Antibody drug research and development has shown explosive growth in the past decade. A total of 31 antibody drugs and 10 antibody biosimilar drugs were approved on the global market from 2013 to 2017, compared with an average of 1.6 antibody drugs per year in 2002-2012, among which no antibody biosimilar drugs were approved, and the field of oncology and hematology was the main area of disease development. At present, most of the antibody drugs on the market are monospecific (mono-specific) antibodies. Monospecific antibodies refer to an antibody that can only recognize one antigen, while multi-specific (multi-specific) antibodies refer to an antibody or molecules similar to antibody structure, which can bind to two or more antigens at the same time, among which bispecific antibodies are the main development type at present. With the help of breakthroughs in molecular biology, immunology and protein structure research, great progress has been made in the research and development of multi-specific antibodies. In this paper, the structures and mechanisms of different multispecific antibodies are discussed, and their advantages and disadvantages are analyzed.

Multi-specific antibodies classified by structures
  • Multi-specific antibody fragments

This structure does not have a Fc region, and the common antibody structures are BiTE (bi-specific T-cell engager), DARTs (dual-affinity re-targeting proteins), and TandAbs (tandem diabodies).

Blinatumomab, which belongs to BiTE type bispecific antibody, targets CD3 and CD19. However, because of its small molecular weight and lack of Fc region, the half-life of blinatumomab in blood is only 1.25 ±0.63h, which is much shorter than that of a complete antibody, and requires continuous intravenous injection for 4 weeks to reach the effective dose. In addition, during the production, it’s of poor stability with easy formation of aggregates. It also affects the quality of products and increases production costs. Another kind of antibody fragment structure, DARTs, is similar to the natural antibody binding to the antigen, through the combination of two Fv fragments to form two binding sites with the antigen, which can maintain the binding ability and action of the antibody to the antigen. Compared with the first two small molecular weight antibodies, TandAbs have a molecular weight of about 105 kDa and can provide a longer half-life. At present, the production of antibody fragment structure can be performed in both eukaryotic and prokaryotic systems. If you choose a prokaryote, such as E. coli, it is easy to produce inclusion body (inclusion body) or abnormal folding, which can be improved by simultaneous expression of chaperon proteins, such as Skp, OmpH, HlPA, FkpA, or with proteins that help to increase solubility on the fragment. For example, MBP, NusA, TRx, etc., form fusion proteins (which need to be removed before forming the final product), which will increase the production cost and should be taken into account when choosing the performance system. In addition, no matter in what system, the output of this structure is only at the mg/L level, which is a major challenge for future mass production.

  • Multi-specific IgG-like antibodies

The multi-specific antibody with this structure can retain the original Fc region and has the pharmacokinetic characteristics and action of IgG antibody. Common techniques include quadroma (hybrid-hybridoma), knobs-into-holes, common light chain, co-culture and other techniques to produce multi-specific antibodies. Catumaxoamab, which has been released from the market, uses quadroma technology to rehybridize two independent hybridomas to form a four-hybrid tumor, and collect bispecific antibodies targeting CD3 and EpCAM. However, the source of hybridoma is mouse origin (rat and mouse), causing serious immunogenicity and side effects in use. Knob-into-holes technique combines the CH3 functional area of the Fc region with the CH3 functional region of another Fc region to achieve accurate matching to form a complete bispecific antibody. Usually, the light chain of the antibody is the same, but the difference is the heavy chain binding to the antigen, and this structure is easy to form the correct bispecific antibody, the purification method is the same as the general antibody purification method. Finally, there is co-culture technology, which allows cells to express half of the molecules, which are combined in vitro to form complete antibodies. The disadvantage is that they are prone to the risk of contamination.

However, no matter what the technology is used, the expression amount on CHO cells is only 1-3 g/L or even lower. Compared with the average production of monospecific antibody on CHO cells above 3 g/L, it is necessary to overcome the difficulties of mass production and production cost in the future.

