A method for designing and engineering pH-sensitive antibodies based on yeast display

Abstract

The application discloses a method for modifying pH sensitivity of an antibody based on yeast display. A degenerate codon VAM (coding H, E, D, K, N, Q) is used to construct a yeast display library for at least two region combinations in LCDR1, HCDR1 and HCDR3, and 1-2 mutations are introduced into each CDR region, taking the antibody to be modified as a template. The library is transformed into Saccharomyces cerevisiae EBY100 to induce expression, and fast dissociation clones are enriched through four rounds of flow sorting (including pH 7.4 binding, pH 6.0 non-binding and'saturated binding-acid dissociation-neutral re-binding' screening). The obtained antibody has a KD ratio at pH 5.8 to pH 7.4 of up to 674.46, an expression amount of up to 263.3 mg / L, excellent physicochemical properties, and can enhance the curative effect of antibody conjugated drugs and improve the therapeutic index.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and antibody engineering technology, specifically to a method for designing and modifying pH-sensitive antibodies based on yeast display technology, as well as the pH-sensitive antibodies, yeast display libraries, vectors, host cells, and their applications prepared by this method. Background Technology

[0002] pH-sensitive antibodies, also known as recycling antibodies, are characterized by their efficient binding to target antigens in a neutral physiological environment (pH 7.4) and rapid dissociation from antigens in an acidic endosomal environment (pH 5.0–6.5). Figure 1 (As shown). This property allows antibodies to return to the cell surface via the FcRn receptor-mediated recycling pathway, which can both prolong the in vivo half-life of antibodies and improve the intracellular uptake efficiency of antibody-drug conjugates (ADCs), making it an important direction for the development of next-generation antibody drugs.

[0003] Currently, the modification of this field mainly relies on the "histidine scanning" strategy. The mainstream strategy for modifying pH-sensitive antibodies is histidine scanning, which involves introducing histidine residues into the antibody complementarity-determining region (CDR) and inducing pH-dependent dissociation by protonation of histidine under acidic conditions. For example, Schröter et al. (mAbs, 2015, 7(1):138-151) disclosed a method using a combination of histidine scanning libraries and yeast surface display technology to modify antibodies, obtaining antibody variants with a dissociation rate increased by 230-780 times.

[0004] However, existing modification technologies have the following drawbacks in practical applications: (1) Poor physicochemical properties: Single-point histidine mutations can easily destroy the natural structure of antibodies, leading to reduced expression, decreased thermal stability, and increased aggregation tendency.

[0005] (2) Low screening efficiency: Conventional methods are difficult to accurately screen out acidic rapid dissociation mutants from complex mutation libraries, resulting in long cycles and low success rates.

[0006] (3) Lack of synergistic effect: Single-point mutations cannot utilize the synergistic effect of charge between amino acids, making it difficult to balance neutral high affinity and acidic high dissociation.

[0007] (4) The expression level is limited, and the expression level of the resulting mutants is often low, which cannot meet the needs of industrial production.

[0008] (5) Single mutation strategy: Existing technologies are mostly limited to histidine mutations, and the CDR mutation region selection is limited, making it difficult to leverage the synergistic effect between regions; the screening logic only focuses on "acidic non-binding", and cannot directly enrich the dual phenotype of "rapid dissociation + neutral rebinding".

[0009] Therefore, developing an antibody engineering method that can simultaneously achieve high pH sensitivity, high expression levels, and excellent physicochemical properties has become an urgent need in the field of antibody drug development. Summary of the Invention

