B-cell lymphoma cell targeting polypeptide, targeting effect conjugate and application of B-cell lymphoma cell targeting polypeptide and targeting effect conjugate

Abstract

The invention relates to the technical field of biological medicines, in particular to a polypeptide targeting B cell lymphoma cells, a targeting effect conjugate and application of the polypeptide and the targeting effect conjugate. Wherein the peptide chain length of the polypeptide targeting the B-cell lymphoma cells is 12 amino acids, and the polypeptide is selectively and specifically combined with the B-cell lymphoma cells. The targeting effect conjugate is formed by connecting polypeptide and pro-apoptotic peptide through a connecting peptide. The pro-apoptotic peptide can induce programmed death of target cells by destroying a mitochondrial membrane structure. The molecular weight of the polypeptide is small, the chemical synthesis process is simple, convenient and efficient, high consistency between different batches of products can be ensured in the synthesis process, and a guarantee is provided for subsequent quality control. The small molecular structure is also convenient for sequence optimization and terminal modification, can create extremely favorable conditions for subsequent structure-function relationship research and pharmaceutical development work, and is helpful for accelerating the research and development process of novel therapeutic drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polypeptide targeting B-cell lymphoma cells, a targeting effector conjugate, and their applications. Background Technology

[0002] B-cell lymphoma (BCL) is one of the most common hematologic malignancies, accounting for over 60% of all lymphomas. Among the various subtypes of BCL, some aggressive subtypes are characterized by insidious onset, rapid disease progression, and high relapse rates, seriously threatening patients' lives and health. Although clinical treatments have improved in recent years, approximately 30% of patients still develop relapsed or refractory BCL, with extremely poor prognosis. Furthermore, some commonly used targets have overlapping expression between tumor cells and normal B cells, easily leading to damage to normal B cells during treatment, resulting in immunosuppression and increased risk of infection, thus affecting long-term drug safety and patient benefit. Therefore, developing novel precision targeted therapies, especially those that can effectively treat the disease in its early stages and reduce side effects, has become an urgent clinical need.

[0003] The key to targeted therapy lies in the specific recognition of tumor cell surface markers. Currently used clinical targets (such as CD20) are usually expressed on both tumor cells and normal B cells. This means that targeted drugs (such as rituximab) can damage normal B lymphocytes while killing tumor cells, leading to side effects such as immunosuppression and increased risk of infection. Furthermore, mainstream antibody drugs have large molecular weights, limited tissue penetration, and uneven distribution within tumor tissue, affecting their therapeutic efficacy. In addition, the complex manufacturing process and high cost of these antibody drugs limit their wider clinical application.

[0004] Small molecule peptides, as targeted molecules, possess advantages over antibody drugs, including small molecular weight, simple structure, good tissue penetration, low immunogenicity, and ease of chemical synthesis and modification, making them potential candidates for next-generation tumor-targeting molecules. However, screening for targeted peptides with high specificity against tumor cells and low binding to normal cells remains a technical challenge to be solved in this field. Apoptotic peptides, such as (KLAKLAK)2, are functional peptides that can induce apoptosis by disrupting mitochondrial membrane structure, and have shown strong cytotoxic effects in in vitro experiments. However, these peptides lack natural selectivity for tumor cells, and direct application can easily cause non-specific damage to normal cells, limiting their in vivo application.

[0005] Therefore, developing a targeting effector molecule that can specifically target B-cell lymphoma cells is an important direction in current targeted therapy research. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide that specifically targets lymphoma cells, capable of specifically binding to binding sites on the surface of B-cell lymphoma cells, while exhibiting low non-specific binding to normal peripheral blood mononuclear cells.

[0007] The present invention also provides a targeting effector conjugate that enables the pro-apoptotic peptide to selectively act on B-cell lymphoma cells under the mediation of the targeting peptide, thereby achieving selective killing of tumor cells and reducing the risk of non-specific toxicity.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a polypeptide targeting B-cell lymphoma cells, wherein the polypeptide chain comprises 12 amino acids, and the polypeptide selectively and specifically binds to the surface of B-cell lymphoma cells.

[0009] Furthermore, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1 or as shown in SEQ ID NO:2.

