A photodegradable protein-targeted chimeric antibody targeting BRD4 protein, and its preparation method and application

Abstract

The present invention relates to the field of biomedicine, and in particular to a photodegradable protein-targeted chimeric antibody targeting BRD4 protein, and a preparation method and application thereof. The present invention provides a photodegradable protein-targeted chimeric antibody targeting BRD4 protein, comprising a targeting ligand JQ1‑aci targeting BRD4 protein and a photosensitizer pyropheophorbide‑a, as well as a linker connecting the two parts. The design of the photodegradable protein-targeted chimeric antibody provided by the present invention can guide PPa to the BRD4 region, and then, under light conditions, the photosensitizer can be excited to produce a local high concentration of singlet oxygen, thereby achieving light-targeted destruction and degradation of the BRD4 protein, and exhibiting anti-tumor activity, which is of great significance for targeted tumor therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a photodegradable protein-targeted chimeric antibody targeting BRD4 protein, and a preparation method and application thereof. Background Art

[0002] Targeted protein degradation (TPD) strategies regulate tumor growth by degrading proteins of interest (POIs) along targeted pathways and are an effective approach for treating cancer. The photodegradation-targeted chimeras (PDTACs) strategy, which utilizes laser irradiation to target sites, can be used for precise and spatiotemporally controllable TPD, which only degrades the targeted protein at the laser irradiation site, ensuring precise treatment. Bromo- and extra-terminal domain (BET) proteins are key activators of oncogenic transcription and are widely studied epigenetic targets in the treatment of lung cancer, ovarian cancer, breast cancer, renal cell carcinoma, and melanoma. Among them, BRD4, a member of the BET protein family, plays an important role in tumor infiltration, metastasis, and malignant progression.

[0003] Currently, studies have reported highly effective BET inhibitors, but BET proteins play an important role in the growth of neural crest and lymphocytes, and therefore may cause neurotoxicity and hematologic toxicity. Furthermore, BET inhibitors still face the problem of drug resistance in tumor treatment, and their pharmacokinetic properties are poor, with limited penetration into deep tumor tissue. Although several PDTACs have been reported, current research remains at the in vitro or intertumoral injection level, and no PDTAC can be systemically administered in vivo. Therefore, it is necessary to develop a PDTAC that is effective in treating tumors. Summary of the Invention

[0004] The present invention aims to provide a photodegradable protein-targeted chimeric antibody targeting the BRD4 protein, as well as its preparation method and application, to address the problems of the above-mentioned prior art. The photodegradable protein-targeted chimeric antibody targeting the BRD4 protein provided by the present invention can significantly inhibit tumor volume, achieving the purpose of tumor treatment.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a photodegradation protein targeting chimeric antibody targeting BRD4 protein, wherein the photodegradation protein targeting chimeric antibody comprises a targeting ligand JQ1-aci and pyropheophorbide-a, and a linker connecting the targeting ligand JQ1-aci and the pyropheophorbide-a; the structural formula of the photodegradation protein targeting chimeric antibody is shown in Formula I,

[0007]

[0008] The present invention provides a method for preparing the above-mentioned photodegradable protein-targeting chimeric antibody, comprising the following steps:

[0009] (1) connecting pyropheophorbide-a and Boc-hexanediamine via an amide condensation reaction to obtain product 1;

[0010] (2) mixing the product 1, dichloromethane and trifluoroacetic acid to react to obtain compound 2;

[0011] (3) Connecting the compound 2 and the targeting ligand JQ1-aci through an amide condensation reaction to obtain the photodegradable protein targeting chimeric antibody.

[0012] Preferably, in step (1), the mass ratio of the pyropheophorbide-a to the Boc-hexamethylenediamine is 50:30.34.

[0013] Preferably, in step (3), the mass ratio of the compound 2 to the targeting ligand JQ1-aci is 40:30.36.

[0014] Preferably, in step (2), the mass volume ratio of the product 1, the dichloromethane and the trifluoroacetic acid is 22 mg:2 mL:0.3 mL.

[0015] Preferably, in steps (1) and (3), the condensing agents used in the amide condensation reaction are HATU and DIPEA.

[0016] The present invention provides the use of the above-mentioned photodegradable protein-targeting chimeric antibody in the preparation of anti-tumor drugs.

