Specific binding cyclic peptide of tiralizumab, cyclic peptide derivative and application of cyclic peptide derivative

Abstract

The invention relates to the field of biological medicine, in particular to a cyclic peptide capable of being specifically combined with tiralizumab, a cyclic peptide derivative and application of the cyclic peptide derivative in recognition and combination of tiralizumab. The invention provides a novel teralizumab specific binding cyclic peptide which can specifically recognize and bind to teralizumab. The polypeptide can be chemically synthesized and labeled with various reporter molecules, has small batch difference, small molecular weight and high stability, and can be stored and used for a long time. According to the present invention, after the teralizumab specific binding cyclic peptide modifies different reporter molecules, the biosensor for detecting the blood concentration of the teralizumab can be constructed;

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a cyclic peptide, a cyclic peptide derivative thereof, and its application in recognizing and binding to tislelizumab. Background Technology

[0002] Tislelizumab is a humanized monoclonal antibody targeting programmed death receptor-1 (PD-1) and is widely used to treat various cancers. Through structural optimization of its Fc fragment, it minimizes binding to macrophage FcγR, eliminating antibody-dependent cell-mediated phagocytosis and exhibiting strong antitumor activity. Simultaneously, its slow dissociation rate from PD-1 contributes to its high antitumor activity, meeting the clinical demand for "highly effective and low-toxicity" drugs.

[0003] However, with the widespread use of tislelizumab, several clinical problems have emerged. First, the efficacy rate is low. Although these drugs are usually used in combination therapy to improve treatment efficacy, when used alone, the efficacy rate is only between 10% and 30%, except for treatment of classical Hodgkin's lymphoma, where it can exceed 60%. Second, there are significant individual variability. Both efficacy and toxicity vary considerably among individuals when using these drugs. Currently, significant individual response differences and low efficacy are believed to be related to factors such as serum drug concentration and immunogenicity after drug use. Therefore, to address these issues, on the one hand, it is necessary to monitor the drug concentration of tislelizumab during treatment; on the other hand, it is necessary to assess the in vivo response of tislelizumab to the specific target PD-1 at the molecular level, in order to accurately evaluate the body's response and therapeutic effect of the antibody drug. However, how to effectively capture tislelizumab in complex biological environments (such as body fluids or within the body), i.e., how to specifically identify tislelizumab, is the primary problem in solving the monitoring of blood drug concentration and the molecular assessment of its target response.

[0004] Peptides are ideal specific recognition units, exhibiting excellent biocompatibility, bioactivity, and safety. Compared to conventional antibodies and other recognition units, they are small in size, easy to immobilize in relatively dense and uniform formations, and can form more uniform biorecognition. Currently, they have been incorporated into biomaterials and are widely used in biomedical fields such as molecular recognition, drug delivery, bioimaging, and disease diagnosis and treatment. Furthermore, peptides possess various active functional groups, such as amino, hydroxyl, carboxyl, and thiol groups, which can be used for various chemical modifications to achieve different research objectives. Therefore, peptides can meet the requirements of high specificity recognition for antibody drugs, providing an effective tool for in vitro and in vivo studies of monoclonal antibody drugs.

[0005] For obtaining peptides, current methods include phage display, protein degradation, and computer prediction. Among these, phage display technology (PDT) utilizes genetic engineering to insert a foreign gene into the coding gene of a phage coat protein, thereby expressing a fusion protein on the phage surface. Peptides that specifically bind to the target molecule are then selected through panning. Compared to other methods, peptides obtained through phage display exhibit high specificity in binding to the target molecule, good biological activity, and small molecular weight, making them suitable for identifying target molecules in complex biological samples.

[0006] Developing peptides that specifically recognize tislelizumab could provide a powerful tool for monitoring tislelizumab blood concentrations and assessing its target response at the molecular level. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing detection methods for tislelizumab, to find a recognition element that can bind to it with high specificity, and to provide a cyclic polypeptide (cyclic peptide), cyclic peptide derivative, and its application that can bind to tislelizumab with high specificity and high affinity.

