OX40 nano antibody, preparation method thereof, OX40 nano antibody derivative and application of OX40 nano antibody derivative and OX40 nano antibody derivative

Abstract

The invention discloses an OX40 nano antibody, a preparation method thereof, an OX40 nano antibody derivative and application of the OX40 nano antibody derivative and the OX40 nano antibody derivative. The OX40 nano antibody comprises a heavy chain variable structural domain, the heavy chain variable structural domain comprises heavy chain complementary determining regions VHCDR1, VHCDR2 and VHCDR 3 as shown in SEQ ID NO.12-14, the VHCDR1 comprises a sequence GYSRGDYC (SEQ ID NO.12), and the VHCDR2 comprises a sequence GYSRGDYC (SEQ ID NO.12). The VHCDR2 comprises a sequence IGT 4 and a sequence GNPGAA (SEQ ID NO.13); and the VHCDR3 comprises a sequence of VAVGPYSGCSADYFLRE GPAGNAY (SEQ ID NO.14), and the sequence of the VHCDR3 is shown in the description. The OX40 nano antibody disclosed by the invention has good specificity of being combined with OX40 through ELISA (Enzyme-Linked Immunosorbent Assay) and flow type verification, and can be used for effectively blocking the combination of the OX40 and OX40L.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to OX40 nano antibody and a preparation method thereof, OX40 nano antibody derivatives and applications thereof. Background Art

[0002] OX40 (CD134) and its binding ligand protein OX40L (CD252) are members of the TNFR / TNF superfamily and are expressed on activated CD4 and CD8 T cells and many other lymphoid and non-lymphoid cells. OX40 molecules are similar to co-stimulatory signaling molecules of traditional T cells, which can promote T cell division and survival and enhance their clonal expansion ability when effector cells and memory cell populations encounter antigen stimulation. OX40 can also inhibit the differentiation and activity of Treg cells. OX40 and OX40L also regulate cytokine production by T cells, antigen-presenting cells, NK cells, and NKT cells, and regulate cytokine receptor signaling. OX40 / OX40L interactions have been found to play a central role in the development of a variety of inflammatory and autoimmune diseases, making them attractive drug development targets for clinical intervention (D.J. Paterson et al., (1987), Molecular Immunology, 24(12), 1281-1290), (Michael Croft et al., (2009). Immunol Rev. May; 229(1): 173-91).

[0003] The interaction between OX40 and OX40L has been extensively studied in most major models of inflammatory or autoimmune diseases. OX40 and OX40L-deficient mice showed a significant reduction in Th2 lung inflammation in an allergic asthma mouse model (Jember AG et al. (J Exp Med. 2001); 193: 387-92), and blocking OX40L inhibited the accumulation of memory effector T cells, thereby eliminating the key features of asthmatic reactions, including eosinophilia, airway hyperresponsiveness, mucus production, and Th2 cytokine production (Salek-Ardakani S et al. (2003); J Exp Med.; 198: 315-24). Recently, it has also been shown that blocking OX40L can prevent TSLP-induced allergic asthma disease, and most importantly, it has also shown therapeutic effects in preclinical monkey studies (Seshasayee D et al. (2007) J Clin Invest.; 117: 3868-78), further indicating the great prospects for the treatment of asthma.

[0004] Emma Guttman-Yassky's team at the Icahn School of Medicine at Mount Sinai in the United States studied the efficacy and safety of OX40 antibodies in the treatment of moderate to severe atopic dermatitis. The relevant paper was published in The Lancet on December 9, 2022. The results showed that the OX40 blocking antibody Rocatinlimab was used clinically to treat patients with atopic dermatitis. Compared with the placebo group, at week 16, the EASI scores of all rocatinlimab groups were significantly reduced. Most patients maintained this improvement after discontinuation of the drug, and the treatment was well tolerated (Guttman-Yassky E et al., (2023) Lancet. Jan 21; 401(10372): 204-214.).

[0005] Therefore, providing highly effective and low-cost OX40 antibodies will be of great significance for reducing treatment costs and improving treatment effects.

[0006] However, the activity and production cost of the existing OX40 antibodies are relatively high. In particular, research on OX40 antibodies in China is still very limited, and the clinical options are very poor. Summary of the invention

[0007] To overcome the above disadvantages, the purpose of the present invention is to provide an OX40 nanobody, which has good binding activity to OX40 molecules, can block the interaction between OX40 and OX4OL, and has good anti-inflammatory activity. The present invention was completed on this basis.

[0008] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0009] The present invention provides an OX40 nanobody comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises heavy chain complementary determining regions VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO.12-14,

[0010] VHCDR1 comprises the sequence GYSRGDYC (SEQ ID NO. 12);

[0011] VHCDR2 comprises the sequence IGTGNPGAA (SEQ ID NO. 13);

[0012] VHCDR3 comprises the sequence VAVGPYSGCSADYFLREGPAGNAY (SEQ ID NO. 14). The OX40 nanobody of the present invention has good specificity for binding to OX40 as verified by ELISA and flow cytometry, and can effectively block the binding of OX40 to OX40L.

[0013] Further, the heavy chain variable domain comprises an amino acid sequence as shown in SEQ ID NO.10. In some embodiments, the amino acid sequence of the heavy chain of the OX40 nanobody of the present invention is as shown in the sequence list SEQ ID NO.10; in other possible embodiments, its amino acid sequence can also be obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO.10, and has more than 90% identity with the amino acid sequence of SEQ ID NO.10 and has the same function. For example, it can have more than 90%, more than 90.5%, more than 91%, more than 92%, more than 93%, more than 94%, more than 94.5%, more than 95%, more than 96%, more than 97%, more than 97.5%, more than 98% or more identity with SEQ ID NO.10.