[0010] The purpose of this invention is to overcome the above-mentioned defects of existing pH-sensitive antibody modification technology, and to provide a method for engineering pH-sensitive antibodies based on yeast display technology. At the same time, it provides pH-sensitive antibodies prepared by this method and their applications, so that the antibodies can maintain neutral high affinity and excellent physicochemical properties, while having acidic rapid dissociation characteristics and increased expression levels.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for designing and modifying pH-sensitive antibodies based on yeast display, comprising the following steps: (1) Construction of yeast display library: Using the antibody sequence to be modified as a template, mutation design is carried out targeting the complementarity-determining region; the mutation adopts the degenerate codon VAM, which encodes the amino acids histidine (H), glutamic acid (E), aspartic acid (D), lysine (K), asparagine (N) and glutamine (Q); the mutation site is located in the combination of at least two regions of LCDR1, HCDR1 and HCDR3, and 1 to 2 mutation sites are introduced in each selected complementarity-determining region; (2) Induced library expression: The yeast display library constructed in step (1) is transformed into yeast cells and induced to express, so that the antibody molecules are displayed on the surface of the yeast cells; (3) Multi-round sorting and screening: The yeast cells displaying antibody molecules obtained in step (2) are screened, and the screening includes the following steps in sequence: In the first round of enrichment of binding molecules, the yeast cells were incubated with biotinylated fluorescently labeled antigen under neutral conditions of pH 7.4, and positive yeast cells that bound the antigen were sorted out. Second round: The yeast cells obtained from the first round of sorting were incubated with biotinylated antigen at pH 6.0. After washing, fluorescently labeled streptavidin was added. Yeast cells that did not show a signal under acidic conditions were collected for amplification, and pH-sensitive clones were enriched. The third round: screening for clones with the characteristic of rapid dissociation, specifically including: a. Under neutral conditions of pH 7.4, yeast cells obtained from the second round of sorting were incubated with 100 nM unlabeled antigen at 4°C for 1 hour to allow the surface-displayed antibodies to bind to the unlabeled antigen until saturation; b. After washing to remove free unlabeled antigen, resuspend the yeast cells in pH 6.0 acidic buffer and incubate at 4°C for 15-20 minutes to allow pH-sensitive antibodies to release the bound unlabeled antigen. c. After washing to remove the released unlabeled antigen, add biotinylated fluorescently labeled antigen, incubate at pH 7.4, and sort out the positive yeast cells that capture the fluorescently labeled antigen; Fourth round: The yeast cells obtained from the third round of sorting were incubated with biotinylated fluorescently labeled antigens under neutral conditions of pH 7.4 to verify antigen binding activity.

[0012] Furthermore, the complementary determination region is selected from a combination of at least two regions of LCDR1, HCDR1, and HCDR3.

[0013] Furthermore, the combined library is HCDR3 and LCDR1, HCDR1 and HCDR3, or HCDR1 and LCDR1.

[0014] Furthermore, the yeast cells are *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ) EBY100 strain.

[0015] Secondly, the present invention provides a pH-sensitive antibody prepared by the above method, wherein the complementarity-determining region contains 1 to 2 mutation sites encoded by degenerate codons VAM, and the mutation sites are located in a combination of at least two regions of LCDR1, HCDR1 and HCDR3.

[0016] Furthermore, the mutation site comprises at least one of histidine, glutamic acid, aspartic acid, lysine, asparagine, or glutamine.

[0017] Thirdly, the present invention provides a recombinant expression vector comprising a nucleotide sequence encoding the pH-sensitive antibody described in the second aspect.

[0018] Fourthly, the present invention provides a host cell containing the recombinant expression vector described in the third aspect, wherein the host cell is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ) EBY100 strain.

[0019] Fifthly, the present invention provides the use of the pH-sensitive antibody described in the second aspect in the preparation of a drug or antibody-drug conjugate with an extended in vivo half-life.

[0020] Furthermore, the pH-sensitive antibody rapidly dissociates from the antigen in an acidic endosomal environment of pH 5.0–6.5 and returns to the cell surface via an FcRn receptor-mediated recycling pathway, thereby enhancing the therapeutic index of the drug.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The expression yield of antibodies is increased, and the pH-sensitive antibodies prepared can reach up to 263.3 mg / L, which effectively solves the defect of low expression of antibody mutants in the existing technology; (2) The antibody possesses precise pH kinetic response characteristics, with an affinity constant (K0) at pH 5.8. D The dissociation rate is reduced by at least 82.7 times compared to pH 7.4, and the dissociation rate is significantly accelerated under acidic conditions, meeting the needs of antibody recycling and antibody-drug conjugate development. (3) The antibody has excellent physicochemical properties and stable structure. Its hydrophobic interaction chromatographic retention time is 13.3~16.1 min. It has moderate hydrophobicity and low aggregation risk, which greatly improves the drug-likeness of the antibody and solves the problem of poor physicochemical properties of the antibody in the existing technology. (4) High screening efficiency and accuracy. It adopts a four-round high-throughput sorting strategy, which rapidly dissociates the target antibody phenotype. The screening process is clear and controllable, which solves the defects of low efficiency and low success rate of traditional screening methods. (5) The modification method is highly versatile and can be adapted to pH sensitivity modification of various antibodies and multiple targets. It has a wide range of applications and provides an efficient and universal technical platform for antibody drug development. Attached Figure Description

