Benzotriazole-modified affinity peptides and their mediated antibody region-directed functionalization modification

Abstract

This invention discloses a benzotriazole-modified affinity peptide and its mediated antibody region-directed functionalization modification. The invention chemically modifies an affinity peptide capable of binding to the Fc domain of an antibody by modifying its lysine side chain with a Dbz structure and a reaction handle. The disulfide bonds of the affinity peptide are further refolded to give it the spatial configuration of an antibody. The Dbz structure is activated in vitro to a Bt structure before antibody binding, and then purified. During antibody activation and transfer, the modified side chain structure of the affinity peptide makes Bt spatially more susceptible to attack by lysine residue 248 of the antibody heavy chain, achieving selective antibody modification through the proximity effect. The modified azide reaction handle can subsequently be bioorthogonally coupled with the payload, providing a synthetic method for constructing homogeneous antibody-drug conjugates.

Description

Technical Field

[0001] This invention relates to the fields of peptide chemical synthesis and selective protein modification technology, specifically to benzotriazole-modified affinity peptides and their mediated antibody region-directed functionalization modification. Background Technology

[0002] Antibody-drug conjugates (ADCs) are one of the most promising research directions in the field of cancer treatment. Traditional cancer treatments often suffer from high recurrence rates and severe side effects due to incomplete treatment of small lesions and lack of targeting. ADCs offer an effective solution to these problems, combining the high targeting of monoclonal antibodies with the tumor-killing properties of cytotoxic payloads. They allow monoclonal antibodies to accurately deliver cytotoxic drugs to the tumor site, effectively avoiding the side effects of chemotherapy on other normal cells. The basic structure of an antibody-drug conjugate consists of a monoclonal antibody (mAb), a cytotoxic payload, and a linker connecting the two. The linker acts as a "bridge" between the two components of the ADC, ensuring its effectiveness. The linker needs to remain stable after the ADC enters the bloodstream to prevent premature drug release, and it also needs to release the drug promptly upon antibody binding to tumor cell surface antigens to exert its therapeutic effect. Therefore, linker design is crucial in ADC development.

[0003] For an ideal ADC, a crucial indicator is the Drug-Antibody Ratio (DAR), which significantly impacts clinical efficacy. A low DAR means each antibody molecule carries a small number of drug molecules, limiting its killing effect on tumor cells; conversely, a high DAR may lead to drug aggregation, reducing the drug's half-life in the body. Furthermore, the DAR value of each antibody molecule in an ADC should be as uniform as possible. If the produced ADC is a mixture of antibodies with varying DAR values, its stability and pharmacokinetic properties will be greatly reduced, affecting efficacy. In the case of IgG antibodies, proteins with an average molecular weight of 150 kDa, numerous reactive amino acid residues on their surface can react with the linker attached to the payload. Without control, this will inevitably result in a heterogeneous mixture. Therefore, the linker needs site-selective modification during covalent coupling with the antibody to form ADCs with uniform and stable DAR values. Current selective modification techniques are mainly based on lysine (Lys) and cysteine ​​(Cys) in natural antibodies, such as the re-bridging of disulfide bonds between light and heavy chains and the modification of antibody glycosyl groups. In addition, genetic engineering techniques can be used to achieve the insertion of engineered Cys and non-natural amino acids, the coupling of specific sequences to the C-terminus of heavy chains, and enzyme-catalyzed selective linkage.

[0004] In recent years, a selective modification method based on affinity peptide labeling has emerged. This method uses small protein domains or affinity peptides that can non-covalently bind to conserved sequences in the antibody Fc region with high affinity. Introducing reactive groups onto the peptide chain can promote selective covalent modification of the antibody in that region. The main advantage of affinity peptide labeling is that it does not require genetic engineering or glycan remodeling, and the same affinity peptide can be applied to almost all IgG antibodies of the same subtype, offering broad applicability. However, considering the potential immunogenicity of non-natural peptides and the possibility that their structural size may hinder the physiological function of the Fc region, a traceless affinity peptide labeling method for selective modification has evolved. Among these, the method developed by Yamada et al. introduces a cleavable disulfide bond between the affinity peptide and an antibody-modifiable NHS ester, allowing the affinity peptide to be removed after modification to restore the original antibody Fc region structure. This method is named Affinity Peptide-Mediated Regioselective Functionalization (AJICAP).

[0005] Easily leaving acyl donor structures are a class of options for modifying proteins and peptides, including thioester structures, NHS esters, acylpyrazoles, and benzotriazole (Bt) structures. Benzotriazole is derived from its precursor o-phenylenediamine through activation. Traditional activation methods involve conversion using NaNO2 at -20°C. More efficient conversion processes can be achieved using DEA NONOate and directly via t-BuONO in the organic phase. These methods are gentler on proteins than traditional methods and have less impact on the converted Bt structure. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide an antibody region affinity peptide with lysine side chain modified o-phenylenediamine and reactive handle.

[0007] A second objective of this invention is to provide a method for activating the aforementioned affinity peptide.

[0008] A third objective of this invention is to provide a benzotriazole-modified affinity peptide obtained by the above method.

[0009] The fourth objective of this invention is to provide a method for targeted and traceless functionalization modification of antibody regions mediated by the above-mentioned benzotriazole-modified affinity peptide.

[0010] The fifth objective of this invention is to provide an IgG antibody with a reactive handle selectively modified by lysine at position 248 of the heavy chain.

[0011] The sixth objective of this invention is to provide the application of the above method in the preparation of antibody-drug conjugates.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] An antibody reactivity peptide modified with o-phenylenediamine (Dbz) and a reactive handle via a lysine side chain, wherein the antibody reactivity peptide is an IgG1 antibody Fc reactivity peptide, and o-phenylenediamine is coupled to the amino group of its lysine side chain; a reactive handle is coupled to one amino group of the o-phenylenediamine; and the reactive handle is a bioorthogonal reactive group.

[0014] Preferably, the IgG1 antibody Fc region affinity peptide is selected from any one of the following: RGNCAYHKGQLVWCTYH, FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC, FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC, FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC; preferably RGNCAYHKGQLVWCTYH.

[0015] Preferably, the bioorthogonal reactive group is an azide group or a polyethylene glycol azide group.

[0016] More preferably, the bioorthogonal reactive group is a tetraethylene glycol azide group.

