Antibody fragment drug conjugate based on targeting p16 single-chain antibody

Abstract

The invention discloses an antibody fragment drug conjugate based on a targeting p16 single-chain antibody. The antibody fragment drug conjugate is composed of a p16 single-chain antibody, a cell-penetrating peptide, a connexon and a carrier drug. The p16 single-chain antibody and the cell-penetrating peptide form a fusion protein through fusion expression, and the fusion protein is further coupled and connected with the carrier drug through a linker to form the antibody fragment drug conjugate. The invention provides a novel treatment strategy through targeting p16, and is especially suitable for tumor types with high expression of p16, and the tumor types lack effective treatment means at present. By targeting the p16 target spot in the cell nucleus, not only is the candidate target spot library of targeted therapy enriched, but also feasibility verification and technical reference are provided for future drug development of the target spot in the nucleus.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and particularly relates to an antibody fragment-drug conjugate based on a p16-targeting single-chain antibody. Background Art

[0002] The p16 protein is a key tumor suppressor that is abnormally highly expressed in a variety of cancers, such as triple-negative breast cancer and cervical cancer, and is mainly distributed in the nucleus and cytoplasm. Due to its specific intracellular localization, traditional treatments are difficult to effectively target and intervene. The development of nuclear targets has opened up a new direction for precision tumor treatment, especially in interfering with cell proliferation mechanisms to treat solid tumors, which has important clinical significance.

[0003] Antibody-drug conjugates (ADCs) combine the high specificity of antibodies with the killing effect of cytotoxic drugs, showing great potential in tumor treatment. However, most existing ADC drugs are based on full-length antibodies, mainly targeting cell membrane-related or cytoplasmic proteins (such as HER2, EGFR), and there is relatively little research on nuclear targets. In addition, due to their large molecular weight, full-length antibodies have limited tumor penetration in solid tumors with high interstitial pressure.

[0004] In contrast, single-chain antibodies (scFv) have the advantages of small molecular weight and strong tumor penetration, and are particularly suitable for delivery and intervention of nuclear targets. At present, some studies focus on regulating p16INK4a expression through RNA interference (such as siRNA, shRNA), but these methods mostly belong to nucleic acid drugs, and FDC drugs (antibody fragment-drug conjugates) based on antibody fragments have not yet been developed. In addition, anti-p16 antibodies used for cancer diagnosis are only used for immunohistochemical analysis and have not been developed as a therapeutic tool. Therefore, the innovative development of FDC targeting p16 nuclear targets is still blank. Summary of the invention

[0005] The purpose of the present invention is to target p16 in the nucleus by fusing the cell-penetrating peptide S4 13 , achieving transmembrane delivery and nuclear retention of antibodies, and conjugating them with the chemotherapy drug doxorubicin (DOX), innovatively designing an antibody fragment drug conjugate (FDC) based on a single-chain antibody (scFv), and verifying its in vitro anti-tumor activity.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An antibody fragment drug conjugate based on a p16-targeted single-chain antibody comprises a p16 single-chain antibody, a cell-penetrating peptide, a linker and a carrier drug; the p16 single-chain antibody and the cell-penetrating peptide are fused and expressed to form a fusion protein, which is then coupled with the carrier drug through the linker to form the antibody fragment drug conjugate based on the p16-targeted single-chain antibody.

[0008] Preferably, the p16 single-chain antibody is humanized before use.

[0009] Preferably, the nucleotide sequence of the p16 single-chain antibody is shown in SEQ ID NO:2.

[0010] Preferably, the p16 single-chain antibody is prepared by monoclonal antibody secreted by hybridoma cells.

[0011] Preferably, the p16 single-chain antibody is prepared by sequencing a monoclonal antibody secreted by hybridoma cells and by genetic engineering.

[0012] Preferably, the p16 single-chain antibody is obtained by extracting RNA from hybridoma cells, obtaining the antibody variable region sequence, and then expressing it through recombinant expression.

[0013] Preferably, the preparation of the p16 single-chain antibody comprises: extracting RNA from the hybridoma cells, reverse transcribing to obtain the antibody variable region sequence, and then recombinantly expressing to obtain the p16 single-chain antibody.

[0014] Preferably, the hybridoma cell comprises mouse hybridoma cell line 3837-04, which is deposited in the Institute of Microbiology, Guangdong Academy of Sciences with a deposit number of GDMCC No: 65754.

[0015] Preferably, the cell-penetrating peptide comprises S4 13 .

[0016] Preferably, the nucleotide sequence of the cell-penetrating peptide is as shown in SEQ ID NO:1.

[0017] Preferably, the linker comprises oxidized Dextran T-10; the preparation of the linker comprises: mixing Dextran T-10 with NaIO 4 The mixture was mixed and protected from light, and the Oxidized Dextran T-10 was obtained after freeze-drying.

[0018] Preferably, the carrier drug comprises doxorubicin.

[0019] It is understood by those skilled in the art that DOX coupling is one of the carrier drugs, and other drug molecules may also be selected for construction, such as monomethyl auristatin E (MMAE), irinotecan (SN38), etc., which also fall within the scope of the present invention.

[0020] An application of the above-mentioned antibody fragment drug conjugate based on the p16-targeting single-chain antibody is used to prepare a drug that specifically targets the p16 target.

