Introduction to antibody structure and function
An antibody molecule composes of an antigen-binding fragment (Fab) and a crystallizable fragment (Fc), forming a Y-shaped structure. The Fab region is a mixed light and heavy chain dimer composed of variable light chain (VL), constant light chain (CL), variable heavy chain (VH), and constant heavy chain 1 (CH1) segments. The Fc region is a heavy chain dimer composed of constant heavy chain 2 (CH2) and constant heavy chain 3 (CH3) fragments.
Figure 1. The structure of an antibody (Chang Yang, 2023)
The Fab region determines the specificity and affinity of an antibody for its target antigen. The Fc region can actively supplement or bind to Fc receptors (FcRs) on the surface of immune effector cells, thereby exerting antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis ( ADCP), and other antibody effector functions.
Antibody-mediated ADCC activity is affected by factors such as tumor target expression abundance, FcR genotype, and the number of effector cells in tumor tissue. If the number of target molecules expressed per cell is less than 104, it is not enough to induce ADCC activity, and only when the number of molecules reaches 105–106 can strong ADCC activity be induced.
Figure 2. Antibody-dependent effector functions(Anupama Samantasinghar, 2023)
What is the difference between the four subtypes of IgG?
Human IgG includes IgG1, IgG2, IgG3, and IgG4. Although all isoforms share more than 90% identity at the amino acid level, each isoform has unique properties in terms of hinge region length, number of interchain disulfide bonds, and Fc effector function. IgG1 has the highest affinity with FcγR and has the strongest ADCC effect, followed by IgG3, IgG2, and IgG4, respectively. FcγRs include six subtypes (FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB), which differ in their cellular distribution and affinity for Fc and the resulting biological activity. Except for FcγRI, which is an activating receptor with high affinity for IgG and can be activated by monomeric IgG, all other FcγRs can be functionally activated by the Fc region of IgG only when in contact with specific antigen complexes. FcγRIIB is the only inhibitory FcγR. Among all FcγR subclasses, FcγRIIIA is mainly expressed in NK cells, so it mainly mediates ADCC, and FcγRIIA is mainly expressed in macrophages, which mainly mediates ADCP.
Selection of IgG subtype—single-target antibody
Single-target anti-tumor antibodies have always been popular. Although there is no strict standard for the selection of IgG subtypes of single-target antibodies, the cellular distribution and biological functions of the target should be considered.
IgG selection targeting tumor cells
When the target is an antigen on tumor cells, IgG1 should be the preferred antibody as it has a higher affinity for activating FcγRs and can produce strong ADCC and/or ADCP activity on tumor cells. When ADCP function is activated, effector cells (macrophages or dendritic cells) can present processed tumor antigens to T cells, thereby further triggering tumor-specific immunity. Most of the anti-tumor antibody drugs approved by the FDA have chosen the IgG1 type. This class of drugs can block tumor growth signals (EGFR), inhibit angiogenesis (VEGFR), activate immune cells (PD-L1), and at the same time kill and eliminate tumor cells through ADCC/CDC, thereby further enhancing the anti-tumor effect.
IgG selection targeting immune cells
The selection of anti-tumor IgG subtypes targeting immune cells is more complicated, and some targets transmit inhibitory/promotive signals to immune cells to regulate immune functions. These inhibitory signals are collectively called inhibitory receptors (such as CTLA-4, PD-1, TIM-3, and LAG-3), and promoting signals (4-1BB, CD40, OX40, CD27, GITR, etc.) are called immunostimulatory receptors.
- When these inhibitory receptor targets are mainly expressed on immune effector cells, IgG4 with the weakest FcγRs affinity should be selected as much as possible. This is because IgG4 does not have ADCC/CDC effect and cannot kill and phagocytize target cells, thereby retaining immune effector cells to exercise anti-tumor effect. This also explains why most antibody drugs targeting PD-1 have chosen the IgG4 subclass.
- The IgG1 class should be preferred when these inhibitory receptor targets are primarily expressed on tumor-promoting or immunosuppressive cells because IgG1 can clear the immunosuppressive cells in the tumor microenvironment through the ADCC/ADCP mechanism.
- The selection of immunostimulatory receptor antibody IgG is more complicated, and more factors need to be considered. Most immunostimulatory receptors belong to the tumor necrosis factor superfamily (such as 4-1BB, CD40, OX40, CD27, and GITR), and the antitumor activity depends on the cross-linking of Fc and FcγR. FcγRIIB, in particular, acts as a scaffold to promote antibody multimerization that promotes receptor aggregation, a prerequisite for the activation of downstream intracellular signalings. An approach to further optimize the antitumor activity of anti-TNFR superfamily member antibodies is to engineer the Fc region of the antibody to improve FcγRIIB engagement.