Nanometer antibody conjugate as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a nano antibody conjugate as well as a preparation method and application thereof. The nano antibody conjugate comprises coupled antibody fusion protein and bortezomib; the antibody fusion protein is a fusion protein of elastin-like polypeptide and an anti-Her2 nano antibody. The nanometer antibody conjugate can improve the stability and enzyme selectivity of bortezomib and reduce the toxic and side effects of drugs, meanwhile, the tumor targeting effect is achieved by combining a tumor specific nanometer antibody targeting technology, and the nanometer antibody conjugate can be used for treating Her2 high-expression cancers and has excellent application and popularization value and potential.

Description

Technical Field

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

[0002] Bortezomib is currently the first-line drug for the treatment of multiple myeloma, but it has the disadvantages of large toxic side effects, a small treatment window, and poor effect on solid tumors. It has the following main characteristics: the stability of this type of compound is poor, the polypeptide mother nucleus is easily degraded by various proteases in the body, and the carbon-boron bond is easily oxidized and broken; in addition, its metabolic stability in the blood is poor, resulting in it often being degraded before it has time to enter tissues and organs, so this type of protease inhibitor is usually not effective in vivo against solid tumors. Second, the enzyme selectivity of this type of compound is low, and it is easy to bind to other enzymes or proteins after entering the body. In addition, it lacks selectivity for tumor cells and normal cells, resulting in off-target and various toxic side effects, including thrombocytopenia, anemia, unknown pain, febrile neutropenia and other side effects.

[0003] How to improve the stability and enzyme selectivity of bortezomib while achieving tumor targeting effect and reducing the toxic side effects of the drug has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0004] In order to solve the above technical problems, the present invention is proposed.

[0005] First, the present invention provides a nano-antibody conjugate, comprising a conjugated antibody fusion protein and bortezomib; The antibody fusion protein is a fusion protein of a nanobody and an elastin-like polypeptide (ELP) sequentially connected from the N-terminus to the C-terminus; The nanobody is an anti-Her2 nanobody; The amino acid sequence of the elastin-like polypeptide is (XPGVG)n, wherein X is any natural amino acid except proline, and 30≤n≤150.

[0006] The present invention achieves the effect of bortezomib targeting tumors through anti-Her2 nano antibodies, and at the same time utilizes the characteristics of elastin-like polypeptides to help the small molecule compound bortezomib form a sustained release effect in vivo, thereby reducing the toxic and side effects of the drug. Among them, the present invention has conducted a large number of design and screening of the sequences of elastin-like polypeptides, and found that the above-mentioned elastin-like polypeptides and anti-Her2 nano antibodies can produce a good coordination effect, and the above-mentioned elastin-like polypeptides can ensure the efficient expression of nano antibody proteins in the carrier, and the connected elastin-like polypeptides will not interfere with the targeting of the nano antibody, ensuring that the nano antibody conjugate has a high targeting antigen activity.

[0007] This nano-antibody conjugate can target the Her2 antigen. After subcutaneous injection, it will undergo a phase change in situ under the triggering of body temperature and precipitate to form a drug reservoir, which helps to increase the safe dose of a single injection of the drug, improve the efficiency of treatment, reduce toxic side effects, significantly inhibit tumor cell proliferation, have a good anti-tumor effect, and improve patient compliance.

[0008] Preferably, the nanobody is an anti-human Her2 nanobody.

[0009] Preferably, the amino acid sequence of the anti-human Her2 Nanobody is as shown in SEQ ID NO.1.

[0010] The anti-human Her2 nanobody of the above amino acid sequence cannot be expressed normally in the protein expression vector. When the nanobody of the above amino acid sequence is used in the art, it is usually optimized and modified to increase its expression amount. However, the present invention unexpectedly found that when the elastin-like polypeptide amino acid sequence of the present invention and the nanobody sequence are expressed in the same vector, the elastin-like polypeptide can greatly promote the expression of the nanobody protein.

[0011] Preferably, X is any one selected from valine, phenylalanine, tryptophan, tyrosine, alanine, glycine, methionine, threonine, serine, leucine, and isoleucine.

[0012] Preferably, in the elastin-like polypeptide, X is valine, and 60≤n≤120.

[0013] More preferably, in the elastin-like polypeptide, X is valine and n is 90.

[0014] The use of the above-mentioned elastin-like polypeptide sequence can better promote the expression of nanoantibodies, and while exerting an excellent sustained-release effect, it can also significantly improve the expression level of nanoantibodies. In addition, the fusion protein also has higher antigen targeting activity.

[0015] Preferably, the antibody fusion protein is constructed by expressing the coding genes of the nanobody and the elastin-like polypeptide into a protein expression vector and obtained by protein expression.

[0016] Furthermore, the present invention provides a method for preparing the nanobody conjugate, comprising: chemically modifying the bortezomib with a five-membered carbocyclic diol protecting group, coupling it to the antibody fusion protein through a maleimide reaction, and connecting the chemically modified bortezomib to the C-terminus of the nanobody.

