WO2018032574A1

WO2018032574A1 - Single-chain antibody and application thereof

Info

Publication number: WO2018032574A1
Application number: PCT/CN2016/100210
Authority: WO
Inventors: 林志国; 殷一博; 黄建萍; 葛海涛; 慕璐岩; 暴洪博; 杨昌霖; 龙宇; 金林春; 胡力; 王佳斌; 夏松松; 唐重阳; 陶海鹏
Original assignee: 林志国
Priority date: 2016-08-17
Filing date: 2016-09-26
Publication date: 2018-02-22
Also published as: CN106220733A

Abstract

A single-chain antibody and an application thereof. The single-chain antibody comprises: an IFNγ secretory signal peptide, an antibody variable region, an IgG1 hinge fragment, and an IgG1 CH2CH3 region connected in that order. An amino acid sequence of the IFNγ secretory signal peptide is shown in SEQ ID NO:1, with a base sequence as shown in SEQ ID NO:2. An amino acid sequence of the IgG1 hinge fragment is shown in SEQ ID NO:3, with a base sequence as shown in SEQ ID NO:4. The single-chain antibody is used mainly in medical and biotechnology fields, and the main uses thereof are: 1, as a coupling tracer for tumor imaging during surgery; 2, as a targeting drug for a targeting treatment; 3, as a neutralizing antibody to a bound target thereof, inhibiting a function of the bound target; and 4, for detecting the expression of an antigen recognized by the variable region. The present invention preserves the functions and stability of an antibody, simplifies a manufacturing process and an antibody structure, and improves a performance of an antibody.

Description

Single-chain antibody and application thereof

Technical field

[0001] The present invention belongs to the field of genetic engineering technology, and relates to a single chain antibody and application thereof.

Background technique

[0002] Today, the use of antibodies is becoming more widespread, including detection of related antigen expression, blocking of corresponding antigens, intraoperative imaging, production of targeted drugs, and the like. Industrial production of antibodies relies mainly on the purification of supernatants of hybridoma cells secreting the corresponding antibodies, but this method is difficult to generalize because hybridoma cells of the corresponding antibodies are not readily available. Using genetic engineering means, the cells express the heavy and light chains of the corresponding antibodies, and after the cells are assembled and secreted by themselves, the extraction method of the purified supernatant is relatively easy to be popularized, but the expression of two amino acid chains involving heavy and light chains is involved. And assembly, low efficiency, and cumbersome procedures. On the other hand, with the increasing application of antibodies in vivo, how to make their functions more efficient while maintaining their functions and stability has become a hot research topic.

technical problem

[0003] The object of the present invention is to provide a single-chain antibody and its use, which not only maintains the function and stability of the antibody, but also simplifies the preparation procedure and antibody structure, and improves the performance of the antibody.

Problem solution

Technical solution

[0004] The object of the present invention is achieved by the following technical solutions:

[0005] A single-chain antibody, which is composed of an IFNY secretion signal peptide, an antibody variable region, an IgG1 hinge fragment, and an IgG1 CH ₂ CH ₃ region, wherein: the amino acid sequence 歹 ij of the IFNY secretion signal peptide is SEQ ID No. The base sequence is shown in SEQ ID No. 2; the amino acid sequence of the IgG1 hinge fragment is shown in SEQ ID No. 3, and the base sequence 歹IJ is shown in SEQ ID No. 4.

[0006] The above single-chain antibodies are mainly used in the fields of medical and biotechnology, and the main uses are as follows: 1. It can be used as a coupled tracer for intraoperative tumor imaging; 2. As a targeted drug for targeted therapy; As a neutralizing antibody to which the target is bound, the function of inhibiting the bound target is achieved; 4. For detecting the expression of the antigen recognized by the variable region. [0007] The funding sources for the research and development process of the present invention are: National International Science and Technology Cooperation Special No. 2014 DFA31630, Heilongjiang Province Applied Technology Research and Development No. GA15C108, Harbin Medical University Graduate Innovation Research Project No. YJSCX2015-20HYD.

Advantageous effects of the invention

Beneficial effect

The present invention has the following advantages:

[0009] 1. The efficiency of antibody production is improved, and the simplified structure of the single chain functions as a double-chain antibody;

[0010] 2. The volume of the antibody is reduced, making it easy to diffuse and penetrate in the body;

[0011] 3. The ADCC and CDC functions of the antibody and the affinity and stability of the antibody are retained;

[0012] 4, retaining the purification mode of the antibody by binding proteins A, G, L;

[0013] 5. The present invention uses an EGFR antibody linked to the variable region sequence of EGFR as an example. The short peptide is used to directly link the variable regions of the heavy and light chains of the antibody, and the front end is linked to the IFN _Y secretion signal peptide. The IgGl hinge fragment was ligated to the IgG1 CH ₂ CH ₃ region, and this structure first guided the secretion of the antibody by the IFN _Y secretion signal peptide, and the connection mode of the antibody was first proposed. In addition to antibodies to EGFR, the variable region sequences of other antibodies can also be ligated into the antibody structures of the invention.

Brief description of the drawing

DRAWINGS

1 is a linked form of a single-chain antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The technical solutions of the present invention are further described below with reference to the accompanying drawings, but are not limited thereto, and all modifications and equivalents of the technical solutions of the present invention should be included without departing from the spirit and scope of the technical solutions of the present invention. Within the scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First, as shown in FIG. 1, the single-chain antibody provided by the present embodiment secretes a signal peptide from IFN _Y.