[0022] Figure 1 The table shows the structural simulation and kinetic parameters of pH-sensitive antibody variants; where YS and SG correspond to variants VL:Y55H / VH:S103H and VH:S55H / G57E, respectively; the table shows the affinity of each antibody under neutral and acidic conditions.

[0023] Figure 2 A schematic diagram of primer design for CDR scanning: mutation sites and primer layout for LCDR1, HCDR1, and HCDR3.

[0024] Figure 3 This is a flowchart of the experimental steps for pH-dependent antibody screening based on yeast libraries.

[0025] Figure 4 This is a schematic diagram of the first round of sorting: the process of enriching antigen-binding positive yeast cells under pH 7.4 conditions.

[0026] Figure 5 This is a schematic diagram of the second round of sorting: the process of enriching pH-sensitive clones under pH 6.0 conditions.

[0027] Figure 6 This is a schematic diagram of the third round of sorting, illustrating the process of screening clones with rapid dissociation characteristics through "saturated binding - acidic dissociation - neutral recombination". Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the following will provide a more detailed description of this application in conjunction with embodiments. However, the scope of protection of this invention is not limited to the following embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0029] This embodiment focuses on a method for designing and modifying pH-sensitive antibodies based on yeast display. It sequentially completes the design of yeast display libraries, library construction, pH-conditional flow cytometry sorting, and in vitro functional verification of pH-sensitive candidate antibody molecules. All experimental operations are based on conventional biological experimental operation specifications in the field. General operations not mentioned can be completed with reference to well-known technical manuals in the field.

[0030] Example 1: Design of a yeast library This embodiment uses a humanized antibody derivative as a template, designs a mutation strategy based on VAM degenerate codons, and constructs a yeast display library with multi-regional combined mutations. The specific design scheme is as follows: 1.1 Antibody template Using known humanized antibody derivatives as templates, which share the same complementarity-determining region (CDR) sequence as their parent antibodies, the study determined that LCDR1, HCDR3, and HCDR1 are key regions mediating antigen binding, with LCDR1 being the longest and making a significant contribution to antigen binding.

[0031] 1.2 Mutation Strategy and Degenerate Codon Design To achieve broad sequence diversity exploration and circumvent stop codons, the VAM degenerate codon (V=A / G / C, M=A / C) was used for yeast library design. This codon encodes six amino acids: histidine (H), glutamic acid (E), aspartic acid (D), lysine (K), asparagine (N), and glutamine (Q). Among them, H, E, D, and K can undergo protonation or charge change in an acidic environment with pH < 6.0, inducing antibody conformational changes through electrostatic interactions to achieve pH-dependent antigen binding / dissociation; N and Q are used to maintain antibody structural stability or introduce polar modifications to the antibody.

[0032] 1.3 Library Construction Scheme Three combined mutant libraries were designed targeting the three key antigen-binding regions of LCDR1, HCDR1, and HCDR3 to cover the synergistic effects between different regions. Figure 2 The specific library design is as follows: Library 1: Combinatorial mutations targeting the HCDR3 and LCDR1 regions.

[0033] Library 2: Combinatorial mutations targeting the HCDR1 and HCDR3 regions.

[0034] Library 3: Combinatorial mutations targeting the HCDR1 and LCDR1 regions.

