A nanoantibody conjugate and its preparation method and application

Abstract

The present invention relates to the field of biomedicine, and in particular to a nanobody conjugate, its preparation method, and application. The nanobody conjugate comprises a conjugated antibody fusion protein and bortezomib; the antibody fusion protein is a fusion protein of an elastin-like polypeptide and an anti-Her2 nanobody. The nanobody conjugate of the present invention can improve the stability and enzyme selectivity of bortezomib, reduce the drug's toxic side effects, and simultaneously achieve tumor targeting effects by combining tumor-specific nanobody targeting technology. It can be used to treat cancers with high Her2 expression, and has excellent value and potential for widespread application.

Description

Technical Field

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

[0002] Bortezomib is currently a first-line drug for the treatment of multiple myeloma, but it suffers from significant toxic side effects, a narrow therapeutic window, and relatively poor efficacy against solid tumors. These drawbacks are primarily due to the following: The compound is relatively unstable, with the polypeptide nucleus easily degraded by various proteases in the body, and the carbon-boron bond is easily oxidatively broken. Furthermore, its metabolic stability in the bloodstream is poor, resulting in its degradation before it can enter tissues and organs. Therefore, this type of protease inhibitor is generally ineffective against solid tumors in vivo. Secondly, the compound has low enzyme selectivity, making it susceptible to binding to other enzymes or proteins after entering the body. Furthermore, its lack of selectivity for tumor cells versus normal cells can lead to off-target effects and various toxic side effects, including thrombocytopenia, anemia, unexplained pain, and febrile neutropenia.

[0003] How to improve the stability and enzyme selectivity of bortezomib while achieving tumor targeting effect and reducing the drug's toxic side effects 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 nanobody conjugate, comprising a conjugated antibody fusion protein and bortezomib;

[0006] 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;

[0007] The nanobody is an anti-Her2 nanobody;

[0008] 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.

[0009] The present invention achieves the tumor-targeting effect of bortezomib through anti-Her2 nanobodies, while simultaneously utilizing the properties of elastin-like polypeptides to facilitate sustained release of the small molecule compound bortezomib in vivo, thereby reducing the drug's toxic side effects. The present invention has conducted extensive design and screening of elastin-like polypeptide sequences and discovered that the elastin-like polypeptides and anti-Her2 nanobodies can produce a good synergistic effect. The elastin-like polypeptides can ensure efficient expression of the nanobody protein in the vector, and the attached elastin-like polypeptide does not interfere with the targeting of the nanobody, ensuring that the nanobody conjugate has high antigen-targeting activity.

[0010] This nano-antibody conjugate can target the Her2 antigen. After subcutaneous injection, it will undergo a phase change in situ triggered by 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 good anti-tumor effects, and improve patient compliance.

[0011] Preferably, the nanobody is an anti-human Her2 nanobody.

[0012] Preferably, the amino acid sequence of the anti-human Her2 Nanobody is as shown in SEQ ID NO.1.

[0013] The anti-human Her2 Nanobody with the above amino acid sequence does not express normally in a protein expression vector. In the art, when using Nanobodies with the above amino acid sequence, they are usually optimized and modified to increase their expression level. However, the present invention unexpectedly discovered 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 significantly promote the expression of the Nanobody protein.

[0014] Preferably, X is any one selected from valine, phenylalanine, tryptophan, tyrosine, alanine, glycine, methionine, threonine, serine, leucine, and isoleucine.

[0015] Preferably, in the elastin-like polypeptide, X is valine, and 60≤n≤120.

[0016] More preferably, in the elastin-like polypeptide, X is valine and n is 90.

[0017] 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 increase the expression level of nanoantibodies. In addition, the fusion protein also has higher targeting antigen activity.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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 oxidizing it to 1-(3-cyclopentenyl)methyl formate using K2OsO4·2H2O as a catalyst; subsequently removing the methyl ester protecting group through a saponification reaction to obtain 1-(3-cyclopentenyl)formic acid, reacting 1-(3-cyclopentenyl)formic acid with N-(2-aminoethyl)maleimide hydrochloride through an amide condensation reaction to obtain an intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine; and finally esterifying the intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine with bortezomib in dichloromethane to obtain chemically modified bortezomib.

[0022] The structural formula of chemically modified bortezomib is:

[0023] .