[0010] Furthermore, the method for screening the polypeptide includes the following steps: S1. The phage-displayed random 12-peptide library is co-incubated with control cells to negatively screen the phage-displayed random 12-peptide library. Then, target cells are obtained and incubated with the screened phage-displayed random 12-peptide library at room temperature to ensure that the peptides displayed on the surface of the phage are in full contact with the cell surface binding sites. S2. After incubation, the phages bound to the surface of the target cells are washed away; S3. The bacteriophages obtained by elution are recovered and subjected to host bacterial amplification culture. After the culture is completed, the titer of the bacteriophages is determined, and the bacteriophages with the determined titer are used as input bacteriophages for the next round of screening. S4. Repeat steps S2 and S3 to gradually enrich the phages with high affinity for the binding sites on the target cell surface; S5. Select multiple bacteriophages, extract their DNA and sequence it, translate the obtained nucleotide sequences into corresponding polypeptide sequences, and screen the polypeptides after homology analysis.

[0011] Furthermore, the target cells are B-cell lymphoma cell lines; before incubating the target cells with a phage-displayed random 12-peptide library, the target cells are cultured to the logarithmic growth phase, and then the target cells are collected and their density adjusted to 1×10⁻⁶. 6 ~5×10 6 Any value in the range of particles / mL.

[0012] This application also provides a targeting effect conjugate, which is composed of the aforementioned polypeptide and apoptosis-promoting peptide linked by a linker peptide, wherein the linker peptide is a flexible linker peptide.

[0013] Furthermore, the polypeptide and the pro-apoptotic peptide are covalently linked.

[0014] Furthermore, the pro-apoptotic peptide is (KLAKLAK)2, and the polypeptide is attached to the N-terminus of the pro-apoptotic peptide.

[0015] Furthermore, the linker peptide is a short peptide, and the peptide chain of the short peptide comprises no more than 5 amino acids.

[0016] Furthermore, the amino acid sequence of the targeting effect conjugate is shown in SEQ ID NO: 3.

[0017] This application also provides the use of the above-mentioned targeting effector conjugate in formulations that induce apoptosis of B-cell lymphoma cells or inhibit their proliferation.

[0018] The beneficial effects of this invention are as follows: The polypeptide provided in this application exhibits high specificity for B-cell lymphoma cells, specifically binding to binding sites on the surface of B-cell lymphoma cells, while showing low non-specific binding to normal peripheral blood mononuclear cells. This improves the selectivity of treatment and significantly reduces its potential impact on normal cells. Compared with traditional antibody molecules, this polypeptide has a smaller molecular weight, a simpler and more efficient chemical synthesis process, and ensures high consistency between different batches of products during synthesis, providing assurance for subsequent quality control. Furthermore, its small molecular structure facilitates sequence optimization and terminal modification, creating highly favorable conditions for subsequent structure-function relationship studies and pharmaceutical development, thus accelerating the development of novel therapeutic drugs.

[0019] The targeted effector conjugate provided in this application organically links a peptide targeting B-cell lymphoma cells with a pro-apoptotic peptide using a linker peptide. This conjugate has a clear mechanism of action and a stable structure, enabling it to exert a stable therapeutic effect in vivo. The targeted peptide acts as a recognition or labeling element, mediating the preferential binding and entry of the conjugate into B-cell lymphoma cells; while the pro-apoptotic peptide, as the effector unit, rapidly exerts its effect after entering the B-cell lymphoma cells, inducing programmed cell death by disrupting the mitochondrial membrane structure, thereby effectively inducing apoptosis and inhibiting the proliferation of B-cell lymphoma cells. Compared with untargeted pro-apoptotic peptides, the targeted effector conjugate of this application can precisely act on target cells with minimal impact on non-target cells, greatly reducing the risk of potential side effects during treatment and exhibiting high safety. Furthermore, the conjugate has a simple structure and can be prepared using conventional peptide chemical synthesis methods, demonstrating good feasibility and reproducibility, providing a feasible solution for large-scale production.

[0020] Furthermore, the peptide platform constructed in this application has strong scalability, enabling the flexible combination of targeted peptides with other functional peptides or effector molecules according to different application needs, thereby developing more novel drugs or treatments with targeted therapeutic effects. This provides technical support for subsequent productization and clinical translation applications, and has broad application prospects and huge market potential, which is expected to bring a major breakthrough in the treatment of B-cell lymphoma and other tumors.

