阅读说明：本技术 一种对抗体pH依赖结合活性的快速改造及筛选方法 (Rapid modification and screening method for pH-dependent binding activity of antibody ) 是由 徐曼 张丽英 刘娟 根纳季·戈洛洛博夫 李竞 顾继杰 陈智胜 于 2019-09-19 设计创作，主要内容包括：本发明公开一种对抗体pH依赖结合活性的改造及筛选的方法,包括步骤：(1)构建野生型抗体scFv质粒模板；(2)对抗体的6个CDR区进行定点突变,用组氨酸替代CDR区域的每个氨基酸；(3)建立Capture-ELISA条件并进行高通量筛选；(4)KD-ELISA和FACS检测：对样品进行梯度稀释后,用不同pH buffer进行KD-ELISA的检测,根据检测数据绘制的曲线,确认目标克隆在抗原表面和细胞表面靶点的pH依赖结合活性。本发明提供的方法能够高通量、高效率、低成本的获得pH依赖结合活性的抗体。(The invention discloses a method for modifying and screening the pH-dependent binding activity of an antibody, which comprises the following steps: (1) constructing a wild type antibody scFv plasmid template; (2) site-directed mutagenesis of 6 CDR regions of an antibody, replacing each amino acid of the CDR regions with histidine; (3) establishing a Capture-ELISA condition and carrying out high-throughput screening; (4) KD-ELISA and FACS detection: after the sample is subjected to gradient dilution, KD-ELISA detection is carried out by using different pH buffers, and the pH-dependent binding activity of the target clone on the antigen surface and the cell surface target spot is confirmed according to a curve drawn by detection data. The method provided by the invention can obtain the antibody with pH-dependent binding activity with high flux, high efficiency and low cost.)

1. A method for modifying and screening pH-dependent binding activity of an antibody, comprising the steps of:

(1) construction of wild-type antibody scFv plasmid template: after connecting the heavy chain and light chain variable regions of the antibody through connecting peptides, utilizing restriction endonuclease sites to be connected into an escherichia coli expression vector to obtain a complete scFv plasmid template, and facilitating the transformation of a variable region sequence;

(2) carrying out site-directed mutagenesis on 6 CDR regions of the antibody, replacing each amino acid of the CDR regions by histidine by a PCR method, then digesting a plasmid template by using restriction enzyme to obtain a mutated PCR product for transforming escherichia coli, and obtaining a soluble scFv antibody after inducing by using an inducer;

(3) establishing Capture-ELISA conditions and carrying out high-throughput screening:

firstly, coating anti-c-Myc antibodies with different concentrations, enabling a c-Myc label to exist on an scFv antibody, adding an induced mutant expression supernatant, adjusting the pH value by using different buffer solutions, screening and incubating under corresponding different pH conditions, and enabling a soluble scFv antibody fragment to be captured on an ELISA plate quantitatively;

then adding antigens with different concentrations and labels, diluting with buffer solutions with different pH values, simultaneously screening under the corresponding different pH conditions, and then incubating;

then adding a secondary antibody for detecting the antigen label, diluting with buffer solutions with different pH values, and screening under corresponding different pH conditions to obtain an antigen-antibody combined affinity signal;

the screening requirements are as follows: ensuring that the unpurified antibody expressed by the escherichia coli keeps stable pH in an experiment and is not influenced by a bacterial culture solution and secretion; the conditions for the screening include: coating antibody concentration, buffer pH and salt concentration, antigen concentration;

(4) KD-ELISA and FACS detection: after the samples are diluted in a gradient way, KD-ELISA and FACS detection are carried out by using different pH buffers, and the pH-dependent binding activity of the target clone on the antigen surface and the cell surface target spot is confirmed according to a curve drawn by detection data.

2. The method of claim 1, wherein in step (2), the restriction enzyme is a DpnI restriction enzyme.

3. The method of claim 1, wherein in step (2), the induction agent is isopropyl thiogalactoside (IPTG).

4. The method of claim 1, wherein in step (3), the tag is an Fc tag.

5. The method of claim 4, wherein in step (3), the tag is an mFc or hFc tag.

6. The method of claim 1, wherein in the step (3), the Escherichia coli is BL21 Escherichia coli.

7. The method of claim 1, wherein the method further comprises: and subsequently screening the induced mutant supernatant by using the screening condition.

8. The method of claim 1, wherein the method further comprises: and (3) performing relative quantitative detection on the induced mutant expression supernatant by adopting Capture-ELISA.

9. The method of claim 1, wherein the method further comprises: coating and screening the label antibody according to the screened condition to obtain single-point mutation clone with a pH-dependent binding signal; selecting a mutation point sensitive to pH, carrying out combined mutation, and then carrying out secondary screening by using Capture-ELISA to obtain the final clone which has pH-dependent binding activity and does not obviously reduce the antigen-antibody affinity.

