCAGAAAATATGGGTTGGGTTCGTCAAGCACCGGGCAAGGGTCTGGAGTGGGTTAGTGCGATTTATCGTCAGGATGGAAGCACCTATTATGCAGATAGTGTGAAAGGCCGTTTTACGATTAGCCGGGACAATAGCAAAAATACCCTGTATCTGCAGATGAATTCACTGCGTGCAGAAGACACCGCCGTTTATTATTGTGCAAGCCGTTTTCTGAGCATTTTTGTTGGCAGCTATCCGAAATTAGAATTTTGGGGGCAGGGCACCCTGGTGACCGTTAGCAGC。

[0201] 3. Identification of sdTNFs recombinant protein

[0202] (1) SDS-PAGE and Western Blot analysis: The sdTNFs recombinant protein was identified by SDS-PAGE Coomassie brilliant blue staining and Western Blot methods. The antibody used in Western Blot was HRP Anti-6xHis antibody, which can specifically recognize the 6XHis tag at the tail of the protein.

[0203] (2) Protein purity analysis: Image j software was used to analyze the SDS-PAGE results and calculate the protein purity. The results showed that after a series of purification steps, the purity of the sdTNFs recombinant protein reached more than 90%.

[0204] The 16 strains of sdTNFs genetic engineering bacteria constructed [sequential number E.coli BL21(DE3) / sdTNF01-E.coliBL21(DE3) / sdTNF16] can effectively express sdTNFs recombinant protein ( Figure 3 A, B); through a series of purification steps such as ultrasonic fragmentation, inclusion body washing, affinity chromatography, dialysis renaturation and desalting, the purity of sdTNFs recombinant protein reached more than 90% ( Figure 3 (C, D).

[0205] Example 5 ELISA analysis of the affinity of sdTNFs to TNF-α

[0206] 1. Experimental preparation and coating

[0207] (1) Coating: Use coating buffer to fix hTNF-α antigen in the wells of ELISA plate. Add 100 μL of coating solution containing 50 ng of hTNF-α antigen to each well. Seal the wells with sealing film and incubate at 4°C overnight to ensure sufficient fixation of the antigen.

[0208] 2. Blocking and antigen-antibody binding

[0209] (1) Blocking: Wash the plate with 0.05% PBST washing solution to remove unfixed antigens and other impurities. Then, add 100 μL of 5% BSA blocking solution to each well to block nonspecific binding sites on the plate surface. Incubate at 37°C for 1 hour.

[0210] (2) Antigen-antibody binding: Wash the plate again with PBST washing solution. Then, add 0.1 mg / mL of sdTNFs recombinant protein and use ATN103 (Ozoralizumab / US20100172894) as a positive control. Incubate at 37°C for 1 hour to allow the antibody to fully bind to the antigen.

[0211] (3) Pour off the blocking solution, add 200 μL 0.05% PBST and let stand for 30 seconds, then pour off the solution, repeat the washing twice, add 0.1 mg / mL sdTNFs recombinant protein, and incubate with ATN103 at 37°C for 1 hour.

[0212] 3. Antibody incubation and color development

[0213] (1) Incubation with primary antibody: After washing the plate, add 100 μL of diluted (1:1000) mouse anti-His antibody (mouse anti-His antibody, targeting the His tag on the sdTNFs recombinant protein) and incubate at 37°C for 30 minutes to detect sdTNFs bound to TNF-α.

[0214] (2) Incubation with secondary antibody: After washing the plate, add 100 μL of diluted (1:2000) anti-mouse IgG1-HRP secondary antibody and incubate at 37°C for 30 min.

[0215] (3) Color development: After washing the plate with PBST, add 100 μL of TMB color development solution and incubate at room temperature away from light. When the blank well begins to turn blue, add 100 μL of 1 M sulfuric acid to terminate the reaction, and the blue turns to yellow. Finally, use an ELISA reader to detect the absorbance of each well at 450 nm.

[0216] Compared with the blank group, sdTNF01, 03, 05, 07, 08 and 15 single domain antibodies specifically bound to TNF-α with statistical significance. Among them, sdTNF05, 08 and 15 had higher affinity ( Figure 4 ).

[0217] Example 6 Determination of cytotoxicity of sdTNFs and hTNF-α on L929 cells (MTT method)

[0218] The sensitivity of mouse fibroblast cell line L929 to TNFα and the property that actinomycin D can increase the sensitivity of L929 cells to TNF were used to determine the ability of sdTNFs to antagonize TNF-α activity. The main methods and steps are as follows:

[0219] 1. Cell inoculation: First, select L929 cells in the logarithmic growth phase, collect the cells by PBS washing and trypsin digestion, then adjust the cell concentration and inoculate them into 96-well plates. Incubate the cells in a 37°C, 5% CO2 incubator until the confluence is close to 80%-90%.