[0021] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0022] Figure 1 The image shows a comparison of fluorescence intensities of FITC-C1, FITC-C2, and the negative control peptide NsG, as detected by flow cytometry in this invention, binding to B-cell lymphoma cell lines OCI-LY3 and TMD-8, and control cells PBMC (peripheral blood mononuclear cells). Figure 2 This is a diagram showing the changes in the activity of B-cell lymphoma cells after treatment with C1-KLA, C2-KLA, and the control pro-apoptotic peptide KLA, as illustrated in this invention, using the CCK-8 assay. Figure 3a and Figure 3b This is a representative scatter plot (four-quadrant analysis plot) of cell apoptosis after treatment with C1-KLA, C2-KLA and control peptides, as shown in this invention, using Annexin V / 7-AAD double staining combined with flow cytometry. Figure 4The apoptosis ratio is a statistical graph obtained by statistically analyzing the flow cytometry results shown in Figure 3. Figure 5 This is a representative schematic diagram of tumor tissue stripping at the end point of the experiment of the B-cell lymphoma nude mouse xenograft model shown in this invention. Figure 6 The graph shows the change in tumor volume over time and the statistical chart of tumor weight at the experimental endpoint in the B-cell lymphoma nude mouse xenograft model shown in this invention. Detailed Implementation

[0023] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Please see Figure 1This application discloses a preferred embodiment of a polypeptide targeting B-cell lymphoma cells. The polypeptide chain comprises 12 amino acids and selectively binds specifically to the binding site of B-cell lymphoma cells, exhibiting low non-specific binding to normal peripheral blood mononuclear cells. Existing tumor-targeted therapy targets are diverse, mainly including tumor cell-associated antigens (TCA), immune regulatory molecules, tumor microenvironment-related molecules, and key molecules in tumor metabolic pathways. Among these, tumor cell surface-associated molecules are the primary source of targets for various treatment strategies due to their ease of recognition and binding. However, most tumor cell surface antigens are not strictly "tumor-specific antigens," being expressed in both tumor and normal cells. For example, commonly used targets in B-cell lymphoma treatment are also expressed in normal B lymphocytes. Clearing tumor cells can easily damage normal B cells, leading to decreased immune function and increased risk of infection. Furthermore, the expression heterogeneity of some targets is significant in different patients or tumor subtypes, limiting the applicable population and long-term efficacy of targeted drugs. To balance efficacy, safety, and broad applicability, it is necessary to explore new forms and mechanisms of action of targeted molecules. Compared to antibody molecules, peptides are easier to synthesize chemically, have controllable structures, and are easier to couple or fuse with other functional molecules, making them suitable for constructing targeted delivery or killing molecules. Therefore, screening peptide sequences with high affinity and selectivity for target tumor cells and low binding to normal cells is crucial in the field of small molecule peptide targeted therapy. The peptide provided in this embodiment can specifically bind to binding sites on the surface of B-cell lymphoma cells, while exhibiting low non-specific binding to normal peripheral blood mononuclear cells. It can be used to construct structurally well-defined and implementable targeted effector molecules, achieving selective killing of tumor cells and reducing the risk of non-specific toxicity. In this embodiment and other embodiments, the amino acid sequence of the peptide is as shown in SEQ ID NO:1 or SEQ ID NO:2. Specifically, SEQ ID NO:1: QMGFMTSPKHSV, SEQ ID NO:2: DHAQRYGAGHSG.

[0025] One embodiment provides a method for screening peptides. This method uses phage display technology to screen peptide sequences that can specifically bind to binding sites on the surface of B-cell lymphoma cells, and includes the following steps: S1. A phage-displayed random 12-peptide library is co-incubated with control cells (PBMCs) to negatively screen the phage-displayed random 12-peptide library. Then, target cells are obtained and incubated with the screened phage-displayed random 12-peptide library at room temperature to ensure that the peptides displayed on the phage surface are in full contact with the cell surface binding sites. S2. After incubation, the phages bound to the surface of the target cells are washed away. S3. Recover the eluted phages and perform host bacterial amplification culture. After the culture is completed, determine the phage titer and use the phages with the determined titer as the input phages for the next round of screening. S4. Repeat steps S2 and S3 to gradually enrich phages with high affinity for binding sites on the target cell surface. S5. Select multiple bacteriophages, extract their DNA and sequence it. Translate the obtained nucleotide sequences into corresponding polypeptide sequences, and screen the polypeptides after homology analysis.

[0026] In step S1, at room temperature, a phage-displayed random 12-peptide library is first co-incubated with PBMC cells to ensure sufficient contact between the peptides displayed on the phage surface and the PBMC cell surface. This negatively screens the phage-displayed random 12-peptide library, excluding phage surface peptides that bind to PBMC cells, thereby improving the screening efficiency for peptides that specifically bind to lymphoma target cells. The supernatant, which is the negatively screened phage-displayed random 12-peptide library, is then incubated with target cells to ensure sufficient contact between the negatively screened phage surface peptides and the cell surface. Incubating the target cells with the negatively screened phage-displayed random 12-peptide library allows for sufficient contact between the peptides displayed on the phage surface and the cell surface binding sites, leading to specific binding. In this and other embodiments, the phage-displayed random 12-peptide library used is a product of New England Biolabs, constructed by embedding 12 amino acid residues into the pIII protein of M13 phage. The selected target cells are B-cell lymphoma cell lines (such as OCI-LY3). Before incubating the target cells with the phage-displayed random 12-peptide library, the target cells need to be pretreated. Specifically, the target cells are first cultured to the logarithmic growth phase, then washed 2-5 times with phosphate-buffered saline (PBS) or other buffers to thoroughly remove culture medium residues. Afterward, the target cells are collected and their density is adjusted to approximately 1 × 10⁻⁶. 6 ~5×10 6 per mL.