[0008] In a first aspect, the present invention provides a specific binding cyclic peptide or cyclic peptide derivative of tislelizumab, the amino acid sequence of which is shown below:

[0009] Sequence 1 is CLS: ACLSIWGTFC, SEQ ID NO.1;

[0010] Sequence 2 is CIS: ACISIWGTFC, SEQ ID NO.2;

[0011] ACLSIWGTFCGGGS,SEQ ID NO.3;

[0012] ACISIWGTFCGGGS,SEQ ID NO.4;

[0013] ACLSIWGTFCKKKKC, SEQ ID NO.5;

[0014] ACISIWGTFCKKKKC, SEQ ID NO.6.

[0015] Furthermore, in the cyclic peptide sequence, the cyclic structure is formed by disulfide bonds between cysteine ​​residues (Cys2-Cys10).

[0016] Furthermore, in the cyclic peptide derivative, other functional groups or molecules are attached or labeled at the 5' or 3' end of the sequence, or other amino acids are attached to the 5' or 3' end of the sequence, and the resulting sequence can also be used for the specific binding of tislelizumab; the other functional groups or molecules are selected from: biotin, amino, thiol, radioisotope, enzyme labeling or nanoluminescent materials.

[0017] Furthermore, this invention modifies sequence 1 or sequence 2 by synthesizing the spacer sequence GGGS (Gly-Gly-Gly-Ser) along with an amidation modification of the C-terminal carboxylic acid, ultimately obtaining the cyclic peptide derivative shown below:

[0018] CLS: ACLSIWGTFCGGGS-CONH2(Cys2-Cys10),

[0019] CIS: ACISIWGTFCGGGS-CONH2(Cys2-Cys10).

[0020] Furthermore, in order to better attach the cyclic peptide to the gold nanoparticles, the spacer sequence of the cyclic peptide was modified in this invention, replacing the aforementioned GGGS (Gly-Gly-Gly-Ser) with KKKC (Lys-Lys-Lys-Cys) (SEQ ID NO.3, SEQ ID NO.4). This modification not only increases the hydrophilicity of the peptide, but also promotes the formation of a relatively stable gold-sulfur bond between the terminal cysteine ​​and the gold nanoparticles, thereby constructing a gold nanoparticle colorimetric sensor based on cyclic peptide modification.

[0021] All the other polypeptides or molecules derived from the above have the same or similar molecular structure and function as the procyclic peptide, meaning they can all be used for the specific binding of tislelizumab.

[0022] In a second aspect, the present invention provides a gold nanoparticle colorimetric sensor based on cyclic peptide modification, which is constructed by forming stable gold-sulfur bonds between the terminal cysteine ​​residues of the cyclic peptide or cyclic peptide derivative as described above and gold nanoparticles.

[0023] A third aspect of the present invention provides the application of the cyclic peptide or cyclic peptide derivative, and the gold nanoparticle colorimetric sensor described above, in the preparation of products for detecting tislelizumab in blood, serum, and plasma.

[0024] In a fourth aspect, the present invention provides the application of the cyclic peptide or cyclic peptide derivative, as described above, and the gold nanoparticle colorimetric sensor in the separation and purification of tislelizumab.

[0025] In a fifth aspect, the present invention provides the application of the cyclic peptide or cyclic peptide derivative, and the gold nanoparticle colorimetric sensor as described above, in the preparation of tislelizumab detection probes and target probes.

[0026] Compared with existing technologies, the advantages of this invention are:

[0027] 1. A novel tislelizumab-specific binding cyclic peptide is provided, which can specifically recognize and bind to tislelizumab;

[0028] 2. The provided specific binding cyclic peptides can be chemically synthesized and labeled with various reporter molecules, with small batch-to-batch variations, small molecular weight, and high peptide stability, allowing for long-term storage and use.

[0029] 3. The provided tislelizumab-specific binding cyclic peptide, after being modified with different reporter molecules, can be used to construct a biosensor for detecting the blood concentration of tislelizumab. Attached Figure Description

[0030] Figure 1 This is a flowchart of the screening process for tislelizumab-specific binding cyclic peptides.

[0031] Figure 2 This is a secondary structure diagram of the tislelizumab-specific binding cyclic peptides CLS and CIS.

[0032] Figure 3 A schematic diagram illustrating the binding dissociation constant of a specific binding cyclic peptide characterized by surface plasmon resonance (SPR) technique.

