An antibody fragment drug conjugate based on a single-chain antibody targeting p16

Abstract

This invention discloses an antibody-drug conjugate based on a p16-targeting single-chain antibody, comprising 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 together to form a fusion protein, which is further conjugated to the carrier drug via a linker to form the antibody-drug conjugate. This invention provides a novel therapeutic strategy by targeting p16, particularly suitable for tumor types that highly express p16, for which effective treatments are currently lacking. By targeting the p16 site within the cell nucleus, this invention not only enriches the candidate target library for targeted therapy but also provides feasibility validation and technical reference for future drug development targeting nuclear targets.

Description

Technical Field

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

[0002] The p16 protein is a key tumor suppressor factor, which is abnormally highly expressed in various 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, it is difficult for traditional treatment methods to effectively target and intervene. The development of nuclear targets has opened up a new direction for precision cancer treatment, especially in interfering with the cell proliferation mechanism 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 relatively few studies have been conducted on nuclear targets. In addition, due to their large molecular weight, full-length antibodies have limited penetration into tumors in solid tumors with high interstitial pressure.

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

[0005] The object of the present invention is to, aiming at the nuclear target p16, by fusing the cell-penetrating peptide S4 13 , achieve transmembrane delivery and nuclear retention of the antibody, conjugate with the chemotherapeutic drug doxorubicin (DOX), innovatively design an antibody fragment drug conjugate (FDC) based on a single-chain antibody (scFv), and verify its in vitro anti-tumor activity.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An antibody-drug conjugate based on a p16 single-chain antibody includes 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 conjugated to the carrier drug through the linker to form the antibody-drug conjugate based on the p16 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 from monoclonal antibodies secreted by hybridoma cells.

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

[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 via recombination.

[0013] Preferably, 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 it to obtain the p16 single-chain antibody.

[0014] Preferably, the hybridoma cells include mouse hybridoma cell line 3837-04, which is deposited at the Institute of Microbiology, Guangdong Academy of Sciences, with accession number GDMCC No: 65754.

[0015] Preferably, the cell-penetrating peptide includes 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 NaIO4 and reacting in the dark, followed by freeze-drying to obtain the Oxidized Dextran T-10.

[0018] Preferably, the carrier drug includes doxorubicin.

[0019] Those skilled in the art will understand that DOX conjugation is one type of drug carrier, and other drug molecules can also be selected for construction, such as monomethyl ozogatine E (MMAE), irinotecan (SN38), etc., which are also within the scope of this invention.

[0020] An application of the above-mentioned antibody fragment drug conjugate based on a single-chain antibody targeting p16, for the preparation of a drug specifically targeting p16.

[0021] A drug obtained through the above-mentioned application.

[0022] This invention can be used to prepare related antibody fragment conjugates, particularly for the treatment of cancers with high p16 target expression.

[0023] Implementing this invention has the following beneficial effects:

[0024] 1. Application of new targets

[0025] Currently, research in the field of targeted therapy mainly focuses on common membrane surface targets such as HER2 and EGFR. While these targets are widely used in clinical practice, they also face challenges such as drug resistance and limited applicability. In contrast, this invention is the first to use p16 protein, a clinically significant intranuclear target, in the design of fragment drug conjugates (FDCs). As a tumor suppressor, the abnormally high expression of p16 protein is closely related to the development and progression of various solid tumors, such as triple-negative breast cancer and cervical cancer. This invention provides a novel treatment strategy by targeting p16, particularly suitable for tumor types that highly express p16, for which effective treatments are currently lacking. By targeting the intranuclear p16 target, this invention not only enriches the candidate target library for targeted therapy but also provides feasibility verification and technical reference for future drug development targeting intranuclear targets.

[0026] 2. Intranuclear targets

[0027] Current antibody-drug conjugates (ADCs) primarily target sites on the cell membrane surface, failing to efficiently deliver to the cell nucleus. In contrast, the FDC developed in this invention utilizes the cell-penetrating peptide S4. 13 This approach enables transmembrane drug delivery and intranuclear retention, allowing for highly efficient and precise targeting of the nuclear p16 target. Intranuclear target therapy offers the advantage of directly interfering with DNA repair, gene transcription, and cell cycle regulation, and compared to membrane surface targets, it can influence tumor cell proliferation mechanisms at a deeper level. Furthermore, by fusing S4… 13Achieving efficient transmembrane and nuclear retention, this design fills the technological gap in nuclear target delivery of existing ADC drugs, providing a novel solution for drugs that are difficult to reach the nucleus. Precise targeting within the nucleus effectively avoids off-target effects from the extracellular or cytoplasmic regions, reducing toxicity and significantly improving therapeutic efficacy, especially in the complex tumor microenvironment.