[0027] In step S2, after incubation for 10-60 minutes, the cells are washed multiple times with TBST buffer containing 0.1% Tween-20 or other buffers to remove unbound or non-specifically bound phages. Subsequently, phages bound to the cell surface are eluted using a specific elution buffer, such as glycine elution buffer (pH 2.2-2.5). Immediately after elution, the eluted cell solution is neutralized with a buffer, such as Tris-HCl buffer, to prevent phage inactivation due to an acidic environment.

[0028] In step S3, the enrichment of phage clones during the screening process is characterized by measuring the titer of the phages.

[0029] In step S4, through multiple rounds of screening, phage clones with high affinity for binding sites on the surface of B-cell lymphoma cells are gradually enriched to improve the accuracy and effectiveness of screening.

[0030] In step S5, approximately 100 phage clones were selected from the screening results, their DNA was extracted and sequenced, and the obtained nucleotide sequences were translated into corresponding polypeptide sequences. Homology analysis was performed using the NCBI database and Clustal software to screen for target polypeptides and further explore their biological characteristics and potential functions.

[0031] This application also provides a targeting effect conjugate, which is formed by linking the selected peptide and the pro-apoptotic peptide through a linker peptide. Preferably, the linker peptide is a flexible linker peptide, which links the selected peptide and the pro-apoptotic peptide capable of inducing programmed cell death together through a chemical reaction to form a conjugate with a targeting effect. The selected peptide acts as a recognition and localization unit, mediating the preferential binding and entry of the conjugate into B-cell lymphoma cells, while the pro-apoptotic peptide acts as an effector unit, inducing programmed cell death by disrupting the mitochondrial membrane structure after entering the target cell. In this embodiment and other embodiments, it is preferable to covalently link the peptide and the pro-apoptotic peptide through a linker peptide to form a high-strength, irreversible chemical bond, thereby constructing a structurally stable conjugate with a targeting effect.

[0032] In one embodiment, the preferred pro-apoptotic peptide is (KLAKLAK)2. (KLAKLAK)2 is a synthetic amphiphilic cationic peptide composed of repeating KLAKLAK units in an α-helix structure. It is rich in positively charged lysine and hydrophobic residues such as leucine and alanine, exhibiting membrane-disrupting capabilities, and its D-amino acid configuration enhances enzyme stability. (KLAKLAK)2 has selective killing effects on tumor cells and bacteria, but low direct toxicity to normal mammalian cells, making it suitable for integration into multifunctional drug platforms as a "killing effector module," synergistically acting with targeting peptides. Furthermore, the mechanism of action of (KLAKLAK)2 is well-defined; it specifically targets mitochondria, inserting into the mitochondrial membrane lipid bilayer through electrostatic and hydrophobic interactions, disrupting membrane integrity, leading to a decrease in membrane potential, increased permeability, and the release of cytochrome c. Cytochrome c activates a caspase cascade reaction (such as caspase-3) in the cytoplasm, ultimately triggering programmed apoptosis. In this and other embodiments, the polypeptide is preferably linked to the N-terminus of the pro-apoptotic peptide via a linker peptide. The N-terminus of the pro-apoptotic peptide is a key region for its function, and the polypeptide can shield this region from easily degradable sites, effectively preventing the pro-apoptotic peptide from being destroyed by proteases. This ensures the precise release of the pro-apoptotic peptide at the target site, thereby enhancing drug stability, achieving precise drug delivery, and optimizing the overall therapeutic effect.

[0033] In one embodiment, the linker peptide is preferably a short peptide, and more preferably, the peptide chain of the short peptide comprises no more than 5 amino acids. In some embodiments, the linker peptide may preferably be a dipeptide or tripeptide sequence such as LP, GG, or GGS. These short peptides, as linker structures, can connect polypeptides and pro-apoptotic peptides to achieve precise delivery, while being efficiently absorbed without digestion and being taken up by the body preferentially over other nutrients.

