Expression and preparation methods for polyvalent multi-specific 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 problems of affecting the half-life of ADCC effect, limiting the possibility of becoming a drug, and losing the structure of the antibody, etc.

Active Publication Date: 2017-02-15
SHANGHAI JIAO TONG UNIV +2
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  • Abstract
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  • Claims
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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 cro

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  • Expression and preparation methods for polyvalent multi-specific antibody and immune hybrid protein
  • Expression and preparation methods for polyvalent multi-specific antibody and immune hybrid protein
  • Expression and preparation methods for polyvalent multi-specific antibody and immune hybrid protein

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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...

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Abstract

The invention discloses expression and preparation methods for a polyvalent multi-specific antibody and an immune hybrid protein and belongs to the technical field of biology. According to the invention, by using characteristics of protein Intein, a novel method for preparing polyvalent specific antibodies, or antibody and cell factor merged hybrid immunoproteins, or antibody and toxin protein merged immunotoxins or antibody and other active protein merged immune hybrid proteins is designed and developed. According to the methods, products, i.e., the polyvalent specific antibody and the immune hybrid protein are prepared through expressing all parts of the hybrid protein separately in appropriate procaryotic or eucaryotic cell systems, carrying out high-performance affinity-chromatography purification and separation, and then, carrying out splicing in vitro under the condition of intein splicing. The methods have high production efficiency, are wide in applicable application range and facilitate the separation and purification of the products. The invention provides a novel method for preparing biological drugs for directional attack on cancers 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

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Application Information

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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 JIAO TONG UNIV
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