Anti-trail nanobodies and uses thereof

Abstract

This invention belongs to the field of biomedicine and relates to nanobodies. The amino acid sequence of the anti-trail nanobody is shown in SEQ ID No. 1. It can also be a tandem antibody composed of two or more repeating anti-trail nanobodies. The nanobody has a small molecular weight, strong tissue penetration, and high affinity. The anti-trail nanobody of this invention has good binding affinity to human trail protein, and it also exhibits good trail-DR5 pathway blocking activity, significantly reducing cerebral ischemia-reperfusion injury after cerebral infarction, and has broad clinical application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a nanobody. BACKGROUND

[0002] Cerebral infarction is mainly a disease of brain tissue ischemia and anoxic necrosis caused by cerebral blood supply disorder, and nerve function defect. The morbidity, disability rate and mortality of cerebral infarction are extremely high. There are about 12 million patients with cerebral infarction in China at present, and the number of people dying of cerebral infarction is close to 2 million per year, which is the "No. 1 killer" threatening healthy life. Restoring cerebral blood perfusion as soon as possible through intravenous thrombolysis and mechanical thrombectomy is the best treatment measure for the disease, but reperfusion will cause rapid cascade reaction damage of neurons, induce apoptosis or necrosis, and the pathological damage of ischemic tissue will be further aggravated, which is called cerebral ischemia / reperfusion injury (IRI). How to reduce cerebral IRI and develop suitable therapeutic drugs are problems to be solved in clinical practice.

[0003] The pathological mechanism of cerebral IRI is very complex, mainly involving energy metabolism disorder, excitotoxicity, Ca 2+ overload, oxidative stress, apoptosis, inflammatory response and blood-brain barrier damage. TRAIL protein is an important apoptosis-inducing molecule in the human body. In addition to inducing cell apoptosis, TRAIL protein also directly participates in the process of inflammatory response when combined with DR5 receptor, for example, the short TRAIL antibody and use method disclosed in the application with publication number CN110494152A. It is found that TRAIL protein is not expressed in normal brain tissue, but the expression of TRAIL protein in astrocytes and activated microglia is up-regulated during cerebral ischemia / reperfusion in mice. At the same time, DR5 is up-regulated in neurons, and has a good correlation with the distribution of apoptotic cells. The use of soluble DR5 protein, anti-TRAIL monoclonal antibody CD253 or sDR5-Fc fusion protein can reduce neuronal damage by blocking the interaction between TRAIL and DR5 through intracerebroventricular pathway. Therefore, blocking the TRAIL-DR5 pathway in the cerebral ischemic penumbra after cerebral infarction may be an ideal strategy for treating cerebral IRI.

[0004] Blood-Brain Barrier (BBB) is a dynamic interface between the central nervous system and the blood circulation system, which is mainly composed of brain microvascular endothelial cells, pericytes, astrocytes and basement membrane tight junction, strictly regulates the material transport between blood and brain, and provides stable internal environment for the realization of neural function. Studies have shown that the BBB permeability of animal models of cerebral ischemia and clinical stroke patients is significantly increased, but the anti-TRAIL monoclonal antibody CD253, soluble DR5 protein and sDR5-Fc fusion protein prepared by the laboratory in the early stage cannot penetrate the blood brain barrier and still need to be injected intracranially, which limits its clinical application. SUMMARY

[0005] The application provides an anti-TRAIL nanobody and a preparation method thereof, which solves the problem that conventional monoclonal antibodies cannot penetrate the blood brain barrier to reduce ischemia-reperfusion injury after cerebral infarction.

[0006] The technical scheme of the application is as follows:

[0007] The primary purpose of the application is to provide a nanobody against Tumornecrosis factor-related apoptosis inducing ligand (TRAIL), and the amino acid sequence of the anti-TRAIL nanobody is selected from any one of the following: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10.

[0008] The second purpose of the application is to provide an antibody, which comprises one or more of the anti-TRAIL nanobodies, and the antibody comprises two or more nanobody units in series, and the amino acid sequence of the nanobody unit is shown in any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10.

[0009] A third object of the present application is to provide a gene fragment encoding the anti-TRAIL nanobody, the nucleotide sequence of which is shown in any one of SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20.