[0014] The present invention also provides a nucleic acid molecule encoding the aforementioned OX40 nanobody.

[0015] Further, the nucleic acid encoding the heavy chain of the nanobody is the nucleic acid shown in SEQ ID NO.11. In some embodiments, the nucleotide sequence of the heavy chain of the OX40 nanobody of the present invention is as shown in SEQ ID NO.11 in the sequence table; in other possible embodiments, the nucleotide sequence of the heavy chain of the OX40 nanobody can also be a nucleic acid that hybridizes with the nucleotide sequence of SEQ ID NO.11 and encodes the same function; or a sequence that has more than 90% homology with the nucleotide sequence of SEQ ID NO.11 and has the same function. For example, it can have a sequence with more than 90%, more than 91%, more than 91.5%, more than 92%, more than 93%, more than 94%, more than 95%, more than 95.5%, more than 96%, more than 97%, more than 97.5%, more than 98% or more homology with SEQ ID NO.11.

[0016] The present invention also provides a vector containing the aforementioned nucleic acid molecule.

[0017] The present invention also provides a fusion or conjugate comprising the aforementioned antibody.

[0018] The present invention also provides an expression box or recombinant bacterium comprising the aforementioned nucleic acid molecule.

[0019] The present invention also provides a composition comprising at least one of the aforementioned OX40 nanobody, nucleic acid molecule, vector or fusion or conjugate, and at least one of its corresponding pharmaceutically acceptable carriers.

[0020] The present invention also provides a method for detecting OX40 in a biological sample, comprising contacting the biological sample with the aforementioned OX40 nanobody, fusion or conjugate.

[0021] The present invention also provides a use of the aforementioned antibody or antigen-binding fragment, fusion or conjugate, or composition in the preparation of a pharmaceutical preparation for blocking the binding of OX40L to OX40 or in a pharmaceutical preparation for treating or delaying cancer; or in the preparation of a pharmaceutical preparation for treating or delaying autoimmune inflammatory diseases such as atopic dermatitis and rheumatoid arthritis.

[0022] The pharmaceutical preparations of the present invention include "pharmaceutically acceptable" and "physiologically acceptable" carriers, diluents or excipients. The pharmaceutical dosage form can be an emulsion, suspension, syrup, elixir, tablet, capsule, powder, granule, crystal or microbead, ointment, salves, gel, cream or patch. Pharmaceutical formulations suitable for specific local, regional or systemic administration or delivery routes can be prepared.

[0023] The present invention also provides a method for preparing OX40 nanobody, which comprises the following steps:

[0024] Preparation of recombinant OX40 antigen: using the amino acid sequence of human OX40 from the Uniprot protein database, the human OX40 sequence is shown in SEQ ID NO.1;

[0025] The screening steps of OX40 nanoantibodies include: animal immunization, RNA extraction, construction of OX40 nanoantibody phage library and enrichment of OX40 positive nanoantibodies.

[0026] The immunized animal used in the animal immunization step is a camel,

[0027] In the step of constructing the phage library, two sets of primers are included: CALL001 / CALL002 are used as primers, the first round of PCR amplification is performed with the cDNA template, and Camel-Phage-F / Camel-Phage-R is used for the second round of PCR amplification;

[0028] OX40 nanobody expression and preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1( a ) is the result of SDS-PAGE electrophoresis (8-20% reducing gel) of OX40 recombinant protein in Example 1;

[0030] Figure 1(b) is the result of SDS-PAGE electrophoresis (8-20% reducing gel) of OX40L recombinant protein in Example 1;

[0031] Figure 2This is a flow cytometry result diagram of the preliminary detection of OX40 blocking nanoantibodies in Example 4, in which the X-axis represents the SA-PE fluorescence intensity and the Y-axis represents the side scatter signal.

[0032] Figure 2 (a) is the flow cytometry result of control 1 without OX40L-biotin.

[0033] Figure 2 (b) is the flow cytometry result of control 2 with the addition of OX40L-biotin.

[0034] Figure 2 (c) is the flow cytometry result of OX40-1 antibody sample.

[0035] Figure 2 (d) is the flow cytometry result of OX40-2 antibody sample.

[0036] Figure 2 (e) is the flow cytometry result of OX40-3 antibody sample.

[0037] Figure 2 (f) is the flow cytometry result of OX40-4 antibody sample.

[0038] Figure 2 (g) is the flow cytometry result of OX40-5 antibody sample.

[0039] Figure 2 (h) is the flow cytometry result of OX40-6 antibody sample.

[0040] Figure 2 (i) is the flow cytometry result of OX40-7 antibody sample,

[0041] Figure 2 (j) is the flow cytometry result of OX40-8 antibody sample.

[0042] Figure 2 (k) is the flow cytometry result of OX40-9 antibody sample.