[0024] By studying the properties of bortezomib, the present invention chemically modifies and protects the boronic acid group through a five-membered carbocyclic diol protecting group, thereby improving its stability and enzyme selectivity. At the same time, 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.

[0025] Preferably, the chemically modified bortezomib is subjected to a maleimide reaction with the antibody fusion protein in a DMSO solution to prepare the nanobody conjugate.

[0026] Furthermore, the present invention provides a pharmaceutical composition containing the nanobody conjugate or the nanobody conjugate prepared by the preparation method.

[0027] 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.

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

[0029] Preferably, the medicament is for treating cancer expressing Her2.

[0030] Preferably, the drug is used to treat cancers with high Her2 expression.

[0031] Preferably, the medicament is used to treat an indication for which bortezomib is effective.

[0032] Preferably, the drug is used to treat ovarian cancer solid tumors.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a nano-antibody conjugate, which can improve the stability and enzyme selectivity of bortezomib, reduce the toxic side effects of the drug, and at the same time combine with tumor-specific nano-antibody targeting technology to achieve tumor targeting effects. It can be used to treat cancers with high Her2 expression and has excellent promotion and application value and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to 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 any creative work.

[0036] Figure 1 It is a schematic diagram of the reverse conversion cycle technology process.

[0037] Figure 2 It is the SDS-PAGE picture of Anti-Her2-ELP and ELP after ITC purification.

[0038] Figure 3 This is a synthetic route map for exploring the chemical modification of the small molecule compound bortezomib using oxygen-containing five-membered ring diol protecting groups.

[0039] Figure 4 This is a synthetic route map for exploring the chemical modification of the small molecule compound bortezomib using a five-membered carbocyclic diol protecting group.

[0040] Figure 5 is the hydrogen spectrum of compound 12.

[0041] Figure 6 is the hydrogen spectrum of compound 13.

[0042] Figure 7 is the hydrogen spectrum of compound 5.

[0043] Figure 8 This is the high-resolution mass spectrum of compound 5.

[0044] Figure 9 This is the synthetic route of the chemically modified small molecule compound bortezomib (PS341).

[0045] Figure 10 Schematic diagram of the purification of elastin-like polypeptide conjugates.

[0046] Figure 11 This is a graph showing the average hydrodynamic radius detection results of Anti-Her2-ELP and Anti-Her2-ELP-PS341.

[0047] Figure 12 This is a graph showing the phase transition temperature measurement results.

[0048] Figure 13 Images of different samples at 100 μM at temperatures below and above their Tt.

[0049] Figure 14 This is the result of circular dichroism analysis of the secondary structure of Anti-Her2-ELP and Anti-Her2-ELP-PS341.

[0050] Figure 15 This is the affinity coefficient result graph of Anti-Her2-ELP.

[0051] Figure 16 This is the affinity coefficient result graph of Anti-Her2-ELP-PS341.

[0052] Figure 17 This figure shows the results of the binding ability assay of Anti-Her2-ELP and Anti-Her2-ELP-PS341 to SKOV3 ovarian cancer cells.

[0053] Figure 18 This is a laser confocal microscopy showing the localization results of Anti-Her2-ELP and Anti-Her2-ELP-PS341 in cells.

[0054] Figure 19 This is a graph comparing the inhibitory activity of bortezomib on SKOV3 ovarian cancer cells before and after chemical modification.

[0055] Figure 20 This figure shows the results of in vitro biological activity determination of Anti-Her2-ELP and Anti-Her2-ELP-PS341 using the CCK8 method. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0057] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0058] Example

[0059] This example provides a nanobody conjugate, the preparation steps of which are as follows:

[0060] 1. Construction of plasmid

[0061] JMD5 (Novagen Inc., Madison, WI) vector plasmids containing the gene sequences encoding ELP and Anti-Her2-ELP were constructed. The amino acid sequences of ELP and Anti-Her2-ELP are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively. For ELP construction, primers were designed at the 5' and 3' ends of the gene as follows:

[0062]

[0063] EcoRI).