[0010] A fourth object of the present application is to provide an expression vector containing the gene fragment.

[0011] A fifth object of the present application is to provide a host cell containing the expression vector, or having the gene fragment integrated into its genome.

[0012] A sixth object of the present application is to provide an immunoconjugate containing:

[0013] (a) the above-mentioned nanobody or antibody; and / or (b) a conjugating moiety selected from any one of the following: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle, a nanorod, a nanomagnetic particle and a viral coat protein.

[0014] A seventh object of the present application is to provide a pharmaceutical composition comprising:

[0015] (i) the above-mentioned anti-TRAIL nanobody, or the above-mentioned antibody, or the above-mentioned immunoconjugate; and

[0016] (ii) a pharmaceutically acceptable carrier.

[0017] An eighth object of the present application is to provide a method for preparing the anti-TRAIL nanobody, which comprises transfecting a competent cell with an expression vector containing the encoding gene, and culturing the host cell to obtain the anti-TRAIL nanobody.

[0018] A ninth object of the present application is to provide the use of the above-mentioned nanobody or the above-mentioned antibody in the development of a therapeutic drug for acute cerebral infarction.

[0019] The present application has the following advantages:

[0020] The application provides an anti-TRAIL nanobody, the amino acid sequence of the anti-TRAIL nanobody is shown in any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10, the molecular weight of the nanobody is small, about 15 kDa, the affinity is high, the tissue penetration is strong, and the nanobody has the function of blocking the combination of human TRAIL protein and death receptor 5 (Death receptor 5, DR5). The nanobody can reduce cerebral ischemia-reperfusion injury after cerebral infarction after intravenous injection, and has a broad clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0022] Figure 1 It is an electrophoresis map of phagemid vector; wherein A is a total RNA extraction gel running map of peripheral blood lymphocytes separated from blood of alpaca after five immunizations; B is a first round of nested PCR electrophoresis map; C is a second round of nested PCR electrophoresis map, lanes 1 and 2 are IgG2c and IgG2b subtype antibody gene fragments respectively; D is an electrophoresis map before and after enzyme digestion of VHH gene, lanes 1 and 3 are IgG2c and IgG2b subtype antibody gene fragments respectively, and lanes 2 and 4 are IgG2c and IgG2b subtype antibody gene fragments after enzyme digestion respectively; E is a pComb3XSS vector electrophoresis map, lanes 1, 2 and 3 are pComb3XSS vector after enzyme digestion, pComb3XSS vector after ligation and original pComb3XSS vector respectively.

[0023] Figure 2 It is a library diversity and capacity determination after electroporation; wherein A is a library transformant number determination plate; B is a library target gene insertion rate determination bacterial liquid PCR electrophoresis map; C is a single clone bacterial liquid VHH amino acid sequence alignment result.

[0024] Figure 3 It is a phage clone ELISA color development result.

[0025] Figure 4 It is an SDS-PAGE gel running map of prokaryotic expression of different TRAIL nanobodies.

[0026] Figure 5 Jurkat cell viability test results after TRAIL protein and TRAIL nanobody treatment.

[0027] Figure 6 pComb3XSS-Nb19 plasmid map.

[0028] Figure 7 Protective effect of TRAIL nanobody Nb19 in a mouse cerebral ischemia-reperfusion model; A is TTC staining of mouse brain; B is the comparison result of mouse brain tissue infarction rate; C is the comparison result of mouse mNSS. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0030] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained through commercial channels unless otherwise specified.

[0031] In the technical solutions claimed in the present application, the amino acid sequence of the TRAIL nanobody is as shown in any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9, and the nucleotide sequence encoding the antibody amino acid is as shown in any one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No. 18, respectively.

[0032] In the following examples, the 1 L 2YT medium contains 16 g of proteose peptone, 10 g of yeast extract, and 5 g of sodium chloride.