[0017] Preferably, the tumor comprises melanoma.

[0018] The present invention provides an anti-tumor drug, the active ingredient of which includes the above-mentioned photodegradable protein-targeting chimeric antibody.

[0019] Further preferably, the tumor comprises melanoma.

[0020] The present invention provides an anti-tumor system, comprising: a) the above-mentioned photodegradable protein-targeting chimeric antibody; and b) an illumination device.

[0021] Further preferably, the tumor comprises melanoma.

[0022] The present invention discloses the following technical effects:

[0023] The present invention provides a photodegradable protein-targeted chimeric antibody targeting BRD4 protein, comprising a targeting ligand JQ1-aci targeting BRD4 protein and a photosensitizer pyropheophorbide-a (PPa), as well as a linker connecting the two parts. Bromo- and extra-terminal domain (BET) proteins are key activators of oncogenic transcription and are widely studied epigenetic targets in the treatment of various cancers. Among them, the BRD4 protein in this protein family is a key protein affecting tumor infiltration, metastasis and malignant progression. The design of the photodegradable protein-targeted chimeric antibody provided by the present invention can guide PPa to the BRD4 region, and then, under light conditions, the photosensitizer can be excited to produce a local high concentration of singlet oxygen, thereby achieving light-targeted destruction and degradation of the BRD4 protein, and exhibiting anti-tumor activity, which is of great significance for targeted tumor therapy. It can be seen that the photodegradable protein-targeted chimeric antibody of the present invention can specifically target the BRD4 protein, shorten the distance between the photosensitizer and the BRD4 protein, and after applying a 660nm laser, it can excite the photosensitizer to produce a local high concentration of singlet oxygen, thereby achieving phototargeted destruction and degradation of the BRD4 protein, and has excellent anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 Flow chart for the synthesis of chimeric antibodies targeting photodegradable proteins;

[0026] Figure 2 This is the H NMR spectrum of the chimeric antibody targeting photodegradable protein;

[0027] Figure 3 This is the UV absorption spectrum of the chimeric antibody targeting photodegradable protein;

[0028] Figure 4 Fluorescence spectroscopy of chimeric antibodies targeting photodegradable proteins;

[0029] Figure 5 The UV absorption spectrum of the singlet oxygen generation ability of the chimeric antibody targeting photodegradable protein detected by DPBF probe;

[0030] Figure 6 The results of the cytotoxicity test on B16 cells after the photodegradable protein-targeted chimeric antibody was prepared into nanoparticles;

[0031] Figure 7The results show the selective degradation of BRD4 protein in living B16 cells by photodegradable protein-targeting chimeric antibodies prepared into nanoparticles at different concentrations; A is HSA@JQ1-acid, B is HSA@PPa-JQ1, and C is HSA@PPa-JQ1+L;

[0032] Figure 8 Figure 2 is the curve of tumor volume changes with treatment time in tumor-bearing mice in different treatment groups. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] Synthesis route of chimeric antibodies targeting photodegradable proteins Figure 1As shown, PPa is first linked to Boc-hexanediamine via an amide condensation reaction, and the BOC (tert-butyloxycarbonyl) protecting group is then removed with trifluoroacetic acid (TFA) to generate an intermediate product containing an -NH3 terminus. The amino-containing intermediate product is then linked to JQ1-acid with a carboxylic acid group via an amide condensation reaction again to generate PPa-JQ1 containing the photosensitizer PPa and the BRD4-targeting ligand JQ1. The specific steps are as follows:

[0040] (1) Accurately weigh PPa (50 mg, 1.0 equiv) and Boc-hexanediamine (30.34 mg, 1.5 equiv) were dissolved in 5 mL of dry dimethylformamide (DMF), and then HATU (42.7 mg, 1.2 equiv, condensing agent) and N,N-diisopropylethylamine (DIPEA, 29 μL, 3.0 equiv, condensing agent) were added and reacted at room temperature for 24 h. After the reaction was completed, water was added to the solution after the reaction was completed, and the mixture was extracted with EA for 5 times. The organic phases were then combined and dried over anhydrous sodium sulfate. After evaporation of the solvent, the resulting residue was purified by silica gel column chromatography (EA:PE = 3:1, v / v) to obtain a black solid 1, whose structural formula is shown below. Figure 1 As shown in 1, a total of 44 mg was obtained, with a yield of 64.0%.