[0033] Figure 4 This is a schematic diagram of a cyclic peptide-modified gold nanoparticle biosensor.

[0034] Figure 5 The UV absorption spectrum for biosensors to identify tislelizumab.

[0035] Figure 6 Fluorescence absorption spectrum for biosensor recognition of tislelizumab. Detailed Implementation

[0036] The specific embodiments of the present invention will be described in detail below with reference to examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Example 1: Screening for tislelizumab-specific binding cyclic peptides

[0038] The phage heptacyclic peptide library (Ph.D.-C7C), Escherichia coli ER2738, and sequencing primer 5´-CCCTCATAGTTAGCGTAACG-3´ (SEQ ID NO.7) were all purchased from New England Biolabs.

[0039] Detailed screening process ( Figure 1 )as follows:

[0040] Step 1: Dilute tislelizumab (Fab')2 with 0.1 M NaHCO3 (pH 8.6) solution to a protein dilution concentration of 100 μg / mL;

[0041] Step 2: Apply 150 μL of the coating to an ELISA plate and incubate overnight at 4 °C;

[0042] Step 3: Add 250 μL of BSA (5 mg / mL) blocking solution to each well and incubate at 4 °C for 1 h;

[0043] Step 4: Discard the sealing solution and rinse quickly 6 times with 0.1% TBST (TBS + 0.1% [v / v] Tween-20) solution;

[0044] Step 5: Dilute 10 μL of phage library (titer 2×10⁻⁶) with 100 μL TBST. 12 PFU), then transfer the diluted phage to an ELISA plate using a pipette and incubate at room temperature for 60 min;

[0045] Step 6: Discard unbound phages and wash the plate rapidly 10 times with 100 μL of 0.1% TBST (TBS + 0.1% [v / v] Tween-20) solution;

[0046] Step 7: Add 100 μL of Glycine-HCl (pH 2.2) buffer, gently shake at room temperature for 15 min, and then elute the bound phage;

[0047] Step 8: Transfer the eluent to a centrifuge tube and add 15 μL of 1 M Tris-HCl (pH 9.0) buffer to neutralize the eluent;

[0048] Step 9: Retain 10 μL of eluent for titer determination, and simultaneously amplify the remaining eluent for the second round of screening;

[0049] Step 10: The second round of screening is the same as the first round, repeating steps 1-9, except that the 0.1% TBST solution used is replaced with a 0.5% TBST solution;

[0050] Step 11: The third round of screening is the same as above, repeating steps 1-9 with some slight changes: ① Replace the 0.1% TBST solution with 0.5% TBST solution; ② Replace the antibody in step 1 with IgG, repeat steps 1-5, and aspirate the unbound phages for screening; ③ Do not amplify the obtained phage elution buffer.

[0051] Step 12: After three rounds of screening, 20 phage blue spots were randomly selected for fully automated sequencing.

[0052] Sequencing yielded three sequences, two of which were verified to have affinity: sequence 1 and sequence 2. The primary structures of sequences 1 and 2 are shown below. Figure 2 As shown.

[0053] Sequence 1 is CLS: ACLSIWGTFC (SEQ ID NO.1);

[0054] Sequence 2 is CIS: ACISIWGTFC (SEQ ID NO.2).

[0055] Structural optimization of specific binding peptides:

[0056] For further research, the selected sequences were synthesized. Since the N-terminus of a polypeptide is free in phages, while the C-terminus is embedded in the phage particle, the C-terminal residues of the selected sequences did not contain carboxylic acid groups during phage panning. Therefore, we modified the synthesized sequences. In the phage library, a spacer sequence GGGS (Gly-Gly-Gly-Ser) exists between the random sequence and the phage. This spacer sequence was synthesized along with the sequences (SEQ ID NO. 3, SEQ ID NO. 4), and the C-terminal carboxylic acid was also amidated. The final sequence obtained was...

[0057] CLS: ACLSIWGTFCGGGS-CONH2(Cys2-Cys10),

[0058] CIS: ACISIWGTFCGGGS-CONH2(Cys2-Cys10).