[0017] Preferably, the five-membered carbocyclic diol protecting group is a cis-1,2-five-membered (hetero)cyclic diol protecting group, and the connecting arm is an ethylenediamine-diamide structure.

[0018] Preferably, the preparation method comprises: using 3-cyclopentene-1-carboxylic acid as a starting material, esterifying it with a methanol / thionyl chloride system, and then using K2OsO4·2H2O as a catalyst to oxidize it to 1-(3-cyclopentenyl)formic acid methyl ester; subsequently removing the methyl ester protecting group through a saponification reaction to obtain 1-(3-cyclopentenyl)formic acid, and subjecting 1-(3-cyclopentenyl)formic acid and N-(2-aminoethyl)maleimide hydrochloride to an amide condensation reaction to obtain an intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine; and finally subjecting the intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine to an esterification reaction with bortezomib in dichloromethane to obtain chemically modified bortezomib.

[0019] The structural formula of chemically modified bortezomib is: .

[0020] The present invention studies the properties of bortezomib and chemically modifies the boronic acid group with a five-membered carbocyclic diol protecting group, thereby improving its stability and enzyme selectivity. Meanwhile, the chemically modified bortezomib can react with the antibody fusion protein and be coupled to the C-terminus of the nanobody through a maleimide reaction.

[0021] Preferably, the chemically modified bortezomib and the antibody fusion protein are subjected to a maleimide reaction in a DMSO solution to obtain the nanobody conjugate.

[0022] Furthermore, the present invention provides a pharmaceutical composition, which contains the nanobody conjugate described, or the nanobody conjugate prepared by the preparation method described.

[0023] Furthermore, the present invention provides the use of the nanobody conjugate or the nanobody conjugate prepared by the preparation method in the preparation of drugs.

[0024] Preferably, the medicament is used for treating tumors or cancer.

[0025] Preferably, the medicament is for treating cancer expressing Her2.

[0026] Preferably, the drug is used to treat cancers with high Her2 expression.

[0027] Preferably, the medicament is used to treat an indication for which bortezomib is effective.

[0028] Preferably, the medicament is used to treat ovarian cancer solid tumors.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a nano-antibody conjugate, which can improve the stability and enzyme selectivity of bortezomib, reduce the toxic and side effects of the drug, and at the same time combine the tumor-specific nano-antibody targeting technology to achieve a tumor targeting effect, can be used to treat cancers with high Her2 expression, and has excellent promotion and application value and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are 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.

[0031] Figure 1 It is a schematic diagram of the process flow of the reverse phase conversion cycle technology.

[0032] Figure 2 It is the SDS-PAGE picture of Anti-Her2-ELP and ELP after ITC purification.

[0033] Figure 3 It is a synthetic route map for exploratory experiments using oxygen-containing five-membered ring diol protecting groups to chemically modify the small molecule compound bortezomib.

[0034] Figure 4 It is a synthetic route map for exploratory experiments on chemically modifying the small molecule compound bortezomib using a five-membered carbocyclic diol protecting group.

[0035] Figure 5 is the hydrogen spectrum of compound 12.

[0036] Figure 6 is the hydrogen spectrum of compound 13.

[0037] Figure 7 is the hydrogen spectrum of compound 5.

[0038] Figure 8 This is the high-resolution mass spectrum of compound 5.

[0039] Fig. 9 This is a synthetic route map for the chemically modified small molecule compound bortezomib (PS341).

[0040] Fig.10 This is a schematic diagram of the purification of elastin-like polypeptide conjugates.

[0041] Fig.11This is a graph showing the average hydrodynamic radius detection results of Anti-Her2-ELP and Anti-Her2-ELP-PS341.

[0042] Fig.12 This is a graph showing the phase transition temperature measurement results.

[0043] Fig.13 are images of different samples at 100 μM at temperatures below and above their Tt.

[0044] Fig.14 This is the result of circular dichroism analysis of the secondary structure of Anti-Her2-ELP and Anti-Her2-ELP-PS341.

[0045] Fig.15 This is the affinity coefficient result graph of Anti-Her2-ELP.

[0046] Fig.16 This is the affinity coefficient result graph of Anti-Her2-ELP-PS341.

[0047] Fig.17 This is a graph showing the results of the binding ability test of Anti-Her2-ELP and Anti-Her2-ELP-PS341 to SKOV3 ovarian cancer cells.

[0048] Fig.18 This is a laser confocal microscopy showing the localization results of Anti-Her2-ELP and Anti-Her2-ELP-PS341 in cells.

[0049] Fig.19 This is a comparison of the inhibitory activity of bortezomib on SKOV3 ovarian cancer cells before and after chemical modification.