[0043] Figure 2 (l) is a flow cytometry result diagram of OX40-10 antibody sample;

[0044] Figure 3 (a) is the result of SDS-PAGE reduction electrophoresis (8-20% gel) of OX40VHH-FC antibody in Example 6;

[0045] Figure 3(b) is the result of SDS-PAGE non-reducing electrophoresis (8-20% gel) of OX40VHH-FC antibody in Example 6;

[0046] Figure 3(c) is the result of SDS-PAGE reduction electrophoresis (8-20% gel) of OX40VHH-HIS antibody in Example 6;

[0047] Figure 3 (d) is the result of SDS-PAGE non-reducing electrophoresis (8-20% gel) of OX40VHH-HIS antibody in Example 6;

[0048] Figure 4 The specific detection result of OX40 nanoantibody identified by ELISA in Example 6;

[0049] Figure 5 This is a graph showing the results of OX40 nanobody inhibiting the binding of OX40 molecule with ligand OX40L molecule in Example 6;

[0050] Figure 6 The specific results of OX40 nanobody identified by flow cytometry in Example 6;

[0051] Figure 7 The results of flow cytometry identification of the blocking activity of OX40 nanobody in Example 6;

[0052] Figure 8 This is the result of identifying the blocking activity of OX40 nanobody based on the cell signaling pathway method in Example 6. DETAILED DESCRIPTION

[0053] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0054] Currently, the activity and production cost of existing OX40 antibodies are relatively high. In particular, research on OX40 antibodies in China is still very limited, and the clinical options are very poor.

[0055] However, the inventors accidentally screened a nano antibody with high affinity and specific blocking effect on the OX40 / OX40L signaling pathway through protein immunization camel and phage screening technology.

[0056] The recombinant OX40 antigen of the present invention may be a natural or artificial OX40 antigen, for example, a human OX40 antigen or an animal OX40 antigen.

[0057] Example 1: Preparation of recombinant OX40 antigen and OX40L

[0058] 1.1 Obtain recombinant OX40 antigen protein plasmids pcDNA3.4-hOX40-hFc and pcDNA3.4-hOX40-his, and OX40L plasmid pcDNA3.4-OX40L-his

[0059] The amino acid sequence of human OX40 (hOX40) was obtained based on the amino acid sequence of human OX40 (P43489) in the Uniprot protein database, and the sequence is shown in SEQ ID NO.1.

[0060] SEQ ID NO.1 Amino acid sequence of human OX40 (P43489)

[0061] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDN QACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0062] The extracellular domain of the OX40 molecule (ie, Met1-Ala216) was selected to construct hOX40-hFC and hOX40-his proteins for animal immunization and antibody screening, and the sequences are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0063] SEQ ID NO.2hOX40-hFC amino acid sequence

[0064] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0065] SEQ ID NO.3 hOX40-his amino acid sequence

[0066] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAHHHHHHHH*

[0067] SEQ ID NO.4 OX40L-HIS amino acid sequence

[0068] HHHHHHGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKV QNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVA SLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL*

[0069] DNA fragments encoding the amino acid sequences of hOX40, hOX40-hFC and hOX40-HIS molecules were obtained by artificial gene synthesis. The synthesized gene sequences were subcloned into the commercial vector pcDNA3.4 by double restriction digestion with HindIII and EcoRI, and the accuracy of the constructed plasmids was verified by sequencing to obtain recombinant plasmid DNA, namely pcDNA3.4-hOX40-hFc and pcDNA3.4-hOX40-his. The DNA fragment with the amino acid sequence of SEQ ID NO.4 was used to construct the pcDNA3.4-OX40L-his plasmid.

[0070] 1.2 Expression and correctness verification of recombinant OX40 antigen protein and OX40L-HIS protein

[0071] pcDNA3.4-hOX40-Fc, pcDNA3.4-hOX40-his and pcDNA3.4-OX40L-his were transiently transfected into HEK293 cells for protein production. The recombinant expression plasmid was diluted with CD05 medium (brand: Aupuma, catalog number: CD05), and PEI (polyethyleneimine) solution was added for transformation; each group of plasmid / PEI mixtures were added to the cell suspension, incubated at 37°C, 7% CO2, and 120rpm. After 6 days, the culture supernatant was collected and purified by ProteinA affinity chromatography to obtain hOX40-Fc; hOX40-HIS and OX40L-HIS protein samples were purified by nickel column and used in the following examples. The obtained protein samples were subjected to SDS-PAGE electrophoresis (8-20% reducing gel) for preliminary detection. As shown in Figure 1, it can be clearly seen that hOX40-Fc recombinant protein, hOX40-HIS recombinant protein and OX40L recombinant protein all formed clear bands, indicating that each recombinant protein was successfully prepared.

[0072] Example 2: Construction of cells for antibody screening (OX40L / 293 cells)

[0073] According to the OX40L amino acid sequence (from Uniprot protein database, P23510), the OX40L gene (OX40L amino acid sequence as SEQ ID NO.5) was synthesized by gene synthesis, and the GFP gene was fused with OX40L for expression, and a T2A shear peptide was introduced in the middle, and it was cloned into the pCDNA3.4 plasmid vector, with restriction sites of XbaI / EcoRI, and the CMV promoter was used to promote the expression of OX40L, and the EF1-a promoter was used to promote the expression of the puromycin resistance gene PuroR. HEK293 cells were transfected, and stable transfection cells OX40L / HEK293 were obtained by antibiotic selection.

[0074] SEQ ID NO.5

[0075] MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL

[0076] Example 3: Construction of OX40-NF-κB-JurkaT cells

[0077] Construction of OX40-NF-κB plasmid: The Luciferase reporter gene was placed behind the NF-κB regulatory sequence element, and the CMV promoter was used to drive the expression of the OX40 gene. At the same time, the EF1a promoter drove GFP and the puromycin resistance gene PuroR for subsequent cell screening. The plasmid was transferred into JurkaT cells by electroporation. After 24 hours of transfection, puromycin was added to the cell culture medium at a final concentration of 2 μg / ml for screening, and positive clone cells OX40-NF-κB / JurkaT cells were obtained.