[0064] The PCR system for constructing the target gene consisted of 1 ng of the master plate, 1 μL of each primer, and 1 μL of each enzyme, for a total volume of 25 μL. The remainder was made up with ddH2O. The annealing temperature was 55°C. After confirming the target gene fragment by DNA electrophoresis gel, the gel was excised and recovered. The vector plasmid was linearized by enzyme digestion, and then the ELP and Anti-Her2-ELP gene sequences were inserted into the JMD5 plasmid using ligase. Sequence size was initially confirmed by DNA electrophoresis, and the fragments were then sent to a sequencing company for testing.

[0065] 2. Expression of antibody fusion protein (Anti-Her2-ELP)

[0066] Transform the sequenced plasmid into E. coli (BL21) for overexpression. Thaw the competent cells on ice. Add the target DNA to the competent cell suspension, ensuring the volume should not exceed one-tenth of the total volume. Gently swirl the centrifuge tube to mix the contents, and place on ice for 30 minutes. Place the centrifuge tube in a 42°C water bath for 60-90 seconds, then quickly transfer to an ice bath for 2-3 minutes. Add 500 μL of sterile, antibiotic-free LB medium to the centrifuge tube and incubate at 37°C with shaking at 180 rpm for 1 hour. This allows the kanamycin resistance marker gene on the plasmid to express and the cells to recover. Then, spread an appropriate amount of the transformed competent cells onto an LB plate containing the appropriate antibiotic and incubate inverted at 37°C for 12-16 hours. Excessive culture medium can inhibit bacterial growth. If a small number of colonies are expected, remove a portion of the culture medium after centrifugation, resuspend the cells, and spread them onto a single plate.

[0067] After 24 hours, well-growing colonies were selected and gradually expanded to 1 L TB (Terrific broth) culture medium. When the OD value was between 0.6-0.8, 500 μL of 1M IPTG was added to inhibit the continued proliferation of E. coli to produce more target proteins, and cultured at low temperature for 16 hours. The cells were collected and resuspended in 50 mM Tris·HCl, pH = 7.4, 150 mM NaCl. After ultrasonic disruption, centrifugation was performed at 4°C and 14000 rpm for 15 min. The collected supernatant was treated with 1% w / v polyethyleneimine (PEI), the nucleic acids were precipitated, and then centrifuged. The precipitate was discarded, and the ELP in the supernatant was 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. The mixture is centrifuged at 14,000 rpm at 37°C for 15 minutes, the supernatant removed, and the aggregated ELP precipitated. The precipitate is then resuspended in pre-chilled 10 mM PBS, pH 7.4, and dissolved on ice at 4°C for 15 minutes or overnight at 4°C with a magnetic stirrer. Once completely dissolved, the mixture is centrifuged at 4°C to remove any insoluble protein precipitate. This cycle is repeated twice. The purified protein concentration is determined using a BCA protein assay kit.

[0068]

[0069] 3. Exploration of the chemically modified small molecule compound bortezomib

[0070] (1) Oxygen-containing five-membered ring diol protecting group

[0071] First, ribitol was used as the raw material to synthesize and purify an oxygen-containing five-membered ring diol protecting group, which was then directly condensed with the free boronic acid group at the C-terminus of bortezomib. Generally speaking, the synthesis of boronic acid and diol protecting groups is relatively simple. 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 the solvent and molecular sieves as the dehydrating medium to stir the reaction at room temperature, but TLC results showed that no reaction occurred. Subsequently, solvents such as acetonitrile (ACN), N,N-dimethylformamide (DMF), acetone (ACT), and dioxane were used, and the reaction temperature was gradually increased. The results showed that only two new product spots 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 generated during the heating of the oxygen-containing ether solvent 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.

[0072] Subsequently, we synthesized an oxygen-containing five-membered ring linker by sequentially reacting the diol protecting group with acetonide protection, ester condensation with 2-maleimidoacetic acid, and dilute acid deacetonide protection in three steps, thereby avoiding the interference of free hydroxyl groups ( Figure 3 b). Specifically, in the second ester condensation reaction, we tried three ester condensation systems: EDC / HOBT / DIPEA, oxalyl chloride / K2CO3, and HATU / DIPEA. The HATU / DIPEA system yielded a distinct main product spot, which was confirmed as the target product after column purification and NMR analysis. However, the yield was low. Similarly, condensation of the oxygen-containing five-membered ring linker with bortezomib under molecular sieve dehydration yielded a faint new product spot, while the majority of the starting material remained unreacted. This product spot disappeared after continued reaction at 75°C. Purification and NMR analysis revealed that it was not the target product. Based on this, we hypothesize that the oxygen atom on the five-membered ring of the diol protecting group also interferes with the boronic 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.