[0028] 3. Antibody fragment conjugates

[0029] Solid tumors, due to their high interstitial pressure and hypoxic environment, exhibit poor drug diffusion and penetration, making it difficult for traditional antibody-drug conjugates (ADCs) to distribute evenly. This invention utilizes a single-chain antibody (scFv) form of FDC, overcoming the drawbacks of full-length antibodies, such as large molecular weight and weak tumor penetration, making it more suitable for the treatment of solid tumors. Single-chain antibodies (approximately 25 kDa) have a small molecular weight and stable structure, exhibiting better tumor penetration and uniform distribution, resulting in stronger therapeutic effects deep within solid tumors. Compared to full-length antibody-drug conjugates, the FDC of this invention is more applicable to solid tumors with high interstitial pressure, particularly showing better therapeutic effects on tumor types that are difficult to penetrate.

[0030] 4. This invention focuses on the precise identification and targeting of a specific target (p16), and integrates the cell-penetrating peptide S4. 13 This invention aims to address the technical challenges of achieving intranuclear target delivery and binding for antibody drugs, and further lay the foundation for the development of antibody-drug conjugates (FDCs). The invention focuses not only on delivery efficiency but also on targeted design (fusion with S4). 13 The screening of high-affinity scFv sequences allows for precise targeting of the p16 protein within the cell nucleus, which can then be used to prepare FDCs. Combined with cytotoxic small molecule drugs, this enhances the therapeutic efficacy against tumors with high p16 expression.

[0031] 5. The scFv of this invention is derived from the extraction of heavy and light chain variable region genes from five hybridoma cell lines. Through molecular modeling and molecular docking screening, sequences with optimal affinity were obtained, and subsequently optimized into variable region fragments linked by flexible peptides, excluding constant regions and thus achieving greater miniaturization. Simultaneously, this invention humanizes the scFv, reducing immunogenicity and making it more suitable for in vivo application. Furthermore, the scFv of this invention incorporates a cell-penetrating peptide (CPP), endowing it with unique cell-penetrating capabilities, allowing it to directly enter cells and exert its effects. Therefore, the scFv of this invention is more innovative in its design concept, particularly suitable for drug delivery or precision treatment of intracellular targets.

[0032] 6. In this invention, the compound portion DOX inserted into the FDC is the core factor for exerting anti-tumor activity. This compound portion is covalently linked to the p16 single-chain antibody fragment 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 a highly efficient killing effect.

[0033] 7. The synthesis process of antibody fragment and small molecule drug conjugation in this invention is based on non-site-directed conjugation of antibody amino groups with oxidized dextran T-10. This method is characterized by its simplicity, good biocompatibility, and high conjugation efficiency. By forming Schiff base bonds between the natural amino groups on the antibody and the aldehyde groups in oxidized dextran T-10, and further stabilizing them into covalent bonds using NaBH4 reduction, this non-site-directed conjugation technique avoids complex genetic engineering modifications to the antibody. Simultaneously, it effectively protects the activity of both the antibody and the small molecule drug under mild reaction conditions (room temperature and low temperature). As a multifunctional linker, dextran T-10 not only provides ample space and flexibility, reducing interference with the antibody's functional regions and ensuring the preservation 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 to determine the optimal purification protocol. Initially, we attempted purification using a nickel affinity column with added His tags, but found that only scFv within inclusion bodies could bind, while the soluble scFv portion could not effectively bind to the nickel column. We then tried increasing the number and position of His tags, but binding remained ineffective, suggesting that the His tag conformation in the soluble scFv was masked, leading to low binding efficiency. We subsequently tried other fusion tags, including GST, MBP, and Strep, and attempted purification using corresponding affinity chromatography columns, but the soluble expression products of these tags also failed to bind effectively to the corresponding affinity columns. Although inclusion bodies can achieve protein recovery through denaturation and renaturation, this method is prone to scFv misfolding or functional loss, especially affecting affinity significantly, and therefore is not the preferred strategy. To maximize the preservation of scFv affinity and functionality, we ultimately chose a DEAE ion exchange column for purification. Ion exchange columns separate proteins based on their charge characteristics, independent of the exposure state of specific tags, thus avoiding the aforementioned problems. By optimizing ion exchange conditions, we successfully achieved efficient purification of scFv and ensured its functional integrity in its native conformation. Attached Figure Description