[0078] The present invention prepares OX40 antigen by self-recombination instead of using the existing OX40 antigen, and utilizes the above-mentioned antigen preparation to make the purity and biological activity of the antigen higher, thereby ensuring that subsequent animal immunization has a good immune result.

[0079] Example 4: Screening of OX40 Nanobodies

[0080] 4.1 Animal immunization

[0081] The screening of OX40 nanoantibodies is mainly completed through camel immunization, camel peripheral blood (PBMC) isolation, phage library construction and phage screening processes.

[0082] A healthy 3-year-old male camel was selected for antigen immunization. Before immunization, the hOX40-Fc recombinant protein prepared in the above example was mixed with an adjuvant, the volume of the antigen and the adjuvant were both 1 ml, and the emulsification of the antigen and the adjuvant was completed by ultrasonic emulsification.

[0083] Four immunizations were performed in total. The adjuvant used in the first immunization was Freund's complete adjuvant, and the adjuvant used in the subsequent three immunizations was Freund's incomplete adjuvant. All adjuvants were purchased from Sigma. The amount of antigen used for each immunization was 1 mg. The interval between each immunization was 2 weeks, and the immunization site was subcutaneous at the back of the neck.

[0084] 4.2 Extraction of OX40 Nanobody RNA

[0085] After the third and fourth immunizations, 50 ml of peripheral blood was collected, and camel PBMCs were isolated using a PBMC isolation kit and stored in a -80°C refrigerator.

[0086] RNA was extracted from PBMC using Trizol reagent (ThermoFisher, Catalog No.: 15596026). The RNA extraction experimental steps are as follows:

[0087] ① After PBMC separation and counting, each 0.25ml PBMC (cell volume: 5-10×10 6 ) add 0.75ml TRIzol TM The reagent is placed at room temperature for 5 minutes to allow it to fully lyse. It cannot be directly frozen before it is fully lysed (the standard for whether it is fully lysed is: take the sample and observe it under a microscope, and all the cells are broken and there are no intact cells). After fully lysed, the sample is placed in -80℃ for long-term storage.

[0088] ② Centrifuge at 12,000 rpm for 5 min and discard the precipitate.

[0089] ③ Add chloroform at a ratio of 200 μl chloroform / ml Trizol, vortex to mix, and place at room temperature for 15 min. Note: Do not use a vortex oscillator to prevent the genomic DNA from breaking.

[0090] ④ Centrifuge at 12,000g for 15 min at 4℃. Pipette the upper aqueous phase into another centrifuge tube.

[0091] ⑤ Add isopropanol at a rate of 0.5 ml isopropanol / ml Trizol, mix well, and leave at room temperature for 5-10 minutes.

[0092] ⑥ Centrifuge at 12,000g at 4℃ for 10 min, discard the supernatant, and the RNA will settle to the bottom of the tube.

[0093] ⑦ Add 75% ethanol at a rate of 1 ml 75% ethanol / ml Trizol, gently shake the centrifuge tube to suspend the precipitate. Centrifuge at 8,000 g for 5 min at 4°C, and discard the supernatant as much as possible.

[0094] ⑧ Air dry at room temperature or vacuum dry for 5-10 minutes. Note: Do not over-dry the RNA sample, otherwise it will be difficult to dissolve.

[0095] ⑨ Dissolve the RNA sample with 50 μl H2O, treat at 55-60℃ for 5-10 min, and then perform subsequent quantitative calculation of RNA.

[0096] ⑩Measure OD value and quantify RNA concentration.

[0097] 4.3 Construction of OX40 nanobody phage library

[0098] The RNA samples in the above step were reverse transcribed using a reverse transcription kit (ThermoFisher, Cat. No. K1622) to obtain C DNA. Two sets of primers were designed to amplify the nanobody fragment VHH by nested PCR. CALL001 / CALL002 were used as primers. C The DNA template was subjected to the first round of PCR amplification, and the amplification system and conditions were as follows:

[0099] 2×buffer Mix 25μl 125 CALL001 (10 mM each) 2μl 10 CALL002 (10 mM each) 2μl 10 cDNA 4μl(400μg) <=20μl dNTP mixture (each 10mM) 1μl 5 High-fidelity polymerase 1μl 5 <![CDATA[H2O]]> 40μl 75μl Total 50μl 250μl

[0100] Reaction conditions: 95°C for 4 min; 95°C for 30 s, 52°C for 30 s, 72°C for 30 s, 5 cycles; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 20 cycles; 72°C for 10 min; stored at 10°C. 1.2% gel (long gel) electrophoresis yielded two bands (about 700 bp and 950 bp, respectively), and the 700 bp band was the VHH fragment.