[0050] Fig. 20 This is a graph showing the in vitro biological activity results of Anti-Her2-ELP and Anti-Her2-ELP-PS341 measured by the CCK8 method. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0053] Example

[0054] This embodiment provides a nanobody conjugate, and the preparation steps are as follows: 1. Construction of plasmid

[0055] JMD5 (Novagen Inc., Madison, WI) vector plasmids containing the gene sequences encoding ELP and Anti-Her2-ELP were constructed respectively. The amino acid sequences of ELP and Anti-Her2-ELP are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively. When constructing ELP, primers were designed at the 5' and 3' ends of the gene as follows:

[0056] EcoRI).

[0057] The PCR system for constructing the target gene was: 1 ng of the master plate, 1 μL of each primer, 1 μL of each enzyme, and a total system of 25 μL. The rest was supplemented with ddH2O, and the annealing temperature was 55 °C. After confirming the target gene fragment by DNA electrophoresis gel, the gel was cut and recovered. The vector plasmid was linearized by enzyme digestion, and then the ELP and Anti-Her2-ELP gene sequences were inserted into the plasmid JMD5 vector respectively by ligase. The sequence size was preliminarily determined by DNA electrophoresis, and then sent to the sequencing company for detection.

[0058] 2. Expression of antibody fusion protein (Anti-Her2-ELP)

[0059] Transform the correctly sequenced plasmid into Escherichia coli (E. coli, BL21) for overexpression. Thaw the competent cells on ice, add the target DNA to the competent cell suspension, the volume of the target DNA should not exceed one tenth of the volume of the competent cell suspension, gently rotate the centrifuge tube to mix the contents, and place it in an ice bath for 30 minutes. Place the centrifuge tube in a 42℃ water bath for 60-90 seconds, then quickly transfer it to an ice bath for 2-3 minutes. Add 500 μL of sterile, antibiotic-free LB medium to the centrifuge tube and culture it at 37℃ 180 rpm for 1 hour. The purpose is to express the kanamycin resistance marker gene on the plasmid and revive the bacteria. Then take an appropriate amount of transformed competent cells and spread them on LB plates containing the corresponding antibiotics, and invert and culture them at 37℃ for 12-16 hours. Too much bacterial liquid can inhibit bacterial growth. If the expected number of clones is small, part of the culture liquid can be removed by centrifugation, and the bacteria can be suspended and spread on a plate.

[0060] After 24 hours, select the colonies with good growth and gradually expand them to 1 L TB (Terrific broth) culture medium. When the OD value is between 0.6-0.8, add 500μL of 1M IPTG to inhibit the continued proliferation of E. coli to produce more target proteins, and culture at low temperature for 16 hours. Collect the cells and resuspend them in 50 mM Tris·HCl, 150 mM NaCl, pH = 7.4. After ultrasonic disruption, centrifuge at 4°C and 14000 rpm for 15 min, treat the collected supernatant with 1% w / v polyethyleneimine (PEI), precipitate the nucleic acids and centrifuge. The precipitate is discarded, and the ELP in the supernatant is further purified by inverse transition cycling (ITC), as shown in the schematic diagram. Figure 1 As shown. ITC begins with the addition of NaCl to a final concentration of 3 M, centrifugation at 37 °C, 14,000 rpm for 15 min, removal of the supernatant, precipitation of aggregated ELP, and re-suspending the precipitate with pre-cooled 10 mM, pH 7.4 PBS buffer, dissolving in an ice bath at 4 °C for 15 min or overnight at 4 °C under a magnetic stirrer, and centrifugation at 4 °C to remove insoluble protein precipitates after complete dissolution. This cycle was repeated twice. The concentration of the purified protein was determined using a BCA protein concentration assay kit.

[0061]

[0062] 3. Exploration of chemically modified small molecule compound bortezomib

[0063] (1) Oxygen-containing five-membered ring diol protecting group

[0064] First, ribitol was used as a raw material to synthesize and purify an oxygen-containing five-membered ring diol protecting group, and then an attempt was made to directly condense it with the free boronic acid group at the C-terminus of bortezomib. In general, the synthesis of boronic acid and diol protecting groups is relatively simple, and the Dean-Stark apparatus can be used for azeotropic distillation or dehydrating media such as anhydrous MgSO4 and molecular sieves to remove the water generated in the reaction system and promote the equilibrium reaction to proceed in the forward direction. Therefore, we initially tried to use DCM as a solvent and molecular sieves as a dehydrating medium to stir the reaction at room temperature, but the TLC results showed that there was no reaction. Subsequently, acetonitrile (ACN), N,N-dimethylformamide (DMF), acetone (ACT), dioxane and other solvents were used, and the reaction temperature was gradually increased. The results showed that only two new product points appeared under dioxane solvent and heating conditions. However, after preparative HPLC separation and nuclear magnetic resonance identification, it was found that the two were oxidative deboronation degradation products of bortezomib. It is speculated that the peroxides produced during the heating of oxygen-containing ether solvents oxidized bortezomib ( Figure 3 a). Based on this, we speculate that the free hydroxyl group on the 2-side chain of the diol protecting group will interfere with the boronic acid-diol ester condensation reaction.