[0017] The method for preparing the antibody region affinity peptide modified with o-phenylenediamine and reactive handle by the above-mentioned lysine side chain includes the following steps:

[0018] (1) The full-length affinity peptide was synthesized according to the amino acid sequence by solid-phase peptide synthesis technology, and then the Fmoc protecting group was removed and the N-terminal amino group of the affinity peptide was blocked; among them, when introducing lysine, Fmoc-L-Lys(Dde)-OH building blocks were used to introduce lysine protected by Dde protecting group.

[0019] (2) Remove the Dde protecting group of the lysine side chain on the solid phase to expose the amino group of the lysine side chain;

[0020] (3) Continue to couple Fmoc2-Dbz-OH on the solid phase and complete the removal of the Fmoc protecting group;

[0021] (4) Continue to couple the reactive handle on the solid phase so that the reactive handle condenses with the amino group of Dbz;

[0022] (5) Cut the peptide with the completed side chain modification from the solid resin;

[0023] (6) The cut crude peptide is refolded in the liquid phase to form a spatial structure that can regionally affinity antibody, thus obtaining the antibody regional affinity peptide with lysine side chain modified o-phenylenediamine and reactive handle.

[0024] More preferably, the removal of the Fmoc protecting group includes the following steps: adding piperidine dissolved in a solution of N,N-dimethylformamide (DMF) at a volume ratio of 20%±2%, reacting at 25℃±5℃ for 7±2 min, washing the resin with N,N-dimethylformamide (DMF) and dichloromethane (DCM), and then repeating the above process once.

[0025] More preferably, the N-terminal amino group of the blocked affinity peptide is defined by the following steps: adding acetic anhydride dissolved in a solution of N,N-dimethylformamide (DMF) at a volume ratio of 20%±2% and reacting at 25℃±5℃ for 20±2 min.

[0026] More preferably, the removal of the Dde protecting group from the lysine side chain includes the following steps: adding hydrazine hydrate (N2H4·H2O) dissolved in N,N-dimethylformamide (DMF) at a volume ratio of 2%±0.5%, and reacting at 25℃±5℃ for 15±2 min.

[0027] More preferably, the coupling step of Fmoc2-Dbz-OH is as follows: based on the actual loading amount of MBHA resin (0.5 mmol / g), 3 ± 0.5 equivalents of 3,4-bis((fluorenylmethoxycarbonyl)amino)benzoic acid (Fmoc2-Dbz-OH), 3 ± 0.5 equivalents of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) and 6 ± 0.5 equivalents of N,N-diisopropylethylamine (DIEA) are added, and the reaction is carried out for 2.5 ± 0.5 h.

[0028] More preferably, the steps for removing the Fmoc protecting group after coupling Fmoc2-Dbz-OH are as follows: Add a solution of piperidine dissolved in DMF at a volume ratio of 20% ± 2%, react at 25℃ ± 5℃ for 7 ± 2 min, wash the resin with N,N-dimethylformamide (DMF) and dichloromethane (DCM), and repeat the above process once more. After removing the Fmoc protecting group from Dbz, to prevent Dbz from oxidizing and deteriorating in air and at room temperature, it should be quickly added to the next reaction step for handle connection.

[0029] Preferably, when the reactive handle is polyethylene glycol azide-based, the steps for coupling the reactive handle are as follows: Based on the actual loading of MBHA resin, add 2 ± 0.5 equivalents of azide-tetraethylene glycol-carboxylic acid, 2 ± 0.5 equivalents of 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), and 4 ± 0.5 equivalents of N,N-diisopropylethylamine (DIEA), and react for 2 ± 0.5 h. To avoid both amino groups of Dbz being connected to the reactive handle and affecting the subsequent activation reaction, attention must be paid to controlling the reaction equivalents and reaction time in this step.

[0030] More preferably, the cleavage step is as follows: 3 mL of cleavage reagent is used per 100 mg of resin. The cleavage reagent is a mixture of trifluoroacetic acid / 3,6-dioxa-1,8-octanedithiol / water with a volume ratio of 95:2.5:2.5, and the reaction is carried out with shaking at room temperature for 2.5 h. The crude peptide after cleavage does not require purification and is directly subjected to one-pot refolding after lyophilization.

[0031] Preferably, the step of refolding disulfide bonds in the liquid phase is as follows: the obtained crude peptide is dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 10±1 mM, followed by the addition of a methanol solution containing 2±0.5 equivalents of 30%±2% hydrogen peroxide and 20±2 equivalents of ammonia, and reacted at 37°C for 15 min; after the reaction, the reaction solution is diluted in guanidine hydrochloride buffer to a final concentration of 1±0.5 mM; the composition of the buffer is: 6±0.5 mol / L GdmCl, 0.2±0.05 mol / L NaH2PO4, pH=3.0±0.2.

[0032] The activation method of the antibody region affinity peptide modified with o-phenylenediamine and reactive handle by the above-mentioned lysine side chain includes the following steps: dissolving the antibody region affinity peptide modified with o-phenylenediamine and reactive handle by the lysine side chain at a final concentration of 5±0.5 mM in ultra-dry N,N-dimethylformamide (DMF); dissolving 7±0.5 equivalents of tert-butyl nitrite (t-BuONO) of the affinity peptide in acetic acid (AcOH) at a volume ratio of 1:(9±1) to N,N-dimethylformamide (DMF); mixing the reaction solution after pre-cooling; and reacting at 0°C for 10±2 min.

[0033] Preferably, the method further includes the following steps: after reacting at 0℃ for 10±2 min, diluting with guanidine hydrochloride buffer and purifying by preparative HPLC; the buffer has the following composition: 6±0.5 mol / L GdmCl, 0.2±0.05 mol / L NaH2PO4, pH= 3.0±0.2.

[0034] Preferably, the activation process needs to be carried out in a strictly anhydrous and low-temperature environment.

[0035] A benzotriazole-modified affinity peptide was obtained by the above method.

[0036] The above-mentioned method for antibody region-directed, traceless functionalization modification mediated by benzotriazole-modified affinity peptide includes the following steps: taking deglycosylated antibody and diluting it in buffer; adding the benzotriazole-modified affinity peptide and reacting; first replacing the pH of the reaction system to 2.5±0.2, then replacing the pH of the reaction system to 7.5±0.2; finally performing functionalization modification based on bioorthogonal reaction to obtain IgG antibody functionalized with lysine at position 248 of the heavy chain.

