Features of  humanized antibodies

According to the structure and function of the constant region of antibodies, natural antibodies in humans can be divided into 9 different types. It is not difficult to understand that IgG1 is the first choice for therapeutic antibodies because it has a long half-life in vivo and can interact strongly with the human immune system. Therefore, most of the constant regions in humanized antibodies currently use the IgG1 subtype.

Applications of humanized antibodies 

In the current research and development of biomedicine, antibodies and their derivatives account for about 25%. Since 1990s, many monoclonal antibodies have been approved by the FDA for clinical use in anti-tumor, anti-graft rejection, anti-coagulation reactions, and treatment of various immune system diseases. Most of the therapeutic antibodies currently on the market are humanized antibodies (chimeric or CDR-grafted antibodies). They contain 10% -30% of mouse-derived proteins, so in clinical applications, there is more or less immunity rejection, so the development target for therapeutic antibodies is fully human antibodies. Of the more than 70 antibodies currently in clinical research, chimeric antibodies and humanized antibodies account for more than 70%, and there are several fully human antibodies. The research field also made phased results in the research of therapeutic antibodies. Many laboratories have successfully obtained human-mouse chimeric antibodies, single-chain antibodies, bispecific antibodies and other antibodies against various antigens, such as VEGF, CEA, TNFα, etc. and other small molecule antibodies. There are many reagent antibodies or diagnostic antibodies on the market, while only a few therapeutic antibodies have entered clinical trials.

Evolution and research progress of humanized antibodies

Humanized antibodies are a transitional form from mouse monoclonal antibodies to fully human antibodies. On the basis of murine monoclonal antibodies, the corresponding regions of the murine antibodies were replaced with human antibody constant regions to form human-mouse chimeric antibodies. About 70% of these antibody molecules are of human origin, and retain the characteristics of the parent antibody in terms of antigen specificity and affinity, while the immunogenicity is reduced to about 12%, and the half-life and effector function in the body are closer to humans. Chimeric antibodies have now entered the market for the treatment of cancer, rheumatoid arthritis, myocardial infarction and transplant rejection.

On the basis of the chimeric antibody, the mouse-derived component is further reduced, and only the CDR region of the mouse antibody is retained, and the rest replaces the corresponding part of the adult antibody. The human-derived component of this modified antibody reaches 90%, which is generally referred to as a humanized antibody. So far, many humanized antibodies have been approved for marketing to date. It is worth noting that CDR transplantation often results in decreased affinity and is not applicable to every murine antibody.

Full human antibodies are the development trend of therapeutic antibodies. At present, the methods for producing fully human antibodies have reached a relatively mature stage, mainly including antibody library technology and transgenic mouse technology. The development of antibody library technology makes it possible to obtain antibodies in vitro without immunization. Classic antibody libraries are displayed on the surface of phages in the form of phage coat protein III and protein VIII fusion single chain antibodies (ScFv), and then single-chain antibodies of completely human origin can be obtained through multiple rounds of antigen panning. There is now an improved way to screen ScFv completely in vitro. This technology starts with the ScFv gene library, and transcribes genes in the acellular system without inserting a terminator into the gene to form an RNA-ribosome-ScFv complex. After obtaining a complex that specifically binds to the target molecule in a similar manner to phage library screening, Isolate RNA for PCR amplification, and can also introduce mutations at the same time of amplification to stimulate the affinity maturation process and obtain a secondary antibody library containing higher affinity.

In transgenic animals, there are several different ways to produce human antibodies. One method is to introduce lymphocytes from donors or cancer patients who have developed an immune response into severe combined immunodeficiency mice (SCID) or Trimera mice. As a temporary human immune system performs limited functions, it is possible to obtain hybridomas that secrete human antibodies by hybridizing mouse spleen cells with human myeloma cells. But such systems must rely on donors that have developed a certain immune response, and immunization with preselected antigens is not possible. So most of the antibodies that are of interest to therapeutic targets cannot be obtained with this technique. Another way to produce human antibodies is to inactivate the mouse's own genes and introduce new genes through gene knockout technology to create transgenic mice that carry the human antibody heavy and light chain gene cluster and inactivate the autoantibody genes. The human DNA fragment carried by this human antibody transgenic mouse (commonly known as HuMab, Human antibody mouse) has complete functions, which can effectively perform isotype conversion and affinity maturation. Any target antigen can be used to immunize the mouse to produce high-affinity human antibodies. The latest development in this field is the creation of so-called "transchromosomic mice". This mouse carries human microchromosomes, which are chromosomal fragments isolated from human chromosomes 14 and 2 and containing all human antibody heavy and light chain germline gene clusters (including all V, D, J fragments and antibody constant regions). The mice carrying microchromosomes can provide almost the same human immunoglobulin gene environment and accurately reproduce the human antibody production process in mice.

Whether through antibody libraries or transgenic mouse technology, ultimately, antibodies still need to be produced at the cellular level. Although there are many research systems for the expression of complete antibodies, such as insects, yeasts, plants, etc., all the antibodies currently on the market are produced by mammalian cell systems, because the antibodies can be correctly folded and glycosylated when expressed in mammalian cell lines. Human antibodies produced by other expression systems may have heterologous glycosylation. For example, non-mammalian cell lines such as yeast and bacteria have not been concerned in the production of antibodies because they cannot provide the correct folding and glycosylation. It may be feasible to produce antibodies. Although plant glycosylation differs from humans, the clinical application of plant antibodies is still problematic, but this obstacle may be overcome by genetic modification in the future.

In clinical treatment, patients generally need to repeat large doses of drugs (hundreds of micrograms at a time), which substantially increases the cost of treatment. It is economical to develop genetically modified animals, such as goats and cattle, to obtain monoclonal antibodies from their milk.

The current technology makes it possible to produce chimeric and human antibodies, which not only have a long half-life, low immunogenicity, but also interact with natural effectors. In the future, human monoclonal antibodies prepared by phage antibody library and human antibody gene transgenic mouse technology are expected to be applied to a series of immune and infectious diseases. At the same time, these antibodies can also be used to target a variety of cytokines, chemical factors, their receptors, and extracellular acceptors involved in cell-cell interactions. As far as cancer is concerned, the application of naked antibodies may progress relatively slowly, because specificity is a key issue restricting their application. A more feasible development direction is to select those factors that can regulate the weak immune response as targets.

Here is a list of examples some FDA-approved monoclonal antibody drugs.

l abciximab (Reopro)

l adalimumab (Humira, Amjevita)

l alefacept (Amevive)

l alemtuzumab (Campath)

l basiliximab (Simulect)

l belimumab (Benlysta)

l bezlotoxumab (Zinplava)

l canakinumab (Ilaris)

l certolizumab pegol (Cimzia)

l cetuximab (Erbitux)

l daclizumab (Zenapax, Zinbryta)

l denosumab (Prolia, Xgeva)

l efalizumab (Raptiva)

l golimumab (Simponi, Simponi Aria)

l inflectra (Remicade)

l ipilimumab (Yervoy)

l ixekizumab (Taltz)

l natalizumab (Tysabri)

l nivolumab (Opdivo)

l olaratumab (Lartruvo)

l omalizumab (Xolair)

l palivizumab (Synagis)

l panitumumab (Vectibix)

l pembrolizumab (Keytruda)

l rituximab (Rituxan)

l tocilizumab (Actemra)

l trastuzumab (Herceptin)

l secukinumab (Cosentyx)

l ustekinumab (Stelara)