[0220] 2. Cell modeling and drug treatment: Then, actinomycin D (final concentration 5 μg / mL) and hTNF-α (final concentration 2 ng / mL) were added to the cells to construct a cell injury model. At the same time, different drug administration groups were set up, including sdTNFs, positive control ATN103, and control group (PBS only). All groups were incubated at room temperature for 30 minutes under complete medium conditions, added to the 96-well plate seeded with cells, and continued to incubate in the incubator for 12 hours.

[0221] 3. MTT detection of cell apoptosis: Finally, the MTT method was used to detect cell apoptosis. After adding the MTT solution and continuing to culture for a period of time, centrifugation, addition of DMSO and shaking were performed, and the absorbance (OD value) of each well was measured at a wavelength of 570nm using an enzyme marker.

[0222] With ATN103 as the positive control, at a concentration of 2 μM, six sdTNFs (sdTNF03, 05, 07, 08, 09 and 15) showed an inhibitory effect on TNF-α killing of L929 cells ( Figure 5 Further microscopic observation revealed that the cells in the normal group showed spindle and flat star morphology, and the cells in the model group showed shrinkage and necrosis-like characteristics. The sdTNFs single domain antibody was able to neutralize hTNF-α and effectively inhibit its killing effect on L929 cells. The cells showed normal spindle and flat star morphology ( Figure 5 (B).

[0223] Example 7 EC of sdTNFs 50 Determination

[0224] To further verify the neutralization potency of sdTNFs against hTNFα, three sdTNFs recombinant proteins with better activity (sdTNF07, 08, 09) were selected and their EC 50 value.

[0225] sdTNFs-EC 50 The determination method and steps are basically the same as those in Example 6, with the main difference being that different concentration gradients are set for sdTNFs. Specifically, the maximum concentration of sdTNF07 is set to 8000nM, diluted at a ratio of 1:3, and a total of 8 concentration points are set; the maximum concentration of sdTNF08 is 166.66nM, diluted at a ratio of 1:1.5, and a total of 9 concentration points; and the maximum concentration of sdTNF09 is also 8000nM, but diluted at a ratio of 1:2, and 8 concentration points are also set. A fixed concentration of actinomycin D (5μg / mL) and hTNF-α (2ng / mL) were added to each dosing group, and only 5μg / mL actinomycin D was added to the blank control group to evaluate the activity of sdTNFs under the same conditions. After adding the sample to each group, mix well and incubate at room temperature for 30min. There are at least 3 replicates in each group, and 100μL per well is added to a 96-well plate. After culturing at 37°C for 12h, MTT detection is performed.

[0226] like Figure 6 As shown in Figure 2, sdTNF07, 08 and 09 single domain antibodies all showed a dose-dependent effect. With the increase of antibody concentration, their protective effect on cells increased. The EC value of sdTNF07 was 50The EC of sdTN08 was 141.9 nM. 50 The EC of sdTNF09 was 25.58 nM. 50 It is 477.1nM.

[0227] Example 8 Determination of cytotoxicity of sdTNFs and hTNFα on L929 cells (Hoechst 33342 / PI double staining / fluorescence microscope observation)

[0228] 1. In this example, Hoechst / PI double staining and fluorescence microscopy were used to observe the effects of sdTNFs neutralization and hTNFα on the state of L929 cells; the cell inoculation scheme was referred to the corresponding part of Example 6 of the present invention, and sdTNF07, 08 and 09 were used as examples to analyze the cytotoxicity of sdTNFs neutralization and hTNFα on L929 cells.

[0229] 2. The dosing concentrations of sdTNF07 were 5nM, 40nM and 320nM, and the positive control was 320nM ATN103; the dosing concentrations of sdTNF08 were 0.5nM, 2nM and 8nM, and the positive control was 128nM ATN103; the dosing concentrations of sdTNF09 were 50nM, 200nM and 800nM, and the positive control was 800nM ATN103; 5μg / mL actinomycin D and 2ng / mL hTNF-α were added to the above dosing groups; 5μg / mL actinomycin and D2 ng / mL hTNF-α were added to the cell model group; only 5μg / mL actinomycin D was added to the control group; after adding the sample to each group, mix well and incubate at room temperature for 30min, with at least 3 replicates for each group, 100μL per well was added to a 96-well plate, and stained after incubation at 37℃ for 12h.