Technical Field

The invention relates to engineering modification of an antibody, in particular to a method for quickly modifying and screening pH-dependent binding activity of an antibody.

Background

Monoclonal antibodies are widely used in disease therapy. Conventional antibodies bind to an antigen without condition dependence, e.g., the antibody exhibits the same antigen binding activity over a certain pH range. And the introduction of pH dependent activity by antibody engineering means has high application value. After binding to the antigen, the antibody without pH-dependent activity extends the half-life of the target antigen by "buffering", does not eliminate the antigen from the plasma, but rather increases the concentration of the antigen in the plasma. While some natural receptors not only bind to ligands, but also dissociate in a pH-dependent manner in acidic endosomes (pH 5.5), continuously clearing ligands from plasma, and then recycling the receptors to the cell surface for reuse. Antibodies can be engineered to have a weak affinity at acidic endosomal pH, but a higher affinity for target binding near neutral pH. The antibody releases antigen in vivo in the cell nucleus to go through a degradation pathway, and the antibody can be recovered to the cell surface for reuse. For example, antibodies directed against IL-6R and PCSK9, have been shown to have pH sensitive binding by histidine mutations, to reduce antibody clearance and increase PK and potency in vivo.

In addition, in order to obtain an antibody specifically binding to a target in a tumor-specific micro-acid microenvironment, the antibody needs to be modified so that the binding in the micro-acid environment is far stronger than that in neutral pH, so as to achieve the effects of specific binding to the tumor and reduction of toxicity, and thus the antibody can achieve a therapeutic effect similar to that of a conventional antibody at a lower dose. However, since the pH of the slightly acidic environment of the tumor is about 6.5, which differs from neutral pH7.4 by less than 1, the sensitivity of the conventional screening method is difficult to achieve. Therefore, there is a need in the art for a more sensitive set of methods for screening and differentiating.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for modifying and screening the pH-dependent binding activity of an antibody, so that the antibody with the pH-dependent binding activity can be obtained with high flux, high efficiency and low cost.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for modifying and screening pH-dependent binding activity of an antibody, comprising the steps of:

(1) construction of wild-type antibody scFv plasmid template: after connecting the heavy chain and light chain variable regions of the antibody through connecting peptides, utilizing restriction endonuclease sites to be connected into an escherichia coli expression vector to obtain a complete scFv plasmid template, and facilitating the transformation of a variable region sequence;

(2) carrying out site-directed mutagenesis on 6 CDR regions of the antibody, replacing each amino acid of the CDR regions by histidine by a PCR method, then digesting a plasmid template by using restriction enzyme to obtain a mutated PCR product for transforming escherichia coli, and obtaining a soluble scFv antibody after inducing by using an inducer;

(3) establishing Capture-ELISA conditions and carrying out high-throughput screening:

firstly, coating anti-c-Myc antibodies with different concentrations, adding induced mutant expression supernatant, adjusting pH by using different buffer solutions, simultaneously screening and incubating under correspondingly different pH conditions, and quantitatively capturing soluble scFv antibody fragments on an ELISA plate;

then adding antigens with different concentrations and labels, diluting with buffer solutions with different pH values, simultaneously screening under the corresponding different pH conditions, and then incubating;

then adding a secondary antibody for detecting the antigen label, diluting with buffer solutions with different pH values, and screening under different pH conditions to obtain an affinity signal of antigen-antibody combination;

the screening requirements are as follows: ensuring that the unpurified antibody expressed by the escherichia coli keeps stable pH in an experiment and is not influenced by a bacterial culture solution and secretion; the conditions for the screening include: coating antibody concentration, buffer pH and salt concentration, antigen concentration;

to ensure that antigen-antibody binding occurs at a predetermined pH, pH buffers of different salt concentrations are selected to achieve the same pH in the induced culture supernatants in order to achieve the same pH in the induced antibody supernatants. This buffer was then used as a buffer to establish screening conditions, and final screening conditions (coating antibody concentration, antigen concentration, etc.) were selected based on the results of the Capture ELISA. Subsequently, screening the induced mutant supernatant by using the selected screening conditions. The Capture ELISA can realize relative quantification of induced supernatant, so that detection signals are comparable and fast, and all points can be detected in 2-3 days. Then combining the screened mutation points with pH-dependent binding activity, and carrying out secondary screening to finally obtain a target clone which has pH-dependent binding activity and does not have obviously reduced antigen-antibody affinity;

(4) KD-ELISA and FACS detection: after the samples are diluted in a gradient way, KD-ELISA and FACS detection are carried out by using different pH buffers, and the pH-dependent binding activity of the target clone on the antigen surface and the cell surface target spot is confirmed according to a curve drawn by detection data.

Specifically, in the step (2), the restriction enzyme is a DpnI restriction enzyme.

Specifically, in the step (2), the inducer is isopropyl thiogalactoside (IPTG).

Specifically, in the step (3), the tag is an Fc tag.