The antibody variable region, the IgG1 hinge fragment and the IgG1 CH ₂ CH ₃ region are sequentially ligated, wherein: the IFN _Y secretion signal peptide portion is used for secretion of the antibody synthesized into the cell structure, and the amino acid sequence thereof is represented by SEQ ID NO. , the base sequence is shown in SEQ ID No. 2; the antigen binding region can make the single-chain antibody play a phase The role of the intact antibody should be; the IgGl hinge fragment is used to ensure the function and stability of the single-chain antibody, the amino acid sequence 歹IJ is shown in SEQ ID No. 3, the base sequence 歹ij is shown as SEQ ID No. 4; CH ₂ region and CH Region ₃ is used to stabilize the antibody structure, and the purification of the antibody is facilitated. The amino acid sequence CH ij of CH ₂ is shown in SEQ ID No. 5, and the base sequence 歹 IJ is shown in SEQ ID No. 6; CH ₃

The amino acid sequence of the region is shown in SEQ ID No. 7, and the nucleotide sequence is shown in SEQ ID No. 8.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0017] This embodiment provides the use of a single-chain antibody which can be used as a coupled tracer for intraoperative tumor imaging, as follows:

[0018] 1, the eluted antibody was concentrated to 10mg / ml of 50mM potassium phosphate solution, pH = 8.5;

[0019] 2. IRDye 800CW NHS ester (LI-COR) to single-chain antibody in a molecular ratio of 2.3: 1, at 20

^ Darkroom coupling 2 small sputum;

[0020] 3. eluting the uncoupled dye;

[0021] 4, the solute is adjusted to pH = 7 PBS, the final concentration of the antibody is 2mg / ml;

[0022] 5, intravenous injection, fluorescence microscope to observe the tumor development.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 3: The present embodiment provides the use of a single-chain antibody, which can be used for the targeted therapy of a drug as a drug, or as a neutralizing antibody to a target, to achieve inhibition. The function of the combined target.

DETAILED DESCRIPTION OF THE INVENTION [0024] This embodiment provides a single-chain antibody, which can be used for detecting the expression of an antigen recognized by a variable region, and the single-chain antibody can be used for immunohistochemistry, Western blot and the like. A qualitative and quantitative method for detecting the expression of an antigen recognized by a variable region, which can be coupled to a fluorescent molecule, biotin or a labeled secondary antibody that targets a human IgG1Fc fragment for identification of a variable Expression of the antigen recognized by the region.

MODE FOR CARRYING OUT THE INVENTION V: This embodiment uses an EGFR antibody linked to the variable region sequence of EGFR as an example, and uses a short peptide to directly link the variable regions of the antibody heavy and light chains, and the front end is linked to the IFN _Y secretion signal. Peptide, back-end via IgGl hinge fragment with IgGlCH ₂ CH ₃

The region is connected (Fig. 1), the amino acid sequence is shown in SEQ ID No. 9, and the base sequence is shown in SEQ ID No. 10

, the preparation method is as follows:

(1) ligating the base sequence represented by SEQ ID No. 10 to an expression vector (which may be a lentiviral vector, a retroviral vector, a sleeping beauty vector or a transient expression vector); (2) transfecting or transforming a mammalian cell into the above vector (the available cells include myeloma cells, C

HO cells, monkey COS cells, human embryonic kidney HEK293 cells, etc.;

[0028] (3) collecting cell culture supernatant;

(4) affinity-purifying the antibody in the supernatant using egg SA, G or L;

(5) eluting a single-chain antibody.

Sequence table free content

Sequence table

[0032] <110> Lin Zhiguo

[0033]

<120>-Single-chain antibody and application thereof

[0035]

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[0037]

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[0041]

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[0044]

<210>2

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<212>DNA

[0048]

<400> 2

CTT GGC TCC CTC GGC TGT TAC TGT

[0051] Us/D/ O siosId s3⁄4siAV

900 inch<> Us/D/ O siosId s3⁄4siAV

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Claims

Claim

A single-chain antibody characterized in that the single-chain antibody is sequentially linked by an IFN _Y secretion signal peptide, an antibody variable region, an IgG1 hinge fragment, and an IgG1 CH ₂ CH ₃ region.

The single-chain antibody according to claim 1, wherein the amino acid sequence of the IFN _Y secretion signal peptide is represented by SEQ ID No. 1, and the base sequence 歹IJ is represented by SEQ ID No. 2.

The single-chain antibody according to claim 1, wherein the amino acid sequence 歹 lj of the IgG1 hinge fragment is represented by SEQ ID No. 3, and the base sequence 歹 lj is represented by SEQ ID No. 4.

The single-chain antibody according to claim 1, wherein the base sequence of the CH ₂ region is as shown in SEQ ID No. 6.

The single-chain antibody according to claim 1, wherein the base sequence of the CH ₃ region is as shown in SEQ ID No. 8.

The single chain antibody of any of claims 1-5 can be used as a coupled tracer for intraoperative tumor imaging.

The single-chain antibody of any of claims 1-5 can be used as a targeted drug for targeted therapy.

The single-chain antibody according to any one of claims 1 to 5 can function as a neutralizing antibody to which a target is bound, thereby inhibiting the binding of the target.

The single-chain antibody of any of claims 1-5 can be used to detect expression of a variable recognized by a variable region.