[0035] Figure 1 A: Electrophoresis diagram of total RNA from five hybridoma cell lines; B: V H PCR amplification results; C:V LPCR 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 The docking results of scFv-p16-01 and p16 antigen (pink) molecules are as follows: A: scFv-p16-01 and p16 antigen; 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 sonication of uninduced bacterial cells; 2: Precipitate after sonication of uninduced bacterial cells; 3: Supernatant after sonication of induced bacterial cells; 4: Precipitate after sonication of induced bacterial cells. D: scFv-p16-S4 13 Western blotting results. (1): Supernatant after ultrasonic disruption of bacterial cells; (2): Precipitate after ultrasonic disruption of bacterial cells. 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 of purified antibody. In the figure, M: Protein Marker; 1: Cell supernatant after sonication; 2: 2M NaCl elution sample; 3: Flow-through sample. C, D, E, F, G: Western blotting to validate 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. 13 ELISA signal.

[0040] Figure 6 .FDC synthesis schematic diagram.

[0041] Figure 7 A: UV-Vis spectral scans (250-600 nm) using Dextran T-10, Oxidized Dextran T-10, scFv, DOX, and FDC. B, C, D: Standard curves at 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. Data with the same superscript letter within the same group indicate no significant difference (p>0.05), while different superscript letters indicate significant differences (p<0.05).

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

[0044] Figure 10 Internalization assessment of DOX and FDC in MDA-MB-231, HeLa, BT-549, LO2, and HEK-293T cells. A: Representative cell image; B, C: Fluorescence intensity. BF indicates bright-field cells, DOX indicates red fluorescence (scale bar, 300 μm). Data with the same superscript letter in the same group indicate no significant difference (p>0.05), while different superscript letters indicate significant differences (p<0.05). DOX: Doxorubicin; FDC: Antibody fragment conjugated drug. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the present invention clearer, 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 stable mouse hybridoma cell lines secreting p16 monoclonal antibodies (3837-01, 3837-03, 3837-04, 3837-07 and 3837-08).

[0049] 2. Test Methods

[0050] 2.1 Amplification of the p16 antibody variable region

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

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

[0053] The variable region sequences of the light and heavy chains obtained above were imported into BIOVIA Discovery Studio 2023 to analyze the antibody's framework region (FR) and complementarity-determining region (CDR). To reduce the immunogenicity of the antibody, the CDR of the non-human antibody was transplanted into the human framework region to achieve antibody humanization. The specific operation is as follows: homology modeling was performed using the Model Antibodies function, and the 3D model with the highest homology was selected as the template. Finally, the Procheck function in the online website (https: / / saves.mbi.ucla.edu / ) was used to evaluate the rationality of the homology model. Subsequently, the PredictHumanizing Mutations function was used to transplant the antibody's CDR region onto the human donor template. Finally, the FR region of the antibody was reverse-mutated to obtain the humanized scFv-p16 sequence. To facilitate the subsequent molecular docking between the humanized scFv-p16 and p16 antigen, homology modeling was performed on the humanized scFv-p16 and p16 antigen (NCBI:NM_000077.5) using the same method as above. Then, molecular docking was performed using the Dock Protein function in BIOVIA Discovery Studio 2023. The specific steps were as follows: the five scFv-p16s obtained from homology modeling were set as receptors and the p16 antigens as ligands, and the Dock Protein (ZDOCK) program was run. The Process Poses (RDOCK) function was used to fix the binding sites selected in the previous step to the CDR3 region of the antibody. Then, the Refine Docked Proteins (RDOCK) function was 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 molecular docking results were visualized using PyMOL 3.0 software.