[0101] The second round of PCR amplification was performed using Camel-Phage-F / Camel-Phage-R using the PCR product from the previous round as a template. The amplification system was as follows:

[0102] 2×buffer Mix 200 μl Camel-Phage-F (10mM each) 16μl Camel-phage-R (10mM each) 16μl The product of the previous PCR was recovered 8μl (>200ng) dNTP mixture (each 10mM) 4μl High-fidelity polymerase 4μl <![CDATA[H2O]]> ~152μl, Up to 200μl Total 400μl

[0103] Reaction conditions: 95℃4min; 95℃30s, 56℃30s, 72℃30s, 10cycles; 95℃20s, 60℃25s, 72℃30s, 20cycles; 72℃10min; 10℃ storage. 1.2% gel electrophoresis was used to detect the product and the target fragment was recovered; the above-mentioned recovered fragment and the vector were digested with SfiI respectively, and the digestion was carried out at 50℃ overnight (about 20h). The digestion system is as follows:

[0104] VHH target fragment 4 μg SfiI enzyme 8μl 10*G Buffer 8μl <![CDATA[ddH2O]]> Up to 80μl

[0105] Phage vector 6μg SfiI enzyme 1.5~2μl 10*G Buffer 5μl <![CDATA[ddH2O]]> Up to 50μl

[0106] The purified VHH and phage vector were connected using the following connection system (fragment: vector = 4:1): first, the fragment and vector were mixed and dissolved at 50°C for 3 minutes (55°C, 5min), and immediately placed on ice for annealing. Then, Solution I (TAKARA) was added for half an hour at room temperature, and then connected at 16°C overnight. The ligase was inactivated at 70°C for 15min, and then the connection product was purified by gel column.

[0107] VHH digestion products 1.2 μg Vector digestion product 3μg Solution I 100μl <![CDATA[H2O]]> Up to 200μl

[0108] The second round PCR products and phage vectors were digested with SfiI and the corresponding fragments were recovered, and the phage library was prepared by electroporation of TG1 (brand: lucigen, catalog number: 60502-2) competent cells. The electroporation parameters and experimental procedures were referred to the instructions for the TG1 competent cell product.

[0109] In the above steps, the nucleotide sequence of primer CALL001 is shown in SEQ ID NO.6:

[0110] GTCCTGGCTGCTCTTCTACAAGG′;

[0111] The nucleotide sequence of primer CALL002 is shown in SEQ ID NO.7:

[0112] GGTACGTGCTGTTGAACTGTTCC′;

[0113] The nucleotide sequence of primer Camel-Phage-F is shown in SEQ ID NO.8:

[0114] CTCGCGGCCCAGCCGGCCATGGCAGATGTGCAGCTGCAGGAGTCTGGRGGAG;

[0115] The nucleotide sequence of primer Camel-Phage-R is shown in SEQ ID NO.9:

[0116] GGTGTTGGCCTCCCGGGCCGCTGGAGACGGTGACCTGGGT.

[0117] 4.4 Enrichment of OX40-positive nanoantibodies

[0118] The hOX40-his protein was biotin labeled. The method of protein biotinylation was referred to the instructions of the biotin reagent. 100 μl of SA magnetic beads were taken and washed three times with 0.05% tween20-PBS (be careful to avoid excessive drying of the magnetic beads).

[0119] Take 100 μl of the phage prepared above (1*10E11 CFU OX40 immune camel phage display library), add hOX40-his (biotin) protein, and incubate at room temperature for 2 hours; take 20 μl SA magnetic beads and add them to the above phage library, mix them by inverting on a rotating instrument, and incubate them at room temperature for 2 hours. Place the magnetic bead-EP tube on a magnetic rack and remove the supernatant; wash 5 times with 0.05% PBS+Tween-20 to wash away non-specific phages; add 100 μl 0.25% trypsin, incubate at 37°C for 20 minutes, dissociate the phages that specifically bind to OX40, and infect Escherichia coli TG1 cells in logarithmic phase growth, culture at 37°C for 1 hour, produce and purify phages for the next round of screening, and repeat the same screening process for 3 rounds to obtain enriched phage clones. From the plate where the titer of the last round of panning eluate was measured, 96 single colonies were randomly picked with a sterilized toothpick and inoculated into a 96-well deep-well plate containing 0.3 mL 2×YT-A, and cultured at 37°C and 220 r / min until the logarithmic growth phase. 0.1 mL of bacterial solution was transferred to a new 96-well plate, which was sealed and placed in a 4°C refrigerator. M13K07 phage was added to the original 96-well plate at a ratio of cell: phage = 1:10, and the plate was left to stand for 15 min at 37°C, and cultured at 220 r / min for 30 min; an equal volume of culture medium was added, and cultured overnight at 30°C with vigorous shaking; the above culture was centrifuged at 4°C and 5000 rpm for 10 min, and the supernatant was collected for ELISA identification.

[0120] 4.5 Preliminary identification of the blocking function of nanoantibodies

[0121] The hOX40L-his protein was labeled using the Biotin labeling kit. The crude lysate of the OX40 nanobody TG1 strain was prepared using the same method as Zhu Min et al., Nanoscale Res Lett., 2014 Sep 26; 9(1): 528; 5×10 6 OX40-NF-κB-JurkaT cells expressing OX40 were resuspended in 0.2% BSA-PBS buffer, and 100 μl of the above crude extract was added. At the same time, a negative control was set up, and hOX40L-his-Biotin with a final concentration of 2 μg / ml was added to each well and incubated at 4°C for 20 min; the cells were washed twice with PBS, and SA-PE fluorescent secondary antibody (Biolegend, catalog number: 405203) was added, incubated at 4°C for 30 min, and the cells were washed twice with PBS and detected by flow cytometry (BD FACS Calibur), and finally 1 nano antibody was obtained. The results are as follows Figure 2 shown.