[0037] Preferably, the antibody can be any type of IgG1 antibody. More preferably, the antibody is trastuzumab.

[0038] Preferably, the functional modification includes any one of fluorescence modification, biotin modification, polyethylene glycol modification, or drug molecule modification.

[0039] Preferably, the deglycosylation step is as follows: dissolve the antibody in a buffer solution, add N-glycosidase F (PNGase F, 83534-39-8), and incubate at 37°C for 20±2 h; the buffer solution has the following composition: 0.5±0.05 mol / L sodium phosphate, pH 7.5±0.2.

[0040] More preferably, the dilution refers to dilution to a final antibody concentration of 18±2 μM.

[0041] More preferably, the composition of the buffer solution is: 50±5 mM NaOAc, pH 5.5±0.2.

[0042] More preferably, the amount of the benzotriazole-modified affinity peptide added is 30 ± 5 times the equivalent of the antibody.

[0043] Before adding the benzotriazole-modified affinity peptide to the antibody, both components need to be sonicated to ensure that the antibody is fully dispersed.

[0044] Preferably, when the functionalization modification is fluorescent modification, the functionalization modification steps are as follows: the pH of the reaction system is replaced by ultrafiltration to 2.5±0.2, followed by a further replacement to 7.5±0.2, 50±5 equivalents of the fluorescent molecule DBCO-FITC are added, and the reaction is carried out at 37°C in the dark for 2±0.5 h. The purpose of first adjusting the pH of the reaction system to 2.5±0.2 is to cause the affinity peptide to lose its affinity at a low pH, thus removing the affinity peptide.

[0045] More preferably, the buffer solution used for the pH of the displacement reaction system has the following components: 137±10 mM NaCl, 2.7±0.5 mM KCl, 10±2 mM Na2HPO4, 1.8±0.2 mM KH2PO4, pH 2.5±0.2; and the buffer solution used for the pH of the re-displacement system has the following components: 137±10 mM NaCl, 2.7±0.5 mM KCl, 10±2 mM Na2HPO4, 1.8±0.2 mM KH2PO4, pH 7.5±0.2.

[0046] More preferably, the fluorescent molecule DBCO-FITC has poor solubility in salt solution, requiring the addition of half a volume of DMF to aid dissolution, followed by dilution with PBS.

[0047] More preferably, the PBS comprises: 137±10 mM NaCl, 2.7±0.5 mM KCl, 10±2 mM Na2HPO4, 1.8±0.2 mM KH2PO4, and pH 7.4±0.2.

[0048] An IgG antibody functionalized with lysine at position 248 of the heavy chain was prepared by the above method.

[0049] The above method is applied in the preparation of antibody-drug conjugates. The Bt structure has a certain degree of stability, but its reactivity is relatively low compared to other acyl donors. This low reactivity ensures better reaction selectivity. When the Bt space is adjacent to the lysine residue of the antibody heavy chain, there is a greater probability of reaction, ensuring a more uniform DAR value when conjugating antibodies.

[0050] The principle of this invention: This invention utilizes the AJICAP strategy to introduce Dbz and a reaction handle (N3-PEG4-COOH) that can be activated into an acyl donor structure onto the side chain of the affinity peptide in the Fc region of the affinity antibody. Through the proximity effect, the lysine residues spatially adjacent to the affinity peptide nucleophilically attack the activated Bt structure, achieving selective modification of the reaction handle on the lysine of the antibody heavy chain. At the same time, the affinity peptide and the Bt structure leave, achieving traceless modification.

[0051] The present invention has the following advantages and effects compared with the prior art:

[0052] The improved AJICAP technology employed in this invention can deliver a mild acyl donor structure modified on the affinity peptide to the desired reaction region of the antibody heavy chain via the Fc region of the affinity peptide with a specific sequence. Due to the proximity effect, the likelihood of reaction between the antibody's amino acid residues and the acyl donor structure of the affinity peptide is greatly increased, making it highly probable that the reactive handle on the affinity peptide will be modified onto specific residues of the antibody, thus achieving selective functionalization of the antibody.

[0053] The antibody-selective modification method proposed in this invention complements the ADC construction strategy. By utilizing the mild reactivity of the activated Bt structure, the selectivity of the coupling reaction is significantly improved. This helps to mitigate off-target phenomena previously observed in the linker-antibody coupling process of ADCs, providing a new approach for producing ADCs with more uniform DAR values.

[0054] The AJICAP technology employed in this invention allows for the removal of affinity peptides bound to the Fc region after selective modification of the antibody region, achieved through pH adjustment, resulting in a residue-free modification. This residue-free modification ensures that the antibody's native structure remains unaffected, guaranteeing its normal physiological activity.

[0055] The present invention offers high flexibility in the selection of reaction handles on affinity peptides. In the aforementioned work of synthesizing affinity peptides and modifying side chains, various bioorthogonal groups or fluorescein and other reaction handles can be coupled, which facilitates subsequent coupling with cytotoxic payloads. This broadens the application scope of this selective modification method.

[0056] The core process involved in this invention is the activation from the Dbz structure to the Bt structure, which is a supplement to the previous selective conjugation of functional groups to antibodies. The Bt structure has relatively mild reactivity and can remain stable for a certain period of time in neutral and weakly acidic aqueous solutions, which facilitates preparation and purification and avoids some side reaction problems caused by excessive reactivity.

[0057] The experimental procedure involved in this invention is the synthesis, modification, refolding, and activation of affinity peptides. This route is simple to operate and has good reproducibility. By paying attention to details such as reaction equivalents and time, the product can be obtained in high yield, which to a certain extent avoids the generation of byproducts and unnecessary loss of intermediates, and improves the conversion efficiency of each reaction step and the cumulative yield of the target affinity peptide.

[0058] In one embodiment of the present invention, using AJICAP technology, an affinity peptide capable of binding to the Fc domain of an antibody is chemically modified by modifying its lysine side chain at position 8 with a Dbz structure and an N3-PEG4 reaction handle. The disulfide bond of the affinity peptide is further refolded to give it the spatial configuration of an antibody. Dbz is then activated in vitro to a Bt structure before binding to the antibody, and the modified structure is purified. During antibody activation and transfer, the modified side chain of the affinity peptide makes Bt spatially more susceptible to attack by lysine residue 248 of the antibody heavy chain, achieving selective modification of the antibody through the proximity effect. The modified azide reaction handle can subsequently be bioorthogonally coupled with the payload, providing a synthetic method for constructing homogeneous antibody-drug conjugates.