[0035] Example 2: Construction of a yeast display library 2.1 Experimental Materials (1) Materials related to yeast library generation: The strain was Saccharomyces cerevisiae EBY100; the buffers and reagents included electroconversion buffer (1 mol / L sorbitol, 1 mmol / L CaCl2), LiAc buffer (0.1 mol / L LiAc, 10 mmol / L dithreitol (DTT), DTT was prepared as a 1M stock solution and stored at -20℃), 1 mol / L sorbitol solution, and PBS buffer (pH 7.4); the culture media included YPD medium, SDCAA medium (pH 4.5), SDCAA medium (pH 6.0), and SGCAA induction medium; the instruments and equipment included an electroporation system (GenePulserXcell, Bio-Rad), a 0.2 cm electroconversion cuvette (Bio-Rad), a 30℃ constant temperature incubator, and a spectrophotometer; other reagents were BactoCasamino acids (BD Biosciences) and ampicillin.

[0036] (2) Materials related to yeast library labeling and sorting: The primary antibody was mouse anti-c-myc monoclonal antibody (clone 9E10, BioLegend); the secondary antibodies included goat anti-human IgG Fc labeled with Brilliant Violet 421 (Jackson), SA-436 (streptavidin-436 fluorescent dye, BioLegend), and anti-mouse IgG1 antibody labeled with PE / Dazzle 594 (BioLegend); the auxiliary reagent was PBSA buffer containing 0.1% BSA (PBS + 0.1% BSA).

[0037] (3) Materials related to library construction and cloning identification: Molecular biology reagents include restriction endonucleases NcoI-HF and NotI-HF (NEB), Prime STAR MAX DNA polymerase (Takara), low melting point agarose, GelRed nucleic acid dye, TAE electrophoresis buffer, DNA loading buffer, and Fast DNA Ladder (NEB); Plasmid manipulation reagents include Zymoprep yeast plasmid miniprep kit, QIAGEN plasmid miniprep kit, Top 10 competent cells, and Frozen-EZ yeast transformation kit (Zymo Research).

[0038] 2.2 Experimental Methods (1) Construction of yeast library plasmids (overlap extension PCR) Based on the VAM mutation strategy, overlapping primers containing degenerate codons were designed in the LCDR1, HCDR1, and HCDR3 regions. A first round of PCR was performed using a high-fidelity enzyme to amplify the DNA fragment containing the mutation site. The first round of PCR materials were mixed, and splicing extension was performed using overlapping sequences. An outer primer was added for a second round of PCR to amplify the full-length antibody gene. A specific band of approximately 1.0 kb was recovered by agarose gel electrophoresis for subsequent transformation experiments.

[0039] (2) Yeast electroconversion Saccharomyces cerevisiae EBY100 strain was inoculated into YPD medium and cultured overnight. The next day, it was transferred to fresh YPD medium and cultured at 30°C until the logarithmic growth phase. OD 600 (≈1.6); Centrifuge to collect cells, wash sequentially with ice water and electroporation buffer, and finally resuspend in LiAc buffer and incubate for 30 minutes. Wash again to prepare competent cells for later use; Mix 400 μL of competent cells with the constructed library DNA, incubate on ice for 10 minutes, and then transfer to a pre-cooled 0.2 cm electroporation cuvette. Set the electroporation parameters to 2.5 kV, 200 Ω, and 25 μF for electroporation; Immediately after electroporation, add YPD / sorbitol mixture and thaw for 1 hour; Spread the thawed cells on SDCAA plates for screening, incubate at 30°C for 72 hours, calculate the library capacity, merge qualified libraries, resuspend in glycerol-containing preservation solution, and aliquot and store at -80°C.

[0040] (3) Monoclonal identification Single clones were selected from the sorted and amplified yeast colonies and inoculated into YPD medium for culture. Plasmids were extracted using the Zymoprep yeast plasmid mini-prep kit. The extracted plasmids were transformed into E. coli Top 10 and amplified. E. coli plasmids were extracted using the QIAGEN plasmid mini-prep kit and Sanger sequencing was performed to confirm the correctness of the sequence and reading frame of the mutation site.

