Bispecific antibody (BsAb) generally refers to an antibody that can specifically bind to two antigens or two different epitopes of the same antigen. Today, let's briefly browse through the knowledge about bispecific antibodies, mainly introducing them from three aspects: configuration, mechanism of action, and future trends.

▉ Configuration of bispecific antibodies

Dual antibody configuration (format), mainly divided into three categories:

The first type is fusion proteins assembled from antigen binding regions (Fab), which can avoid problems such as light and heavy chain mismatch. At the same time, due to the absence of glycosylated Fc segments, they can be produced using relatively simple prokaryotic or lower mammalian cells, with high yield and low cost. The disadvantages of not containing Fc segment are also obvious. Due to the inability to bind with FcRn, it is quickly cleared in plasma, with a very short half-life. Stability and easy aggregation are also issues that need to be considered during development. At present, a dual antibody (Blinatumomab) has been launched for this configuration.

The second type is symmetrical, which fuses antibody fragments on the basis of naked antibodies. Symmetric bispecific antibodies differ in molecular weight and structure from naked antibodies, which may lead to stability and solubility issues. Symmetrical bispecific antibodies generally use tetravalent 2+2. Due to the close proximity of the binding sites of the two antigens in this antibody, it may affect the simultaneous approach of the two targets, resulting in a decrease in potency. Optimization of this molecule may be necessary in the later stage.

The third type is asymmetric configuration, which is also the configuration adopted by most pharmaceutical companies in development to preserve the properties of natural antibodies as much as possible. This configuration mimics natural antibodies and lacks non natural antibody domains or linkers, making it considered the least immunogenic configuration. Typically, asymmetric bispecific antibodies are bivalent 1+1, meaning that the antibody binds to two antigens in a monovalent form. This configuration accounts for about 50% of bispecific antibodies, with one Emicizumab already on the market, one delisted due to commercialization reasons (cartumaxomab), and several others in different stages of clinical development.

structure of BsAb: a mechanistic revies of the pipeline, Nature Reviews, 2019

Bispecific antibodies: a mechanistic review of the pipeline

TIPS：What is valence?

Potency refers to the number of antigen binding sites in an antibody molecule. The design of bispecific antibody configurations will affect the number of binding sites for each target. The divalent bsAb with one binding site per target is represented as 1+1. Incorporating other binding sites can lead to trivalent (2+1) and tetravalent (2+2 or 1+3) designs.

antibody format, biying

Bispecific antibodies: a mechanistic review of the pipeline

▉ The mechanism of action of bispecific antibodies

At present, the mechanism of action of dual antibodies mainly includes two categories: bridging two cell types (in trans binding) and binding two molecules to one cell (in cis binding). At present, the mainstream treatment fields of dual antibodies are mainly divided into three categories: anti-tumor, autoimmune diseases, and other diseases. Among them, anti-tumor products are the most common, and CD3T cell adaptor dual antibodies account for half of the dual antibodies.

Examples of obligate mechanisms of action of bsAbs.

Bridged cells - T cell recruitment

Initially, bispecific antibodies were used for T cell redirection, bridging T cells with tumor cells. By binding to CD3e in TCR complexes, T cells can bypass MHC molecules and be directly activated. Activated T cells kill tumor cells by releasing perforin, granzyme, and other substances. Although this idea has been validated in vitro, there has been no sustained clinical effect. Due to complications and the instability of the bispecific antibody itself, the subsequent development of bispecific antibodies is hindered. It is necessary to mention the blinatumomab developed by Anjin, which greatly promoted the development of the entire field. This bispecific antibody is a single chain antibody without Fc segment, with only 55kDa and a plasma half-life of 1.25 ± 0.63 hours. When the trough level is reached through continuous intravenous injection, NHL patients show good response, with a complete sustained release rate of 43% for relapsed and refractory ALL.

There are several factors that affect T cell killing, and the distance between tumor targets and T cells directly affects the formation of immune synapses, which is directly related to the effective killing of T cells in the body. The bivalent target binding enhances the affinity for target binding, which can enhance T cell killing against tumors. Currently, Roche has a 1+2 bispecific antibody that has shown excellent results in phase I clinical trials. When designing this type of bispecific antibody, full consideration should be given to the affinity of one end of CD3. Excessive affinity will allow the bispecific antibody to occupy the CD3 antigen of the cell, continuously activate the cell, and ultimately lead to T cell exhaustion. Low affinity will cause the bispecific antibody to continuously bind and dissociate with CD3 of different cells, and the repeated binding with T cells can produce a waterfall effect, which can cluster T cells and ultimately activate a large number of T cells, ultimately killing tumor cells. In addition, a high affinity for CD3 can cause dual antibodies to be more concentrated in tissues such as the spleen, making it difficult to reach tumor tissues, which is equivalent to dispersing firepower.

Bi-specific T-cell engager，wikipedia

treatment effect: 1( antibody for cancer cell)+1(antibody for T cell)>2

Double antibodies that bind to two different antigens: Unlike double antibodies that bind to two different antigens, this type of double antibody selects to bind to two different epitopes of the same antigen, enhances binding strength through antigen cross-linking and aggregation, and mimics the effects of antibody mixtures and polyclonal antibodies. At present, the dual antibody ZW25 (Zanidatamab) introduced by BeiGene in China has this mechanism of action. By binding to two non overlapping HER2 epitopes, it can double block HER2 signaling, enhance binding and remove HER2 protein on the cell surface, and have strong antibody effector functions to enhance anti-tumor activity in patients.

Simulated cofactor: Roche's dual antibody simulates coenzyme factor III (FVIII) to bring the enzyme (FIXa) closer to the substrate (FX), thereby activating the enzyme to form an activated substrate, which is used to treat the rare disease hemophilia A caused by FVIII deficiency and coagulation disorders. This drug was launched in the United States in 2017 and can effectively reduce the amount and frequency of bleeding in hemophilia. As a preventive treatment, the annual treatment cost of this drug is as high as $400000. Currently, it has been on the market for only four years and its sales have reached $2.4 billion. Foreign institutions predict that its annual sales will reach $4.5 billion by 2022.

(https://link.springer.com/article/10.1007/s12185-018-2545-9；Emicizumab, a humanized bispecific antibody to coagulation factors IXa and X with a factor VIIIa-cofactor activity)

▉ Bispecific antibodies already on the market or in phase III clinical trials

▉Future development trend of bispecific antibodies

Looking ahead to the future, many promising concepts in their early stages of development have emerged.

1、 IGM Biosciences utilized natural structures with J-chain antibodies (such as IgM and IgA) to connect effector cell targeting arms to the J-chain, allowing for the assembly of 1+10 bispecific antibodies. Currently, one bispecific antibody is in clinical phase I, with 9 out of 14 patients showing significant tumor shrinkage, demonstrating good clinical efficacy.

2、 Another novel concept of bispecific antibodies is the expression of bispecific antibodies after delivering mRNA and DNA to the body, both of which exhibit good tumor clearance in animals. In industrial preparation, the production of mRNA and DNA is faster than that of protein, which is likely to accelerate the clinical development of new bispecific antibodies. Especially compared to mRNA or antibody proteins, DNA not only has low production costs and high stability, but also provides great convenience for long-term storage and transportation. This targeted dual antibody DNA delivery can be used for antiviral and bacterial infections, and is highly likely to become a safe alternative to viral vaccines.

3、 Another new concept is to add dual antibodies or receptor proteins to oncolytic viruses or CAR-T to achieve targeted delivery and function in the tumor microenvironment.The above article is excerpted from the internet

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