[0021] A medicine obtained by the above application.

[0022] The invention can be used for preparing relevant antibody fragment-coupled drugs, and can be particularly used for treating relevant cancers with high expression of p16 target.

[0023] The implementation of the present invention has the following beneficial effects:

[0024] 1. Application of new targets

[0025] At present, research in the field of targeted therapy is mainly focused on common membrane surface targets such as HER2 and EGFR. Although these targets have been widely used in clinical practice, they also face the problems of drug resistance and limited scope of application. In contrast, the present invention uses p16 protein, a nuclear target with important clinical significance, for the first time in the design of fragment drug conjugates (FDCs). As a tumor suppressor, the abnormally high expression of p16 protein is closely related to the occurrence and development of various solid tumors (such as triple-negative breast cancer and cervical cancer). The present invention provides a new treatment strategy by targeting p16, which is particularly suitable for tumor types with high expression of p16, and these tumor types currently lack effective treatment methods. By targeting the p16 target in the cell nucleus, the present invention not only enriches the candidate target library for targeted therapy, but also provides feasibility verification and technical reference for the future drug development of nuclear targets.

[0026] 2. Targets in the cell nucleus

[0027] Current antibody-drug conjugates (ADCs) mainly target the cell membrane surface and cannot be efficiently delivered into the cell nucleus. In contrast, the FDC developed by the present invention uses the cell-penetrating peptide S4 13 The drug is delivered across the membrane and retained in the nucleus, allowing the p16 target in the nucleus to be acted on efficiently and accurately. Nuclear target therapy has the advantage of directly interfering with DNA repair, gene transcription and cell cycle regulation, and can affect the proliferation mechanism of tumor cells at a deeper level compared to membrane surface targets. At the same time, by fusing S4 13This design achieves efficient transmembrane and nuclear retention functions, filling the technical gap in the delivery of existing ADC drugs to nuclear targets, and provides a new solution for drugs that are difficult to reach the nucleus. The precise action of nuclear targets effectively avoids off-target effects in the extracellular or cytoplasmic areas, reduces toxic side effects, and greatly improves the therapeutic effect, especially in the complex tumor microenvironment.

[0028] 3. Antibody fragment conjugates

[0029] Due to the high interstitial pressure and hypoxic environment of solid tumors, the diffusion and permeability of drugs are poor, making it difficult for traditional ADC drugs to be evenly distributed. The present invention uses FDC drugs in the form of single-chain antibodies (scFv), which overcome the shortcomings of large molecular weight and weak tumor penetration ability of full-length antibodies, and are more suitable for the treatment of solid tumors. Single-chain antibodies (about 25kDa) have small molecular weight and stable structure, better tumor permeability and uniform distribution, and can exert stronger therapeutic effects in the deep layers of solid tumors. Compared with full-length antibody conjugates, the FDC drugs of the present invention are more applicable in solid tumors with high interstitial pressure, especially showing better therapeutic effects on tumor types that are difficult to penetrate.

[0030] 4. The present invention focuses on the precise identification and targeting of specific targets (p16) and integrates the cell-penetrating peptide S4 13 This is just to solve the technical problem of how antibody drugs can achieve nuclear target delivery and binding, and further lay the foundation for the preparation of antibody-drug conjugates (FDC). The present invention not only focuses on delivery efficiency, but also through targeted design (fusion S4 13 ) and high-affinity scFv sequences were screened to precisely target the p16 protein in the cell nucleus, which was further used in the preparation of FDC and combined with cytotoxic small molecule drugs to enhance the therapeutic efficacy against tumors with high p16 expression.

[0031] 5. The scFv of the present invention is obtained by extracting heavy chain and light chain variable region genes from 5 hybridoma cells, and then the sequence with the best affinity is obtained by molecular modeling and molecular docking screening, which is then optimized into a variable region fragment form connected by a flexible peptide, does not contain a constant region, and is more miniaturized. At the same time, the present invention has humanized the scFv, which can reduce immunogenicity and make it more suitable for in vivo application. In addition, the scFv of the present invention also incorporates a cell-penetrating peptide (CPP), which gives it a unique cell-penetrating ability and can directly enter the cell to play a role. Therefore, the scFv of the present invention is more innovative in design concept, and is particularly suitable for precise treatment of drug delivery or intracellular targets.

[0032] 6. In the present invention, the compound part inserted into FDC, namely DOX, is the core factor in exerting anti-tumor activity. This compound part is connected to the p16 single-chain antibody fragment through a covalent bond, and is specifically delivered to tumor cells with high expression of the p16 gene under the guidance of the targeting antibody. After being released at a specific location, it achieves an efficient killing effect.

[0033] 7. The synthetic process of coupling antibody fragments and small molecule drugs in the present invention is a method based on non-specific coupling of antibody amine groups with oxidized dextran T-10, which has the characteristics of simple operation, good biocompatibility and high coupling efficiency. The natural amine groups on the antibody form Schiff base bonds with the aldehyde groups in the oxidized dextran T-10, and further NaBH 4 Reduction-stabilized covalent bonds, this non-site-specific coupling technology avoids complex genetic engineering modifications to antibodies, while effectively protecting the activity of antibodies and small molecule drugs under mild reaction conditions (room temperature and low temperature). As a multifunctional linker, Dextran T-10 not only provides ample space and flexibility, reduces the interference of coupling on the functional area of ​​the antibody, ensures the retention of antibody targeting, but also reduces potential immunogenicity due to its biocompatibility and degradability.