[0059] This invention provides a novel affinity peptide tagging-based traceless modification technique for selective modification of specific antibody sites. This technique, mediated by the Dbz structure, results in a Bt structure after activation, which is a mild acyl donor structure with good reaction selectivity. Both affinity peptide synthesis and modification can be performed in the solid phase, offering simple operation and high yields. The Dbz structure is activated before affinity bonding with the antibody, avoiding potential side reactions during activation, and the high activation-conversion yield is evident in the liquid chromatography. Following modification, a click reaction selectively modifies primarily the antibody heavy chain, mitigating off-target effects associated with modifications on the light chain.

[0060] Furthermore, the modification method of the present invention can flexibly modify the reaction handle, which to some extent enriches the types of antibody-drug conjugates that can be constructed. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the synthetic route for lysine side chain modification of o-phenylenediamine and reactive handle affinity peptide in Example 1.

[0062] Figure 2 The reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram and ESI-MS characterization chromatogram are shown for the solid-phase synthesis of affinity peptides and the removal of the Dde protecting group from the lysine side chain in Example 1.

[0063] Figure 3 The reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram and ESI-MS characterization chromatogram of Dbz and N3-PEG4-COOH modified with lysine side chains coupled on the solid phase in Example 1 are shown.

[0064] Figure 4 The image shows the reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS characterization of the N3-PEG4-modified affinity peptide refolded in liquid phase in Example 1.

[0065] Figure 5 The image shows the reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and ESI-MS characterization of the refolded affinity peptide in Example 2.

[0066] Figure 6 Selective modifications were made to the antibody activation and transfer in Examples 3 and 4, followed by gel electrophoresis (SDS-PAGE) characterization after click reaction with fluorescent molecules.

[0067] Figure 7 This is a schematic diagram of the method of the present invention.

[0068] Figure 8 This is a schematic diagram of the route for comparison with the present invention.

[0069] Figure 9 Reversed-phase high-performance liquid chromatography (RP-HPLC) characterization of the solid-phase synthesis of affinity peptides modified with azidoacetic acid, followed by refolding after cleavage.

[0070] Figure 10 The figure shows the experimental results of Comparative Example 2; the left figure shows the results of reversed-phase high-performance liquid chromatography characterization after screening the SNP usage equivalents and activation time, respectively; the right figure shows the gel electrophoresis characterization of the supernatant and precipitate during activation after affinity antibody.

[0071] Figure 11 The graph shows the screening results of activation conditions for affinity peptides by DEA NONOate; the left graph shows the corresponding equivalent reversed-phase high-performance liquid chromatography characterization, and the right graph shows the activation percentage obtained from the peak area integration of the liquid chromatography.

[0072] Figure 12 The image shows the gel electrophoresis results of the whole antibody after the click reaction and the reduced antibody. Detailed Implementation

[0073] This invention discloses a method for selectively modifying specific regions of an antibody to achieve its functionalization. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. The method and product of this invention have been specifically described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods described herein without departing from the content, spirit, and scope of this invention to achieve the technical results of this invention.

[0074] To further understand the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0076] Unless otherwise specified, all materials and reagents used in the following examples are commercially available products and can be purchased through commercial channels.

[0077] The structural formula of Fmoc-L-Lys(Dde)-OH (CAS No.: 150629-67-7) used in the following examples is shown below:

[0078] .

[0079] The trastuzumab used in the following examples was purchased from Shanghai Henlius Biotech Co., Ltd., under the trade name Hanquyou. The antibody purchased directly has glycosylation modification and requires subsequent deglycosylation pretreatment.

[0080] The reagent names and abbreviations used in the following examples are as follows:

[0081] DMF: N,N-dimethylformamide;

[0082] DCM: Dichloromethane;

[0083] TBTU: 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (CAS No.: 125700-67-6).

[0084] DIEA: N,N-Diisopropylethylamine (CAS No.: 7087-68-5);

[0085] N2H4·H2O: hydrazine hydrate;

[0086] The structural formula of Fmoc2-Dbz-OH: 3,4-bis((fluorenylmethoxycarbonyl)amino)benzoic acid (CAS No.: 345958-22-7) is shown below:

[0087] ;

[0088] N3-PEG4-COOH: Azide-tetraethylene glycol-carboxylic acid (CAS No.: 1257063-35-6) has the following structural formula:

[0089] ;

[0090] TFA: Trifluoroacetic acid;

[0091] DODT: 3,6-dioxa-1,8-octanedithiol (CAS No.: 14970-87-7).

[0092] ACN: Acetonitrile;

[0093] NH3·MeOH: Ammonia in methanol;

[0094] GdmCl: Guanidine hydrochloride;

[0095] NaH2PO4: Sodium dihydrogen phosphate;

[0096] AcOH: Acetic acid;

[0097] t-BuONO: tert-butyl nitrite (CAS No.: 540-80-7);

[0098] NaOAc: Sodium acetate;

[0099] DBCO-FITC: Dibenzocyclooctylene-fluorescein isothiocyanate;

[0100] SNP: Sodium nitroferricyanide;

[0101] DEA NONOate: 2-(N,N-diethylamino)-diazepine-2-oxodiethylamine salt;

[0102] NaOH: Sodium hydroxide;

[0103] DTT: Dithiothreitol.

[0104] The following examples use RGNCAYHKGQLVWCTYH as an example to demonstrate the synthesis of lysine-modified o-phenylenediamine and reactive handle affinity peptides, as well as fluorescein modification.

[0105] Example 1: Synthesis of o-phenylenediamine and reactive handle affinity peptide modified with lysine side chain.

[0106] Includes the following steps:

[0107] (1) The full-length affinity peptide was synthesized according to the amino acid sequence by solid-phase peptide synthesis technology, and then the Fmoc protecting group was removed and the N-terminal amino group of the affinity peptide was blocked; wherein, when introducing lysine, Fmoc-L-Lys(Dde)-OH building blocks were used to introduce lysine protected by the Dde protecting group.