[0073] (2) Five-membered carbocyclic diol protecting group

[0074] Considering the potential interference effect of the oxygen atom on the ring of the aforementioned 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 this condensation reaction. First, we used the -COOH-protected five-membered carbocyclic diol protecting group to explore its condensation conditions with bortezomib ( Figure 4 a), in order to avoid the interference effect of the aforementioned -OH protons, 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 relatively 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-processing 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:

[0075] (A) Synthesis route of oxygen-containing five-membered ring diol protecting groups

[0076]

[0077] (2 R , 3 S , 4 S )-2-Hydroxymethyltetrahydrofuran-3,4-diol (10)

[0078] Ribitol (2.0 g, 13.2 mmol) and pyridine hydrochloride (2.4 g, 21.0 mmol) were uniformly mixed and heated in an oil bath at 150°C for 8 h. TLC confirmed the completion of the reaction (EA:MeOH = 5:1). After completion, the product was purified by normal-phase silica gel column chromatography (EA:MeOH, 8:1 → 5:1, v / v) to yield a bright yellow viscous liquid, which transformed into an off-white, lumpy solid upon refrigeration. The yield was 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).

[0079]

[0080] 1,4-Anhydro-2,3-O-isopropylidene-D-ribitol (11)

[0081] Compound 10 (4.0 g, 29.8 mmol) and 2,2-dimethoxypropane (14.62 ml, 119.2 mmol) were dissolved in 100 ml of acetone. p-Toluenesulfonic acid (1.03 g, 6.0 mmol) was added and stirred at room temperature for 1.5 h. TLC confirmed the reaction was complete (EA:PE = 4:1). The reaction was quenched with 150 ml of saturated sodium bicarbonate solution, and most of the acetone was removed by rotary evaporation. The product was transferred to a 500 ml separatory funnel and extracted with 200 ml of ethyl acetate. The product was then washed with 200 ml of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a pale yellow oil in a yield of 55.8%.

[0082]

[0083] 2-(Maleimid-1-yl)-O-(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D-ribitol-5-yl)acetate (12)

[0084] 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. The mixture was stirred and activated at room temperature for 1 h. Compound 11 (409 mg, 2.35 mmol) was added and the reaction was stirred at room temperature. TLC (EA:PE = 1:1) showed a clear product spot, but prolonged heating and heating at 50°C did not lead to complete reaction. After 28 h, the reaction solution was spin-dried to dryness and thoroughly suspended in 110 ml of ethyl acetate and a small amount of water. The suspension was then 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. The product was dried over anhydrous sodium sulfate and then spin-dried. The product was 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 shown in Figure 2. Figure 5 shown.

[0085]

[0086] 2-(Maleimid-1-yl)-O-(1,4-anhydro-5-deoxy-D-ribitol-5-yl)acetate (13)

[0087] Compound 12 (361 mg, 1.16 mmol) was dissolved in 5 ml of methanol. 5 ml of 1 M dilute hydrochloric acid was added under an ice bath. The mixture was removed from the ice bath and allowed to cool to room temperature. Stirring was continued for 5 h. The reaction was complete as monitored by TLC (EA:PE = 4:1). The pH was adjusted to 7 with saturated sodium bicarbonate solution. After removing most of the solvent, the mixture was extracted with 110 ml of ethyl acetate and washed with 110 ml of saturated sodium chloride solution. After drying over anhydrous sodium sulfate and drying by spin drying, the mixture was purified on a normal phase silica gel column (EA:PE, 1:2 → 1:1, v / v) to obtain a yellow viscous liquid in 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 shown in Figure 2. Figure 6 shown.

[0088] (B) Synthesis route of five-membered carbocyclic diol protecting groups

[0089]

[0090] 3-Cyclopentene-1-carboxylate (2), methyl cyclopent-3-ene-1-carboxylate

[0091] A 50 mL eggplant-shaped flask was filled with 20 mL of anhydrous methanol. After ice-bathing for 20 min, 2 mL of thionyl chloride was added dropwise. The reaction was vigorous. The system was kept in the ice-bath for 30 min, and 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 until the reaction was complete. All volatiles were removed by vacuum distillation to obtain crude methyl 3-cyclopentene-1-carboxylate as a colorless oil, which was used directly in the next reaction without purification.