[0034] 8. During the purification of scFv, we conducted multiple experiments and optimizations for the best purification scheme. At first, we tried to purify it using a nickel affinity column by adding a His tag, but found that only the scFv in the inclusion body could bind, while the soluble expressed scFv part could not effectively bind to the nickel column. Then we tried to increase the number and position of the His tag, but it still could not bind. It is speculated that it may be because the conformation of the His tag in the soluble scFv is shielded, resulting in low binding efficiency. Subsequently, other fusion tags, including GST, MBP and Strep, were replaced and tried to be purified through the corresponding affinity chromatography column, but the soluble expression products of these tags also failed to effectively bind to the corresponding affinity column. Although inclusion bodies can achieve protein recovery through denaturation and renaturation, this method is prone to scFv folding errors or functional loss, especially the impact on affinity cannot be ignored, so it is not the preferred strategy. In order to maximize the retention of the affinity and functionality of scFv, DEAE ion exchange columns were finally selected for purification. Ion exchange columns are separated based on protein charge characteristics and do not depend on the exposure state of specific tags, avoiding the above problems. By optimizing the ion exchange conditions, we successfully achieved efficient purification of scFv and ensured its functional integrity in its native conformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 .A: Total RNA electrophoresis of five hybridoma cell lines; B: V H PCR amplification results; C: VL PCR amplification results; D: V H Colony PCR electrophoresis results; E: V L Colony PCR electrophoresis results. In the figure, M: marker; 1: 3837-01; 2: 3837-03; 3: 3837-04; 4: 3837-07; 5: 3837-08.

[0036] Figure 2 .Humanized scFv-p16 (V L : Purple; V H : Molecular docking results of scFv-p16-01 and p16 antigen (green); B: scFv-p16-03 and p16 antigen; C: scFv-p16-04 and p16 antigen; D: scFv-p16-07 and p16 antigen; E: scFv-p16-08 and p16 antigen.

[0037] Figure 3 .A, B, C: scFv-p16-S4 13 SDS-PAGE results. In the figure, M: Protein Marker; 1: Supernatant after ultrasonic disruption of uninduced bacteria; 2: Precipitate after ultrasonic disruption of uninduced bacteria; 3: Supernatant after ultrasonic disruption of induced bacteria; 4: Precipitate after ultrasonic disruption of induced bacteria. D: scFv-p16-S4 13 Western-Blot results. (1): supernatant after bacterial ultrasonic disruption; (2): precipitate after bacterial ultrasonic disruption. 1: scFv-p16-S4 13 -01;2:scFv-p16-S4 13 -03;3:scFv-p16-S4 13 -04;4:scFv-p16-S4 13 -07;5:scFv-p16-S4 13 -08.

[0038] Figure 4 .A, B: scFv-p16-S4 13 SDS-PAGE after antibody purification. In the figure, M: Protein Marker; 1: Cell supernatant after ultrasonic disruption; 2: 2M NaCl elution sample; 3: Flow-through sample. C, D, E, F, G: Western Blot verification of recombinant scFv-p16-S4 13 Antibody expression.

[0039] Figure 5 .A: Schematic diagram of indirect Elisa. B: Recombinant scFv-p16-S4 at different dilution ratios 13Elisa signals.

[0040] Figure 6 .Schematic diagram of FDC synthesis.

[0041] Figure 7 A: UV-visible spectra of Dextran T-10, Oxidized Dextran T-10, scFv, DOX and FDC (250-600 nm). B, C, D: Standard curves of BSA (280 nm), DOX (480 nm) and DOX (280 nm).

[0042] Figure 8 Effects of DOX, scFv and FDC on the viability of MDA-MB-231, Hela, BT-549, LO2 and HEK-293T cells. The same letters in the same group of data indicate no significant difference (p>0.05), and different letters indicate significant difference (p<0.05).

[0043] Fig. 9 .Representative images of scFv and FDC in MDA-MB-231, Hela, and BT-549 cells. A: Representative cell images; B, C: Fluorescence intensity. FITC: Green fluorescence, indicating scFv; DOX: Red fluorescence, indicating DOX; DAPI: Blue fluorescence, indicating cell nuclei. FDC: Antibody fragment drug conjugate; scFv: Single-chain antibody.