[0122] Depend on Figure 2(a) It can be seen that the fluorescence signal of control 1 is very weak because OX40L-biotin is not added. Figure 2 (b) It can be seen that in control 2, OX40L-biotin was added, and OX40L-biotin combined with OX40 overexpressing cells (OX40-NF-κB-JurkaT), so the fluorescence signal was stronger; subsequently, 10 OX40 antibody samples were analyzed by flow cytometry (the results are shown in Figure 2 (c)- Figure 2 (l)), the results showed that the fluorescence intensity of OX40-5 sample was significantly weaker than that of other samples, indicating that OX40-5 sample could effectively block the binding of OX40 and OX40L-biotin, thereby screening out a strain of OX40 nanoantibody with good blocking performance.

[0123] Example 5OX40 Nanobody Expression and Preparation

[0124] Through the phage screening of the above example, a Nanobody with blocking activity was obtained, and its amino acid sequence and nucleotide sequence were obtained as follows:

[0125] The amino acid sequence of the heavy chain variable domain (OX40VHH) of the OX40 nanobody of the present invention is shown in SEQ ID NO.10.

[0126] DVQLQESGGGSVQAGGSLRVACEASGYSRGDYCTGWFRQAPGKEREGVAAI

[0127] GTGNPGAAYADSVKGRFTISRDSANNTLYLQMNSLKPDDTAMYYCVAVGPY

[0128] SGCSADYFLREGPAGNAYWGQGTQVTVSS

[0129] The variable domain sequences of the OX40 nanobody of the present invention are shown in Table 1, and the complementarity determining regions (CDRs) based on Kabat numbering are underlined.

[0130] Table 1 Amino acid sequences of the variable domains of the OX40 nanobodies of the present invention

[0131]

[0132] The sequences of the heavy chain complementary determining regions VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable domain of the present invention are respectively: VHCDR1 comprises the sequence GYSRGDYC (SEQ ID NO. 12);

[0133] VHCDR2 comprises the sequence IGTGNPGAA (SEQ ID NO. 13);

[0134] VHCDR3 comprises the sequence VAVGPYSGCSADYFLREGPAGNAY (SEQ ID NO. 14).

[0135] The nucleotide sequence of the heavy chain variable domain (OX40VHH) of the OX40 nanobody of the present invention is shown in SEQ ID NO.11.

[0136] GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGT

[0137] CTCTGAGAGTCGCCTGTGAAGCCTCTGGATACAGTCGCGGTGACTACTGC

[0138] ACGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGTCGCAG

[0139] CGATTGGTACTGGTAATCCTGGCGCAGCCTATGCCGACTCCGTGAAGGGCC

[0140] GATTCACCATCTCCCGAGACAGCGCCAACAACACGCTATATCTGCAAATGA

[0141] ACAGCCTGAAAACCTGACGACACTGCCATGTACTACTGTGTGGCAGTCGGG

[0142] CCGTATAGCGGTTTGTAGTGCTGATTACTTCCTGCGGGAGGGACCTGCCGGC

[0143] AATGCTTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA

[0144] In order to prepare the nanobody, the nanobody sequence gene was fused with the FC gene of human IgG1 to obtain the OX40VHH-FC fusion gene (the amino acid sequence of the corresponding antibody is shown in SEQ ID NO.15). In addition, the nanobody gene was fused with the HIS tag to obtain the OX40VHH-HIS gene (the amino acid sequence of the corresponding antibody is shown in SEQ ID NO.16). The OX40VHH-FC fusion gene and the OX40VHH-HIS gene were cloned into the pcDNA3.4 vector for subsequent antibody expression.

[0145] SEQ ID NO.15 Amino acid sequence of nanobody OX40VHH-FC

[0146] DVQLQESGGGSVQAGGSLRVACEASGYSRGDYCTGWFRQAPGKEREGVAAIGTGNPGAAYADSVKGRFTISRDSANNTLYLQMNSLKPDDTAMYYCVAVGPYSGCSADYFLREGPAGNAYWGQGTQVTVSSGAPGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0147] SEQ ID NO.16 Amino acid sequence of nanobody OX40VHH-HIS

[0148] DVQLQESGGGSVQAGGSLRVACEASGYSRGDYCTGWFRQAPGKEREGV AAIGTGNPGAAYADSVKGRFTISRDSNNTLYLQMNSLKPDDTAMYYCVAVG PYSGCSADYFLREGPAGNAYWGQGTQVTVSSHHHHHHHH*

[0149] pcDNA3.4-OX40VHH-Fc and pcDNA3.4-OX40VHH-his were transiently transfected into HEK293 cells for protein production. The recombinant expression plasmid was diluted with CD05 medium and PEI (polyethyleneimine) solution was added for transformation; each group of plasmid / PEI mixtures were added to the cell suspension and incubated at 37°C, 7% CO2, and 120rpm. After 6 days, the culture supernatant was collected and purified by ProteinA affinity chromatography to obtain OX40VHH-Fc; the OX40VHH-HIS protein sample was purified by nickel column for OX40 nanoantibody activity identification. The obtained protein sample was subjected to preliminary SDS-PAGE detection. The results are shown in Figure 3. The target band can be clearly seen, indicating that the OX40 antibody proteins OX40VHH-FC and OX40VHH-HIS are both successfully expressed.

[0150] Example 6OX40 Nanobody Activity Detection

[0151] 6.1 ELISA to identify the specificity of OX40 nanoantibodies

[0152] ① Dilute the OX40L-HIS prepared in the above example to 1 μg / ml, coat the plate with 100 μl / well, incubate at 4°C overnight, wash the plate three times with PBS containing 0.1% Tween20, and block the plate with PBS containing 0.1% Tween20 and 1% BSA (200 μl / well) at 4°C overnight.

[0153] ② Wash the plate three times with PBS containing 0.1% Tween20.