[0041] (2) The black solid 1 (44 mg, 1 equiv) was dissolved in 4 mL of dry dichloromethane, 600 μL of trifluoroacetic acid was added, and the mixture was stirred on ice for 4 h. After the reaction was completed, the dichloromethane and most of the trifluoroacetic acid were removed under reduced pressure to obtain compound 2, whose structural formula is shown below: Figure 1 As shown in 2, a total of 40 mg was obtained.

[0042] (3) Compound 2 (40 mg, 1.0 equiv) and JQ1-acid (30.36 mg, 1.2 equiv, manufactured by Shanghai Bid Pharmaceutical Technology Co., Ltd., product number: BD630518-5g) were dissolved in 5 mL of dry DMF, and then HATU (36.08 mg, 1.5 equiv) and DIPEA (65.40 μL, 10.0 equiv) were added and reacted at room temperature for 24 h. After the reaction was completed, the residue was purified by silica gel column chromatography (EA: methanol = 20:1, v / v) to obtain a photodegradable protein-targeted chimeric antibody, also known as a photodegradable protein-targeted chimeric antibody PPa-JQ1, with a total yield of 35 mg and a yield of 54.6%. The H NMR spectrum of the product is as follows: Figure 2 As shown, the H NMR spectrum data are as follows:

[0043] 1H NMR(400MHz,Chloroform-d)δ9.55(s,1H),9.44(s,1H),8.54(s,1H),8.02(s,1H),7.22(s,4H),6.53(s,1H), 6.28(s,1H),6.18(s,1H),5.85(s,1H),5.25(s,1H),5.14(s,1H),4.55(s,1H),4.38(s,1H),4.32(s,1H),3.67 (s,3H),3.39(s,3H),3.27(s,3H),2.22(s,3H),2.13(s,1H),2.08(s,1H),2.06(s,3H),1.77(s,4H),1.72(s, 2H),1.70(s,3H),1.68(s,3H),1.40(s,4H),1.33(s,2H),1.28(s,2H),1.25(s,4H),1.14(s,3H),0.88(s,2H).

[0044] (4) The structural formula of the photodegradable protein-targeting chimeric antibody PPa-JQ1 is shown in Formula I.

[0045]

[0046] Example 2 Characterization of the Photodegradable Protein Targeting Chimeric Antibody Prepared in Example 1

[0047] 1. UV spectrum characterization

[0048] The photodegradable protein targeting chimeric antibody was dissolved in DMSO solution (containing 10% water) and prepared into solutions of different concentrations (2.5, 5, 10, 20 and 30 μM), and the UV absorption spectra of different solutions were measured. Figure 3 As shown. Figure 3 It can be seen that PPa-JQ1 exhibits a maximum characteristic absorption peak at 668 nm, which is similar to PPa, and its absorbance value gradually increases with the increase of the concentration of the photodegradable protein targeting chimeric antibody.

[0049] 2. Fluorescence characterization spectrum

[0050] The photodegradable protein-targeted chimeric antibody was dissolved in DMSO solution, and its fluorescence emission (Emission) and excitation (Excitation) spectra were measured. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the maximum emission wavelength of PPa-JQ1 is 675 nm and the maximum excitation wavelength is 660 nm.

[0051] 3. Use singlet oxygen indicator fluorescent probe (DPBF probe) to detect singlet oxygen produced by photodegradable protein targeting chimeric antibody

[0052] Use DPBF as 1 As an O2 capture reagent, the photodegradable protein-targeting chimeric antibody PPa-JQ1 (20 μM, dissolved in 300 μL ion-exchanged water) was added to DPBF (20 μM, dissolved in 700 μL DMSO). The UV absorption of PPa-JQ1 at 425 nm was recorded by UV-visible spectrophotometer under 660 nm laser irradiation at different time intervals. The experimental results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that with the increase of illumination time, DPBF 1 After the interaction with O2), it undergoes an irreversible oxidation process, and its absorbance intensity at 425nm in the UV-visible spectrum decreases rapidly, thus confirming the 1 O2 production capacity.