[0044] Fig.10 . Internalization evaluation of DOX and FDC in MDA-MB-231, Hela, BT-549, LO2 and HEK-293T cells. A: Representative cell images; B, C: Fluorescence intensity. BF indicates bright field cells, DOX indicates red fluorescence, (scale bar, 300 μm). The same letters in the same group of data indicate no significant difference (p>0.05), and different letters indicate significant difference (p<0.05). DOX: doxorubicin; FDC: antibody fragment conjugate drug. DETAILED DESCRIPTION

[0045] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0046] Experimental Example 1

[0047] Prokaryotic expression and purification of scFv-p16

[0048] 1. Experimental materials: Five mouse hybridoma cell lines that stably secrete p16 monoclonal antibodies (3837-01, 3837-03, 3837-04, 3837-07 and 3837-08)

[0049] 2. Test methods

[0050] 2.1p16 antibody variable region amplification

[0051] Five hybridoma cells were cultured at 1x10 5 The cells were inoculated in a 6-well plate at a concentration of 10 cells / mL, and total RNA was extracted using TRIZOL reagent. The total RNA concentration was measured using Nanodrop and then reverse transcribed into cDNA. Subsequently, 2×Rapid Taq MasterMix was used to amplify the variable regions of the antibody light chain and heavy chain. The amplification primer sequences were referenced from the monograph "Recombinant Antibodies" (Shen Beifen et al., 2005). The amplification system was: 5μL cDNA, 2μL of upstream and downstream primers, 25μL of 2×Rapid Taq Master Mix, and 16μL of DEPC water. The amplification procedure was as follows: pre-denaturation at 98℃ for 2min; denaturation at 98℃ for 7s, annealing at 55℃ for 30s, and extension at 72℃ for 15s, for a total of 30 cycles; complete extension at 72℃ for 10min. The PCR product was electrophoresed on a 1% agarose gel at 120V for 30min, and the target band was recovered after gel cutting. Subsequently, the recovered target band was connected to the pMD18-T vector using the TA cloning method and transformed into E. coli DH 5α competent cells, spread on a solid plate containing 100 mg / mL Amp, and cultured overnight at 37°C. The light and heavy chain positive clones were picked and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0052] 2.2 Homology modeling, humanization modification and molecular docking of scFv-p16

[0053] The variable region sequences of the light chain and heavy chain obtained above were imported into BIOVIA Discovery Studio 2023 to analyze the framework region (FR) and complementary determining region (CDR) of the antibody; in order to reduce the immunogenicity of the antibody, the CDR of the non-human antibody was transplanted to the human framework region to achieve the humanization of the antibody. The specific operation is as follows: Use the Model Antibodies function to perform homology modeling, and select the 3D model with the highest homology as the template; finally, use the Procheck function in the online website (https: / / saves.mbi.ucla.edu / ) to evaluate the rationality of the homology model. Subsequently, the PredictHumanizing Mutations function was used to transplant the CDR region of the antibody to the human donor template, and finally the FR region of the antibody was reversely mutated to obtain the humanized scFv-p16 sequence; in order to facilitate the subsequent molecular docking between the humanized scFv-p16 and the p16 antigen, the humanized scFv-p16 and the p16 antigen (NCBI: NM_000077.5) were homology modeled according to the same method as above. Then, the Dock Protein function in BIOVIADiscovery Studio 2023 was used for molecular docking. The specific operations were as follows: the five scFv-p16 obtained by homology modeling were set as receptors, the p16 antigen was set as a ligand, and the Dock Protein (ZDOCK) program was run. The Process Poses (RDOCK) function was used to fix the binding sites screened in the previous step to the CDR3 region of the antibody. The Refine Docked Proteins (RDOCK) function was then used to flexibly optimize the docking results, and the binding mode with the lowest E.RDOCK value was selected as the docking result. Finally, the PyMOL 3.0 software was used to visualize the molecular docking results.

[0054] 2.3 Construction of scFv-p16 prokaryotic expression system

[0055] According to the above molecular docking results, V L and V H The V L -Linker-V H The single-chain antibody was then cloned into the pET-28a(+) vector. To achieve efficient internalization of scFv-p16, the cell-penetrating peptide S4 was used. 13 Fusion expression with scFv-p16, cell-penetrating peptide S4 13 The nucleotide sequences of the two proteins are shown in SEQ ID NO: 1 and are named scFv-p16-S4. 13-01 (corresponding to hybridoma cell line 3837-01), scFv-p16-S4 13 -03 (corresponding to hybridoma cell line 3837-03), scFv-p16-S4 13 -04 (corresponding to hybridoma cell line 3837-04, wherein hybridoma cell line 3837-04 was deposited in the Institute of Microbiology, Guangdong Academy of Sciences on January 10, 2025, with the deposit number GDMCC No: 65754), scFv-p16-S4 13 -07 (corresponding to hybridoma cell line 3837-07) and scFv-p16-S4 13 -08 (corresponding to hybridoma cell line 3837-08). S4 13 Connect to the N segment of scFv-p16, the primer sequences are shown in Table 1. The first round of amplification system is: 2 μL of plasmid, 2 μL of upstream and downstream primers (F1, R), 2× Primer MAX DNA Polymerase 25μL, DEPC water 16μL; the first round of amplification procedure is as follows: 98℃ denaturation for 10s; 60℃ annealing for 30s, 72℃ extension for 1min, a total of 30 cycles; the first round of PCR products were subjected to 1% agarose gel electrophoresis at 120V for 30min, and then the gel was cut to recover the target band. The recovered target band was used as a template for the second round of PCR. The upstream primer was replaced with F2. The rest of the reaction system and procedures were the same as above. The PCR products were subjected to 1% agarose gel electrophoresis at 120V for 30min, and then the gel was cut to recover the target band; XhoⅠ and BamHⅠ were used to double-digest the target band with the pET-28a(+) empty vector at 37℃ for 3h, and after cleaning and recovery, T4 DNA ligase was used to connect at 16℃ overnight, and then the plasmid was transformed into E.coli DH by heat shock method. Among the 5α competent cells, positive clones were picked the next day and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmids were transformed into E. coli BL21 (DE3) by heat shock method.