[0041] Example 3: Yeast-based pH-conditional flow cytometry sorting This embodiment utilizes the difference in antigen binding capacity under different pH conditions, and uses four-stage flow cytometry (FACS) to sort and enrich yeast clones that bind to the target antigen under neutral conditions (pH 7.4) and rapidly dissociate the target antigen under acidic conditions (pH 6.0). Figure 3 The specific experimental procedure is as follows: 3.1 Experimental Principle By designing antigen binding and dissociation steps under different pH conditions, flow cytometry was used to sort antibodies displayed on the surface of yeast and enrich antibody molecules that can bind to the target antigen at physiological pH 7.4 and rapidly dissociate from the target antigen in an acidic simulated endosomal environment at pH 6.0. This characteristic enables antibody recycling and prolongs the half-life of antibodies in vivo.

[0042] 3.2 Preparation of Experimental Materials and Buffer Solutions Key reagents: biotinylated antigen and fluorescently labeled streptavidin, used to detect primary and secondary antibodies that display tags on the yeast surface.

[0043] Buffer system: PBSA buffer is PBS (pH 7.4) + 0.1% BSA; acidic buffer is citrate buffer or PBS buffer, with a pH range of 4.5~6.0.

[0044] First round of sorting: Enrichment of all combiners Yeast clones that did not bind to the target antigen were removed from the original yeast display library, retaining all clones that recognized the target antigen (Ag1). Yeast cells were incubated with fluorescently labeled biotinylated antigen 1 (Ag1) at neutral pH 7.4 to allow the antigen to fully bind to the antibody on the yeast surface. Flow cytometry was used for detection, with the sorting gate set to capture cell populations that were positive for both c-myc tag expression and antigen signal. The sorting rate for library 1 in this round was approximately 0.3795% (e.g., ...). Figure 4 As shown in the figure, a sub-library rich in antigen-binding capacity was obtained.

[0045] Second round of sorting: Screening for clones that do not bind under acidic conditions (pH 6.0). Yeast cells enriched after the first round of sorting were incubated with biotinylated antigen at pH 6.0. After washing, fluorescently labeled streptavidin was added for detection. Cells showing significantly weakened or absent antigen-binding signals under acidic conditions were screened by flow cytometry. The sorting gate was set to capture populations that were c-myc tag positive but had negative (or very low) antigen signals. The sorting rate for library 1 in this round was approximately 6.45% (e.g., ...). Figure 5 As shown in the figure, a sub-library with pH-sensitive screening characteristics was initially enriched.

[0046] Third round of sorting: Screening clones with rapid dissociation characteristics. This round employs a kinetic screening strategy of "neutral saturation binding - acidic forced dissociation - neutral labeling rebinding." Only yeast cells that can rapidly dissociate from the unlabeled antigen under acidic conditions will have their surface binding sites released, allowing them to rebind to the fluorescently labeled antigen in subsequent steps. The specific operation is as follows: (1) Saturated bonding: take about 1×10 6 The yeast cells amplified after the second round of screening were washed once with 0.1% PBSA at pH 7.4; the yeast cells were resuspended in 100 nM unlabeled antigen in 0.1% PBSA solution at pH 7.4 (100 μL / tube) and incubated at 4°C for 1 hour to saturate the antigen-binding sites of the yeast surface antibody with unlabeled antigen. (2) Acidic dissociation: Wash twice with 0.1% PBSA at pH 7.4 to remove free unlabeled antigen; resuspend yeast cells in 0.1% PBSA at pH 6.0 and incubate at 4°C for 15-20 minutes to allow the antibody with rapid dissociation properties to release bound unlabeled antigen and expose antigen binding vacancies; (3) Labeling overlap and sorting: Wash twice again with 0.1% PBSA at pH 7.4 to elute the dissociated unlabeled antigen; resuspend yeast cells in a 50 μL / well staining system, add 50-100 nM biotinylated antigen and 0.5 μg / mL PE-Anti-kappa, incubate at 4℃ for 30 minutes, then add SA-436 and incubate for 15-20 minutes; after washing, resuspend the cells, and sort the positive yeast cells that captured the fluorescently labeled antigen by flow cytometry. Figure 6 ).