[0230] 3. Take out the 96-well plate that has been incubated for 12 hours, discard the supernatant, gently add 100 μL PBS to each well along the tube wall for washing, and discard the PBS; add 200 μL of 4% paraformaldehyde fixative to fix the cells, and place them at room temperature away from light for 15 minutes.

[0231] 4. Dilute the original concentration of 10 mg / ml Hoechst at a ratio of 1:1000 using serum-free medium to a final concentration of 10 μg / mL; dilute the original concentration of 50 μg / ml PI at a ratio of 1:5 using serum-free medium to a final concentration of 10 μg / mL, add 100 μL of staining solution to each well, cover with tin foil to avoid light and place at 4°C for staining for 15 minutes; discard the staining solution, add 100 μL PBS to wash once, discard PBS after 5 minutes, and add 100 μL PBS again; observe under an inverted fluorescence microscope, blue-labeled cells are total cells, red-labeled cells are dead or necrotic cells, and use Image J software for cell counting analysis.

[0232] like Figure 8-Figure 10 As shown, most cells in the low-dose group of the three single-domain antibodies sdTNF07, sdTNF08 and sdTNF09 died, some cells in the medium-dose group showed apoptosis, and most cells in the high-dose group survived, further indicating that the protective effects of these three sdTNFs on TNF-α-damaged L929 cells are dose-dependent.

[0233] Example 9 Determination of cytotoxicity of sdTNFs and hTNFα on L929 cells (Annexin V-FITC / PI flow cytometry)

[0234] 1. This example uses Annexin V-FITC / PI flow cytometry to analyze the effect of sdTNFs neutralizing hTNFα on L929 cell apoptosis

[0235] Cells in the logarithmic growth phase were taken and 5×10 5 Inoculated in 96-well plates, 100 μL per well was placed in a cell culture incubator for 24 h. sdTNF07, sdTNF08 and sdTNF09 single domain antibodies were selected for detection. Under the conditions of 5 μg / mL actinomycin D and 2 ng / mL hTNF-α, the concentrations of sdTNF07 in the experimental group were 5 nM, 40 nM and 320 nM, the concentrations of sdTNF08 were 0.05 nM, 2 nM and 80 nM, and the concentrations of sdTNF09 were 10 nM, 100 nM and 1000 nM; the positive control group ATN103 was the highest concentration of each single domain antibody, and 5 μg / mL actinomycin D and 2 ng / mL hTNF-α were added; the negative control group only added culture medium containing 5 μg / mL actinomycin D; the model group added 5 μg / ml actinomycin D and 2 ng / mL hTNF-α. After incubation at room temperature for 30 min, 2 mL was added to each well of a 6-well plate, with at least 2 replicates per group. In addition, 3 wells were set up with only 5 μg / mL actinomycin D culture medium for single staining and blank control tubes.

[0236] 2. Label the 5mL centrifuge tubes to collect the supernatant culture medium from each well, add 500μL EDTA-free trypsin to each well and digest for 2 minutes at room temperature, add the supernatant of MEM complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics, mix well by pipetting, collect cells, centrifuge at 1000g for 5 minutes, and discard the supernatant; add 1mL PBS to each tube to wash away the remaining complete culture medium, centrifuge at 1000g for 5 minutes, and discard the supernatant; take 100μL Annexin V Binding Buffer to resuspend the cells, add 5μL 7-aminoactinomycin D (7-AAD) and 5μL APC Annexin V to each tube of the experimental group, add only 5μL 7-AAD or 5μl APC Annexin V to each of the two single-staining tubes, and do not add dye to the blank control tube. Incubate the above dyes at room temperature for 15 minutes and add 400μL Annexin V Binding Buffer. The buffer was filtered through a 300-mesh filter cloth, and the sample was analyzed on a flow cytometer FACSVerse (BD, USA).

[0237] Annexin V-FITC / PI staining was used to detect the protective effect of sdTNFs recombinant protein on hTNF-α-induced apoptosis of L929 cells. The upper left quadrant is the cell fragments of non-programmed death, the lower left quadrant is the healthy living cells, the lower right quadrant is the early apoptotic cells, and the upper right quadrant is the late apoptotic cells. Figure 11-13 As shown, most of the cells in the normal group are healthy living cells, and most of the cells treated with hTNF-α are in the late stage of apoptosis. The addition of sdTNFs recombinant proteins sdTNF07, sdTNF08 and sdTNF09 can effectively neutralize hTNF-α and have a protective effect on L929 cells, showing a dose-dependent effect as in Examples 7 and 8.