[0056] Table 1 scFv-p16-S4 13 Primers used to construct

[0057]

[0058] Note: The underline indicates the restriction site

[0059] 2.4scFv-p16-S4 13 Expression and SDS-PAGE identification

[0060] The bacterial solution was inoculated into 100 mL LB liquid medium at 1.5% inoculum, and cultured at 37°C, 220 rpm until OD600 was between 0.6 and 0.8. IPTG was added at a final concentration of 0.5 mM, and induced at 30°C, 220 rpm for 5 h. The pET-28a (+) empty vector was induced under the same conditions as the control. After the induction, the cells were centrifuged at 4°C, 8000 rpm for 10 min, and the cells were collected and washed twice with PBS. The obtained cells were dissolved in PBS and lysed using an ultrasonic cell disruptor. The ultrasonic power was 180 W, 2 s on, 6 s off, and the total time was 15 min. The broken bacterial solution was centrifuged at 4°C, 8000 rpm for 30 min, and the supernatant and precipitate after the break were collected and stored at 4°C. The supernatant and precipitate collected above were added to 5× Loading Buffer respectively, boiled in a boiling water bath for 10 minutes, centrifuged at 10000rpm for 10 minutes, and electrophoresed at 80V for 30 minutes after loading, followed by electrophoresis at 120V for 1 hour. After the electrophoresis, the separation gel was removed, stained with a rapid gel stain for 2 hours, and photographed using a gel imaging system.

[0061] 2.5 Western Blot Verification of scFv-p16-S4 13 Express

[0062] Perform SDS-PAGE as described above. After electrophoresis, transfer the gel containing the target protein to a PVDF membrane at 280 mA for 1 h. After transfer, block with 5% BSA at room temperature for 1 h, then wash with TBST 5 times, incubate with Mouse Anti-His tag antibody at 4°C overnight, wash the membrane with TBST 5 times the next day, incubate with HRP-labeled goat anti-mouse IgG at room temperature for 2 h, wash the membrane with TBST 5 times, develop the PVDF membrane with ECL developer, and finally take pictures with a gel imaging system.

[0063] 2.6 Recombinant scFv-p16-S4 13 Antibody Purification and Western Blot

[0064] 2.6.1 Recombinant scFv-p16-S4 13 Antibody purification

[0065] DEAE Sepharose FF was used to purify the target protein. The specific operation was as follows: First, the filler was loaded into the column and rinsed with 2 column volumes of ultrapure water to remove the residual ethanol in the filler. Next, the resin was equilibrated with 10 column volumes of equilibration solution (20mMTris-HCl, pH 8.0). Then, the sample was loaded at a flow rate of 1mL / min and the flow-through was collected. After the loading was completed, the column was washed again with the equilibration solution until the absorbance at 280nm was close to the baseline. Subsequently, the impurities were eluted with the eluent (20mM Tris-HCl+2MNaCl, pH 8.0) until the absorbance at 280nm was close to the baseline again. After the elution was completed, the filler was equilibrated with 5 column volumes of equilibration buffer, and then the filler was washed with 5 column volumes of ultrapure water. Finally, 20% ethanol was added for preservation and placed in a 4°C refrigerator. The flow-through and eluent were collected separately for SDS-PAGE analysis. The purified target protein was concentrated to 1 mg / mL using a 3 kDa ultrafiltration tube, glycerol was added to a final concentration of 10%, and the aliquots were stored at -20°C.

[0066] 2.6.2 Identification of recombinant scFv-p16-S4 by Western Blot 13 Binding of antibodies to p16 antigen

[0067] The p16 antigen was subjected to SDS-PAGE, the sample volume was unified as 10 μL, and the membrane was transferred as described above. Five recombinant scFv-p16-S4 were used with a dilution ratio of 1:1000. 13 The primary antibody was incubated at 4°C overnight, and the secondary antibody was HRP-labeled anti 6×His at a dilution ratio of 1:6000. After development with ECL developer, the gel was photographed using a gel imaging system.

[0068] 2.7 Validation of recombinant scFv-p16-S4 by indirect Elisa 13 Binding ability of antibodies to p16 antigen

[0069] The p16 antigen was diluted to 3 μg / mL using carbonate buffer (pH 9.6), and 100 μL of p16 antigen was added to each well of the ELISA plate and incubated overnight at 4°C. The next day, the plates were washed three times with PBST solution, and then 100 μL of 1% BSA was added to each well and blocked at 37°C for 2 h to remove nonspecific adsorption. After blocking, 100 μL of the dilution of recombinant scFv-p16-S4 was added to each well. 13Antibody, add PBS as negative control, and incubate at 37℃ for 1h. After incubation, wash again with PBST solution 3 times, add 1:2000 diluted HRP-labeled anti 6×His antibody to each well, and incubate at 37℃ for 1h. After incubation, wash 3 times with PBST solution, then add 100μL color development solution to each well, and color at 37℃ in the dark for 10min. After color development, add 100μL stop solution to each well to stop color development. Finally, use an enzyme reader to measure the absorbance at 450nm. The absorbance value of the sample must be greater than 2.1 times that of the negative control to be considered a positive result.