[0108] (2) Remove the Dde protecting group of the lysine side chain on the solid phase to expose the amino group of the lysine side chain;

[0109] (3) Continue to couple Fmoc2-Dbz-OH on the solid phase and complete the removal of the Fmoc protecting group;

[0110] (4) Continue to couple the reactive handle on the solid phase so that the reactive handle condenses with the amino group of Dbz;

[0111] (5) Cut the peptide with the completed side chain modification from the solid resin;

[0112] (6) The cut crude peptides are refolded in the liquid phase to form a spatial structure that can regionally affinity antibodies.

[0113] Specifically:

[0114] Step 1: Solid-phase peptide synthesis of Ac-affinity peptides:

[0115] (1) Take 1.0 g of MBHA resin (Rink Amide-MBHA Resin, theoretical loading capacity of 0.798 mmol / g) in a peptide synthesis tube and swell it with DMF solvent for 30 min. Wash the resin repeatedly with DMF and DCM three times, drain the solvent, add 20% piperidine solution and shake at room temperature for 7 min, wash the resin and drain the solvent, and then add 20% (v / v) piperidine solution to repeat the above operation once. Add Fmoc-L-His-OH containing 297 mg (0.48 mmol), 151 mg (0.47 mmol) TBTU, and 165 μL (0.96 mmol) DIEA dissolved in 6 mL DMF, and shake at room temperature for 2.5 h for coupling reaction; wash the resin and drain the solvent, add 10 mL of DMF solution containing 20% ​​(v / v) acetic anhydride, and shake at room temperature for 20 min to block the uncoupled reaction sites on the resin. After washing and drying the resin, UV testing was performed to determine the actual resin loading, yielding Fmoc-His-MBHA resin with an actual loading of approximately 0.5 mmol / g.

[0116] (2) The amino acid sequence of Ac-affinity peptide-K (Dde) is: Ac-RGNCAYHK(Dde)GQLVWCTYH-CONH2. Except for the lysine K (Dde) at position 8, the coupling of other amino acids follows the standard amino acid coupling procedure.

[0117] (3) The coupling procedure for introducing lysine K (Dde) is as follows: The building block used is Fmoc-L-Lys(Dde)-OH; 6 mL of DMF reaction solution containing 3 equivalents of Fmoc-L-Lys(Dde)-OH (799 mg, 1.5 mmol), 2.94 equivalents of TBTU (472 mg, 1.47 mmol), and 6 equivalents of DIEA (516 μL, 3 mmol) was dissolved, activated by shaking at room temperature for 5 min, and then added to 1.0 g MBHA resin, and the coupling reaction was carried out by shaking at room temperature for 2.5 h.

[0118] (4) Capping of the N-terminus of the affinity peptide: After the last amino acid Arg is coupled, the resin is washed with DMF and DCM, the solvent is drained, and 20% piperidine solution is added and the reaction is shaken at room temperature for 7 min. This is repeated twice. After washing the resin and draining the solvent, 10 mL of DMF solution containing 20% ​​acetic anhydride is added and the reaction is shaken at room temperature for 20 min to block the N-terminal amino group of the affinity peptide.

[0119] (5) Removal of the Dde protecting group from Ac-affinity peptide-K (Dde): Measure 120 μL of N2H4·H2O and dilute it with DMF to a total volume of 6 mL to prepare a mixed solution with a volume ratio of 2% N2H4·H2O. Add the mixture to 1.0 g of MBHA resin and react with shaking at room temperature for 15 min.

[0120] (6) The procedure for introducing Fmoc2-Dbz-OH into the Ac-affinity peptide side chain is as follows: Fmoc2-Dbz-OH is used as the building block. 3 equivalents of Fmoc2-Dbz-OH building block, 2.94 equivalents of TBTU and 6 equivalents of DIEA are dissolved in 6 mL of DMF and added to 1.0 g MBHA resin. The coupling reaction is carried out at room temperature with shaking for 2.5 h.

[0121] (7) The procedure for removing Fmoc from Ac-affinity peptide-Dbz is as follows: Add 20% piperidine solution to MBHA resin and shake at room temperature for 7 min. Wash the resin and drain the solvent. Then add 20% piperidine solution and repeat the above operation once.

[0122] (8) The procedure for introducing N3-PEG4-COOH into the Ac-affinity peptide side chain is as follows: N3-PEG4-COOH is used as the building block, 2 equivalents of N3-PEG4-COOH building block, 1.98 equivalents of TBTU and 4 equivalents of DIEA are dissolved in 6 mL of DMF and added to 1.0 g MBHA resin, and the coupling reaction is carried out at room temperature with shaking for 2 h.

[0123] (9) Cleavage of Ac-affinity peptide-Dbz-PEG4-N3: 1.0 g of synthesized MBHA resin was dried and transferred to a glass reaction flask. 30 mL of cleavage reagent (TFA: DODT: H2O, 95: 2.5: 2.5, v / v / v, mL / mL / mL) was added. The mixture was shaken at room temperature for 2.5 h. The cleavage solution was filtered, the solvent was evaporated by rotary evaporator, and excess frozen ether was added for extraction, resulting in a large amount of white precipitate. After centrifugation, the precipitate was dried under nitrogen. The crude peptide was dissolved in 20 mL of solution (ACN: H2O, 1: 1, v / v) and freeze-dried into crude peptide powder.

[0124] (10) Refolding of Ac-affinity peptide-Dbz-PEG4-N3 crude peptide: 58 mg of the cleaved crude peptide was dissolved in 2.34 mL of DMSO to a final concentration of 10 mM. Then, 2 equivalents of 30% H2O2 (3.7 μL) and 20 equivalents of NH3·MeOH (7.8 μL) were added, and the reaction was carried out at 37 °C for 15 min. After the reaction, 21 mL of GdmCl buffer (6 mol / L GdmCl, 0.2 mol / L NaH2PO4, pH= 3.0) was added to dilute to a final concentration of 1 mM. The diluted reaction solution was characterized by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS, and purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC).

[0125] Figure 2 The figures show the reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS characterization chromatograms of the solid-phase peptide synthesis of affinity peptides and the removal of the Dde protecting group from the lysine side chain in Example 1. As can be seen from the figures, in the liquid-phase characterization process, each step—from the termination of affinity peptide coupling, the removal of Fmoc and the blocking of the N-terminal amino group, to the removal of Dde—yielded the corresponding product in a high yield (the main peak in the HPLC chromatogram).