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

[0055] Based on the above molecular docking results, V L and V H V was constructed by linking via an intermediate flexible peptide linker (GGGSGGGSGGGS). 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 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, which was deposited at the Institute of Microbiology, Guangdong Academy of Sciences on January 10, 2025, with accession number GDMCCNo: 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 was extended using two primer extensions. 13 The N segment of scFv-p16 was linked, and the primer sequences are shown in Table 1. The first-round amplification system consisted of: 2 μL plasmid, 2 μL each of upstream and downstream primers (F1 and R), and 2× Primer. MAX DNA Polymerase 25 μL, DEPC water 16 μL; the first round amplification program was as follows: denaturation at 98℃ for 10 s; annealing at 60℃ for 30 s, extension at 72℃ for 1 min, for a total of 30 cycles; after the first round of PCR, the PCR products were electrophoresed on a 1% agarose gel at 120V for 30 min, and the target band was then excised and recovered. The recovered target band was used as a template for the second round of PCR, with the upstream primer replaced by F2. The remaining reaction system and program were the same as above. The PCR products were electrophoresed on a 1% agarose gel at 120V for 30 min, and the target band was then excised and recovered. The target band was double-digested with the pET-28a(+) empty vector using XhoⅠ and BamHI at 37℃ for 3 h. After cleaning and recovery, ligation was performed overnight at 16℃ using T4 DNA ligase. The plasmid was then transformed into E. coli DH4 using the heat shock method. The next day, positive clones were picked from 5α competent cells and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmids with correct sequencing were transformed into E. coli BL21(DE3) by heat shock.

[0056] Table 1 scFv-p16-S4 13 Primers used in construction

[0057]

[0058] Note: Underlined areas indicate enzyme cleavage sites.

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

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

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

[0062] SDS-PAGE was performed as described above. After electrophoresis, the gel containing the target protein was transferred to a PVDF membrane at a current of 280 mA for 1 hour. After the transfer, the membrane was blocked with 5% BSA at room temperature for 1 hour, followed by 5 washes with TBST, and incubation overnight with Mouse Anti-His tag antibody at 4°C. The next day, the membrane was washed 5 times with TBST, incubated with HRP-labeled goat anti-mouse IgG at room temperature for 2 hours, washed 5 times with TBST, and developed with ECL developing solution. Finally, the membrane was photographed using 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] The target protein was purified using a DEAE Sepharose FF column. The specific procedure was as follows: First, the packing material was loaded into the column and washed with 2 column volumes of ultrapure water to remove residual ethanol. Next, the resin was equilibrated with 10 column volumes of equilibration buffer (20 mM Tris-HCl, pH 8.0). Then, the sample was loaded at a flow rate of 1 mL / min, and the flow-through was collected. After loading, the column was washed again with equilibration buffer until the absorbance at 280 nm was close to the baseline. Subsequently, impurities were eluted with elution buffer (20 mM Tris-HCl + 2 M NaCl, pH 8.0) until the absorbance at 280 nm was again close to the baseline. After elution, the packing material was equilibrated with 5 column volumes of equilibration buffer, then washed with 5 column volumes of ultrapure water. Finally, 20% ethanol was added for storage at 4°C. 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 mixture was aliquoted and stored at -20°C.

[0066] 2.6.2 Western Blot method for identifying recombinant scFv-p16-S4 13 Antibody binding to p16 antigen

[0067] The p16 antigen was subjected to SDS-PAGE with a uniform loading volume of 10 μL. Transfer to a membrane was performed as described above, using five 1:1000 dilutions of recombinant scFv-p16-S4. 13 The antibody was the primary antibody, which was incubated overnight at 4°C. The secondary antibody was HRP-labeled anti 6×His at a dilution of 1:6000. After development with ECL developing solution, the gel imaging system was used to take pictures.

[0068] 2.7 Indirect ELISA method to verify recombinant scFv-p16-S4 13 The ability of antibodies to bind 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. The plate was incubated overnight at 4°C. The next day, the plate was washed three times with PBST solution, and then 100 μL of 1% BSA was added to each well. The plate was blocked at 37°C for 2 h to remove non-specific adsorption. After blocking, 100 μL of serially diluted recombinant scFv-p16-S4 was added to each well. 13Antibody was added, along with PBS as a negative control, and incubated at 37°C for 1 hour. After incubation, the sample was washed three times with PBST solution. HRP-labeled anti-6×His antibody diluted 1:2000 was added to each well, and the sample was incubated at 37°C for 1 hour. After incubation, the sample was washed three times with PBST solution, and then 100 μL of chromogenic solution was added to each well. The sample was incubated at 37°C in the dark for 10 minutes. After chromogenic development, 100 μL of stop solution was added to each well to stop the development. Finally, the absorbance was measured at 450 nm using a microplate reader. A positive result was considered to be 2.1 times greater than that of the negative control.