[0053] Example 3 Cytotoxicity Experiment of the Photodegradable Protein Targeting Chimeric Antibody Prepared in Example 1

[0054] B16 cells were incubated in a 96-well plate for 24 h, and the photodegradable protein-targeting chimeric antibody HSA@PPa-JQ1 or the control HSA@JQ1-acid (the only difference from HSA@PPa-JQ1 is that albumin only coats JQ1-acid, and JQ1-acid is not connected to PPa, i.e., it does not contain PPa, and the preparation method is the same as that of albumin-coated PPa-JQ1) was added, and the cells were incubated for another 12 h. The cells were illuminated with a 660 nm laser (0.1 W / cm 2 ) The light-treated group (HSA@PPa-JQ1+L) was exposed to light for 3 minutes and then incubated for 24 hours. The dark-treated group (HSA@PPa-JQ1) or the HSA@JQ1-acid group was incubated for 36 hours after drug addition without light treatment. The CCk8 kit was then used to detect cell death in each group. The experimental results are shown in Figure 2. Figure 6 As shown. Figure 6It can be seen that within the given test concentration range, the cell activity of the HSA@JQ1-acid group and the HSA@PPa-JQ1 group remained above 80%, and their cytotoxicity was negligible. This shows that HSA@JQ1-acid alone and HSA@PPa-JQ1 without laser irradiation do not significantly affect cell survival. On the contrary, under the irradiation of 660nm laser, the survival rate of B16 cells in the HSA@PPa-JQ1+L group decreased sharply with increasing concentration, showing a concentration-dependent phototoxicity trend. HSA@PPa-JQ1+L showed significant cytotoxic effects on B16 cells, IC 50 The value was 0.77 μM. This demonstrates that the PDTAC strategy can activate the photoswitch on demand, selectively induce cytotoxicity at the tumor irradiation site, and achieve spatiotemporal control of antitumor effects. This can prevent systemic protein degradation and greatly improve the safety of therapies based on target protein degradation.

[0055] Example 4 Protein degradation experiment of photodegradable protein-targeting chimeric antibody

[0056] The photodegradable protein-targeting chimeric antibody was analyzed by western blot. The specific steps are as follows:

[0057] In six-well plates (5 × 10 5 B1 cells were incubated in the presence of 400 μg / mL of 5% paraformaldehyde (PNA) for 24 h, and the photodegradable protein-targeting chimeric antibody HSA@PPa-JQ1 or the control group HSA@JQ1-acid, which was coated with albumin and prepared in different concentrations (0, 0.5, 1, and 2 μM), was added and the cells were incubated for 12 h. The cells were then illuminated with a 660 nm laser (0.1 W / cm 2 ) The illumination group (HSA@PPa-JQ1+L) was illuminated for 3 minutes and the cells were incubated for 12 hours. The HSA@PPa-JQ1 and control groups HSA@JQ1-acid were incubated for 12 hours after drug addition without illumination treatment.

[0058] On ice, cells were lysed for 30 min in RIPA lysis buffer (Beyotime-Biotechnology) containing a protease-phosphatase cocktail inhibitor cocktail (Beyotime Biotechnology) and collected using a cell scraper. Protein concentrations were quantified and normalized using a BCA protein concentration assay kit (Beyotime Biotechnology).

[0059] The harvested total protein was then separated by SDS-PAGE on a 12% polyacrylamide gel and transferred to a PVDF membrane (Beyotime Biotechnology). The PVDF membrane was incubated in a 5% BSA solution in TBST (TBS + 0.1% Tween 20) for 2 h to block nonspecific binding of the antibody.

[0060] The membrane was incubated with primary antibodies against BRD4 and β-actin at 4°C overnight: the BRD4 primary antibody (catalog number AB128874, sourced from rabbit, diluted 1:1000, produced by Abcam, dissolved in TBST containing 5% BSA); the β-actin primary antibody (catalog number AF7018, sourced from rabbit, diluted 1:5000, produced by Affinity Biosciences, dissolved in TBST containing 5% BSA).