[0034] In one embodiment, the amino acid sequence of the targeting effect conjugate is shown in SEQ ID NO: 3, specifically: QMGFMTSPKHSV-LP-(KLAKLAK)2, wherein the polypeptide is located at the N-terminus of the pro-apoptotic peptide.

[0035] This application also provides the application of this targeting effector conjugate, including its use in formulations that induce apoptosis or inhibit the proliferation of B-cell lymphoma cells. This conjugate enables selective recognition and precise killing of B-cell lymphoma cells, effectively improving therapeutic selectivity while reducing toxic side effects.

[0036] S1. Human OCI-LY3 cells were selected as the target cells for screening. After culturing the OCI-LY3 cells to the logarithmic growth phase, they were collected and washed with phosphate-buffered saline (PBS) to remove culture medium residues. After washing three times, the cell density of the collected OCI-LY3 cells was adjusted to approximately 3 × 10⁻⁶ cells / year. 6 1 x 10^9 cells / mL, for later use. Take 1 x 10^9 cells from a normal individual. 7 One PBMC cell was resuspended in 1 ml of PBS-1% BSA and mixed with approximately 4 × 10⁻⁶ cells. 10 PFU phage display of a random 12-peptide library was incubated at room temperature for 1 hour, followed by centrifugation to collect the supernatant, which was the negatively selected library. Then, at room temperature, the collected OCI-LY3 cells were incubated with the negatively selected library for 30 minutes to ensure sufficient contact between the peptides displayed on the phage surface and the cell surface binding sites.

[0037] S2. After incubation, the cells were washed multiple times with TBST buffer containing 0.1% Tween-20 to remove unbound or non-specifically bound phages. Then, glycine elution buffer at pH 2.5 was added to elute the phages bound to the cell surface, and they were immediately neutralized with Tris-HCl buffer to obtain phages bound to the target cell surface.

[0038] S3. The eluted phages were recovered and cultured for host bacterial amplification. After the culture was completed, the phage titer was measured. The results are shown in Table 1. The phages with measured titers were used as input phages for the next round of screening.

[0039] S4. Repeat steps S2 and S3 three times, for a total of four rounds of screening, to gradually enrich phages with high affinity for the OCI-LY3 cell surface binding sites.

[0040] S5. Approximately 100 phage clones were selected, their DNA was extracted and sequenced, and the obtained nucleotide sequences were translated into corresponding polypeptide sequences. Homology analysis was performed using the NCBI database and Clustal software. The results showed that the selected polypeptide sequences were mainly enriched in the following two sequences: SEQ ID NO:1: QMGFMTSPKHSV and SEQ ID NO:2: DHAQRYGAGHSG. Among them, the polypeptide shown in SEQ ID NO:1 appeared more frequently during the screening process, indicating that it has a higher affinity for the binding site on the surface of B-cell lymphoma cells.

[0041] Table 1. Titer determination results of recovered phages after each round of panning and screening during the phage display screening process. Table 1 shows the titer data of the recovered phages after each round of screening. These data can be used intuitively and accurately to characterize the enrichment of phage clones during the screening process. The titer after the first round of screening was 5 × 10⁻⁶. 2 The titer increased significantly after three rounds of screening, with the concentration of Pfu / 10μL, showing a trend of gradual enrichment of phage clones with high affinity for the OCI-LY3 cell surface binding sites as the screening rounds increased.

[0042] To verify the binding ability of the peptide sequences screened in Example 1 to B-cell lymphoma cells, the in vitro binding characteristics of the peptides shown in SEQ ID NO:1 and SEQ ID NO:2 obtained in Example 1 to B-cell lymphoma cells were analyzed. Specifically, the peptides shown in SEQ ID NO:1 and SEQ ID NO:2 were chemically synthesized and conjugated with the fluorescent label FITC at their ends. After purification, FITC-labeled peptide molecules were obtained. Human B-cell lymphoma cell lines OCI-LY3 and TMD-8 were selected as target cells, while peripheral blood mononuclear cells (PBMCs) were used as control cells to comprehensively evaluate the binding specificity of the peptides. All cell types were collected, washed thoroughly with PBS, and adjusted to appropriate cell densities. Subsequently, FITC-labeled peptides shown in SEQ ID NO:1 and SEQ ID NO:2 were added to the cell suspension at a final concentration of 20 μM. The cells were incubated at room temperature for 30 min to ensure sufficient interaction between the peptides and the cell surface binding sites. Simultaneously, an unrelated sequence peptide NsG at the same concentration was used as a negative control to eliminate interference from non-specific binding. After incubation, cells were washed with PBS to remove unbound peptide molecules. Subsequently, flow cytometry was used to accurately detect the fluorescence signal intensity of each group of cells, and the average fluorescence intensity was used as a key indicator of peptide-cell binding ability. The detection results are shown below. Figure 1 As shown. Figure 1 The study presents fluorescence intensity data of FITC-labeled peptides (as shown in SEQ ID NO:1, denoted as FITC-C1), FITC-labeled peptides (as shown in SEQ ID NO:2, denoted as FITC-C2), and negative control peptide NsG, respectively, after binding to OCI-LY3 cells, TMD-8 cells, and control cells PBMC, to illustrate the specific binding ability of the peptides to the binding sites on the surface of B-cell lymphoma cells.

