Expression and preparation method of multivalent multispecific antibody and immune hybrid protein

A multi-specific antibody and specific technology, which is applied in the field of expression and preparation of multivalent multi-specific antibodies and immune hybrid proteins, can solve the problem of affecting the half-life of ADCC effect, the inability to obtain the yield of immunotoxin, and the inability to solve the mismatch phenomenon, etc. question

Active Publication Date: 2020-07-14
SHANGHAI JIAOTONG UNIV +2
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production of immunotoxin by genetic engineering also has its limitations: the fusion gene is restricted to be expressed in a single host, and the directing part and the toxic part that constitute the immunotoxin often require different host expression environments. This contradiction often leads to the use of a single host for expression The target immunotoxin cannot obtain good yield, yield, purity and consequent increase in cost
By co-expressing two heavy chains and two light chains (must be suitable for both heavy chains) heterodimers (knob-hole) are produced in higher yields than homodimers (knob-knob) (hole-hole) ( Ridgway, J.B., Protein Eng. (Protein Engineering) 9 (1996) 617-621; and WO96 / 027011) Although this format is very attractive, no clinical application data currently exist, and an important limitation of this strategy is that two The light chains of each parental antibody must be identical to prevent light chain mismatches and the formation of impurity molecules
Aiming at the light chain mismatch problem, the specificity of antibody binding is changed by mutation to form a "Two-in-One" bivalent bispecific antibody, so that the specific binding domain of the same antibody can bind to two antigens. The binding of each target is bivalent. Although the desired effect can be obtained in the reconnection and activation target, there are certain deficiencies in blocking the effect of the antigen, and this method requires a large number of antibodies for each two antibody sequences. Genetic engineering such as mutations cannot achieve simple and general purposes (Bostrom, J., Scince (Science) 323 (2009) 1610-1614; Schaefer, G., Cancer Cell (Cancer Cell) 20 (2011) 472-486)
In addition, the crossmab (hybrid antibody) method can optimize the light chain mismatch problem, but it can be well solved by exchanging the light chain and heavy chain part domains of one of the Fabs to form a crossmab (hybrid antibody), but the hybrid antibody contains non- Native domain linkage, loss of native antibody structure (Schaefer, W., Pro.Natl.Acad.Sci.USA (Proceedings of the American Academy of Sciences) 108(2011) 1187-1192)
[0018] American Genentech (Gene Tech) company uses the method of co-cultivation of E. coli expressing two half-antibodies (half antibodies) to obtain bispecific antibodies, but the antibodies expressed by this method have no glycosylation modification, which will affect Its ADCC effect and half-life in blood limit its possibility of becoming a drug (Spiess, C., Nature Biotechnol (Natural Technology) 31 (2013) 753-758)
In order to produce bispecific antibodies that are similar to the natural structure and contain glycosylation modifications, the interface of the Fab undergoes structural analysis for targeted gene mutations, and at the same time adopts the "Knobs-into-Holes" technology through homeopathy Transfection of 293E cells to solve the problem of light chain mismatch and heavy chain mismatch has been greatly improved, but this method must design a suitable mutation screening site through the establishment of a crystal model for each antibody, which cannot be used universally Construction of bispecific antibodies (Levis, S.M., Nature Biotechnol (Natural Technology) 32(2014) 191-198)
In addition, cFAE "half-antibody exchange technology" can direct half-antibody recombination by introducing mutations in the CH3 region, reducing the antibody to a half-antibody through in vitro reduction, and then oxidizing it to a complete antibody, which solves the heavy chain mismatch and light chain mismatch. problem, but there will be 5% mismatches that cannot be resolved, and cannot be removed by purification methods. The existence of heterogeneous components greatly limits the possibility of using cFAE as a drug (Labrijin, A.F., Nature protocol (natural operation method) 9 (2014)2045-2463)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Expression and preparation method of multivalent multispecific antibody and immune hybrid protein
  • Expression and preparation method of multivalent multispecific antibody and immune hybrid protein
  • Expression and preparation method of multivalent multispecific antibody and immune hybrid protein

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0114] The expression and preparation method of the novel bivalent bispecific antibody hybrid protein involved in the present invention comprises the following steps:

[0115] 1. Expression vector construction.