[0070] 3. Test results

[0071] 3.1p16 antibody variable region amplification

[0072] First, the total RNA of five hybridoma cells was extracted by Trizol method, and appropriate amount of product was subjected to nucleic acid electrophoresis to test the quality of the extracted RNA. Figure 1 A, from top to bottom are 28S, 18S and 5S, and the A280 / A260 measured by Nanodrop are all between 1.8-2.0, indicating that the extracted RNA is of good quality and can be used for the next step of reverse transcription; then the RNA is used as a template for reverse transcription to obtain cDNA, and then the cDNA is used as a template to amplify the light and heavy chain variable regions of the antibody using universal primers. The electrophoresis results show that V L The size is 380-400 bp, V H The size of the band was 400-430 bp, which was consistent with the expected size, indicating that V L and V H Successfully amplified ( Figure 1 B and Figure 1 C) Then, the V of the five hybridoma cells were L and V H Gel excision was performed to recover the cloned fragments and cloned into the PMD18-T vector. L and V H The colony PCR verification was performed and the results were as follows Figure 1 D and 1E, and then the positive clones were sent to Beijing Qingke Biotechnology for sequencing. The sequencing results were analyzed using the IMGT (https: / / www.imgt.org / ) online website. L and V H The results obtained were consistent with the characteristics of the variable regions of the antibody light and heavy chains, that is, they all contained 3 CDR regions and 4 FR regions, indicating that the V region of the p16 antibody was successfully amplified. L and V H sequence;

[0073] 3.2 Molecular docking results of humanized scFv-p16 and p16 antigen

[0074] The molecular docking results are as follows Figure 2 As shown in A to E, the binding regions of the five antibodies to the p16 antigen are all in the CDR region, and the key amino acid residues of the specific binding sites are shown in Table 2; and the antibodies obtained from the five hybridoma cells have different binding abilities to the p16 antigen, and the specific E.RDOCK values ​​are shown in Table 3, among which scFv-p16-08 has the lowest E.RDOCK value of -31.70, and Figure 2 E also shows that scFv-p16-08 forms the most hydrogen bonds with the p16 antigen, indicating that the theoretical prediction shows that scFv-p16-08 has the highest affinity with the p16 antigen, but further verification is needed in subsequent experiments.

[0075] Table 2 Key amino acid residues for the interaction between five scFv-p16 and p16

[0076]

[0077]

[0078]

[0079] Table 3 The model with the highest matching degree in humanized scFv-p16 homology modeling and the E.RDOCK value after docking with p16 antigen molecule

[0080]

[0081] 3.3scFv-p16-S4 13 SDS-PAGE and Western-Blot analysis of antibodies

[0082] scFv-p16-S4 13 The cells were induced with 0.5 mM IPTG at 30 °C for 5 h. After ultrasonic disruption, the supernatant and precipitate were subjected to SDS-PAGE. Figure 3 A. Figure 3 B. Figure 3 C, the target protein was found to be expressed in both the supernatant and the precipitate; to further verify whether the target protein was successfully expressed, Western-Blot analysis was performed, and the results showed that the recombinant scFv-p16-S4 13 The proteins were able to bind to anti-His antibodies (see Figure 3 D), further demonstrated that recombinant scFv-p16-S4 13 The antibody was successfully expressed.

[0083] 3.4 Recombinant scFv-p16-S4 13 Antibody purification and Western-Blot analysis

[0084] After SDS-PAGE analysis (see Figure 4 A and 4B), the results showed that the recombinant scFv-p16-S4 with high purity was successfully obtained 13 The antibodies were further quantitatively analyzed using Image J software, and the results showed that the purity of the five recombinant antibodies was more than 70%. At the same time, Western-Blot results showed (see Figure 4 C. Figure 4 D. Figure 4 E. Figure 4 F and Figure 4 G), indicating that all recombinantly expressed single-chain antibodies can bind to the p16 antigen.

[0085] 3.5 Indirect Elisa assay for recombinant scFv-p16-S4 13 Antibody activity

[0086] The BCA assay was used to determine the expression of five purified scFv-p16-S4 13 The concentration of the antibody was adjusted to 1 mg / mL. The sample was then diluted 2048 times by the serial dilution method. The indirect ELISA method was used to evaluate the activity of scFv-p16-S4 13 The antibody's recognition activity for p16 antigen, the reaction principle is as follows Figure 5 A. The experimental results are shown in Figure 5 B. The OD450 value of the negative control was 0.156, and the OD450 value of the sample was 2.1 times greater than the OD450 value of the negative control (i.e., greater than 0.327) for a positive result. The results showed that all recombinantly expressed scFv-p16-S4 13 All antibodies can effectively recognize p16 antigen. Among them, scFv-p16-S4 13 -04 still showed binding activity when diluted to 0.488 μg / mL, indicating that the expressed scFv-p16-S4 13 The -04 antibody retains the specific binding activity of the original monoclonal antibody to the antigen, so in order to achieve efficient and specific targeting, scFv-p16-S4 was selected 13 -04 was used for subsequent FDC synthesis. The nucleotide sequence of scFv-p16-04 is shown in SEQ ID NO: 2.