[0033] Example 1: Construction of TRAIL nanobody library

[0034] (1) 0.2 mg human TRAIL antigen (purchased from Suzhou Jinan Protein Technology Co., Ltd.) was mixed with an equal volume of Freund's complete adjuvant (priming) / incomplete adjuvant (purchased from Sigma Company, USA) and stirred and emulsified at 4°C overnight, and then subcutaneously injected into the neck of a two-year-old male alpaca (purchased from Shenghong Breeding Farm in Jiuxiang County, Shandong Province) to stimulate B cells to express nanobodies specific to the antigen;

[0035] (2) One week after the fifth immunization, the alpaca was bled 100 mL from the jugular vein three days later, and peripheral blood lymphocytes were separated and total RNA was extracted, and reverse transcribed into cDNA;

[0036] (3) The variable domain of heavy chain of heavy-chain antibody (VHH) gene was amplified by nested PCR;

[0037] (4) 10 μg pComb3XSS phage display vector (purchased from Biovector) and 4 μg VHH were digested by restriction endonuclease Sfi I (purchased from NEB Company, Beijing) and ligated by T4 ligase (purchased from NEB Company, Beijing) to construct phagemid;

[0038] (5) The ligation product was electroporated into TG1 competent cells (purchased from Lucigen Company, USA), gradient diluted and plated, and the library size was measured to be 3.28×10 9 pfu, and 48 clones were randomly picked for colony PCR detection.

[0039] The variable domain of heavy chain of heavy-chain antibody gene is as shown in Figure 1 The nanobody gene insertion is as shown in Figure 2 It can be seen that the nanobody gene insertion rate is 100%. After the bacteriophage library was infected by helper phage and shaken overnight, the phage display TRAIL nanobody library was obtained by PEG precipitation.

[0040] Example 2: Screening and identification of TRAIL nanobodies

[0041] Antibody screening:

[0042] (1) 100 μL of 10 μg / mL human TRAIL antigen was coated on an enzyme-labeled plate (purchased from Corning Company, USA) in 4 wells at 4°C overnight;

[0043] (2) After washing 4 times with PBST (PBS + 0.05% Tween-20), 300 μL of 5% skimmed milk powder was added and blocked at 37°C for 1 h;

[0044] (3) After washing the plate, 100 μL of phage library (2 x 10 11 cfu) was added to each well, and the plate was shaken at room temperature for 2 h;

[0045] (4) The plate was washed with PBST for 10 times to remove the non-specifically bound phages;

[0046] (5) 100 μL of 10 μg / mL sDR5-Fc antibody fusion protein (self-made in the laboratory) was added to the enzyme-labeled wells, and the plate was shaken for 2 h. The supernatant was collected and served as the output of this round. The output was used to infect TG1 E. coli (purchased from Beijing Huayueyang Biological Company), and the helper phage was allowed to infect overnight after shaking culture. The phage was precipitated by PEG and served as the input of the next round. The same screening process was repeated for 4 rounds, and the screening pressure was increased during the process. The antigen coating concentrations of the second, third and fourth rounds were 5, 2 and 1 μg / mL, respectively.

[0047] Identification of positive phage clones:

[0048] (1) A total of 192 single colonies were picked from the four-round output titer plates and inoculated into 1 mL of 2YT medium containing 100 μg / mL ampicillin. The medium was shaken at 37°C and 250 rpm until the OD 600 was about 0.6. After the helper phage was allowed to infect at room temperature, kanamycin was added and the medium was shaken overnight. The next day, the medium was centrifuged at 4°C and 10,000 rpm for 15 min, and the supernatant was used for phage enzyme-linked immunosorbent assay (ELISA) detection;

[0049] (2) 5 μg / mL human TRAIL protein and BSA protein were coated overnight at 4°C. 50 μL of sample diluent and 5 μg / mL sDR5-Fc antibody fusion protein were added to two TRAIL protein-coated wells, respectively. 50 μL of sample diluent was added to one BSA protein-coated well. Then, 50 μL of single colony phage supernatant was added to each of the three wells, and the plate was incubated at 37°C for 30 min. After washing the plate, 100 μL of phage secondary antibody (purchased from Chengdu Apark Company) was added, and the plate was reacted at 37°C for 30 min. After washing the plate, 100 μL of TMB color developing solution (purchased from Solabio Biological Technology Co., Ltd.) was added, and the plate was developed at room temperature for 10 min. After adding 50 μL of 1 M HCl to terminate the reaction, the plate was read at 450 nm;