[0126] Figure 3 The figures show the reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS characterization of Dbz and N3-PEG4-COOH modified with lysine side chains in solid phase in Example 1. The figures demonstrate that the main products were obtained with high conversion rates in the HPLC characterization.

[0127] Figure 4 The figures show the reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and ESI-MS characterization of the N3-PEG4-modified affinity peptide refolded in liquid phase in Example 1. As can be seen from the figures, a shorter envelope peak appears to the right of the main peak after refolding, which is actually a column efficiency issue. ESI-MS characterization shows that the main peak and the envelope peak are the same product.

[0128] Example 2: Activation of Ac-affinity peptide-Dbz-PEG4-N3 peptide.

[0129] 1.47 mg of the refolded and purified affinity peptide was dissolved in 108 μL of ultra-dry DMF and placed in a 2 mL centrifuge tube. A small magnetic stir bar was added, and the tube was pre-cooled on ice. To ensure accurate dispensing, a 10-fold increase was made: 120 μL of AcOH was added to 5.9 μL of t-BuONO. To prevent the liquid from freezing in the ice, the mixture was thoroughly mixed and pre-cooled on ice. 12.6 μL of the AcOH / t-BuONO mixture was taken and added to the DMF-dissolved affinity peptide, bringing the total amount of t-BuONO to 7 times the equivalent amount, resulting in a final peptide concentration of 5 mM. The reaction was carried out at 0°C for 10 min with stirring. After dilution with GdmCl buffer (6 mol / L GdmCl, 0.2 mol / L NaH2PO4, pH = 3.0), the peptide was purified by preparative HPLC.

[0130] Figure 5 The figures show the reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS characterization of the refolded affinity peptides in Example 2. As can be seen from the figures, the activated HPLC chromatogram represents the unpurified original reaction solution, demonstrating that the activation process proceeded with extremely high conversion rates and almost no byproduct formation.

[0131] Example 3: Region-directed selective modification of antibodies.

[0132] Weigh 2 mg of trastuzumab (Hanquyou) and dissolve it in 200 μL of buffer solution. Add 5 μL of N-glycosidase F (PNGase F, 83534-39-8) and incubate at 37°C for 20 h to deglycosylate the antibody. The buffer solution used for the reaction consisted of 0.5 mol / L sodium phosphate and pH 7.5. The coupling process of this invention is chemically modified, and no enzyme quenching is required after the reaction; subsequent reactions can proceed directly.

[0133] 5 μL of deglycosylated trastuzumab was diluted in 27.5 μL of 50 mM NaOAc (pH 5.5) to a final antibody concentration of 18 μM. The antibody solution was sonicated to ensure redispersibility of aggregated antibodies. 10 μL of ultradry DMF was added to dissolve 0.057 mg of the activated affinity peptide (30 equivalents of the antibody). To ensure accurate dosage, the activated affinity peptide was weighed in advance and aliquoted into lyophilized centrifuge tubes. After sonicating the affinity peptide, the antibody solution was added to the affinity peptide tubes, vortexed, and allowed to react overnight at room temperature (20°C).

[0134] Example 4: Click reaction of azide-modified antibody with DBCO-FITC.

[0135] First, a buffer solution of 137±10 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and pH 2.5 was added. The pH of the reaction system was then changed to acidic under ultrafiltration to remove the modified components and excess affinity peptides. Subsequently, the system was changed to neutral by passing the buffer solution at pH 7.5. 50 equivalents of the fluorescent small molecule DBCO-FITC (0.117 mg) were added, and the volume was increased 10-fold before weighing. 1.17 mg of the reagent was weighed and dissolved in 150 μL of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and 90 μL of ultra-dry DMF. 8 μL of this solution was added to the antibody-activated conversion reaction solution overnight, and the reaction was carried out in the dark. After the reaction was complete, 10 μL of the reaction solution was taken, diluted with 6 μL of 8 M Urea, and 4 μL of loading buffer was added. After mixing, the mixture was denatured in boiling water for 10 min. SDS-PAGE characterization was then performed.

[0136] Figure 6 Selective modifications were performed on the antibodies used for activation and transfer in Examples 3 and 4, followed by gel electrophoresis (SDS-PAGE) characterization after click reaction with fluorescent molecules. In the figure, ①, ②, and ③ represent the equivalent amounts of affinity peptides added during antibody activation and transfer: 10 eq., 20 eq., and 30 eq., respectively. The left image shows the fluorescent bands of the gel electrophoresis under 490 nm blue light illumination. It can be seen that the 10 eq. lane has almost no fluorescence; the 20 eq. lane has a fluorescent band, but the fluorescence of the light chain is stronger than that of the heavy chain, indicating a significant off-target effect; the 30 eq. lane has a fluorescent band, and the heavy chain band is significantly brighter than the light chain, indicating that the modification mainly occurred on the heavy chain with less off-target effect. The right image shows the bands after staining with Coomassie Brilliant Blue, showing distinct heavy and light chain bands.

[0137] Figure 7 This is a schematic diagram of the method of the present invention. The core concept is to utilize the affinity peptide to bind to the Fc region of the IgG1 antibody, and to modify the heavy chain lysine at position 248 by a benzotriazole structure modified on the affinity peptide through the spatial proximity effect. The modified reaction handle can be flexibly modified on the side chain of the affinity peptide according to the actual situation.

[0138] Figure 8 This is a schematic diagram of the comparative example of the present invention. The difference from the embodiments is that the modified reaction handle is azidoacetic acid, and in the antibody-selective modification, an affinity-then-activation approach is adopted.

[0139] Comparative Example 1: Solid-phase peptide synthesis of Ac-azidoacetic acid-affinity peptide.

[0140] Referring to Example 1, the difference lies in step (7), where the coupling reaction handle is azidoacetic acid. The procedure for introducing the side chain is as follows: the building block used is N3-CH2-COOH; 2 equivalents of N3-CH2-COOH building block, 1.98 equivalents of TBTU, and 4 equivalents of DIEA are dissolved in 6 mL of DMF and added to 1.0 g of MBHA resin; the coupling reaction is carried out at room temperature with shaking for 2 h. After cutting, the refolded structure is characterized by reversed-phase high-performance liquid chromatography.