[0047] Fourth round of sorting: Final confirmation of neutral pH binding capacity Following the method of the first round in this embodiment, the yeast cells sorted in the third round were subjected to antigen staining and flow cytometry sorting under neutral conditions of pH 7.4. Antigen-positive yeast clones were sorted out, confirming that the obtained clones still maintained good antigen binding ability under physiological neutral conditions.

[0048] Example 4: Validation of pH-sensitive candidate antibody molecules using the Octet method 4.1 Sample and buffer preparation The pH-sensitive candidate antibodies obtained by flow cytometry were purified and diluted to an appropriate concentration as the analyte molecules; biotinylated antigens were prepared as the detection antigens; two running buffers were prepared: the neutral buffer was 0.01M PBS + 0.1% BSA + 0.02% Tween 20 (pH 7.4), and the acidic buffer was a buffer of the same composition adjusted to pH 6.0 with HCl.

[0049] 4.2 Sensor Loading Using a streptavidin (SA) sensor as the detection carrier, the SA sensor was first pre-wetted and equilibrated in a neutral buffer solution at pH 7.4; biotinylated antigen was loaded onto the surface of the SA sensor; and then the sensor was immersed in a neutral buffer solution to establish a baseline.

[0050] 4.3 Binding and Dissociation Detection The SA sensor loaded with antigen was immersed in a neutral buffer containing candidate antibodies to complete the antibody-antigen binding reaction at pH 7.4. Subsequently, the sensor was switched to an acidic buffer at pH 6.0 (without antigen), and the dissociation curves of antibody and antigen were observed and recorded. The pH-dependent antibody molecules showed a sharp increase in dissociation rate after the buffer was switched.

[0051] 4.4 Data Fitting and Analysis Using Octet analysis software, the binding and dissociation curves of the antibody and antigen were fitted, and the binding rate constant (k) was obtained through analysis. a ), dissociation rate constant (k d ), affinity constant (K) D ); calculate and compare the k values ​​of the candidate antibody and the control antibody under pH 6.0 and pH 7.4 conditions. a value, k d Value, K D Value, where K D =k d / k a K D A higher ratio indicates a stronger pH sensitivity.

[0052] 4.5 Experimental Results Several antibody molecules exhibiting pH-dependent binding properties were obtained through screening, and the results are shown in Table 1. Experimental data indicate that all candidate molecules, under neutral conditions at pH 7.4, exhibited a binding rate constant (kbinding). a The results were comparable to the control antibody (mAb control). However, compared to the control antibody (K at pH 5.8 / pH 7.4), the results were not comparable. DCompared to a ratio of 4.01, the candidate molecules all exhibited a more significant decrease in affinity under acidic conditions, and this change was mainly due to the dissociation rate (k). d The significant acceleration (k) d The ratio was 11.02-21.85, rather than a change in the binding rate.

[0053] Among these representative preferred clones, the representative antibody mAb-1-PH showed the most outstanding performance, with its K... D The ratio was as high as 674.46, meaning that the affinity at pH 5.8 was nearly 700-fold lower than at pH 7.4, and the expression yield was the highest (263.3 mg / L), approximately 8 times that of the control group. Representative antibodies mAb-4-PH, mAb-2-PH, and mAb-3-PH also exhibited excellent pH sensitivity (K0.05). D The ratios were 213.83, 92.28, and 82.70, respectively, and the yield was also significantly higher than that of the control group. The HIC retention time of all representative antibodies was slightly lower than that of the control group, with moderate hydrophobicity and good physicochemical properties.

[0054] Table 1. Binding kinetic parameters and expression yield of representative preferred clones

[0055] Note: k a k is the binding rate constant. d K is the dissociation rate constant. D R is the affinity constant, HIC is hydrophobic interaction chromatography, and R is the affinity constant. T For retention period.

[0056] In summary, this invention successfully establishes a method for engineering pH-sensitive antibodies based on yeast display technology. Through ingenious library design and rigorous functional screening, this method efficiently obtains antibody molecules that possess high pH sensitivity, high expression levels, and excellent drug-like properties, providing a powerful technological platform for the development of next-generation antibody drugs, especially ADC drugs.