[0043] Depend on Figure 1As shown in (a) and (b), compared to the negative control peptide NsG, the peptides shown in SEQ ID NO:1 and SEQ ID NO:2 exhibited significantly enhanced binding signals with OCI-LY3 and TMD-8 cells, with average fluorescence intensity much higher than that with PBMC cells. This fully demonstrates that the screened peptides can specifically bind to binding sites on the surface of B-cell lymphoma cells. Figure 1 As shown in (c) and (d), the peptide shown in SEQ ID NO:1 has a significantly better binding ability to B-cell lymphoma cell lines OCI-LY3 and TMD-8 than the peptide shown in SEQ ID NO:2, thus indicating that the peptide shown in SEQ ID NO:1 is a peptide with a better targeting effect.

[0044] Example 2 Using the polypeptide shown in SEQ ID NO:1 obtained in Example 1 as the target recognition part, LP is the linker peptide. The polypeptide and the pro-apoptotic peptide (KLAKLAK)2 are chemically synthesized through the linker peptide, and the polypeptide and the pro-apoptotic peptide are covalently linked to obtain the target effect conjugate, denoted as C1-KLA.

[0045] Example 3 The difference between this embodiment and Example 2 is that the polypeptide shown in SEQ ID NO:2 obtained from Example 1 is used as the target recognition part, and the target effect conjugate is finally obtained and denoted as C2-KLA.

[0046] Comparative Example 1 A pro-apoptotic peptide (KLAKLAK)2, denoted as KLA, was synthesized and used as a control peptide.

[0047] The in vitro bioactivity of the targeting conjugates obtained in Examples 1 and 2, as well as the control peptide in Comparative Example 1, was evaluated using cell viability assays. B-cell lymphoma cell lines such as OCI-LY3, TMD-8, and TOLEDO, as well as HUVEC (umbilical vein endothelial cells), were randomly selected as research subjects. These cells were seeded into cell culture plates and placed under suitable culture conditions. After cell adhesion or reaching a stable state, C1-KLA, C2-KLA, and the control peptide KLA were added to each well for treatment. To ensure the reliability and comparability of the experimental results, all treatment groups were incubated for 24 hours under the same experimental conditions. After incubation, an appropriate amount of CCK-8 assay reagent was added to each well, and its final volume was strictly controlled to be approximately 10% of the culture system volume. Subsequently, the culture plates were placed in a constant temperature incubator at 37°C and 5% CO2 for 1-4 hours. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader. Simultaneously, wells containing no cells but with the same volume of culture medium and CCK-8 reagent were set up as blank controls to effectively eliminate the influence of background absorbance on the experimental results. Then, the absorbance values ​​of each treatment group were compared with the control group to scientifically evaluate the effect of different peptide treatments on cell viability, using this as a key indicator for evaluating cell viability. The detection results are as follows: Figure 2 As shown, this illustrates the effect of the targeted pro-apoptotic peptide conjugate on reducing the activity / inhibiting the proliferation of target cells.

[0048] Depend on Figure 2 As shown in (a), (b), and (c), compared to the use of the pro-apoptotic peptide (KLAKLAK)2 alone, the activity of B-cell lymphoma cells in the C1-KLA treatment group was significantly reduced, and the activity of B-cell lymphoma cells in the C2-KLA treatment group also decreased to some extent. This result fully demonstrates that the targeting effect conjugates synthesized in Examples 1 and 2 both possess the ability to inhibit tumor cell growth. Furthermore, the inhibitory effect of C1-KLA on tumor cell activity is significantly better than that of C2-KLA, indicating that C1-KLA has a superior targeting effect. Importantly, from Figure 2 As shown in (d) and (e), under the same experimental conditions, C1-KLA did not significantly affect the activity of human epidermal cancer cells A431 or normal human umbilical vein endothelial cells. This phenomenon strongly suggests that C1-KLA has excellent targeting selectivity, and can precisely inhibit the activity of B-cell lymphoma cells while minimizing toxic side effects on other cells.