[0065] Subsequently, we synthesized the oxygen-containing five-membered ring linker by successively performing three steps of acetonide protection, ester condensation with 2-maleimidoacetic acid, and dilute acid deacetonide protection, thereby avoiding the interference of free hydroxyl groups ( Figure 3 b). In particular, in the second step of the ester condensation reaction, we tried three ester condensation agent systems: EDC / HOBT / DIPEA, oxalyl chloride / K2CO3, and HATU / DIPEA. We found that the HATU / DIPEA system could obtain an obvious main product point, and it was confirmed as the target product after column purification and NMR, but there was a problem of low yield. Similarly, the oxygen-containing five-membered ring connecting chain was condensed with bortezomib under the action of molecular sieve dehydration, and a weak new product point was obtained, while most of the raw materials were unreacted. After continuing the reaction for a period of time under 75°C heating conditions, the product point disappeared. It was purified and separated and identified by NMR, and it was found that it was not the target product. Based on this, we speculate that the oxygen atom on the five-membered ring of the diol protecting group also has an interference effect on the boric acid-diol ester condensation reaction. In summary, the condensation reaction between oxygen-containing five-membered ring protecting groups and free boronic acid is difficult to carry out. Accordingly, the stability of this type of protecting group is expected to be relatively poor, and the plasma stability is poor. Therefore, it is not suitable for the coupling of bortezomib with SH-peptide-COOH.

[0066] (2) Five-membered carbocyclic diol protecting group

[0067] Considering the potential interference effect of the oxygen atom on the oxygen-containing five-membered ring diol protecting group on the boronic acid-diol ester condensation reaction, we redesigned the five-membered carbocyclic diol protecting group and verified its feasibility for the condensation reaction. First, we used the -COOH-protected five-membered carbocyclic diol protecting group to explore its condensation conditions with bortezomib ( Figure 4 a) to avoid the interference effect of -OH protons mentioned above. It was found that the condensation reaction could proceed rapidly at room temperature and without molecular sieves. Subsequently, a five-membered carbocyclic linker was synthesized and attempted to condense with the free boronic acid group at the C-terminus of bortezomib ( Figure 4 b) The reaction also proceeds rapidly at room temperature. These two experiments show that the five-membered carbocyclic protecting group is suitable for the coupling of bortezomib with SH-peptide-COOH. The coupling of the five-membered carbocyclic linker with bortezomib and SH-peptide-COOH involves two orthogonal reaction types: boronic acid-diol ester condensation reaction and thiol-maleimide click chemistry reaction. The reaction conditions of both are mild and the reactions are rapid. The former does not require any catalyst while the latter requires a catalytic amount of triethylamine. On the other hand, the stability of the boronate ester bond formed by the former is much weaker than the thioether bond of the latter. Taking these factors into consideration, in the synthetic route we designed, the linker is first condensed with SH-peptide-COOH to avoid the influence of the boronate ester bond during the post-treatment process. After purification, it is condensed with bortezomib. It is expected that the final product can be obtained by directly spinning the solvent dry. The chemical synthesis steps of the above different strategies are as follows: (A) Synthesis route of oxygen-containing five-membered ring diol protecting group

[0068] (2 R , 3 S , 4 S )-2-Hydroxymethyltetrahydrofuran-3,4-diol (10)

[0069] After ribitol (2.0 g, 13.2 mmol) and pyridine hydrochloride (2.4 g, 21.0 mmol) were uniformly mixed, heated in an oil bath at 150°C for 8 h, and the reaction was complete as monitored by TLC (EA:MeOH = 5:1). After the reaction, it was purified by normal phase silica gel column (EA:MeOH, 8:1 → 5:1, v / v) to obtain a bright yellow viscous liquid, which turned into an off-white block solid after refrigeration, with a yield of 86.2%. 1H NMR (400 MHz, D2O): δ 4.19 (dd, J = 7.3, 4.5 Hz, 1H), 4.04 – 3.94 (m, 2H),3.80 – 3.68 (m, 3H), 3.56 (dd, J = 12.4, 5.1 Hz, 1H).

[0070] 1,4-Anhydro-2,3-O-isopropylidene-D-ribitol (11)

[0071] Compound 10 (4.0 g, 29.8 mmol) and 2,2-dimethoxypropane (14.62 ml, 119.2 mmol) were dissolved in 100 ml acetone, p-toluenesulfonic acid (1.03 g, 6.0 mmol) was added, and the mixture was stirred at room temperature for 1.5 h. The reaction was complete (EA:PE = 4:1) after TLC monitoring. The reaction was quenched with 150 ml saturated sodium bicarbonate solution, and most of the acetone was removed by rotation. The mixture was transferred to a 500 ml separatory funnel and extracted with 200 ml ethyl acetate, then washed with 200 ml saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a light yellow oil with a yield of 55.8%.