[0050] (3) When the PBS well OD value is higher than the sDR5-Fc well OD value, and more than 3 times of the BSA well OD value, the monoclonal colony is determined as a positive clone. All the positive clone bacteria are sequenced and analyzed, and 10 different nanobody amino acid sequences are obtained, numbered as 16, 18, 19, 76, 119, 146, 147, 151, 155 and 175, and the results are shown in Table 1. Figure 3

[0051] Example 3: Expression and purification of TRAIL nanobody

[0052] (1) The positive clone strain is coated on a 2YT culture plate containing 100 μg / mL ampicillin, and incubated at 37°C overnight;

[0053] (2) A single colony is selected and inoculated in 3 mL of 2YT culture solution containing 100 μg / mL ampicillin and 0.05% glucose, and incubated at 37°C, 250 rpm overnight;

[0054] (3) 1 mL of the overnight seed is inoculated into 500 mL of 2YT culture solution containing 100 μg / mL ampicillin and 0.05% glucose, and incubated until the OD value reaches 0.6-0.8, then IPTG (purchased from Solabio Biotechnology Co., Ltd.) is added, and incubated at 30°C, 220 rpm overnight; 600

[0055] (4) The bacteria are centrifuged, and the crude antibody solution is obtained by ultrasonic bacterial disruption method;

[0056] ​​(5) The nickel column ion affinity chromatography is used to prepare nanobodies Nb16 (amino acid sequence as shown in SEQ ID No. 1, nucleotide sequence as shown in SEQ ID No. 11), Nb18 (amino acid sequence as shown in SEQ ID No. 2, nucleotide sequence as shown in SEQ ID No. 12), Nb19 (amino acid sequence as shown in SEQ ID No. 3, nucleotide sequence as shown in SEQ ID No. 13), Nb76 (amino acid sequence as shown in SEQ ID No. 4, nucleotide sequence as shown in SEQ ID No. 14), Nb119 (amino acid sequence as shown in SEQ ID No. 5, nucleotide sequence as shown in SEQ ID No. 15), Nb146 (amino acid sequence as shown in SEQ ID No. 6, nucleotide sequence as shown in SEQ ID No. 16), Nb147 (amino acid sequence as shown in SEQ ID No. 7, nucleotide sequence as shown in SEQ ID No. 17), Nb151 (amino acid sequence as shown in SEQ ID No. 8, nucleotide sequence as shown in SEQ ID No. 18), Nb155 (amino acid sequence as shown in SEQ ID No. 9, nucleotide sequence as shown in SEQ ID No. 19) and Nb175 (amino acid sequence as shown in SEQ ID No. 10, nucleotide sequence as shown in SEQ ID No. 20) with purity of more than 95%, and the purification results are shown in Figure 4 .

[0057] Example 4: Cell viability assay (MTS) for detecting the blocking function of TRAIL nanobodies

[0058] (1) 20 μL of 1 μg / mL human TRAIL protein is added to a 96-well cell culture plate, and then an equal volume of cell culture solution or different concentrations of TRAIL nanobodies are added, and the control group is directly added with 40 μL of cell culture solution;

[0059] (2) 1.25×10 5 Jurkat cells (purchased from ATCC) are added to each cell culture well, and the number of cells added to each well is 60 μL / well. The cells are placed in a 37°C, 5% CO2 incubator for 24 hours;

[0060] (3) 10 μL of MTS is added to each well, and after 3 hours of continuous culture, the absorbance value is detected at 490 nm, and the cell viability calculation formula is: cell viability (%) = (OD 对照组 - OD 实验组 ) / OD 对照组 × 100%.

[0061] The determination results of cell viability are shown in Figure 5As shown, 10 strains of TRAIL nanobodies can block the TRAIL-DR5 pathway to inhibit cell apoptosis, among which Nb19 is the best, and 5 μg / mL of Nb19 can increase the cell viability of 16% treated by TRAIL protein alone to 96%, and the TRAIL-DR5 pathway blocking effect is the best, so Nb19 is used in the following examples.

[0062] The pComb3XSS-Nb19 plasmid map is shown in Figure 6 .

