Current powerful tools for cancer immunotherapy include monoclonal antibodies, tumor vaccines, immune checkpoint inhibitors, CAR-T cell immunotherapy and bispecific antibodies (BsAb). In particular, CAR-T and BsAb have received more and more attention as new strategies for anti-tumor immunotherapy. Although BsAb has met technical difficulties, it is regarded as a prospective drug for tumor and cancer treatment.

Introduction to Bispecific Antibodies

Monoclonal antibodies mainly play a certain biological role by binding a single specific epitope, such as blocking protein-related effects, activating or regulating receptor function. The Fc fragments of antibodies can also induce antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity. However, it also limits its application in some fields, such as the treatment of tumors that need to block multiple signaling pathways, autoimmune diseases, and the treatment of infectious diseases that require multiple antigen sites to prevent virus escape due to the high mutation rate of the virus. Therefore, bispecific antibodies came into being.

Bispecific antibodies are artificially engineered antibodies that can simultaneously bind two specific epitopes or proteins of interest. They have the ability to bind two different epitopes simultaneously and can play some special biological functions. For example, targeting effector cells directly to tumor cells, enhancing their cytotoxicity, improving antibody selectivity and functionality, co-stimulating or inhibiting receptors, avoiding immune escape mechanisms and thereby improving the therapeutic effect, combined with two monoclonal antibody drugs In comparison, BsAb also reduces the cost of drug development and clinical trials.

Bispecific antibody classification

Bispecific antibodies are prepared using DNA recombination and protein engineering techniques. Depending on the components, there can be multiple construction methods, so there will be multiple classifications. According to the left-right symmetry of the structure, it can be divided into symmetric structure and asymmetric structure, according to the integrity of the IgG molecule, it can be divided into intact antibodies and antibody-like fragments, and according to the number of antigen-binding regions, the configuration can be divided into divalent, trivalent, tetravalent or more configurations, etc.

In this article, we will only briefly introduce BsAb (IgG-like BsAb) with Fc fragments and BsAb (non-IgG-like BsAb) without Fc fragments.

IgG-like BsAb means that the IgG of the antibody has an Fc part and has Fc-mediated effector functions, such as ADCC, CDC cytotoxicity and ADCP cell phagocytosis. The relative molecular weight of this type of antibody is relatively large, and the Fc fragment is helpful for the purification of the antibody at a later stage, and improves its solubility and stability. Of course, the Fc part will bind to the receptor FcRn, increasing the antibody serum half-life.

Non-IgG-like BsAb means that the antibody lacks Fc fragments and only exerts a therapeutic effect through antigen binding. It has the characteristics of low immunogenicity, easy production, small molecular weight, etc. It has high tumor tissue permeability and has a stronger treatment effect.

In the development and design of bispecific antibodies, it is necessary to ensure the correct coupling or pairing of two different pairs of light and heavy chains based on the purpose of clinical treatment. It is also necessary to maintain the independence of the binding domain of each monoclonal antibody to ensure the binding of different tables. There will be no steric hindrance when it is placed, and it also requires antibody molecules to be easily expressed in mammalian cells without complicated protein modification processes.

Preparation method of bispecific antibody

The preparation of BsAb mainly includes chemical coupling, double hybridoma cell method, recombinant gene preparation and other methods.

The chemical coupling method is the earliest BsAb preparation technology. Two complete IgG or antibody fragments are coupled into one BsAb through chemical coupling reagents. This technique is fast and simple, but it is easy to destroy the antigen binding site and affect the activity of the antibody, and the cross-linking agent used in the preparation process is uncertain in terms of safety and carcinogenicity.

Two-hybridoma cell method is to synthesize two-hybridoma cell strains by cell fusion of two different hybridoma cells, and then obtain cloned cells through conventional hybridoma screening. Because the two types of hybridoma cells produce two heavy chains and two light chain molecules, these light and heavy chains can be randomly combined. The BsAb produced by this technology is random and low in efficiency, but has good biological activity and stable structure. With the development of technology, Konck-in-hole technology can effectively solve the problem of correct pairing of heavy chains of heterologous antibodies.

Recombinant gene preparation method uses genetic engineering technology to transform antibodies to form various forms of bispecific antibodies. This technique can reduce the random combination of light and heavy chains and achieve mammalian cell expression.

The antibody prepared by chemical coupling method and two-hybridoma fusion method is mouse-derived, has strong immunogenicity, and is difficult to purify, which limits its clinical application. Therefore, recombinant DNA technology is currently commonly used to prepare BsAbs Technology.

To be continued in Part II…

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