[0072]

[0073] 2-(Maleimidyl-1-yl)-O-(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D-ribitol-5-yl) acetate (12)

[0074] 2-Maleimidoacetic acid (364 mg, 2.35 mmol) was dissolved in 15 ml of dichloromethane, and HATU (1.07 g, 2.82 mmol) and DIPEA (820 μl, 4.69 mmol) were added and stirred at room temperature for 1 h. Compound 11 (409 mg, 2.35 mmol) was added and stirred at room temperature for reaction. TLC showed that (EA:PE = 1:1) there was an obvious product point in the reaction, but the reaction could not be completed by extending the time and heating at 50°C. After 28 h, the reaction solution was spin-dried, 110 ml of ethyl acetate and a small amount of water were added to fully suspend, and then the suspension was transferred to a 250 ml separatory funnel and washed twice with 110 ml of water, 0.01 M dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution. After drying over anhydrous sodium sulfate, the product was spin-dried and purified by a normal phase silica gel column (EA:PE, 1:6 → 1:4, v / v) to obtain a yellow viscous liquid with a yield of 65.7%. 1H NMR (400 MHz,CDCl3): δ 6.81 (s, 2H), 4.82 (t, J = 4.6 Hz, 1H), 4.59 (d, J = 5.8 Hz, 1H),4.32 (s, 2H), 4.28 – 4.21 (m, 2H), 4.16 (t, J = 7.1 Hz, 1H), 4.01 (d, J =10.6 Hz, 1H), 3.91 (dd, J = 10.7, 4.3 Hz, 1H), 1.53 (s, 3H), 1.36 (s, 3H). The hydrogen spectrum of compound 12 (solvent: CDCl3) is as follows: Figure 5 shown.

[0075]

[0076] 2-(Maleimidyl-1-yl)-O-(1,4-anhydro-5-deoxy-D-ribitol-5-yl) acetate (13)

[0077] Compound 12 (361 mg, 1.16 mmol) was dissolved in 5 ml methanol, and 5 ml of 1 M dilute hydrochloric acid was added under ice bath. The ice bath was removed and the mixture was naturally restored to room temperature and stirred for 5 h. The reaction was complete under TLC monitoring (EA:PE = 4:1). The pH was adjusted to 7 with saturated sodium bicarbonate solution, and most of the solvent was removed by vortexing. The mixture was extracted with 110 ml of ethyl acetate and washed with 110 ml of saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the mixture was vortexed and purified by normal phase silica gel column (EA:PE, 1:2 → 1:1, v / v) to obtain a yellow viscous liquid with a yield of 70.2%. 1H NMR (400 MHz, CDCl3): δ 6.85 (s, 2H), 4.46 (dd, J = 11.7, 3.4 Hz,1H), 4.38 (s, 2H), 4.36 – 4.28 (m, 2H), 4.15 (dd, J = 10.1, 4.7 Hz, 1H), 4.00(dd, J = 14.4, 6.3 Hz, 2H), 3.86 (dd, J = 10.1, 3.0 Hz, 1H). The hydrogen spectrum of compound 13 (solvent: CDCl3) is as follows Figure 6 shown.

[0078] (B) Synthesis route of five-membered carbocyclic diol protecting groups

[0079] 3-Cyclopentene-1-carboxylate (2) Methyl cyclopent-3-ene-1-carboxylate

[0080] 20 mL of anhydrous methanol was injected into a 50 mL eggplant-shaped flask. After ice bathing for 20 min, 2 mL of dichlorothionyl was added dropwise. The reaction was vigorous. After the system was kept in ice bath for 30 min, 1.12 g (10 mmol) of 3-cyclopentene-1-carboxylic acid (1) was added. The ice bath was removed and the reaction was continued for 24 h. After the reaction was complete, all volatiles were evaporated under reduced pressure to obtain crude 3-cyclopentene-1-carboxylic acid methyl ester as a colorless oil, which was directly used in the next step without purification.

[0081]

[0082] 1-(3,4-cis-cyclopentanediol)carboxylic acid methyl ester (3), methyl (3R,4S)-3,4-dihydroxycyclopentane-1-carboxylate

[0083] Methyl 1-(3-cyclopentenyl)carboxylate (1.26 g, 10 mmol) was dissolved in a mixed solvent of 40 mL acetone and 10 mL H2O, and N-methylmorpholine-N-oxide (NMO, 1.17 g, 10 mmol) and K2OsO4·2H2O (33 mg, 0.1 mmol) were added. The mixture was stirred in an oil bath at 40°C for 14 h. The reaction was complete as monitored by TLC (EA:PE = 3:1). After cooling to room temperature, sodium metabisulfite (150 mg, 0.8 mmol) was added and stirred for another 30 min. The black residue was obtained by rotary evaporation and purified by silica gel column (EA:PE, 1:2→ 1:1, v / v). After cooling, an off-white solid was obtained with a yield of 73.5%.