[0092]

[0093] 1-(3,4-cis-cyclopentanediol)carboxylic acid methyl ester (3), methyl (3R,4S)-3,4-dihydroxycyclopentane-1-carboxylate

[0094] Methyl 1-(3-cyclopentenyl)carboxylate (1.26 g, 10 mmol) was dissolved in a mixture of 40 mL of acetone and 10 mL of H₂O. 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 by TLC (EA:PE = 3:1). After cooling to room temperature, sodium metabisulfite (150 mg, 0.8 mmol) was added and stirred for an additional 30 min. The black residue was evaporated and purified on a silica gel column (EA:PE, 1:2 → 1:1, v / v) to obtain an off-white solid upon cooling in a yield of 73.5%.

[0095]

[0096] 1-(3,4-cis-cyclopentanediol)carboxylic acid (4), (3R,4S)-3,4-dihydroxycyclopentane-1-carboxylic acid

[0097] Methyl 1-(3,4-cis-cyclopentanediol)formate (1.6 g, 10 mmol) was dissolved in a mixture of tetrahydrofuran and water (20 mL / 20 mL). Sodium hydroxide (0.5 g, 12.5 mmol, 1.25 equiv) was added with vigorous stirring. The mixture was allowed to react at room temperature for 12 h. TLC confirmed the completion of the reaction (color development using potassium permanganate). The system was acidified by the addition of dilute hydrochloric acid (4 mL, 4 M). 100 mL of tetrahydrofuran was added, and 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 of the solid in various solvents was low, so DMF was used as the solvent in the next step.

[0098]

[0099] 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

[0100] Compound 4 (approximately 0.146 g, 1 mmol) was dissolved in 5 mL of DMF. HOBt (0.149 g, 1.1 mmol) was added and stirred until homogeneous. EDC (0.23 g, 1.2 mmol) was added and activated at room temperature with stirring for 5 min. N-(2-aminoethyl)maleimide hydrochloride (0.177 g, 1 mmol) and DIPEA (0.15 mL, 1.2 mmol) were added and stirred at room temperature for 4 h. The reaction was monitored for completion by TLC (DCM:MeOH = 20:1). After adding 100 mL of ethyl acetate and 20 mL of water and shaking thoroughly, the suspension was transferred to a separatory funnel and separated to obtain an organic phase. The aqueous phase was extracted twice with 50 mL of ethyl acetate, and the combined organic phases were washed sequentially with 0.01 M dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution twice. After drying over anhydrous sodium sulfate, the product was purified by column chromatography, with pure EA replaced by EA:MeOH = 100:1, to give 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.

[0101]

[0102]

[0103] Compound 5 (approximately 1 mmol) was dissolved in 50 mL of dichloromethane, and bortezomib (0.38 g, <1 mmol) was added, followed by stirring at room temperature. The reaction progress was monitored by TLC. The reaction was complete in approximately 12 h. After evaporation of the solvent, the crude product (6) (i.e., the modified PS341) was directly used in the next coupling reaction.

[0104] The synthetic route of the small molecule compound bortezomib (PS341) is chemically modified by a five-membered carbocyclic diol protecting group. Figure 9 shown.

[0105] 4. Antibody fusion protein coupled with chemically modified small molecule compound bortezomib

[0106] ELP or Anti-Her2-ELP at an appropriate concentration was dissolved in PBS buffer (pH 8.0) at 25°C to a final protein concentration of 50 μM. Modified PS341 was dissolved in DMSO solution (5% of the total volume of the reaction system) and added to the reaction system containing protein molecules after complete dissolution. The final concentration of PS341 was 250 μM. The reaction was incubated at room temperature in the dark for 1 hour. To remove DMSO and unreacted PS341, the reaction product was further purified by dialysis ( Figure 10 ), and the nanoantibody conjugate (Anti-Her2-ELP-PS341) and the elastin-like polypeptide conjugate ELP-PS341 were prepared.