[0087] Example 2

[0088] Synthesis and characterization of FDC

[0089] 1. Test materials: scFv-p16-S4 purified from Test Example 1 13

[0090] 2. Test methods:

[0091] 2.1 Synthesis of FDC

[0092] Select the scFv-p16-S4 with the best affinity obtained from the above optimization 13 Antibodies, using the active amine groups of antibodies themselves, are coupled with cytotoxic small molecule compounds through linkers for non-fixed quantitative coupling to prepare antibody fragment drug conjugates (FDC). See the reaction flow for details. Figure 6 The specific operation method is as follows: first, take 1g Dextran T-10 and add 10mL NaIO4 with a concentration of 0.35mM, react at 150rpm in the dark for 20h, and freeze-dry to obtain Oxidized Dextran T-10; take 2mg DOX and dissolve it in 2mL ultrapure water, add 30mg Oxidized Dextran T-10, and react at 150rpm in the dark for 20h. After the reaction, add 5mg scFv-p16-S413, react at 4℃ and 150rpm in the dark for 20h, then add 100μL NaBH4 with a concentration of 0.13mM, react at 37℃ and 150rpm in the dark for 2h, and finally use a 3kDa ultrafiltration tube to ultrafilter and replace the buffer with PBS (0.01M, pH 7.4).

[0093] 2.2 Determination of DAR value of FDC by UV-visible spectrophotometry

[0094] Dextran T-10, Oxidized Dextran T-10, DOX, scFv and FDC were scanned by UV-visible spectra at 200-600 nm to obtain the UV absorption spectrum and maximum absorption wavelength of each sample, and the spectral differences were compared.

[0095] Prepare 0-1 mg / mL BAS standard solution and 0-200 μg / mL DOX standard solution, scan at 280 nm and 280 nm, 480 nm to obtain absorbance values ​​and draw standard curves, then measure the prepared FDC at 280 nm and 480 nm to obtain absorbance values. Calculate the DAR value according to the following formula: DAR = (DOX concentration / DOX molar mass) / (scFv concentration / scFv molar mass)

[0096] 3. Test results

[0097] 3.1. Through UV-visible light full-band scanning, Figure 7As can be seen from A, the characteristic absorption peak of DOX is located at 480nm, while the absorption peak of DOX in FDC is red-shifted to 490nm, indicating that DOX is successfully coupled to scFv. The absorbance of FDC at 480nm is 2.514 and at 280nm is 3.623. According to the standard curve of DOX and BSA ( Figure 7 B and 7C), the concentrations of DOX and scFv in FDC were calculated to be 0.257 mg / mL and 9.85 mg / mL, respectively. Since DOX also absorbs at 280 nm, the absorbance of DOX at a concentration of 0.257 mg / mL at 280 nm is 0.0615 ( Figure 7 D), so the actual absorbance of scFv is 3.562, and the corresponding concentration is 9.68 mg / mL. Finally, the DAR value of FDC is calculated according to the formula to be 1.5, that is, the average number of DOX molecules loaded on each antibody molecule is 1.5.

[0098] Example 3

[0099] In vitro antitumor activity of FDC

[0100] 1. Test materials: FDC prepared in Test Example 2

[0101] 2. Experimental subjects: Hela, cervical cancer cell line; MDA-MB-231 and BT-549, human breast cancer cell lines; LO2, human liver cancer cell line; HEK-293T, human kidney cancer cell line;

[0102] 3. Test methods

[0103] 3.1 CCK8 assay to determine the cytotoxicity of FDC

[0104] MDA-MB-231, Hela, BT-549, LO2 and HEK-293T were respectively cultured at 5×10 3 Cells were inoculated into 96-well plates and cultured to the logarithmic growth phase. DOX, scFv and FDC were added at concentrations of 0, 10, 15 and 20 μg / mL to treat the cells for 24 h. After incubation, the cell viability was determined using Cell Counting Kit-8 assay.

[0105] 3.2 Immunofluorescence

[0106] MDA-MB-231, Hela, BT-549, LO2 and HEK-293T were respectively cultured at 2×10 5Cells were inoculated into six-well plates, cultured to the logarithmic growth phase, and then scFv and FDC were added at a concentration of 20 μg / mL and incubated for 2 hours. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed with PBS, and incubated with 0.1% Triton X-100 at room temperature for 15 minutes. After washing with PBS, 2% BSA was used to incubate at 37°C for 30 minutes. After the incubation, FITC-labeled anti-6×His antibody was added, incubated at 37°C in the dark for 1 hour, and then DAPI was added and incubated at room temperature for 5 minutes. Finally, the intelligent cell imaging system EVOSM5000 was used to capture the image.

[0107] 3.3 Drug entry into cells

[0108] MDA-MB-231, Hela, BT-549, LO2 and HEK-293T were respectively cultured at 2×10 5 Each well was inoculated into a six-well plate, and after culturing to the logarithmic growth phase, the cells were treated with DOX and FDC (20 ug / mL) for 6 h, 12 h, and 24 h, respectively. Subsequently, the cells were observed and images were taken using the EVOSM5000 imaging system.