[0084]

[0085] 1-(3,4-cis-cyclopentanediol)carboxylic acid (4), (3R,4S)-3,4-dihydroxycyclopentane-1-carboxylic acid

[0086] Methyl 1-(3,4-cis-cyclopentanediol)formate (1.6 g, 10 mmol) was dissolved in a mixed solvent of tetrahydrofuran / water (20 mL / 20 mL). Sodium hydroxide (0.5 g, 12.5 mmol, 1.25 equiv) was added under vigorous stirring. After reacting at room temperature for 12 h, the reaction was completed as monitored by TLC (potassium permanganate color development). Dilute hydrochloric acid (4 mL, 4 M) was added to the system for acidification. 100 mL of tetrahydrofuran was added. The aqueous layer was dried over anhydrous sodium sulfate and filtered. The filter cake was rinsed with 50 mL of tetrahydrofuran. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a colorless glassy solid. The solubility in various solvents was low. DMF was used as the solvent in the next step.

[0087]

[0088] 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine (5), (3R,4S)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3,4-dihydroxycyclopentane-1-carboxamide

[0089] Compound 4 (about 0.146 g, 1 mmol) was dissolved in 5 mL DMF, HOBt (0.149 g, 1.1 mmol) was added and stirred evenly, EDC (0.23 g, 1.2 mmol) was added and stirred at room temperature for 5 min, N-(2-aminoethyl)maleimide hydrochloride (0.177 g, 1 mmol) and DIPEA (0.15 mL, 1.2 mmol) were added, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC (DCM:MeOH = 20:1). 100 ml ethyl acetate and 20 mL water were added to shake the mixture thoroughly, and then the suspension was transferred to a separatory funnel. The organic phase was separated and the aqueous phase was extracted twice with 50 mL ethyl acetate, and the organic phases were combined. The organic phase was washed twice with 0.01 M dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution. After drying over anhydrous sodium sulfate, column chromatography, pure EA was replaced with EA:MeOH=100:1 to obtain a white solid (5). 1H NMR (400 MHz, DMSO) δ 7.87 (t, J= 5.7 Hz, 1H, NH), 7.02 (s, 2H, HC=CH), 4.36 (d, J = 4.2 Hz, 2H, OH), 3.45(t, J = 5.7 Hz, 2H, N-CH2), 3.18 (dd, J = 11.4, 5.9 Hz, 2H, N-CH2), 2.73 (m,1H, CO-CH), 1.71 (m, 2H, CH2), 1.66 – 1.58 (m, 2H, CH2). MS ES—: 267.0999(MH). Compound 5 1H spectrum (solvent: DMSO) Figure 7 As shown in the high-resolution mass spectrometry Figure 8 shown.

[0090]

[0091] Compound 5 (about 1 mmol) was dissolved in 50 mL of dichloromethane, and bortezomib (0.38 g, <1 mmol) was added and stirred at room temperature. The reaction progress was monitored by TLC. The reaction was completed in about 12 h, and the crude product (6) (i.e., modified PS341) was directly used for the next coupling reaction after evaporation of the solvent.

[0092] The synthetic route of the small molecule compound bortezomib (PS341) chemically modified by a five-membered carbocyclic diol protecting group is shown in Fig. 9 shown.

[0093] 4. Antibody fusion protein coupled with chemically modified small molecule compound bortezomib

[0094] ELP or Anti-Her2-ELP of appropriate concentration was dissolved in PBS buffer (pH 8.0) at 25°C to a final protein concentration of 50 μM. The modified PS341 was dissolved in a DMSO solution accounting for 5% of the total volume of the reaction system. After complete dissolution, it was added to the reaction system with protein molecules. The final concentration of PS341 was 250 μM. The reaction was carried out at room temperature in the dark for 1 h. To remove DMSO and unreacted PS341, the reaction product was further purified by dialysis ( Fig.10 ), and the nanoantibody conjugate (Anti-Her2-ELP-PS341) and the elastin-like polypeptide conjugate ELP-PS341 were prepared.

[0095] Based on the principle that boron and azomethine form a brown-yellow complex under weak acid conditions and have a characteristic absorption peak at 420nm, a standard curve method was established to quantify the concentration of PS341. The standard boric acid powder was dissolved in water and gradiently diluted to 600, 500, 250, 125, 62.5, 31.25, 15.625, and 0μM. The concentration of ELP was determined using a BCA protein concentration kit. The ratio of PS341 concentration to protein molecule concentration is the number of PS341 molecules coupled to each ELP molecule. The results showed that the drug loading efficiency of the nanoantibody conjugate (Anti-Her2-ELP-PS341) was 77.7%.

[0096] 5. Physicochemical properties of nanoantibody conjugates

[0097] The average hydrodynamic radius of Anti-Her2-ELP and Anti-Her2-ELP-PS341 was measured by DLS method. The specific method is as follows: After the sample was filtered (0.22 μm pore size, Millipore Corp.), the hydrodynamic radius of Anti-Her2-ELP and Anti-Her2-ELP-PS341 was measured by DLS method using a nanoparticle size and potential analyzer (Zetasizer Nano-zs90, Worcestershire, UK). The measurement temperature was 25 °C and the scattering angle was 90 °. The average hydrodynamic radius distribution of the sample was analyzed using Zetasizer 6.32 software. The results are shown in Fig.11 The results showed that the average hydrodynamic radius of Anti-Her2-ELP was 12.16 nm, and the average hydrodynamic radius of Anti-Her2-ELP-PS341 was 14.62 nm.