[0107] A standard curve method was established to quantify PS341 concentration, based on the principle that boron and azomethine form a brown-yellow complex under weak acid conditions, with a characteristic absorption peak at 420 nm. Boric acid powder was dissolved in water and diluted to 600, 500, 250, 125, 62.5, 31.25, 15.625, and 0 μM. ELP concentration was determined using a BCA protein concentration kit. The ratio of PS341 concentration to protein concentration represents the number of PS341 molecules conjugated to each ELP molecule. Results demonstrated a drug loading efficiency of 77.7% for the nanobody conjugate (Anti-Her2-ELP-PS341).

[0108] 5. Physicochemical properties of nanoantibody conjugates

[0109] The average hydrodynamic radius of Anti-Her2-ELP and Anti-Her2-ELP-PS341 was measured by DLS. The specific method was as follows: After sample filtration (0.22 μm pore size, Millipore Corp.), the hydrodynamic radius of Anti-Her2-ELP and Anti-Her2-ELP-PS341 was determined by DLS 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 samples was analyzed using Zetasizer 6.32 software. The results are shown in Figure 2. Figure 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.

[0110] The phase transition temperatures of Anti-Her2-ELP and Anti-Her2-ELP-PS341 were analyzed using a microplate reader. The turbidity curves of the samples at optical density (OD) of 350 nm were measured at different temperatures using a SpectraMax M3 microplate reader (Molecular Devices). The samples were dissolved in PBS at a concentration of 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 was defined as the temperature at which the sample reached 50% of its maximum turbidity. The results are shown in Figure 2. Figure 12 The images of different samples at 100 μM at temperatures below and above their Tt are shown in Figure 13 As shown in the figure, samples 1 to 4 are Anti-Her2-ELP, Anti-Her2-ELP-PS341, ELP, and ELP-PS341, respectively. The results showed that since the phase transition temperature, Tt, of Anti-Her2-ELP-PS341 at concentrations above 100 μM is approximately 33°C, which is lower than normal body temperature, it can precipitate from body fluids after subcutaneous administration, forming a reservoir that can be used for sustained drug release.

[0111] 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 spectra were recorded at 190-260 nm on a Pistarπ-180 (Applied Photophysics Ltd). The results are shown in Figure 2. Figure 14 The results showed that there was no significant change in the secondary structure of the protein before and after the coupling reaction.

[0112] 6. In vitro activity of nanobody conjugates

[0113] The affinity coefficients of Anti-Her2-ELP and Anti-Her2-ELP-PS341 were determined by biofilm optical interferometry (BLI) instrument based on fiber optic sensor at 30°C. The results were as follows: Figure 15 and Figure 16 The results showed that the affinity coefficient (Kd) of Anti-Her2-ELP was 1.61×10 -9 , has good binding ability to 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.

[0114] 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 the conjugated protein drug were reacted in a sodium bicarbonate buffer at pH 9.0 at a feed ratio of 2:1. A magnetic stirrer was used to gently stir to prevent CY5 precipitation, and the reaction was allowed to proceed overnight at 4°C. Excess CY5 dye was then removed by dialysis. SKOV3 ovarian cancer cells were plated into 6-well plates with 1×10^6 cells per well in a 1 ml volume and allowed to adhere overnight. After 24 hours, the culture medium was discarded and culture medium containing 2μM CY5-labeled protein was added. The cells were then placed in a 37°C incubator for 6 hours. The control groups included 1) blank cells, 2) Anti-her2-ELP, and 3) Anti-Her2-ELP-PS341. The proportion of cells containing the cy5 label was then detected using the ECD channel on a flow cytometer. The results are shown in the figure below. Figure 17 The results showed that the binding ratio of Anti-Her2-ELP and Anti-Her2-ELP-PS341 to cells exceeded 96%, and compared with the control group, they had good targeting to SKOV3 ovarian cancer cells that highly expressed Her2.

[0115] 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 of Cy5-labeled protein was added and incubated at 37 degrees for 1 hour. Then rinsed 3 times with pre-cooled PBS and fixed with 4% paraformaldehyde. Subsequently, the cell nucleus and cell membrane were labeled with DAPI (diluted to 1 μg / 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 follows. Figure 18 The results showed that the drug labeled with CY5 red was mainly distributed in the cytoplasm.

