What is ADCC in immunology？ADCC is the antibody dependent cell mediated cytotoxicity, which means that the Fab segment of the antibody binds to the antigenic epitope of the virus-infected cell or tumor cell, and the FcR of the Fc segment and the killer cell (NK cell, macrophage, etc.) surface. Binding, mediating killer cells directly kills target cells.

IgG antibodies mediate the role of these cells in ADCC, which is the main cell that can function as ADCC. In the process of antibody-mediated ADCC action, the antibody can only specifically bind to the corresponding antigenic epitope on the target cell, and effector cells such as NK cells can kill any target cells that have bound to the antibody, so the antibody and the target cell The antigen binding on it is specific, and the killing effect of NK cells and the like on target cells is non-specific.

The mechanism of action of ADCC:

1. Perforin, granzyme action pathway Perforin is a cytotoxic substance stored in cytoplasmic granules, and its biological effect is similar to that of complement membrane attack complex. In the presence of calcium ions, a polypermein "hole" can be formed on the target cell membrane, allowing the water electrolyte to rapidly enter the cell, causing cell collapse and destruction. Granzyme is a serine protease that enters the cell by the "pore" formed by perforin on the target cell membrane, and causes apoptosis of the target cell by activating an apoptosis-related enzyme system.

2. The surface of NK cells activated by Fas and FasL pathway can express FasL. When FasL expressed by NK cells binds to the corresponding receptor Fas (CD94) on the surface of target cells, Fas trimer can be formed on the surface of target cells, thus The death domains in the cytoplasm are clustered together, and the latter binds to the Fas-associated death domain protein (FADD), which in turn leads to death of target cells by recruiting and activating caspase8 through the caspase cascade.

3, TNF-α and TNFR-I pathway TNF-α and FasL similarly, they bind to the corresponding receptor on the surface of the target cell, namely type I TNF receptor (TNFR-I), can form TNF- R trimers, which cause the death domains in the cytoplasm to cluster into clusters, recruiting death domain protein (FADD) binding, and then recruiting and activating caspase8, ultimately killing the cells.

So how does the antibody structure increase ADCC and CDC activity?

With the development of modern biotechnology, antibody drugs have become the fastest growing therapeutic drugs, occupying an important position in the biotechnology pharmaceutical field. An antibody, also called immunoglobulin (Ig), is a glycoprotein that specifically binds to an antigen. There are five main types of Ig expressed in humans: IgM, IgA, IgD, IgG, and IgE, of which IgG is the most abundant. While IgG has four subclasses, the anti-polysaccharide antigen activity mainly depends on IgG1 and IgG2, while the anti-protein and anti-viral antibodies are involved in IgG1, IgG3, and IgG4. Among them, IgG1 is most expressed in humans, and IgG1 has the longest half-life in human serum for up to 21 days. IgG1 has a Y-shaped structure and consists of two identical heavy chains (about 55 kDa) and two identical light chains (about 25 kDa), which are joined together by hydrophobic interaction and linked by a disulfide chain. The heavy chain is composed of VH, CH1, hinge region, CH2 and CH3, and the light chain is composed of VL and CL. In addition, N-terminal glycosylation of the antibody occurs at the asparagine Asn297 position in the CH2 region of the antibody, consisting of N-GlcNAc, mannose, fucose and galactose, respectively. In terms of the mechanism of action of the antibody, the antibody consists of an antigen binding site (Fab) and a crystallizable site (Fc). The Fab segment can bind to a specific antigen, thereby determining the specificity and affinity of the antibody; the Fc segment can bind to the Fc receptor (FcγRI, FcγRII, FcγRIII) expressed on the surface of the immune cell, complement (C1q) and FcRn in the blood. Thereby activating the immune effect to remove foreign matter and the like.

