By fusing fragments of antibody molecules with other proteins, antibody fusion proteins with multiple biological functions can be obtained. The fusion protein of recombinant antibody and cytokine combines the specificity of antibody and multifunctional activity of cytokine, which is also called immune cytokine. At present, the cytokines fused with antibody include: IL-2, IL-12 and GM-CSF.
Basic structure of antibody cytokine fusion protein
At present, there are many kinds of antibody cytokine fusion proteins, most of which are composed of IgG antibody and cytokine. The antibody part can be whole antibody molecule, F(ab) 2, Fab and scFv. The whole antibody cytokine fusion protein can be constructed by fusing the cytokine directly or through the appropriate connecting peptide at the C-terminal of the third constant region (CH3) of the antibody heavy chain or the N-terminal of the antibody heavy chain, which is characterized by the ability to maintain the activity of the functional regions of the antibody and the connected cytokine.By fusing cytokines directly into the end of the antibody hinge region or the C-terminal of the first constant region of the heavy chain, F(ab) 2 and Fab cytokine fusion proteins can be produced, but these two kinds of antibody cytokine fusion proteins lack the effect function of the antibody Fc segment.The scFv Fc fusion protein can also be constructed by connecting scFv with the Fc segment of human antibody, and then fusing the cytokines with the Fc segment with the appropriate connecting peptide. It is characterized by moderate molecular weight, bivalent binding activity, good stability and good penetration.
Some antibody cytokine fusion protein
Antibody IL-2 fusion protein
The study of antibody cytokine fusion protein fused with IL-2 is the most extensive, and many kinds of antibody IL-2 fusion proteins have been reported. IL-2 is an important mediator of immune response and one of the important anti-tumor cytokines, which has a wide range of biological activities. In vitro, IL-2 can promote the proliferation of cytotoxic cells, NK cells and helper T cells, induce cytotoxic cytokines, and induce the proliferation of LAK cells. In the clinical treatment of animal models and tumor patients, IL-2 alone or combined with LAK cell adoptive therapy can make the tumor shrink.However, IL-2 with nanomolar concentration can cause serious side effects and become one of the main obstacles for its large-scale application.
AntibodyIL-12 fusion protein
The fusion protein IL-12 is an important cytokine that mediates innate and cellular immunity. It has a strong activity of inhibiting tumor and preventing tumor metastasis. IL-12 has no direct effect on tumor cells in vitro, but IL-12 can activate T cells and NK cells, stimulate CD4+ T cells to differentiate into Th1 cells, and cytokines produced by the latter can activate CTL and macrophages.IL-12 is also a powerful inhibitor of angiogenesis, which can inhibit angiogenesis and tumor growth by inducing IFN - γ secretion. IL-12 is a heterodimer composed of p35 and p40 subunits, so it is very difficult to construct a fusion protein with both antibody activity and IL-12 biological activity. It is generally believed that the accessibility of p40 N-terminal is very important for the activity of IL-12.
Antibody GM-CSF fusion protein
GM-CSF can stimulate the proliferation and differentiation of hematopoietic cells, and it is also a multifunctional immune regulator. GM-CSF can improve the antigen presentation of various APCs, promote monocyte to express MHC-II molecules, granulocyte to express adhesion molecules and stimulate T cell proliferation.Animal experiments show that the protective effect of anti-tumor vaccine can be improved by enhancing T cell immunity after GM-CSF injection. Now there are many kinds of antibodies fused with GM-CSF. After fusion, the antibodies retain the ability to bind to the corresponding antigens and have the same affinity as the free antibodies.At the same molar concentration, there was no significant difference between the fusion protein containing GM-CSF and the proliferation response mediated by recombinant GM-CSF. It has been reported that the fusion of GM-CSF into the C-terminal of the antibody will not interfere with the antibody mediated ADCC effect, but also enhance the ADCC effect.Compared with the parent antibody, the fusion protein can trigger macrophage mediated cytotoxicity at low concentration. GM-CSF is a key stimulator of dendritic cells, which can stimulate monocytes, macrophages and antigen-presenting cells to express MHC -II antigens. Some research results show that the GM-CSF fusion protein of antibody can greatly enhance the immunogenicity of tumor antigen, induce the immune response of anti idiotype in mice, and produce high level specific antibody without adjuvant. Therefore, the antibody GM-CSF fusion protein has a good prospect as a vaccine.
In many cases, antibody cytokine fusion protein showed stronger antitumor activity than free antibody plus cytokine. Antibody can effectively guide cytokines to the tumor site. Compared with the systemic administration of cytokines, the cytokines targeted by antibody can make the local tumor reach a higher concentration, and can effectively inhibit the primary and metastasis of tumor. Therefore, it will have a bright future in the treatment of tumor.