[0154] ③ Add 100 μl of OX4OVHH-FC samples of different concentrations (the highest sample concentration was 20 μg / ml, 3-fold dilution, a total of 11 concentrations), and incubate at room temperature for 2 hours.

[0155] ④ Wash the plate three times with PBS containing 0.1% Tween 20, dilute Antihuman IgG-HRP secondary antibody at 1:5000, add 100 μl to each well, and incubate at room temperature for 1 hour.

[0156] ⑤ Wash the plate three times with PBS containing 0.1% Tween20, add 100μl TMB to each well for color development for 15 minutes. ⑥ Add 50μl Stopsolution to each well to stop color development, read the absorbance at 450nm, and the results are as follows: Figure 4 As shown, the experimental results showed that the OX40VHH-FC antibody could significantly bind to the OX40L protein, with an EC50 value of 0.005492 μg / ml.

[0157] 6.2 ELISA to identify the blocking activity of OX40 nanoantibodies

[0158] ① Dilute OX40-HIS to 1 μg / ml, coat the plate with 100 μl / well, incubate at 4°C overnight, and wash the plate three times with PBS containing 0.1% Tween20.

[0159] ② Block the plate with PBS containing 0.1% Tween20 and 1% BSA (200 μl / well) at 4°C overnight, and wash the plate three times with PBS containing 0.1% Tween20.

[0160] ③ Add 50 μl of OX40VHH-HIS samples of different concentrations (the highest sample concentration was 20 μg / ml, 3-fold dilution, a total of 11 concentrations) and 50 μl of 3 μg / ml OX40L-HIS biotin protein, and incubate at room temperature for 2 hours.

[0161] ④ Wash the plate three times with PBS containing 0.1% Tween20, dilute streptavidin-HRP 1:5000, add 100 μl to each well, and incubate at room temperature for 1 hour.

[0162] ⑤ Wash the plate three times with PBS containing 0.1% Tween 20, and add 100 μl TMB to each well for color development for 15 minutes.

[0163] ⑥ Add 50 μl Stopsolution to each well to stop color development and read the absorbance at 450 nm. The experimental results are as follows: Figure 5 shown.

[0164] The experimental results showed that OX40VHH-HIS antibody could significantly inhibit the binding of OX40 protein and OX40L protein, with an EC50 of 0.08137 μg / ml.

[0165] 6.3 Flow cytometry identification of OX40 nanoantibody specificity

[0166] ① Wash OX40-NF-κB-JurkaT cells twice with FACS buffer (PBS solution containing 0.5% BSA), and then incubate the cells at 2*10 6 Resuspend in FACS buffer at a concentration of 10 cells / ml.

[0167] ②Take 100μl solution (2*10 5 cells) were transferred to a test tube, 100 μl of OX40VHH-FC antibody samples of different concentrations were added (the highest concentration of the sample was 20 μg / ml, 3-fold dilution, a total of 8 concentrations), and incubated at 4°C for 60 minutes.

[0168] ③ Add 1 mL of FACS buffer, mix gently with a vortex mixer, centrifuge at 3500 rpm for 4 minutes, aspirate and discard the supernatant.

[0169] ④Repeat step ③.

[0170] ⑤ Resuspend the pellet in 100 μl LFACS buffer and incubate with 10 μg / ml secondary antibody (Antihuman IgG-APC) at 4°C for 60 min.

[0171] ⑥Add 1 mL of FACS buffer, mix gently with a vortex mixer, and centrifuge at 3500 rpm for 4 minutes. Aspirate and discard the supernatant.

[0172] ⑦Repeat step ⑥.

[0173] ⑧Resuspend the pellet in 200 μl FACS buffer and analyze on a flow cytometer.

[0174] The experimental results show that the OX40 antibody prepared by the present invention can specifically bind to JurkaT cells that overexpress OX40 molecules, while the control antibody (Isotype antibody) cannot bind. As the concentration of OX40 antibody increases (the horizontal axis value), the signal of antibody binding to cells gradually increases (the vertical axis value). When the antibody concentration reaches a certain concentration, the binding of the antibody to the cells reaches saturation and the value is stable, which proves that the antibody has good specificity.

[0175] 6.4 Flow cytometry identification of OX40 nanobody blocking activity

[0176] ① Wash JurkaT cells twice with FACS buffer (PBS solution containing 0.5% BSA), and then 6 Resuspend in FACS buffer at a concentration of 10 cells / ml.

[0177] ② 100 μl solution (2*10 5 Cells were transferred into test tubes, and 50 μl of OX40VHH-FC antibody samples of different concentrations (the highest concentration of the sample was 20 μg / ml, 3-fold dilution, a total of 11 concentrations) and 50 μl of 3 μg / ml OX40L-HIS biotin protein were added, and incubated at 4°C for 60 minutes.

[0178] ③ Add 1 mL of FACS buffer, mix gently with a vortex mixer, centrifuge at 3500 rpm for 4 minutes, aspirate and discard the supernatant.

[0179] ④Repeat step ③.

[0180] ⑤ Resuspend the pellet in 100 μl FACS buffer and incubate with 10 μg / ml secondary antibody (streptavidin-PE) at 4°C for 60 minutes.

[0181] ⑥Add 1 mL of FACS buffer, mix gently with a vortex mixer, centrifuge at 3500 rpm for 4 minutes, aspirate and discard the supernatant.

[0182] ⑦Repeat step ⑥.

[0183] ⑧Resuspend the pellet in 200 μl FACS buffer and analyze on a flow cytometer.