[0238] Example 10 Evaluation of the in vivo efficacy of sdTNFs in the treatment of CIA model mice

[0239] 1. Construction of CIA arthritis model mice

[0240] Six-week-old male DBA1 / J mice (weight 20±2g) were randomly divided into a normal group and a collagen-induced arthritis (CIA) group. The CIA group received a subcutaneous injection of 100 μL of emulsion (containing bovine type II collagen with a final concentration of 0.5 mg / mL and fluorinated complete adjuvant) at 2 cm from the base of the tail for the first immunization (recorded as day 0), and a subcutaneous injection of 100 μL of emulsion (containing bovine type II collagen with a final concentration of 0.5 mg / mL and fluorinated incomplete adjuvant) at 3 cm from the base of the tail for the second immunization (day 21). The mice in the normal group were injected with 100 μL of normal saline at the same position. After the second immunization, the mice were observed every 3 days, and the mice were kept until the morphological changes of the ankle joints and toes appeared, and photographed and recorded. The changes in the thickness of the mice's feet were measured using an electronic vernier caliper, and the arthritis index (arthritis index) was used to calculate the arthritis index. The severity of arthritis was assessed by the AI ​​(arthritis index, AI), which was scored by three observers and the average value was taken. The scoring criteria were as follows: the front and rear paws of each rat were scored in a double-blind method. Each paw was scored from 0 to 4 points, and the total score was 0 to 16 points. No redness or swelling of the toes was scored as 0 points; slight swelling with erythema was observed in the toes, which was scored as 1 point; obvious swelling and redness of the toes was scored as 2 points; severe swelling and redness from the ankle joint to the toes was scored as 3 points; and the ankle joint and toes had the greatest swelling and deformity as 4 points.

[0241] 2. Experimental groups and dosing regimen

[0242] On the 30th day of animal modeling, the DAB / 1J mice without modeling were the normal group, and the CIA mice were randomly divided into 6 groups using the random number table method, with 3 mice in each group; subcutaneous injection was used for drug administration, and adalimumab was selected as the positive control, and subcutaneous injection was given once every 3 days, for a total of 6 times; the specific grouping and drug administration scheme are as follows: normal group, CIA model group (CIA mice + 50μl PBS), adalimumab group (CIA mice + 2.0mg·kg-1 adalimumab), sdTNF07 group (CIA mice + 2.0mg·kg -1 sdTNF07), sdTNF08 group (CIA mice + 2.0 mg kg -1 sdTNF08) and sdTNF09 groups (CIA mice + 2.0 mg kg -1 sdTNF09).

[0243] 3. Sample collection and processing

[0244] During the administration period, the severity of arthritis was assessed and the body weight was measured; mice were killed on the 48th day, and serum, spleen and joints were collected; ankle joints were fixed with formalin, embedded in paraffin and sliced ​​for histopathological H&E staining; spleen was weighed after being blotted with filter paper, and spleen index was calculated according to the following formula: spleen index = spleen weight (g) / body weight (g). ELISA analysis was used to detect the effect of sdTNFs protein on serum IL-6, IL-1β and TNFα of CIA mice.

[0245] Scoring criteria for synoviocyte proliferation: 0 = no proliferation, 1 = mild proliferation (2 to 4 layers of synoviocytes), 2 = moderate proliferation (more than 4 layers of synoviocytes), 3 = excessive proliferation of synoviocytes.

[0246] Pannus scoring criteria: 0 = no change, 1 = pannus in 2 sites, 2 = pannus in 4 sites with erosion of the cartilage surface, 3 = pannus in more than 4 sites or extensive pannus in 2 sites.

[0247] On day 30, the paws of the mice that had already been modeled were red and swollen, and the symptoms gradually worsened between day 30 and 33. From day 33, the adalimumab group began to reduce the CIA arthritis index. From day 36 to 42, the sdTNFT07 and sdTN09 administration groups could significantly reduce the joint index of CIA mice. Compared with the CIA model group, the CIA mice injected with sdTNFT07, sdTN09 and adalimumab had significantly reduced joint severity ( Fig.14 , Fig.15 (A).

[0248] The spleen is an important immune organ, and the spleen index reflects the body's immune status to a certain extent. Compared with the CIA model group, the sdTNF07, sdTNF08, sdTNF09 and adalimumab groups all had a significant improvement in the spleen index ( Fig.15 In addition, there was no significant improvement in the body weight of mice between the various drug groups ( Fig.15 Middle C).