[0116] For the construction of expression vectors, general information on the nucleotide sequences of human immunoglobulin light and heavy chains is found in Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service , National Institutes of Health (Public Health Service, National Institutes of Health), Bethesda, MD. (1991)) and drugbank databases. Amino acids of antibody chains are numbered and referred to according to EU numbering (Edelman, G.M., et al., Proc. , Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Desired gene segments are prepared from oligonucleotides prepared by chemical synthesis. 600-1800bp long gene segments we...

Embodiment 1

[0135] Example 1. Construction of CD3×Her2 bispecific antibody

[0136] 1.1. Expression vector construction

[0137] For the construction of expression vectors, general information on the nucleotide sequences of human immunoglobulin light and heavy chains is found in Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service , National Institutes of Health (Public Health Service, National Institutes of Health), Bethesda, MD. (1991)) and drugbank databases. Amino acids of antibody chains are numbered and referred to according to EU numbering (Edelman, G.M., et al., Proc. , Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The CD3 antibody sequence is derived from the humanized OKT3 drug sequence, and the desired gene segment is prepared by oligonucleotides prepared by chemical synthesis. 600-1800bp long gene segments were assembled by annea...

Embodiment 2

[0166] Example 2. Using the trans-splicing function of Npu DnaE to prepare the immunotoxin Herceptin-PE38KDEL

[0167] 2.1. Construction of the Her HC expression vector pCEP4-Her HC-Nn fused with the N-terminus of the split intein Npu DnaE

[0168] Using the primers in Table 1, use the synthesized Herceptin heavy chain nucleic acid molecule containing the gene encoding the signal peptide as a template and use the primers in the table to clone the gene encoding the Herceptin heavy chain Using the synthetic nucleic acid molecule containing Npu DnaE as a template, clone the N-terminal of Npu DnaE The gene was amplified by TaKaRa’s PrimerStar Max, and the PCR conditions were 94°C for 10s, 55°C for 10s, 72°C for 10s, and 30 cycles. The obtained fragments were recovered by agarose gel electrophoresis and overlapped PCR was used to encode the heavy chain of Herceptin. The gene and the N-terminal gene Npu DnaE encoding Npu DnaE were synthesized in sequence at the C-terminus of the f...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses an expression and preparation method of a multivalent and multispecific antibody and an immune hybrid protein in the field of biotechnology; the invention uses the characteristics of the protein Intein to design and develop a new method for preparing a multivalent specific antibody, or A method for a hybrid immune protein fused to an antibody and a cytokine, or an immunotoxin fused to an antibody to a toxin protein, or an immune hybrid protein fused to an antibody and other active protein. In the present invention, each part of the hybrid protein is expressed in a suitable prokaryotic or eukaryotic cell system, purified and separated by high-efficiency affinity chromatography, and then spliced ​​in vitro under the condition of intein splicing to achieve the preparation of multivalent specificity Products of antibodies and immunohybrid proteins. The method has the advantages of high production efficiency, wide applicability and favorable product separation and purification. The present invention provides new methods for preparing biopharmaceuticals targeted to attack cancer or other diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for expressing and preparing a multivalent multispecific antibody and immune hybrid protein. Background technique [0002] Bispecific antibody refers to an antibody molecule that can recognize two antigens or two epitopes at the same time, such as bispecific or multispecific antibodies that can bind more than two antigens are known in the art, and can be passed through cells Fusion method, chemical modification method, gene recombination and other methods, obtained in eukaryotic expression system or in prokaryotic expression system. Similar to this, a multispecific antibody refers to an antibody molecule that can recognize two or more antigens or multiple epitopes at the same time, and an immunohybrid protein refers to one or more specific antibodies combined with cytokines or polypeptide toxins or other Biologically active polypeptide molecule hybrid immunohybr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46
CPCC07K16/18C07K16/28C07K16/2809C07K16/2866C07K16/468C07K2317/51C07K2317/515C07K2317/35C07K2317/31C07K16/46C12N15/62C07K2317/24C07K2319/55C07K2319/92A61K47/6803A61K47/6813A61K47/6849C07K2317/55C07K2317/92C07K2319/00C12N5/16C07K1/16C12N15/63C07K16/32C07K2317/41A61K47/6855C07K1/22C12N15/11C12N5/10
Inventor 朱建伟韩雷陈俊生丁凯谢跃庆江华路慧丽张宝红张蕾
Owner SHANGHAI JIAOTONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products