[0059] Affinity verification of the cyclic peptide sequence with tislelizumab:

[0060] Non-specific binding is unavoidable during phage display, so we need to verify the affinity of the obtained peptide sequence for tislelizumab. Simultaneously, considering that IgG is the isotype antibody of tislelizumab and also the biggest interfering substance in subsequent applications, IgG was selected as a negative protein to verify the specificity of the cyclic peptide sequence. Surface plasmon resonance (SPR) technology was used to verify and analyze the affinity between the cyclic peptide sequence and the tislelizumab antibody. SPR results showed that both sequence 1 (CLS) and sequence 2 (CIS) had good affinity for tislelizumab, with affinity constants of 5.88 μM and 7.93 μM, respectively, both greater than the affinity for IgG. Figure 3 Meanwhile, the binding curves show a rapid binding and dissociation process, indicating that both cyclic peptide sequences specifically bind to tislelizumab.

[0061] Example 2: Construction and application of a cyclic peptide-modified gold nanoparticle colorimetric sensor

[0062] To better attach the cyclic peptide to gold nanoparticles, the spacer sequence of the cyclic peptide was modified in this invention, replacing the original GGGS (Gly-Gly-Gly-Ser) with KKKC (Lys-Lys-Lys-Cys). This modification increases the hydrophilicity of the peptide and promotes the formation of a more stable gold-sulfur bond between the terminal cysteine ​​and the gold nanoparticles, thereby constructing a cyclic peptide-modified gold nanoparticle colorimetric sensor (CPEP1.1@AuNPs). CPEP1.1 (ACLSIWGTFCKKKC, SEQ ID NO.5) was dissolved in DMSO to prepare a 10 mM stock solution. Then, 50 μL of this solution was added to 0.025 mg / mL AuNPs and stirred for 4 h. After dialysis purification, the cyclic peptide-modified gold nanoparticle solution was obtained, and the cyclic peptide-modified gold nanoparticle sensor (CPEP1.1@AuNPs) was successfully constructed. In this solution, the addition of tislelizumab caused the gold nanoparticles to aggregate and eventually settle due to the increased distance between them, resulting in a color change. Figure 4 The process of recording the change in the UV absorption peak of gold nanoparticles with varying tislelizumab concentration using a UV spectrophotometer was analyzed. Figure 5 The process of changing fluorescence emission peaks of gold nanoparticles with varying tislelizumab concentration was recorded using a fluorescence spectrophotometer. Figure 6 Both methods can quantify the concentration of tislelizumab within a certain concentration range.

[0063] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A specific binding cyclic peptide or cyclic peptide derivative of tislelizumab, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1-6.

2. The cyclic peptide or cyclic peptide derivative according to claim 1, characterized in that, In the described cyclic peptide sequence, the cyclic structure forms Cys2-Cys10 through disulfide bonds between cysteine ​​residues.

3. The cyclic peptide or cyclic peptide derivative according to claim 1, characterized in that, In the cyclic peptide derivatives described herein, other functional groups or molecules are attached or labeled at the 5' or 3' end of the sequence, or other amino acids are attached to the 5' or 3' end of the sequence; the other functional groups or molecules are selected from: biotin, amino, thiol, radioactive isotopes, enzyme labeling, or nanoluminescent materials.

4. The cyclic peptide or cyclic peptide derivative according to claim 1, characterized in that, The cyclic peptide derivatives are selected from: ACLSIWGTFCGGGS-CONH2(Cys2-Cys10), ACISIWGTFCGGGS-CONH2(Cys2-Cys10).

5. A gold nanoparticle colorimetric sensor based on cyclic peptide modification, characterized in that, It is constructed by forming a stable gold-sulfur bond between the terminal cysteine ​​residue of the cyclic peptide or cyclic peptide derivative as described in any one of claims 1-4 and gold nanoparticles.

6. The use of the cyclic peptide or cyclic peptide derivative as described in any one of claims 1-4, and the gold nanoparticle colorimetric sensor as described in claim 5, in the preparation of products for detecting tislelizumab in blood, serum, and plasma.

7. The application of the cyclic peptide or cyclic peptide derivative as described in any one of claims 1-4, and the gold nanoparticle colorimetric sensor as described in claim 5, in the separation and purification of tislelizumab.

8. The application of the cyclic peptide or cyclic peptide derivative as described in any one of claims 1-4, and the gold nanoparticle colorimetric sensor as described in claim 5, in the preparation of tislelizumab detection probes and target probes.

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