Multi-specific antibodies classified by mechanisms of action
  • Bridging immune cells and target cells

For example, T cells and tumor cells, one end of the antibody binds to the surface molecules of T cells, and the other end binds to the surface molecules of tumor cells to activate T cells, so that T cells can attack tumor cells nearby. Typical drugs such as catumaxomab and blinatumomab, bind to T cell surface molecules (CD3) and the other end to tumor cell surface molecules EpCAM or CD19 to achieve the effect of inhibiting tumor cells. Similar mechanisms can be derived to different immune cell surface molecules, such as CD16 (NK cells), CD40, 4-1BB, etc., while a large number of surface molecules expressed on tumor cells, such as CD20, CD33, CD38, HER2, PSMA, mesothelin, MET, PD-L1, etc., are also the main targets. However, some studies have pointed out that targeting CD3 may over-activate T cells and other immune cells, resulting in the release of too many cytokines, so there will be safety considerations in the use of CD3. Therefore, among the antibodies targeting CD3, if the Fc region is preserved, attention should be paid to reduce the binding of Fc and FcgR, so as to reduce the side effects.


  • Inhibiting cellular signal transduction

Some ligands or cytokines can induce the activation of intracellular signaling pathways, and then promote cellular functions such as cell growth, angiogenesis, inflammation and so on. These ligands or cytokines, such as VEGF, ANG2, DLL4, TNF, IL-23, IL-17, can be inhibited by multi-specific antibodies at the same time, so that they can not cause subsequent intracellular information transmission and cellular function, and then achieve the therapeutic effect. In addition, they can also target the main receptors of tumor cells, such as HER2, EGFR, MET, etc., and inhibit the receptors so that they can not activate downstream signal transduction.

  • Binding to multiple immune checkpoints

With the launch of Yervoy® (ipilimumab) and Keytruda® (pembrolizumab) in 2011 and 2014, cancer therapy has undergone unprecedented changes, not only proving the importance and efficacy of immune checkpoints in many tumors, but also bringing a new page to antibody drugs. The current direction of research and development is to target multiple immune checkpoints at the same time, either antagonists (activating suppressed immune cells) or agonists (enhancing immune cell activation), or one end targeting tumor cells, one end targeting checkpoints to enhance immune cell activation. No matter what strategy it is, the purpose is to enhance the immune response. However, too many immune reactions will also increase the possibility of adverse reactions. Therefore, it is necessary not only to observe the efficacy of the drug, but also to focus on its safety. In addition, the given dose and cycle also require more data support. Recently, there are also studies that use a single antibody to carry three antigen-recognizing sites, namely trispecific (tri-specific) antibodies, one of which targets tumor cells (CD38), and two sites at the other end that combine CD3 and CD28 to activate T cells to attack tumor cells.

  • Other mechanisms

In addition, there are different research and development strategies, for example, emicizumab, which was launched in 2017, uses multispecific antibodies to act as cofactor, and binds both FIXa and FX-activated clotting mechanisms to treat patients with hemophilia A who lack coagulation factor 8. For example, CD47 is a “don’t eat me” signal, and when this signal is suppressed, cells will be swallowed by macrophages, but normal cells will also show side effects of a single monospecific antibody that inhibits CD47. The recent research and development strategy is to make use of the characteristics of bispecific antibodies, with one end targeting the surface molecules of tumor cells, and the other end binding CD47, thus to increase the specificity of the antibody, leading to the result that it will only attack tumor cells and reduce the occurrence of side effects. In order to increase the half-life in the blood, one end of the multi-specific antibody can be combined with human serum albumin to increase its molecular weight and thus prolong the half-life.

The research and development of specific antibodies can basically be divided into structural development, structural shortcomings improvement, structural advantages strengthening, and mechanism innovation, such as targeting different immune checkpoints or tumor surface cellular molecules, trispecific antibodies and so on. In addition to the two multi-specific antibodies on the market, most of the antibodies are still in the early stage of clinical research and development, and their effectiveness and safety still need more data to support. In the aspect of production, the problems of multi-specific antibodies mainly focus on the output, recovery rate, stability and aggregate formation, which will directly affect the product characteristics and production cost. As a result, the pricing strategy of the final product in the future may be much higher than that of the general monospecific antibody, and it will also have an impact on the future marketing strategy and layout, which are the challenges to be faced in the future research and development of multispecific antibody.


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