[0098] The phase transition temperature of Anti-Her2-ELP and Anti-Her2-ELP-PS341 was analyzed by using a microplate reader. The specific method is as follows: the turbidity curve of the sample at different temperatures at an optical density (OD) of 350 nm was measured by a SpectraMax M3 microplate reader (Molecular devices). The sample was dissolved in PBS at 1.0 mg / mL, and the temperature was increased from 4°C to 70°C at a constant rate of 0.5°C / min. Tt is defined as the temperature at which the maximum turbidity of the sample is 50%. The results are shown in Fig.12 The images of different samples at 100 μM below and above their Tt temperatures are shown in Fig.13 As shown, samples 1 to 4 are Anti-Her2-ELP, Anti-Her2-ELP-PS341, ELP, and ELP-PS341, respectively. The results show that since the phase transition temperature Tt of Anti-Her2-ELP-PS341 above 100 μM is about 33°C, which is lower than normal body temperature, it can be precipitated from body fluids after subcutaneous administration to form a reservoir, which can be used for sustained drug release.

[0099] The secondary structures of Anti-Her2-ELP and Anti-Her2-ELP-PS341 were analyzed by circular dichroism (CD). The specific method was as follows: the sample was diluted with water to 0.15 mg / mL, and the CD spectrum was recorded at 190-260 nm on a Pistarπ-180 (Applied Photophysics Ltd). Fig.14 The results showed that there was no significant change in the secondary structure of the protein before and after the coupling reaction.

[0100] 6. In vitro activity of nanoantibody conjugates

[0101] The affinity coefficients of Anti-Her2-ELP and Anti-Her2-ELP-PS341 were determined using a biofilm optical interferometry (BLI) instrument based on an optical fiber sensor at a temperature of 30°C. The results were as follows: Fig.15 and Fig.16 The results showed that the affinity coefficient (Kd) of Anti-Her2-ELP was 1.61×10 -9 , has good binding ability with Her2 antigen; the affinity coefficient (Kd) of Anti-Her2-ELP-PS341 is 1.44×10 -8 , after purification by coupling reaction, the binding ability of Anti-Her2-ELP-PS341 to Her2 antigen was slightly reduced.

[0102] The binding ability of Anti-Her2-ELP and Anti-Her2-ELP-PS341 to SKOV3 ovarian cancer cells was determined by flow cytometry. The specific method is as follows: CY5-NHS ester and coupled protein drug are reacted in a sodium bicarbonate buffer at pH 9.0 at a feed ratio of 2:1, gently stirred with a magnetic stirrer to prevent CY5 precipitation, and placed at 4°C for overnight reaction. Then the excess CY5 dye is removed by dialysis. SKOV3 ovarian cancer cells are plated on 6-well plates, 1×10^6 cells per well, 1 ml volume, and adhered overnight. After 24 hours, the culture medium is discarded, and the culture medium containing 2μM protein labeled with CY5 is added, and placed in a 37-degree incubator for 6 hours. The control group 1) blank cells, 2) Anti-her2-ELP, 3) Anti-Her2-ELP-PS341, and then the proportion of cells containing cy5 markers is detected using the ECD channel on the flow cytometer. The results are as follows Fig.17 The results showed that the ratio of Anti-Her2-ELP and Anti-Her2-ELP-PS341 binding to cells was more than 96%, and compared with the control group, they had good targeting to SKOV3 ovarian cancer cells that highly expressed Her2.

[0103] The localization of Anti-Her2-ELP and Anti-Her2-ELP-PS341 in cells was shown by laser confocal microscopy. The specific method is as follows: SKOV3 cells were plated on glass plates, with 1×10^5 cells per well and a volume of 1 ml, and allowed to adhere overnight. After 24 hours, the culture medium was discarded, and 2 μM Cy5-labeled protein was added respectively, and incubated at 37 degrees for 1 hour. Then rinsed three times with pre-cooled PBS and fixed with 4% paraformaldehyde. Subsequently, the cell nucleus and cell membrane were labeled with DAPI (diluted to 1μg / ml) and WGA (diluted 200X), respectively, placed at 37 degrees for about 10 minutes, and observed and photographed under a laser confocal microscope. The cell membrane is green, the cell nucleus is blue, and CY5 as a conjugated drug marker is red. The results are as shown in the figure. Fig.18 The results showed that the drug labeled with CY5 red was mainly distributed in the cytoplasm.