[0070] 3. Test Results

[0071] 3.1 Amplification of the p16 antibody variable region

[0072] First, total RNA was extracted from five hybridoma cell lines using the Trizol method. A suitable amount of the product was then subjected to nucleic acid electrophoresis to verify the quality of the extracted RNA. The results are shown below. Figure 1 A, from top to bottom, consists of 28S, 18S, and 5S, and the A280 / A260 ratio, measured by Nanodrop, is between 1.8 and 2.0, indicating that the extracted RNA is of good quality and suitable for the next step of reverse transcription. Subsequently, this RNA was used as a template for reverse transcription to obtain cDNA. Then, using the cDNA as a template, universal primers were used to amplify the light and heavy chain variable regions of the antibody. Electrophoresis results showed V... L Size is 380-400bp, V H The size is 400-430bp, which is in line with expectations, indicating that V L and V H Successfully amplified ( Figure 1 B and Figure 1 C). Subsequently, the V strains of five hybridoma cells were respectively... L and V H The gel was cut, recovered, and cloned into the PMD18-T vector. V samples were then picked up. L and V H Colony PCR verification was performed, and the results were as follows: Figure 1 D and 1E were then sent to Beijing Qingke Biotechnology for sequencing. The sequencing results were analyzed using the IMGT (https: / / www.imgt.org / ) online website for each V. L and V H The results obtained all conformed to the characteristics of the variable regions of the light and heavy chains of the antibody, that is, 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 with p16 antigen

[0074] Molecular docking results as follows Figure 2 As shown in Tables A through E, the binding regions of the five antibodies to the p16 antigen are all located in the CDR region. The key amino acid residues at the specific binding sites are listed in Table 2. Furthermore, the binding abilities of the antibodies obtained from the five hybridoma cell lines to the p16 antigen differ, as shown in Table 3. The E.RDOCK values ​​are the lowest for scFv-p16-08, at -31.70. Figure 2 E also indicates that scFv-p16-08 forms the most hydrogen bonds with the p16 antigen, suggesting that theoretically, scFv-p16-08 has the highest affinity for the p16 antigen, but further verification is needed through subsequent experiments.

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

[0076]

[0077]

[0078]

[0079] Table 3. Models with the highest homology matching degree for humanized scFv-p16 and their E.RDOCK values ​​after docking with p16 antigen molecules.

[0080]

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

[0082] scFv-p16-S4 13 Bacterial cells were induced with 0.5 mM IPTG at 30℃ for 5 h. After ultrasonic disruption, the supernatant and precipitate were subjected to SDS-PAGE, and the results are shown below. 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 blotting analysis was performed, and the results showed that the recombinant scFv-p16-S4... 13 All proteins can bind to anti-His antibodies (see...) Figure 3 D) Further evidence of 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 high purity of recombinant scFv-p16-S4 was successfully obtained. 13 The antibodies were further quantitatively analyzed using ImageJ software, and the results showed that the purity of all five recombinant antibodies exceeded 70%. Meanwhile, Western blotting results showed (see...) Figure 4 C Figure 4 D、 Figure 4 E, Figure 4 F and Figure 4 G) indicates that all recombinantly expressed single-chain antibodies can bind to the p16 antigen.

[0085] 3.5 Indirect ELISA method for determining recombinant scFv-p16-S4 13 antibody activity