[0116] The in vitro biological activity of Anti-Her2-ELP and Anti-Her2-ELP-PS341, as well as the inhibitory activity of bortezomib before and after modification, against SKOV3 ovarian cancer cells were determined using the CCK8 assay. The following method was used: SKOV3 ovarian cancer cells were digested with 1-2 mL of trypsin for 1-3 minutes, then digested with 3 volumes of complete culture medium, resuspended, and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded. The cells were then resuspended in 3 mL of culture medium. 20 μL of cells were added to 20 μL of trypan blue dye, and 20 μL of the cells were collected to measure cell density and viability. Cell viability should be above 90%.

[0117] Plate 5,000 cells per well and seal the surrounding wells with 200 μL of PBS to prevent evaporation. Incubate overnight at 37°C, 5% CO₂. The next day, prepare the drugs. Unfiltered samples should be sterilized by 0.22 μm filtration. The highest concentration sample should be diluted 10-fold and added to the corresponding wells. Incubate at 37°C, 5% CO₂, and assay after 48 hours. On the day of assay, prepare a CCK8-medium mixture in advance: 10 μL of CCK8 reagent and 90 μL of PBS per well. Remove the original medium with a pump and add the CCK8-medium mixture to the sample reservoir. Mix gently. Add 100 μL of the CCK8-PBS mixture to each well and incubate at 37°C. Measure the OD450 value on a microplate reader every 30 minutes; an absorbance value between 1 and 2 is acceptable. 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.

[0118] Comparison of the inhibitory activity of bortezomib before and after chemical modification on SKOV3 ovarian cancer cells Figure 19 As shown in the figure, PS341-idol is the chemically modified bortezomib, and PS341 is the bortezomib before chemical modification. The results showed that the half-maximal inhibitory concentration (IC 50 ) were 4.6 nM (PS341) and 6.7 nM (PS341-idol), respectively. The modified bortezomib showed a slight decrease in cytotoxicity, which is consistent with experimental expectations. To improve drug stability, the active groups were blocked, which theoretically reduced the drug activity slightly. The results of the comparison of the inhibitory activity of Anti-Her2-ELP-PS341 and ELP-PS341 against SKOV3 ovarian cancer cells are shown in the figure. Figure 20 As shown, the half-maximal inhibitory concentration (IC 50 ) were 66.0 nM and 865.6 nM, respectively.

[0119] 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 various embodiments of the present invention.

Claims

1. A method for preparing a nanobody conjugate, characterized in that: include: Bortezomib is chemically modified with a cis-1,2-pentadiol protecting group and then coupled to an antibody fusion protein through a maleimide reaction. 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, and the chemically modified bortezomib is connected to the C-terminus of the nanobody; 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; The preparation method of the chemically modified bortezomib includes: using 3-cyclopentene-1-carboxylic acid as a starting material, esterifying it with a methanol / thionyl chloride system, and then oxidizing it to 1-(3-cyclopentenyl)methyl formate using K2OsO4·2H2O as a catalyst; then removing the methyl ester protecting group through a saponification reaction to obtain 1-(3-cyclopentenyl)formic acid; carrying out an amide condensation reaction between 1-(3-cyclopentenyl)formic acid and N-(2-aminoethyl)maleimide hydrochloride to obtain an intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine; and finally esterifying the intermediate 1-(3,4-cis-cyclopentanediol)formyl-N-maleimide-2-ethylamine with bortezomib in dichloromethane to obtain the chemically modified bortezomib.

2. The method for preparing the nanobody conjugate according to claim 1, wherein The nanobody is an anti-human Her2 nanobody.

3. The method for preparing the nanobody conjugate according to claim 2, wherein: The amino acid sequence of the anti-human Her2 nanobody is shown in SEQ ID NO.

1.

4. The method for preparing the nanobody conjugate according to claim 1, wherein In the elastin-like polypeptide, X is valine, and 60≤n≤120.

5. The method for preparing the nanobody conjugate according to claim 1, wherein 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 through protein expression.

6. The method for preparing the nanobody conjugate according to claim 1, characterized in that: The structural formula of the chemically modified bortezomib is: 。 7. A pharmaceutical composition, characterized in that It contains a nanobody conjugate prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nanobody conjugate prepared by the preparation method according to any one of claims 1 to 6 in the preparation of a drug for treating ovarian cancer.

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