[0104] The in vitro biological activities of Anti-Her2-ELP and Anti-Her2-ELP-PS341, as well as the inhibitory activity of bortezomib before and after modification on SKOV3 ovarian cancer cells were determined by CCK8 method. The specific method is as follows: SKOV3 ovarian cancer cells were taken, 1-2 mL of trypsin was added for 1-3 min, 3 times the volume of complete medium was added to terminate, resuspended, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. Then 3 mL of culture medium was added to resuspend the cells, 20 μL of cells were added to 20 μL of trypan blue dye, and 20 μL was taken to measure the cell density and viability. The cell viability should be above 90%.

[0105] Plate 5000 cells per well, and seal the plate with 200 uL PBS in the surrounding wells to avoid evaporation. Incubate overnight at 37°C, 5% CO2. The next day, prepare the drug, and sterilize the unfiltered sample by 0.22 μm filtration. The highest concentration sample is diluted 10 times and added to the corresponding well. Then incubate at 37°C, 5% CO2, and test after 48 hours of reaction. On the day of the test, prepare the CCK8-culture medium mixture in advance, that is, 10 μL CCK8 reagent and 90 μL PBS per well. Remove the original culture medium with a suction pump, add the CCK8-culture medium mixture to the sample tank, and mix gently. Add 100 μL of CCK8-PBS mixture to each well, incubate at 37°C, and detect OD450 on the microplate reader every 30 minutes. The absorbance value should be between 1-2. GraphPad Prism 5.0 software was used to analyze and fit the data and calculate the half maximal inhibitory concentration (IC 50 ) and compared the cell activities.

[0106] Comparison of the inhibitory activity of bortezomib before and after chemical modification on SKOV3 ovarian cancer cells Fig.19 As shown in the figure, PS341-idol is bortezomib after chemical modification, and PS341 is bortezomib before chemical modification. The results showed that the maximum inhibitory concentration (IC 50 ) were 4.6 nM (PS341) and 6.7 nM (PS341-idol), respectively. The cytotoxicity of the modified bortezomib was slightly reduced, which was consistent with the experimental expectation. In order to improve the stability of the drug, the active groups were blocked, and the drug activity was slightly reduced in theory. The results of the comparison of the inhibitory activity of Anti-Her2-ELP-PS341 and ELP-PS341 on SKOV3 ovarian cancer cells are shown in the figure. Fig. 20 As shown, the half maximum inhibitory concentration (IC 50 ) were 66.0 nM and 865.6 nM, respectively.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanobody conjugate, characterized in that: including antibody fusion proteins conjugated to bortezomib; The antibody fusion protein is a fusion protein of a nanobody and an elastin-like polypeptide sequentially connected from the N-terminus to the C-terminus; The nanobody is an anti-Her2 nanobody; The amino acid sequence of the elastin-like polypeptide is (XPGVG)n, wherein X is any natural amino acid except proline, and 30≤n≤150.

2. The nanobody conjugate according to claim 1, characterized in that The nanobody is an anti-human Her2 nanobody; Preferably, the amino acid sequence of the anti-human Her2 Nanobody is as shown in SEQ ID NO.

1.

3. The nanobody conjugate according to claim 1, characterized in that In the elastin-like polypeptide, X is valine, and 60≤n≤120.

4. The nanobody conjugate according to claim 1, characterized in that The antibody fusion protein is constructed by expressing the coding genes of the nanobody and the elastin-like polypeptide into a protein expression vector and obtained by protein expression.

5. The method for preparing the nanobody conjugate according to any one of claims 1 to 4, characterized in that: include: The bortezomib is chemically modified with a five-membered carbocyclic diol protecting group and then coupled to the antibody fusion protein through a maleimide reaction. The chemically modified bortezomib is connected to the C-terminus of the nanobody.

6. The preparation method according to claim 5, characterized in that: The five-membered carbocyclic diol protecting group is a cis-1,2-five-membered (hetero)cyclic diol protecting group.

7. The preparation method according to claim 6, characterized in that: include: 3-cyclopentene-1-carboxylic acid is used as a starting material, after esterification in a methanol / thionyl chloride system, K2OsO4·2H2O is used as a catalyst to oxidize to 1-(3-cyclopentenyl)carboxylic acid methyl ester; subsequently, the methyl ester protecting group is removed by saponification reaction to obtain 1-(3-cyclopentenyl)carboxylic acid; 1-(3-cyclopentenyl)carboxylic acid and N-(2-aminoethyl)maleimide hydrochloride are subjected to an amide condensation reaction to obtain an intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine; finally, the intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine is subjected to an esterification reaction with bortezomib in dichloromethane to obtain chemically modified bortezomib.

8. The preparation method according to claim 7, characterized in that: The structural formula of chemically modified bortezomib is: 。 9. A pharmaceutical composition, characterized in that It contains the Nanobody conjugate according to any one of claims 1 to 4, or the Nanobody conjugate prepared by the preparation method according to any one of claims 5 to 8.

10. Use of the Nanobody conjugate according to any one of claims 1 to 4, or the Nanobody conjugate prepared by the preparation method according to any one of claims 5 to 8 in the preparation of a drug.

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