[0141] The results are as follows Figure 9 As shown in the figure, solid-phase coupled azidoacetic acid was obtained in high yield; refolding after solid-phase cleavage also yielded high yield. The shorter peak to the right of the main peak in the figure is due to column efficiency issues and is actually the same product.

[0142] Comparative Example 2: Screening for SNP activation conditions of Ac-affinity peptide-azidoacetic acid.

[0143] The Dbz structure was activated using SNPs, and optimal activation conditions were screened. 0.1 mg of Ac-azidoacetic acid-affinity peptide was dissolved in 50 mM PBS buffer (pH 5), and corresponding equivalent amounts of SNP were added. The reaction was carried out at 4°C for the corresponding time. The activation was characterized by reversed-phase high-performance liquid chromatography.

[0144] The results are as follows Figure 10 As shown in the figure, the green area represents the reactants before activation, and the red area represents the product peaks after activation. After screening the SNP equivalent and activation time, 1 equivalent of SNP activation for 5 min was found to be the optimal reaction condition. However, under these conditions, when attempting to activate and transfer the antibody after affinity bonding, significant antibody aggregation occurred. The gel electrophoresis in the figure shows that the two bands are the same substance (the light and heavy chains of the antibody). After confirming that the optimal activation conditions were 1 equivalent of SNP and 5 min of reaction, the affinity peptide was added to the antibody for affinity bonding, followed by activation with SNP. However, a large amount of precipitation was observed in the reaction solution. The supernatant and precipitate were characterized by gel electrophoresis. The precipitate was found to be antibody, which underwent denaturation and aggregation under the SNP activation conditions, indicating that SNP is not suitable for activation after affinity bonding between the affinity peptide and the antibody.

[0145] Comparative Example 3: Screening for DEA NONOate activation conditions of Ac-affinity peptide-azidoacetic acid.

[0146] Using DEA NONOate as a mild activating agent, 0.1 mg of Ac-azidoacetic acid-affinity peptide was dissolved in 50 mM PBS buffer (pH 5), and a corresponding equivalent amount of DEA NONOate dissolved in 0.1 M NaOH was added. The reaction was carried out with shaking at room temperature for 5 min. The activation was characterized by reversed-phase high-performance liquid chromatography.

[0147] Figure 11 This figure shows the screening results of activation conditions for DEA NONOate on affinity peptides. The left figure shows the corresponding equivalents of reversed-phase high-performance liquid chromatography (RP-HPLC) characterization, and the right figure shows the activation percentage obtained from peak area integration in the HPLC chromatogram. The equivalent with the highest activation efficiency was selected for the activation reaction after affinity antibody treatment. As can be seen from the figure, when screening the activation equivalents of DEA NONOate, the green area indicates the reactant peak before activation, and the red area indicates the product peak after activation. After integrating the peak areas before and after activation, 50 and 100 equivalents showed better activation effects. Given that the overall results were not significantly different, selecting the activation condition with a smaller equivalent was a more economical choice.

[0148] Comparative Example 4: Antibody activation and transfer and click reaction with DBCO-FITC.

[0149] Take 5 μL of antibody and dilute it in 56 μL of NaOAc (pH 5.5) to a final antibody concentration of 4 μM. Weigh out 10 equivalents of the activated affinity peptide, dissolve it in PBS (pH 5), add the antibody, and incubate at 37°C for 1 h. Then add 50 equivalents of DEA NONOate relative to the affinity peptide, shake at room temperature, and react at room temperature for 1 h.

[0150] Add 100 mM PBS (pH 2.5) and use ultrafiltration to change the pH of the reaction system to acidity, removing modified and excess affinity peptides. Then, use a pH 7.5 buffer and ultrafiltration to neutralize the system. Add 10 equivalents of DBCO-FITC and react overnight at room temperature in the dark. Reduce the reacted antibody with DTT to break the disulfide bonds between the light and heavy chains, and characterize by gel electrophoresis.

[0151] Figure 12 The images show the gel electrophoresis results of the intact antibody after the click reaction and the reduced antibody. ①-④ represent: after activation and transfer (integrity antibody), after activation and transfer and reduction (integrity antibody), after activation and transfer and clicking (integrity antibody), and after activation and transfer and clicking and reduction (integrity antibody), respectively. It can be seen that after clicking the fluorescent molecule, the antibody is modified with the fluorescent molecule. However, after reduction, both the light and heavy chain bands show bright fluorescent bands, indicating that selective modification of the heavy chain was not achieved, resulting in significant off-target effects.

[0152] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An antibody regioaffinity peptide with a lysine side chain modified with o-phenylenediamine and a reactive handle, characterized in that: The antibody region affinity peptide is an IgG1 antibody Fc region affinity peptide, wherein an o-phenylenediamine is coupled to the amino group of its lysine side chain; a reactive handle is coupled to one amino group of the o-phenylenediamine; the reactive handle is a bioorthogonal reactive group. The IgG1 antibody Fc region affinity peptide is selected from any one of the following: RGNCAYHKGQLVWCTYH, FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC, FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC, FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC.

2. The antibody regio affinity peptide with lysine side chain modified o-phenylenediamine and reactive handle according to claim 1, characterized in that: The affinity peptide of the Fc region of the IgG1 antibody is RGNCAYHKGQLVWCTYH; The bioorthogonal reactive group is an azide group or a polyethylene glycol azide group.

3. The method for preparing the antibody regioaffinity peptide with lysine side chain modified o-phenylenediamine and reactive handle as described in claim 1 or 2, characterized in that: Includes the following steps: (1) The full-length affinity peptide was synthesized according to the amino acid sequence by solid-phase peptide synthesis technology, and then the Fmoc protecting group was removed and the N-terminal amino group of the affinity peptide was blocked; among them, when introducing lysine, Fmoc-L-Lys(Dde)-OH building blocks were used to introduce lysine protected by Dde protecting group. (2) Remove the Dde protecting group of the lysine side chain on the solid phase to expose the amino group of the lysine side chain; (3) Continue to couple Fmoc2-Dbz-OH on the solid phase and complete the removal of the Fmoc protecting group; (4) Continue to couple the reactive handle on the solid phase so that the reactive handle condenses with the amino group of Dbz; (5) Cut the peptide with the completed side chain modification from the solid resin; (6) The cut crude peptide is refolded in the liquid phase to obtain the antibody region affinity peptide with lysine side chain modified o-phenylenediamine and reactive handle.