[0109] 4. Test results

[0110] 4.1 CCK8 assay for FDC cytotoxicity

[0111] The cytotoxicity results showed that the synthesized FDC had targeting properties to cells with high p16 expression and also had certain cytotoxicity, with low cytotoxicity to the p16 low-expressing cell line MDA-MB-231 and normal cells LO2 and HEK-293T cells (p<0.05).

[0112] The results of the cytotoxicity experiment indicated that when doxorubicin (DOX) was used alone to treat MDA-MB-231, HeLa and BT-549 cells at concentrations of 10, 15 and 20 μg / ml, no significant differences were observed in cytotoxicity among the groups (p>0.05; Figure 8 A). Similarly, at the same dose, scFv alone did not show significant cytotoxicity after treating the above cells ( Figure 8 B). However, when the same dose of FDC was used to treat each cell line, FDC significantly inhibited the proliferation of HeLa and BT-549 cells with high p16 expression compared with MDA-MB-231 cells and LO2 and HEK-293T cells with low p16 expression (p<0.05; Figure 8C), and this inhibitory effect showed a certain dose dependence. These results indicate that the prepared FDC has specific targeting activity and exhibits selectivity for p16 high-expressing cells when exerting its toxic effect.

[0113] 4.2 Immunofluorescence

[0114] To further verify whether FDC drugs have specific targeting to p16, we chose to use FITC-labeled fluorescent secondary antibodies to bind to scFv to achieve the role of localization. Fig. 9 As shown in the figure, compared with MDA-MB-231 cells with low expression of p16, Hela and BT-549 cells showed strong green fluorescence, and the fluorescence intensity of BT-549 cells was higher than that of Hela cells, which also explained that after administration of FDC drugs, the cytotoxicity of BT-549 cells was stronger than that of Hela cells, verifying the specificity of scFv for p16 targets. At the same time, significant red fluorescence was also observed in Hela and BT-549 cells treated with FDC drugs, which once again proved the successful coupling of FDC.

[0115] 4.3 The process of FDC entering cells

[0116] To investigate whether FDC can be internalized by p16-overexpressing cell lines, we treated each cell with DOX and FDC for 6, 12, and 24 hours, and then observed the changes in red fluorescence in the cells using a fluorescence microscope. The results showed that after each cell was co-incubated with DOX, the red fluorescence gradually increased over time (p<0.05, Fig.10 A and B), and after FDC treatment, obvious red fluorescence was observed in Hela and BT-549 cells with high p16 expression compared with MDA-MB-231 cells with low p16 expression (p<0.05, Fig.10 A and C), and the fluorescence intensity gradually increased with time.

[0117] The above description is only a preferred embodiment of the present invention, which cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. An antibody fragment drug conjugate based on a single-chain antibody targeting p16, characterized in that: The invention comprises a p16 single-chain antibody, a cell-penetrating peptide, a linker and a carrier drug; the p16 single-chain antibody and the cell-penetrating peptide are fused and expressed to form a fusion protein, which is then coupled with the carrier drug through the linker to form the antibody fragment drug conjugate based on the targeted p16 single-chain antibody.

2. The antibody fragment drug conjugate based on the p16-targeting single-chain antibody according to claim 1, characterized in that: The p16 single-chain antibody was humanized before use.

3. The antibody fragment drug conjugate based on the p16-targeting single-chain antibody according to claim 1, characterized in that: The nucleotide sequence of the p16 single-chain antibody is shown in SEQ ID NO:

2.

4. The antibody fragment drug conjugate based on the p16-targeting single-chain antibody according to claim 1, characterized in that: The p16 single-chain antibody is prepared by sequencing the monoclonal antibody secreted by hybridoma cells and by genetic engineering transformation.

5. The antibody fragment drug conjugate based on the p16-targeting single-chain antibody according to claim 4, characterized in that: The preparation of the p16 single-chain antibody includes: extracting RNA from the hybridoma cells, reverse transcribing to obtain the antibody variable region sequence, and then recombinantly expressing to obtain the p16 single-chain antibody; the hybridoma cells include mouse hybridoma cell line 3837-04, which is preserved in the Institute of Microbiology, Guangdong Academy of Sciences, with a preservation number of GDMCC No: 65754.

6. The antibody fragment drug conjugate based on the p16-targeting single-chain antibody according to claim 1, characterized in that: The cell-penetrating peptides include S4 13 .

7. The antibody fragment drug conjugate based on the p16-targeting single-chain antibody according to claim 1, characterized in that: The nucleotide sequence of the cell-penetrating peptide is shown in SEQ ID NO:

1.

8. The antibody fragment drug conjugate based on the p16-targeting single-chain antibody according to claim 1, characterized in that: The linker includes Oxidized Dextran T-10; the preparation of the linker includes: mixing Dextran T-10 and NaIO4 in the dark for reaction, and freeze-drying to obtain the Oxidized Dextran T-10; the carrier drug includes doxorubicin.

9. An application of the antibody fragment drug conjugate based on the p16-targeting single-chain antibody as claimed in claim 1, characterized in that: Used to prepare drugs that specifically target the p16 target.

10. A medicine obtained by the use as claimed in claim 9.

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