[0061] After washing the PVDF membrane with TBST, the membrane was incubated with a secondary antibody (AB2839429, S0001, goat, 1:5000, Affinity Biosciences, dissolved in 5% BSA in TBST) for 1 hour at room temperature and then washed with TBST. Protein bands were detected using an enhanced chemiluminescence kit (FDbio-Pico-Ecl, Hangzhou Fude Biotechnology Co., Ltd.). Figure 7 The experimental results are shown in Figure 7 As shown, it was shown that the photodegradable protein-targeting chimeric antibody can successfully achieve targeted and selective degradation of BRD4 protein in vitro.

[0062] Example 5 Experimental study on the tumor growth inhibition effect of the photodegradable protein-targeted chimeric antibody prepared in Example 1

[0063] The photodegradable protein-targeting chimeric antibody prepared in Example 1 was coated with albumin to prepare nanoparticles, and its tumor growth inhibitory effect on B16 tumor-bearing mice was tested as follows:

[0064] 100 μL containing 6 × 10 5 A subcutaneous xenograft melanoma tumor model was established by injecting PBS containing 100 B16 cells into the right lower back of female BALB / c nude mice (5-6 weeks old).

[0065] When the tumor volume of BALB / c nude mice grows to 80-100 mm 3Afterwards, the mice were randomly divided into five groups: (I) PBS, (II) PBS+L, (III) HSA@JQ1-acid+L, (IV) HSA@PPa-JQ1, and (VI) HSA@PPa-JQ1+L, with 5 biological replicates in each group. Subsequently, each mouse in each group was administered with a dose of 2.4 mg / kg of the drug (which had been coated with albumin to form nanoparticles) via tail vein injection every day for three consecutive days. 12 hours after administration, the mice were irradiated with a 660 nm laser (0.1 W / cm 2 The tumor site was irradiated with irradiation for 10 min. Tumor size and mouse body weight were recorded daily to evaluate the therapeutic effect.

[0066] The changes of tumor volume in each group of mice are as follows Figure 8 As shown, only the photodegradable protein-targeting chimeric antibody injection plus light irradiation treatment (HSA@PPa-JQ1+L) showed a significant tumor inhibitory effect.

[0067] The present invention verifies for the first time the systemic administration of PDTAC molecules for tumor treatment, providing a promising approach for the treatment of cancer with photodegradable protein-targeted chimeras.

[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A photodegradable protein targeting chimeric antibody targeting BRD4 protein, characterized in that: The photodegradable protein targeting chimeric antibody comprises a targeting ligand JQ1-aci and pyropheophorbide-a, and a linker connecting the targeting ligand JQ1-aci and the pyropheophorbide-a; the structural formula of the photodegradable protein targeting chimeric antibody is shown in Formula I, 2. The method for preparing the photodegradable protein-targeting chimeric antibody according to claim 1, characterized in that: The following steps are involved: (1) connecting pyropheophorbide-a and Boc-hexanediamine via an amide condensation reaction to obtain product 1; (2) mixing the product 1, dichloromethane and trifluoroacetic acid to react to obtain compound 2; (3) Connecting the compound 2 and the targeting ligand JQ1-aci through an amide condensation reaction to obtain the photodegradable protein targeting chimeric antibody.

3. The preparation method according to claim 2, characterized in that In step (1), the mass ratio of the pyropheophorbide-a to the Boc-hexanediamine is 50:30.

34.

4. The preparation method according to claim 2, characterized in that In step (3), the mass ratio of the compound 2 to the targeting ligand JQ1-aci is 40:30.

36.

5. The preparation method according to claim 2, characterized in that In step (2), the mass volume ratio of the product 1, the dichloromethane and the trifluoroacetic acid is 22 mg:2 mL:0.3 mL.

6. The preparation method according to claim 2, characterized in that In steps (1) and (3), the condensing agents used in the amide condensation reaction are HATU and DIPEA.

7. Use of the photodegradable protein-targeting chimeric antibody according to claim 1 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The tumor includes melanoma.

9. An anti-tumor drug, characterized in that: The active ingredient of the drug includes the photodegradable protein-targeting chimeric antibody according to claim 1.

10. An anti-tumor system, characterized in that: The anti-tumor system comprises: a) the photodegradable protein-targeting chimeric antibody according to claim 1; and b) a light irradiation device.

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