[0063] Example 5: TRAIL nanobody affinity assay

[0064] Using Sartorius Octet® R4 system to detect:

[0065] (1) The HIS1K sensor (purchased from Sartorius Company in Germany) was pre-wetted in PBST buffer for 10 min, and then Nb19 was solidified for 180 s, and the solidification concentration was 10 ug / ml;

[0066] (2) Human TRAIL protein and mouse TRAIL protein (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) were respectively gradient diluted to 500 nM, 250 nM, 125 nM, 62.5 nM and 0 nM and added to the sample hole, and the binding and dissociation time was set to 180 s;

[0067] (3) When determining different concentrations, 10 mM Glycine (purchased from Sigma Company in the United States) was used to flush the sensor for regeneration;

[0068] (4) The equilibrium dissociation constant (K D ) was calculated by real-time monitoring of the thickness of the biological membrane layer, and the smaller the K D value, the stronger the affinity, and the calculation formula is: K D =Kd / Ka.

[0069] The determination results are shown in Table 1:

[0070] Table 1: Affinity assay results of Nb19 and mouse and human TRAIL proteins

[0071]

[0072] From Table 1, it can be seen that the binding affinity of nanobody Nb19 to human TRAIL protein is higher than that to mouse TRAIL protein, which is 2.4×10 -9 M and 2.425×10 -7 M, respectively.

[0073] Example 6: Study on the protective effect of TRAIL nanobody on cerebral ischemia-reperfusion injury

[0074] (1) Construct a mouse middle cerebral artery occlusion / reperfusion (MCAO / R) model, select 6-8 weeks, body weight 23 g or so C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), intraperitoneal injection of sodium pentobarbital anesthesia, use a feedback heating pad to monitor the body temperature of the mouse and maintain at 36.5-37.5℃. Dissect and expose the left common carotid artery, internal carotid artery and external carotid artery of the mouse. The distal end of the external carotid artery and the proximal end of the common carotid artery are ligated, and then the nylon thread plug is inserted into the anterior cerebral artery through the oblique opening of the external carotid artery to block the blood flow of the middle cerebral artery. After occluding the middle artery for 2 hours, pull out the nylon thread plug for reperfusion. 5 min before reperfusion, inject PBS, 30 mg / kg Nb19 or 90 mg / kg Nb19 into the mouse body through the tail vein, 200 μL per injection, 5 in each group.

[0075] (2) TTC staining. After 24 hours of reperfusion, the mouse brain was taken out, quickly frozen and cut into thin slices, and the slices were placed in a preheated 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) solution for 30 min, washed with PBS, and placed with the front face up and photographed. Finally, calculate the infarction rate using Image J software.

[0076] (3) mNSS. After 24 h of reperfusion, evaluate the motor, sensory, reflex and balance ability, and the maximum defect score is 18, the higher the score, the more serious the neurological dysfunction.

[0077] As Figure 7 shown, 30 and 90 mg / kg Nb19 reduced the MCAO / R model mouse brain tissue infarction rate by 63% and 82%, respectively, and the mNSS value was significantly reduced. The experimental results show that Nb19 plays a protective role on cerebral ischemia-reperfusion injury.

[0078] In summary, the present application provides an anti-TRAIL nanobody, which has a small molecular weight and strong tissue penetration. The anti-TRAIL nanobody has good binding affinity to human TRAIL protein, and the anti-TRAIL nanobody has good TRAIL-DR5 pathway blocking activity, can significantly reduce ischemia-reperfusion injury after cerebral infarction, and has a broad clinical application prospect.

[0079] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. An anti-TRAIL nanobody, characterized in that, The amino acid sequence of the anti-TRAIL nanobody is shown in SEQ ID No.

3.

2. The gene encoding the anti-TRAIL nanobody of claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

13.

3. An expression vector, characterized in that: The expression vector contains the gene as described in claim 2.

4. A host cell, characterized in that: The host cell contains the expression vector of claim 3 or the gene of claim 2 integrated into its genome.

5. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises: (1) The anti-TRAIL nanobody according to claim 1; (2) The anti-TRAIL nanobody of claim 1 and its pharmaceutically acceptable carrier.

6. A method for preparing anti-TRAIL nanobodies, characterized in that: By culturing the host cells described in claim 4 and purifying their expression products, anti-TRAIL nanobodies can be obtained.

7. The use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for the treatment of acute cerebral infarction.

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