Claims

1. A method for designing and modifying pH-sensitive antibodies based on yeast display, characterized in that, Includes the following steps: (1) Construction of yeast display library: Using the antibody sequence to be modified as a template, mutation design is carried out for the complementarity-determining region. The mutation adopts the degenerate codon VAM, which represents A / G / C and M represents A / C, encoding histidine, glutamic acid, aspartic acid, lysine, asparagine and glutamine. The mutation site is located in the combination of at least two regions in the light chain CDR1, heavy chain CDR1 and heavy chain CDR3, and 1 to 2 amino acid mutation sites are introduced in each selected complementarity-determining region. (2) Induced library expression: The yeast display library constructed in step (1) was transformed into yeast cells and induced to express antibody molecules on the surface of yeast cells. (3) Multi-round sorting and screening: Yeast cells displaying antibody molecules were sequentially sorted in four rounds to enrich pH-sensitive antibody clones: In the first round, yeast cells were incubated with biotinylated fluorescently labeled antigens at pH 7.4, and positive yeast cells that bound the antigens were sorted out and the conjugates were enriched. In the second round, the yeast cells obtained from the first round of sorting were incubated with biotinylated antigens at pH 6.

0. After washing, fluorescently labeled streptavidin was added, and yeast cells without signal under acidic conditions were collected for amplification to enrich pH-sensitive clones. The third round involves screening clones with the characteristic of rapid dissociation, specifically including: a. Under pH 7.4 conditions, yeast cells obtained from the second round of sorting were incubated with 100 nM unlabeled antigen at 4°C for 1 hour to allow the surface-displayed antibodies to bind to the unlabeled antigen until saturation; b. After washing to remove free unlabeled antigen, resuspend the yeast cells in pH 6.0 acidic buffer and incubate at 4°C for 15-20 minutes to allow pH-sensitive antibodies to release the bound unlabeled antigen. c. After washing to remove the released unlabeled antigen, add biotinylated fluorescently labeled antigen, incubate at pH 7.4, and sort out the positive yeast cells that capture the fluorescently labeled antigen; Fourth round: The yeast cells obtained from the third round of sorting were incubated with biotinylated fluorescently labeled antigens at pH 7.4 to verify antigen binding activity.

2. The method for designing and modifying pH-sensitive antibodies based on yeast display according to claim 1, characterized in that, In step (1), three combined mutant libraries are constructed, namely, the combined libraries of HCDR3 and LCDR1, HCDR1 and HCDR3, and HCDR1 and LCDR1.

3. The method for designing and modifying pH-sensitive antibodies based on yeast display according to claim 1, characterized in that, The yeast cells mentioned in step (2) are Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ) EBY100 strain.

4. A pH-sensitive antibody, characterized in that, Prepared by the method according to any one of claims 1 to 3, wherein the complementarity-determining region contains 1 to 2 amino acid mutation sites encoded by degenerate codons VAM, and the mutation sites are located in a combination of at least two regions of LCDR1, HCDR1 and HCDR3.

5. The pH-sensitive antibody according to claim 4, characterized in that, The affinity constant of the antibody to the antigen decreased by at least 82.7 times at pH 5.8 compared to pH 7.4, and its hydrophobic interaction chromatographic retention time was 13.3–16.1 min.

6. A recombinant expression vector, characterized in that, Nucleotide sequences comprising the pH-sensitive antibody of claim 4 or 5.

7. A host cell, characterized in that, The recombinant expression vector according to claim 6 is wherein the host cell is Saccharomyces cerevisiae strain EBY100.

8. The use of the pH-sensitive antibody according to claim 4 or 5 in the preparation of a drug or antibody-drug conjugate with an extended in vivo half-life.

9. The application according to claim 8, characterized in that, The pH-sensitive antibody can rapidly dissociate from the antigen in an acidic endosomal environment of pH 5.0 to 6.5 and return to the cell surface through an FcRn receptor-mediated recycling pathway, thereby improving the intracellular uptake efficiency of antibody-drug conjugates.

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