[0049] To further clarify the specific mechanism by which targeting conjugates induce decreased cell activity, Annexin V and 7-AAD double staining methods, combined with flow cytometry, were used to detect cell apoptosis.

[0050] TMD-8, TOLEDO (human cutaneous T-lymphocytoma suspension cell line), and OCI-LY3 cells were selected as research subjects and incubated with C1-KLA, C2-KLA, or control peptides under the same experimental conditions for a certain period of time. After incubation, the cells were collected and washed twice thoroughly with pre-cooled PBS, and the supernatant was discarded to remove as much residual culture medium and free peptides as possible to avoid interference with subsequent experimental results. Subsequently, the cells were resuspended using the binding buffer provided with the kit, and the cell suspension concentration was adjusted to 1.5 × 10⁻⁶. 5 Cells / mL, ready for use. Under light-protected conditions, add an appropriate amount of Annexin V staining reagent to the cell suspension, mix gently and thoroughly, and incubate at room temperature for approximately 10 minutes. Then, add an appropriate amount of 7-AAD staining reagent and mix again, continuing incubation at room temperature in the dark for approximately 10 minutes to perform staining. After staining, add an appropriate amount of binding buffer to terminate the reaction and dilute the reaction system. Then, perform flow cytometry detection within a short time. To ensure the reliability and comparability of the detection results, multiple control experiments were simultaneously set up, including an unstained blank control, Annexin V single-stain control, 7-AAD single-stain control, and a negative control group, such as cells treated only with the solvent or cells treated with the control peptide. These control experiments were used for instrument voltage setting, fluorescence compensation, gating strategy establishment, and background signal determination.

[0051] During flow cytometry analysis, the four-quadrant analysis method is used to accurately interpret cell state: Annexin V - / 7-AAD - Cells are defined as living cells; Annexin V + / 7-AAD - The cells were early apoptotic cells; Annexin V + / 7-AAD + The cells in question belong to the late-stage apoptotic / necrotic cells. Simultaneously, the proportion of cells in each quadrant was statistically analyzed, and the results are shown in Figure 3. Figure 4 As shown, this method is used to distinguish between surviving cells, early apoptotic cells, and late apoptotic / necrotic cells, and to quantify and compare the effects of different treatment groups on inducing apoptosis.

[0052] Depend on Figure 3a and Figure 3b and Figure 4As shown in (a), (b), and (c), the apoptosis rates in the C1-KLA and C2-KLA treatment groups were significantly increased compared to the control group. The apoptosis-inducing effect of C1-KLA was particularly pronounced. This indicates that when the above-mentioned peptide conjugates were co-incubated with A431 cells, HUVEC cells, and PBMCs, respectively, and analyzed using the same staining and detection procedures, the apoptosis rate was significantly increased. Figure 4 As shown in (d), (e), and (f), no significant increase in the apoptosis rate was observed. This result fully demonstrates that the targeting effect conjugate provided in this application possesses good targeting selectivity. To improve the reproducibility and reliability of the experimental results, the above detection experiments can be independently repeated multiple times under the same experimental conditions, and the detection results can be presented in the form of average values. When necessary, conventional statistical methods in this field can also be used to compare and analyze the differences between different treatment groups to obtain more scientifically valuable conclusions.

[0053] To evaluate the antitumor efficacy of this targeting conjugate in mice, 6-week-old BALB / c nude mice were used as experimental animals to construct a nude mouse xenograft model of B-cell lymphoma.

[0054] Specifically, TOLEDO cells were collected and prepared into a homogeneous cell suspension. Subsequently, 1×10⁶ cells were injected subcutaneously into the subcutaneous tissue on the lateral side of each nude mouse. 7 TOLEDO cells were used to establish a xenograft tumor model. After inoculation, the experimental animals were fed and closely observed according to routine practices until tumors formed and the subcutaneous tumor volume in nude mice grew to approximately 100 mm. 3 During the experiment, the experimental animals were randomly divided into experimental and control groups to ensure comparability between the two groups at the initial stage. The experimental group was administered the target-effect conjugate C1-KLA via tail vein injection, with the dosage adjusted flexibly according to specific experimental needs. In this embodiment, approximately 250 μg / animal was administered. Control group mice received the same dose of KLA. During the administration period, routine observations were conducted on each group of animals to assess their condition, including activity level, food and water intake, and coat condition, to promptly monitor their health status.