[0086] The five purified scFv-p16-S4 cells were determined using the BCA method. 13 The antibody concentration was uniformly adjusted to 1 mg / mL. Subsequently, the sample was serially diluted to 2048-fold. The scFv-p16-S4 was evaluated using an indirect ELISA method. 13 The antibody's recognition activity against the p16 antigen, and the reaction mechanism are as follows: Figure 5 As shown in Figure A. The experimental results are as follows: Figure 5 As shown in B. The OD450 value of the negative control was 0.156, and the OD450 value of the sample needed to be 2.1 times greater than the negative control's OD450 value (i.e., greater than 0.327) to be considered a positive result. The results indicate that all recombinantly expressed scFv-p16-S4... 13 All antibodies effectively recognize the p16 antigen. Among them, scFv-p16-S4 13 -04 still exhibited 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; therefore, to achieve highly efficient and specific targeting, scFv-p16-S4 was selected. 13 -04 was used for the subsequent synthesis of FDC, and 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. Experimental material: scFv-p16-S4 purified from Experiment 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, utilizing their inherent active amino groups, are non-site-specifically and quantitatively conjugated to cytotoxic small molecule compounds via a linker to prepare antibody fragment conjugates (FDCs). The reaction procedure is described below. Figure 6 The specific operation method is as follows: First, take 1g of Dextran T-10 and add 10mL of 0.35mM NaIO4. React at 150rpm in the dark for 20h. After freeze-drying, Oxidized Dextran T-10 is obtained. Take 2mg of DOX and dissolve it in 2mL of ultrapure water. Add 30mg of Oxidized Dextran T-10. React at 150rpm in the dark for 20h. After the reaction is completed, add 5mg of scFv-p16-S413. React at 4℃ and 150rpm in the dark for 20h. Then add 100μL of 0.13mM NaBH4. React at 37℃ and 150rpm in the dark for 2h. Finally, use a 3kDa ultrafiltration tube for ultrafiltration and replace the buffer with PBS (0.01M, pH 7.4).

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

[0094] UV-Vis spectral scans were performed at 200-600 nm using Dexran T-10, Oxidized Dextran T-10, DOX, scFv, and FDC to obtain the UV absorption spectra and maximum absorption wavelengths of each sample, and the spectral differences were compared.

[0095] Prepare 0-1 mg / mL BAS standard solutions and 0-200 μg / mL DOX standard solutions. Obtain absorbance values ​​at 280 nm and 280 nm, and 480 nm, respectively, and plot standard curves. Then, measure the absorbance values ​​of the prepared FDC at 280 nm and 480 nm. Calculate the DAR value using the following formula: DAR = (DOX concentration / DOX molar mass) / (scFv concentration / scFv molar mass).

[0096] 3. Test Results

[0097] 3.1. By scanning the entire ultraviolet-visible wavelength range, from Figure 7As can be seen from Figure A, the characteristic absorption peak of DOX is located at 480 nm, while the DOX absorption peak in FDC is red-shifted to 490 nm, indicating that DOX is successfully coupled with scFv. The absorbance of FDC is 2.514 at 480 nm and 3.623 at 280 nm. According to the standard curves of DOX and BSA (… Figure 7 Based on 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 (B and 7C). Figure 7 Therefore, the actual absorbance of scFv is 3.562, corresponding to a concentration of 9.68 mg / mL. Finally, the DAR value of FDC is calculated to be 1.5 according to the formula, meaning that the average number of DOX molecules loaded on each antibody molecule is 1.5.

[0098] Example 3

[0099] FDC's in vitro antitumor activity

[0100] 1. Experimental materials: FDC prepared in Experiment 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 renal cancer cell line;

[0102] 3. Test methods

[0103] 3.1 CCK8 assay for FDC cytotoxicity

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

[0105] 3.2 Immunofluorescence

[0106] MDA-MB-231, Hela, BT-549, LO2, and HEK-293T were respectively loaded with 2×10 5Cells were seeded per well in six-well plates and cultured to the logarithmic growth phase. Then, scFv and FDC at concentrations of 20 μg / mL were added and incubated for 2 h each. Subsequently, cells were fixed with 4% paraformaldehyde at room temperature for 15 min, washed with PBS, and incubated with 0.1% Triton X-100 at room temperature for 15 min. After washing with PBS, cells were incubated with 2% BSA at 37°C for 30 min. After incubation, FITC-labeled anti-6×His antibody was added, and cells were incubated at 37°C in the dark for 1 h. DAPI was then added and incubated at room temperature for 5 min. Finally, images were captured using the EVOSM5000 intelligent cell imaging system.

[0107] 3.3 The process of drug entry into cells

[0108] MDA-MB-231, Hela, BT-549, LO2, and HEK-293T were respectively loaded with 2×10 5 Cells were seeded per well in six-well plates and cultured to the logarithmic growth phase. Cells were then treated with DOX and FDC (20 μg / mL) for 6 h, 12 h, and 24 h, respectively. Subsequently, the cells were observed and images were captured using an EVOSM5000 imaging system.