[0184] The experimental results are as follows Figure 7 As shown in the results, it can be seen that as the added concentration of the OX40 antibody prepared in this example gradually increases, the fluorescence level decreases, indicating that the OX40 antibody of this example can effectively bind to OX40 and has a good blocking effect on OX40L and OX40. The fluorescence intensity of the control antibody (Isotype antibody) always remains at a high level, indicating that the fluorescence intensity of OX40L remains high, indicating that OX40L binds to OX40 and the control antibody has no blocking effect.

[0185] The OX40 antibody prepared by the present invention can block the binding of OX40L molecules to OX40 overexpressing cell lines. As the antibody concentration increases (the horizontal axis is the antibody concentration), the binding strength of OX40L protein to OX40 overexpressing cell lines gradually decreases (the vertical axis is the MFI value) and is inhibited by the OX40 antibody, which shows that the OX40 antibody prepared by the present invention has a good effect of blocking the binding of OX40L molecules to OX40.

[0186] 6.5 Identification of OX40 Nanobody Blocking Activity by Reporter Cells

[0187] Mix OX40-NF-κB-JurkaT cells and 293-OX40L cells and seed them into 96-well culture plates, 30,000 cells per well. Add samples, the highest concentration of each sample is 20μg / mL, 3-fold dilution, 8 concentrations. After 6 hours of cell culture, take out the cell culture plate to be tested from the incubator and place it at room temperature for 30 minutes to allow the culture plate temperature to equilibrate to room temperature. Add One-Lite detection reagent (brand: Nanjing Novozyme, item number: DD1203-01) with the same volume as the cell culture to be tested and equilibrated to room temperature, and draw 100μl One-Lite detection reagent into 100μl cell culture to be tested. Place at room temperature for at least 3 minutes to allow the cells to fully lyse, and place them on a chemiluminescent microplate reader to detect Luciferase signals.

[0188] When OX40-NF-κB-JurkaT cells and 293-OX40L cells are co-cultured, the OX40L molecules and OX40 molecules interact with each other, which can induce Jurkat cells to express luciferase enzymes. After adding the substrate, the chemiluminescent signal (vertical coordinate value RLU) can be detected by a chemiluminescent microplate reader. The experimental results are shown in Figure 2. Figure 8 As shown in the figure, it can be seen that when the OX40 antibody VHH-HIS is added to the culture system, as the concentration of the OX40 antibody increases, the vertical coordinate RLU value is significantly inhibited, indicating that the OX40 antibody effectively blocks the interaction between the OX40L molecule and the OX40 molecule, thereby blocking the expression of the luciferase enzyme. Therefore, the experimental results once again confirm that the OX40 VHH antibody prepared by the present invention can bring strong blocking activity to the interaction between the OX40 / OX40L molecules.

[0189] Therefore, both the flow cytometry results and the reporter cell results indicate that the OX40 antibody prepared by the present invention can significantly inhibit the binding of OX40 and OX40L, and can be used in drugs that block the binding of OX40 and OX40L.

[0190] The above implementation modes are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An OX40 nanobody, characterized in that: comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises heavy chain complementary determining regions VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO.12-14, VHCDR1 comprises the sequence GYSRGDYC (SEQ ID NO. 12); VHCDR2 comprises the sequence IGTGNPGAA (SEQ ID NO. 13); VHCDR3 comprises the sequence VAVGPYSGCSADYFLREGPAGNAY (SEQ ID NO. 14).

2. The OX40 nanobody according to claim 1, characterized in that: The heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO.

10.

3. A nucleic acid molecule encoding the OX40 nanobody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid encoding the heavy chain of the nanobody is the nucleic acid shown in SEQ ID NO.

11.

5. A vector containing the nucleic acid molecule according to claim 3 or 4.

6. A fusion or conjugate comprising the isolated antibody of claim 1 or 2.

7. An expression cassette or recombinant bacterium comprising the nucleic acid molecule according to claim 3 or 4.

8. A composition comprising at least one of the isolated antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, or the fusion or conjugate of claim 5, and at least one of their corresponding pharmaceutically acceptable carriers.

9. A method for detecting OX40 in a biological sample comprising contacting the biological sample with the isolated antibody of claim 1 or 2 or the fusion or conjugate of claim 5.

10. Use of the antibody or antigen-binding fragment of claim 1 or 2, the fusion or conjugate of claim 6, or the composition of claim 8 in the preparation of a pharmaceutical preparation for blocking the binding of OX40L to OX40 or in a pharmaceutical preparation for treating or delaying cancer; or in the preparation of a pharmaceutical preparation for treating or delaying autoimmune inflammatory diseases such as atopic dermatitis and rheumatoid arthritis.

11. The method for preparing the OX40 nanobody according to claim 1 or 2, characterized in that: The steps include: Preparation of recombinant OX40 antigen: using the amino acid sequence of human OX40 from the Uniprot protein database, the human OX40 sequence is shown in SEQ ID NO.1; The screening steps of OX40 nanoantibodies include: animal immunization, extraction of OX40 nanoantibody RNA, construction of OX40 nanoantibody phage library and enrichment of OX40 positive nanoantibodies. The immunized animal used in the animal immunization step is a camel, In the step of constructing the phage library, two sets of primers are included: CALL001 / CALL002 are used as primers, the first round of PCR amplification is performed with the cDNA template, and Camel-Phage-F / Camel-Phage-R is used for the second round of PCR amplification; OX40 nanobody expression and preparation.

Images