[0055] Following drug administration, the long and short diameters of the tumor were measured every other day during the experimental period, and changes in tumor volume were calculated using standard formulas. Simultaneously, tumor volume data at each time point were recorded to assess the impact of different treatments on tumor growth. The tumor volume was calculated when it reached a predetermined endpoint (e.g., approximately 2000 mm²). 3 The experiment was terminated when the tumor tissue was removed. At the end of the experiment, the experimental animals were treated by removing the tumor tissue, weighing it, and photographing it to visually compare tumor growth among different treatment groups. The results are as follows: Figure 5 , Figure 6As shown, this illustrates the inhibitory effect of targeting-effect conjugates on tumor growth in vivo.

[0056] Depend on Figure 5 and Figure 6 As shown in (a) and (b), compared with the control group, the tumor volume growth rate of mice treated with C1-KLA was significantly slowed, and the tumor weight was significantly reduced at the endpoint. This result demonstrates that this targeting-effect conjugate has excellent inhibitory effects on B-cell lymphoma in vivo, providing solid experimental evidence for its application in the treatment of B-cell lymphoma.

[0057] In summary, this application utilizes phage display technology to screen for peptides with high specific binding, and then organically combines these peptides as target molecules with pro-apoptotic peptides to innovatively construct a targeted effector conjugate. This targeted effector conjugate not only possesses excellent targeting selectivity, specifically recognizing and binding to specific binding sites on the surface of B-cell lymphoma cells while exhibiting extremely low non-specific binding to normal peripheral blood mononuclear cells, but also demonstrates outstanding in vivo stability, maintaining structural integrity and functional activity in complex physiological environments. These characteristics enable precise targeted killing of B-cell lymphoma cells, effectively solving the key challenge in the field of targeted therapy: "how to improve the specificity of target molecules." During treatment, this targeted effector conjugate minimizes damage to normal cells, thereby significantly improving treatment efficacy and ensuring treatment safety. Therefore, combining targeted peptides with pro-apoptotic peptides can significantly improve treatment selectivity, ensure precise drug action on diseased cells, and effectively reduce toxic effects on normal cells, minimizing adverse reactions. This strategy has broad clinical application prospects and is expected to play a key role in the future treatment of B-cell lymphoma and other tumors, bringing new hope and better treatment options to cancer patients.

[0058] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0059] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A polypeptide targeting B-cell lymphoma cells, characterized in that, The polypeptide chain comprises 12 amino acids, and the polypeptide selectively and specifically binds to the surface of B-cell lymphoma cells.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. The polypeptide according to claim 1 or 2, characterized in that, The method for screening the polypeptide includes the following steps: S1. The phage-displayed random 12-peptide library is co-incubated with control cells to negatively screen the phage-displayed random 12-peptide library. Then, target cells are obtained and incubated with the screened phage-displayed random 12-peptide library at room temperature to ensure that the peptides displayed on the surface of the phage are in full contact with the cell surface binding sites. S2. After incubation, the phages bound to the surface of the target cells are washed away; S3. The bacteriophages obtained by elution are recovered and subjected to host bacterial amplification culture. After the culture is completed, the titer of the bacteriophages is determined, and the bacteriophages with the determined titer are used as input bacteriophages for the next round of screening. S4. Repeat steps S2 and S3 to gradually enrich the phages that have a high affinity for the binding sites on the surface of the target cells; S5. Select multiple bacteriophages, extract their DNA and sequence it, translate the obtained nucleotide sequences into corresponding polypeptide sequences, and screen the polypeptides after homology analysis.

4. The polypeptide according to claim 3, characterized in that, The target cells were B-cell lymphoma cell lines; before incubating the target cells with a random 12-peptide library displayed by phage, the target cells were cultured to the logarithmic growth phase, and then the target cells were collected and their density adjusted to 1×10⁻⁶. 6 ~5×10 6 Any value in the range of particles / mL.

5. A targeting effect conjugate, characterized in that, The polypeptide and the pro-apoptotic peptide described in any one of claims 1-4 are linked together by a linker peptide, wherein the linker peptide is a flexible linker peptide.

6. The targeting effect conjugate as described in claim 5, characterized in that, The polypeptide is covalently linked to the pro-apoptotic peptide.

7. The targeting effect conjugate as described in claim 6, characterized in that, The pro-apoptotic peptide is (KLAKLAK)2, and the polypeptide is attached to the N-terminus of the pro-apoptotic peptide.

8. The targeting effect conjugate as described in claim 6, characterized in that, The linker peptide is a short peptide, and the peptide chain of the short peptide includes no more than 5 amino acids.

9. The targeting effect conjugate as described in claim 7, characterized in that, The amino acid sequence of the targeting effect conjugate is shown in SEQ ID NO:

3.

10. The use of the targeting effect conjugate according to any one of claims 5-9 in an agent for inducing apoptosis of B-cell lymphoma cells or inhibiting their proliferation.

Images