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

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...

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)

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