Preferably, in the step (3), the tag is an mFc or hFc tag.

Specifically, in the step (3), the escherichia coli is BL21 escherichia coli.

Preferably, in the step (3), the incubation condition is incubation at 37 ℃ for 1 hour.

Preferably, in the step (3), the buffer solution is a PBS buffer solution.

In a specific embodiment, the method further comprises: and subsequently screening the induced mutant supernatant by using the screening condition.

In a specific embodiment, the method further comprises: and (3) performing relative quantitative detection on the induced mutant expression supernatant by adopting Capture-ELISA.

In a specific embodiment, the method further comprises: coating and screening the label antibody according to the screened condition to obtain single-point mutation clone with a pH-dependent binding signal; selecting a mutation point sensitive to pH, carrying out combined mutation, and then carrying out secondary screening by using Capture-ELISA to obtain the final clone which has pH-dependent binding activity and does not obviously reduce the antigen-antibody affinity.

In order to accelerate the research and development speed and reduce the cost, the invention adopts a faster screening means: Capture-ELISA is a sensitive detection method for picogram to microgram quantification of trace substances (such as hormones, cell signaling chemicals, antigens, cytokines, etc.). After histidine mutation scanning is carried out on single amino acid in CDR region of single-chain antibody (scFv), the supernatant is directly expressed by escherichia coli, an ELISA method specially aiming at the tiny pH difference is established, the scFv binding activity is compared under different pH conditions, and pre-purification or enrichment is not needed.

Because the pH of the unpurified antibody supernatant is inconsistent and can be influenced by the culture conditions of escherichia coli and secretion, in the experiment, the stability of the experiment is ensured by different buffer combinations, the pH of the antibody in the supernatant and the periplasm can be accurately controlled even if no purification is carried out, the flux and the stability of the experiment are ensured, and the experiment period is accelerated; after the information of single-point histidine mutation is obtained, the invention combines some mutation points sensitive to pH, and establishes a rapid Capture-ELISA method for screening again, and new Capture-ELISA conditions (optimized for coating concentration, antigen amount and the like) can be established every time, thereby ensuring the sensitivity and stability of the experiment.

In the method for modifying the pH-dependent binding activity of the antibody, most laboratories currently use a modeling analysis to perform histidine mutation on a site with potential pH-dependent activity, and perform pH-dependent binding screening after cell transfection and purification, so that the method has the defects of low mutation site flux and large workload of cell transfection and purification. The invention carries out histidine scanning based on the scFv segment, covers all sites of 6 CDR regions, simultaneously combines with Capture-ELISA to carry out rapid screening, can complete screening within 3 days, does not need to carry out sample expression and purification, and can use soluble scFv antibody induced by colon bacillus. High flux and low cost.

The modification and screening method for the pH-dependent binding activity of the antibody provided by the invention can rapidly and successfully modify the pH-dependent binding activity of the scFv antibody by using histidine dot scanning and Capture-ELISA technologies, can well obtain the antibody with the pH-dependent activity, can distinguish very small pH differences with high sensitivity, is a high-throughput and effective antibody modification method, and can be applied to modification of all condition-dependent antibodies (scFv, Fab, VHH and the like).

Compared with the modification methods of other researchers, the rapid modification and screening method of the pH-dependent binding activity provided by the invention has the following advantages:

(1) can directly use a prokaryotic expression system, and has short culture period, low cost and easy operation.

(2) The method for establishing the precise Capture-ELISA screening saves the complex steps of protein purification and enrichment, reduces the cost and increases the flux.

(3) This method was verified and the finally obtained clones still maintained a good pH dependent binding activity in IgG format.

Drawings

FIG. 1 is a flow chart of a method for engineering and screening pH-dependent binding activity of antibodies.

FIG. 2 is a graph showing the results of example 1 of modification of pH-dependent binding activity of scFv antibodies; of these, the affinity is much greater at pH7.4 than at pH 5.5.

FIG. 3 is a graph of the results of example 2 of engineering for pH-dependent binding activity of scFv antibodies; of these, the affinity is much greater at pH6.5 than at pH 7.4.

FIG. 4 is a graph showing the results of Capture-ELISA screening of scFv samples after single point mutation; among them, samples 1 to 9, 11, 23, 27 to 29, etc. had significant pH-dependent binding activity, while the wild-type sample (WT) had no pH-dependent binding activity.

FIG. 5 is a graph showing the results of the Capture-ELISA screening of scFv samples after combinatorial mutation; wherein, the samples 42, 43, 46, 47,48, 51-55, 58-65 and the like have obvious pH-dependent binding activity.

FIG. 6 is a graph showing the results of confirmation that the final clone has pH-dependent binding activity by KD-ELISA; only part of the experimental data is shown in the figure, wherein the mutant clones (Lead clone) 1-4 show better pH-dependent binding activity in the KD-ELSIA experiment, while the wild type sample (WT) does not show pH-dependent binding activity.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.