The structure of the antibody determines its mechanism of action, and its Fab segment can recognize free molecules (VEGF, TNF, etc.) targets and cell surface receptors (CD20, CD19, etc.), and determine the specific recognition of antibodies to foreign invaders such as cancer cells. The Fc segment determines the effector function of the antibody, including antibody-dependent cytotoxicity and complement-dependent cytotoxicity. When an antibody binds to a tumor cell surface antigen through an antigen binding site and an Fc site binds to an immune effector cell surface Fc receptor, the immune effector cell is activated to kill the tumor cell, and this process is called ADCC. There are three main types of Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), of which the latter two can be further divided into: FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. Different immune cells express specific Fc receptors, such as neutrophils, which usually express FcγRI, FcγRII, and FcγRIIIb, while NK cells express only FcγRIIIa. FcγRIIIa is generally considered to be a key receptor for ADCC. Therefore, NK cells are considered to be the most important cell population.

Clinical studies have demonstrated that ADCC action is the primary means by which tumor antibodies work, such as trastuzumab and alemtuzumab. The strength of ADCC antibody is related to many factors, such as the affinity of the antibody to the antigen, the affinity of the antibody to the Fc-segment receptor, and the characteristics of the immune effector cells. In general, the stronger the antibody-mediated ADCC effect on antigen or Fc receptor affinity. Modification of antibodies to increase their affinity for Fc receptors is a direct, rapid and effective way to increase the effects of ADCC.

Studies have shown that glycosylation and amino acid sequence modification of the Fc portion of the antibody can increase ADCC activity. Stavenhagen's team used the yeast presentation system to screen a large number of Fc mutants and found that a mutant with five mutations (F243L, R292P, Y300L, V305I and P396L) increased the affinity for FcγRIIIa by a factor of 10, while also having more Strong ADCC activity. In the glycosylation modification of antibodies, fucose is considered to be the most important sugar affecting ADCC activity, and de-fucosylation can significantly increase the affinity of the antibody to FcγRIIIa and ADCC activity (up to 100-fold). The Tsugu Kubota team knocked out the FUT8 gene of CHO cells by genetic means. The antibody produced by this cell does not contain fucose residues, has strong ADCC activity, and cell growth and protein expression are not affected.

Complement is a group of thermolabile, activated, enzymatically active proteins present in human or vertebrate serum and tissue fluids, including more than 30 soluble proteins and membrane-bound proteins. CDC refers to the cytotoxic effect of complement involvement, that is, complement (C1-C9) binds to the corresponding antigen on the cell membrane surface by a specific antibody to form a complex and activates the classical pathway of complement to form a attack-membrane complex to exert a cleavage effect on target cells.

The CDC action is caused by the first binding of the antibody to the complement C1q, and then C2-C9 is activated to form a attacking complex that exerts a cleavage effect on the target cells. Many anti-tumor antibodies can cause CDC effects, such as anti-CD20, CD52, CEA and other antibodies. Scholars have done a lot of research on how to improve CDC activity. Idusogie EE et al. used the alanine scanning mutation technique to study the CDC function of the anti-CD20 antibody rituximab, and found the sites that bind to C1q in the CH2 domain, Asp270, Lys322, Pro329 and Pro331, which can be modified to increase binding to C1q and CDC. active. In addition to modification of the amino acid of the CH2 domain, amino acid adjustment in the hinge region can also significantly increase C1q binding and CDC activity. The classical pathway of IgG3 activates the highest complement activity. The Tsuguo Kubota team replaced the heavy chain constant region sequences of IgG1 and IgG3 to construct an IgG1/IgG3 mixed antibody by genetic mutation. Surprisingly, this antibody exhibited strong binding and CDC activity of C1q. In vitro, the CDC activity of the anti-CD20 IgG1/IgG3 mixed antibody is several tens of times that of wild-type rituximab.

It is worth mentioning that if an antibody is increased in its ADCC activity by defucosylation and the CDC activity is increased by adjusting the heavy chain constant region sequence, the activity can be obtained without affecting ADCC activity without affecting CDC activity. Strong antibodies.