[0109] 4. Test Results

[0110] 4.1 CCK8 assay for FDC cytotoxicity

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

[0112] The results of the cytotoxicity assays indicated that no significant differences in cytotoxicity were observed among the MDA-MB-231, HeLa, and BT-549 cells when treated with doxorubicin (DOX) alone at concentrations of 10, 15, and 20 μg / ml (p>0.05). Figure 8 A). Similarly, at the same dose, scFv alone did not exhibit significant cytotoxicity in the aforementioned cells. Figure 8 B). However, when the cell lines were treated with the same dose of FDC, compared with the p16-low-expressing MDA-MB-231 cells and LO2 and HEK-293T cells, FDC significantly inhibited the proliferation of p16-high-expressing HeLa and BT-549 cells (p<0.05). Figure 8C), and this inhibitory effect exhibits a dose-dependent relationship. These results indicate that the prepared FDCs possess specific targeting activity, while also demonstrating selectivity for p16-overexpressing cells when exerting their toxic effects.

[0113] 4.2 Immunofluorescence

[0114] To further verify whether FDC drugs have specific targeting properties for p16, we chose to use FITC-labeled fluorescent secondary antibodies to bind to scFv, thereby achieving localization. Figure 9 As shown, compared to MDA-MB-231 cells with low p16 expression, HeLa and BT-549 cells exhibited strong green fluorescence, with BT-549 cells showing higher fluorescence intensity than HeLa cells. This explains why BT-549 cells showed stronger cytotoxicity than HeLa cells after FDC administration, validating the specificity of scFv for the p16 target. Simultaneously, significant red fluorescence was also observed in HeLa and BT-549 cells treated with FDC, further demonstrating the successful conjugation of FDC.

[0115] 4.3 The process of FDC entering cells

[0116] To investigate whether FDC could be internalized in p16-overexpressing cell lines, we treated cells with DOX and FDC for 6, 12, and 24 hours, respectively, and then observed the changes in intracellular red fluorescence using fluorescence microscopy. The results showed that after co-incubation with DOX, the red fluorescence of all cells gradually increased over time (p<0.05). Figure 10 In contrast to MDA-MB-231 cells with low p16 expression (A and B), significant red fluorescence was observed in HeLa and BT-549 cells with high p16 expression after FDC treatment (p<0.05). Figure 10 (A and C), and the fluorescence intensity gradually increases with time.

[0117] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. An antibody drug conjugate based on a targeting p16 single chain antibody, characterized in that, The p16 single-chain antibody, a cell-penetrating peptide, a linker and a carrier drug; the p16 single-chain antibody and the cell-penetrating peptide form a fusion protein through fusion expression, and then the fusion protein is coupled with the carrier drug through the linker to form the antibody drug conjugate based on the targeting p16 single-chain antibody; the p16 single-chain antibody is humanized before use; the nucleotide sequence of the p16 single-chain antibody is shown as SEQ ID NO: 2; the cell-penetrating peptide comprises S4 13 ; the nucleotide sequence of the cell-penetrating peptide is shown as SEQ ID NO: 1; and the linker comprises Oxidized Dextran T-10. 2.The antibody drug conjugate based on a single chain antibody targeting p16 according to claim 1, wherein, The p16 single-chain antibody is prepared by sequencing and genetic engineering of a monoclonal antibody secreted by a hybridoma cell.

3. The antibody drug conjugate based on the targeting p16 single chain antibody according to claim 2, characterized in that, The preparation of the p16 single-chain antibody comprises the following steps: extracting RNA of the hybridoma cell, reverse transcription to obtain an antibody variable region sequence, and recombination expression to obtain the p16 single-chain antibody; and the hybridoma cell comprises a mouse hybridoma cell strain 3837-04, which is preserved in the Guangdong Provincial Academy of Microbiology, and the preservation number is GDMCC No: 65754.

4. The antibody drug conjugate based on the targeting p16 single chain antibody according to claim 1, wherein, The preparation of the linker comprises the following steps: mixing Dextran T-10 and NaIO4 in dark, and obtaining the Oxidized Dextran T-10 after freeze-drying; and the carrier drug comprises doxorubicin.

5. The use of the antibody drug conjugate based on the single chain antibody targeting p16 according to claim 1 in the preparation of a medicament. The application comprises one or more of the following: treating cervical cancer, human breast cancer, human liver cancer, and human kidney cancer.

6. A medicament obtained by the use according to claim 5 for the manufacture of a medicament.

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