[0249] H&E staining results of joint tissues Fig.16 As shown in A: The joint cavity of the normal group was normal, with no synovial hyperplasia and pannus formation, and the articular cartilage structure was intact and clear; the joint cavity of the CIA model group was narrow, the synovial hyperplasia in the joints, and a large amount of pannus formation; compared with the CIA model group, the adalimumab, sdTNF07, sdTNF08, and sdTNF09 administration groups had a significant effect on the synovial hyperplasia ( Fig.16 Middle B) and pannus ( Fig.16 The formation of C) is significantly improved.

[0250] like Fig.17As shown in the figure, compared with the normal group, the expression levels of IL-6, IL-1β and TNF-α in the CIA model group were significantly increased, with significant differences; compared with the model group, the expression level of TNF-α in the sdTNF08, sdTNF09 and adalimumab groups was significantly decreased, and it was statistically significant; in addition, for IL-6 in serum, compared with the CIA model group, the expression level of IL-6 in the sdTNF09 and adalimumab groups was significantly decreased, and it was statistically significant; compared with the model group, the expression level of IL-1β in the sdTNF09 and adalimumab groups was significantly decreased, and it was statistically significant.

[0251] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A humanized single domain antibody targeting TNFα, characterized in that: The amino acid sequence of the single-domain antibody includes three complementary determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are any one of 1) to 16): 1) CDR1: VTFNAKDMS, CDR2: TITNPS, CDR3: SSLDKSSSRCAREADVPY; 2) CDR1: VRLIYEVMS, CDR2: TILNND, CDR3: SSDMESEYELLPQTLKS; 3) CDR1: VRVITESMA, CDR2: GILITN, CDR3: TMYYFKRREVETTMRF; 4) CDR1: VMFSYEDMA, CDR2: AIRNHD, CDR3: TCSDKPKEDAIRY; 5) CDR1: DTITNETMA, CDR2: SIISNS, CDR3: SWLLECDIHTWVAKDLEY; 6) CDR1: DTVNYKAMS, CDR2: SISNPD, CDR3: TDCMARASRPPALHY; 7) CDR1: DMVSHEDMG, CDR2: TINSQD, CDR3: TVSSDQTAYEPVAF; 8) CDR1: YSFTSEYMG, CDR2: TINTRN, CDR3: AGRVAEKNMGY; 9) CDR1: FIISNEVMA, CDR2: AIEMPN, CDR3: GVASDNVDSKQISS; 10) CDR1: DSVSAEVMT, CDR2: AILNTN, CDR3: GSMQSIVPRGQKAVYY; 11) CDR1: FTFNDDSMG, CDR2: GIPEHN, CDR3: SRKPAMTQPMKF; 12) CDR1: DSLSSENMS, CDR2: GIEEPD, CDR3: SVGEDLEGDTTFGY; 13) CDR1: DTVINEAMG, CDR2: SIVVEG, CDR3: ACVAMGQESFSY; 14)CDR1: YIFSYENMS, CDR2: AIGDHD, CDR3: TYAWLMSVNYLTVAHEMDY; 15)CDR1: FMVSDEDMA, CDR2: GINTTN, CDR3: RDYGSRVDQHLES; 16) CDR1: YRLTAENMG, CDR2: AIYRQD, CDR3: SRFLSIFVGSYPKLEF.

2. The single domain antibody according to claim 1, characterized in that The single domain antibody further comprises four framework regions FR1, FR2, FR3 and FR4, and the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 are respectively as follows: FR1: QVQLLESGGGLVQPGGSLRLSCAASG, FR2: WVRQAPGKGLEWVS, FR3:GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA, FR4:WGQGTLVTVSS.

3. A recombinant protein, characterized in that The recombinant protein comprises the single domain antibody according to any one of claims 1 to 2.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single domain antibody according to any one of claims 1 to 2 or the recombinant protein according to claim 3.

5. A vector comprising the nucleic acid molecule of claim 4.

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

7. A method for preparing the single domain antibody according to any one of claims 1 to 2 or the recombinant protein according to claim 3, characterized in that: The method comprises the steps of culturing the host cell according to claim 6, and isolating and purifying the single domain antibody or recombinant protein from the culture.

8. A pharmaceutical composition, characterized in that It comprises the single domain antibody according to any one of claims 1 to 2, the recombinant protein according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, or the host cell according to claim 6.

9. Use of the single domain antibody according to any one of claims 1 to 2, the recombinant protein according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, or the host cell according to claim 6 in the preparation of a drug for treating immune diseases targeting TNFα.

10. A kit for detecting TNFα, characterized in that: It comprises the single domain antibody according to claims 1-2 or the recombinant protein according to claim 3.

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