4. The method for preparing the antibody regioaffinity peptide modified with o-phenylenediamine and a reactive handle according to claim 3, characterized in that: The removal of the Fmoc protecting group includes the following steps: adding piperidine dissolved in a solution of N,N-dimethylformamide at a volume ratio of 20%±2%, reacting at 25℃±5℃ for 7±2 min, washing the resin with N,N-dimethylformamide and dichloromethane, and then repeating the above process once. The blocking of the N-terminal amino group of the affinity peptide includes the following steps: adding acetic anhydride dissolved in a solution of N,N-dimethylformamide at a volume ratio of 20%±2% and reacting at 25℃±5℃ for 20±2 min. The removal of the Dde protecting group from the lysine side chain includes the following steps: adding hydrazine hydrate dissolved in N,N-dimethylformamide at a volume ratio of 2%±0.5%, and reacting at 25℃±5℃ for 15±2 min. The cutting steps are as follows: 3 mL of cutting reagent is used for every 100 mg of resin. The cutting reagent is trifluoroacetic acid / 3,6-dioxa-1,8-octanedithiol / water with a volume ratio of 95:2.5:2.

5. The reaction is carried out at room temperature with shaking for 2.5 h. The steps for disulfide bond refolding in the liquid phase are as follows: The obtained crude peptide is dissolved in dimethyl sulfoxide at a final concentration of 10±1 mM, followed by the addition of a methanol solution containing 2±0.5 equivalents of 30%±2% hydrogen peroxide and 20±2 equivalents of ammonia, and reacted at 37°C for 15 min; after the reaction, the reaction solution is diluted in guanidine hydrochloride buffer to a final concentration of 1±0.5 mM; the composition of the buffer is: 6±0.5 mol / L GdmCl, 0.2±0.05 mol / L NaH2PO4, pH= 3.0±0.2; The steps for coupling Fmoc2-Dbz-OH are as follows: Based on the actual loading of MBHA resin, add 3±0.5 equivalents of 3,4-bis((fluorenylmethoxycarbonyl)amino)benzoic acid, 3±0.5 equivalents of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, and 6±0.5 equivalents of N,N-diisopropylethylamine, and react for 2.5±0.5 h; The steps for removing the Fmoc protecting group after coupling Fmoc2-Dbz-OH are as follows: Add a solution of piperidine dissolved in DMF at a volume ratio of 20%±2%, react at 25℃±5℃ for 7±2 min, wash the resin with N,N-dimethylformamide and dichloromethane, and then repeat the above process once; When the reactive handle is polyethylene glycol azide-based, the steps for coupling the reactive handle are as follows: based on the actual loading of MBHA resin, add 2 ± 0.5 equivalents of azide-tetraethylene glycol-carboxylic acid, 2 ± 0.5 equivalents of 2-(1H-benzotriazoL-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate, and 4 ± 0.5 equivalents of N,N-diisopropylethylamine, and react for 2 ± 0.5 h.

5. The activation method of the antibody region affinity peptide modified with o-phenylenediamine and reactive handle as described in claim 1 or 2, characterized in that: The method includes the following steps: dissolving the lysine side-chain modified o-phenylenediamine and reactive handle antibody affinity peptide at a final concentration of 5±0.5 mM in ultra-dry N,N-dimethylformamide; dissolving 7±0.5 equivalents of tert-butyl nitrite of the affinity peptide in acetic acid at a volume ratio of 1:(9±1) to N,N-dimethylformamide; mixing the reaction solution after pre-cooling and reacting at 0℃ for 10±2 min; after reacting at 0℃ for 10±2 min, diluting with guanidine hydrochloride buffer and purifying by preparative HPLC; the composition of the buffer is: 6±0.5 mol / L GdmCl, 0.2±0.05 mol / L NaH2PO4, pH= 3.0±0.

2.

6. A benzotriazole-modified affinity peptide, characterized in that: Obtained by the method described in claim 5.

7. A method for targeted, traceless functionalization modification of antibody regions mediated by benzotriazole-modified affinity peptides as described in claim 6, characterized in that: Includes the following steps: The deglycosylated antibody was taken and diluted in buffer; the benzotriazole-modified affinity peptide was added and reacted; the pH of the reaction system was first replaced to 2.5±0.2, and then replaced to 7.5±0.2; finally, functionalization modification was carried out based on bioorthogonal reaction to obtain IgG antibody with lysine functionalization at position 248 of the heavy chain.

8. The method according to claim 7, characterized in that: The antibody is selected from any IgG1 antibody; The functional modification is any one of fluorescence modification, biotin modification, polyethylene glycol modification, or drug molecule modification; The deglycosylation step is as follows: Dissolve the antibody in a buffer solution with the composition of 0.5±0.05 mol / L sodium phosphate and pH 7.5±0.2, add N-glycosidase F, and incubate at 37°C for 20±2 h; The dilution refers to dilution to a final antibody concentration of 18±2 μM; The buffer solution has the following composition: 50±5 mM NaOAc, pH 5.5±0.2; The amount of the benzotriazole-modified affinity peptide added is 30 ± 5 times the equivalent of the antibody; When the functionalization modification is fluorescent modification, the functionalization modification steps are as follows: the pH of the reaction system is replaced by ultrafiltration to 2.5±0.2, followed by a re-replacement reaction to 7.5±0.2, 50±5 equivalents of the fluorescent molecule DBCO-FITC are added, and the reaction is carried out at 37°C in the dark for 2±0.5 h; the buffer solution used for the pH replacement reaction system is composed of: 137±10 mM NaCl, 2.7±0.5 mM KCl, 10±2 mM Na2HPO4, 1.8±0.2 mM KH2PO4, pH 2.5±0.2; the buffer solution used for the pH re-replacement system is composed of: 137±10 mM NaCl, 2.7±0.5 mM KCl, 10±2 mM Na2HPO4, 1.8±0.2 mM KH2PO4, pH 7.5±0.

2.

9. An IgG antibody functionalized with lysine at position 248 of the heavy chain, characterized in that: It is prepared by the method described in claim 7 or 8.

10. The use of the method of claim 7 or 8 in the preparation of antibody-drug conjugates.

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