Today, more than 40 therapeutic antibodies in the United States and Europe have been approved for clinical use, demonstrating its value in cancer, autoimmunity, transplantation and other indications. Most of them are monospecific full-length IgG1 antibodies. However, at least 100 bispecific antibodies are in preclinical and clinical development. When developing therapeutic antibodies, scientists must not only ensure that it has the highest affinity with the target, other factors are also critical, including antibodies that can elicit an immune response and avoid off-target effects.
What is an antibody?
It is clear to everyone that a typical IgG antibody is a Y-type structure consisting of two arms (Fab region) and a bottom region (Fc region). An IgG consists of four antibody sequencing amino acid chains, a pair of identical heavy chains extending from the bottom to two arms; a pair of identical light chains, one on each arm. The complementarity determining regions (CDRs) are at the front end of the two arms; they are responsible for recognizing and binding to the antigen. Both CDRs recognize the same antigen, which makes the antibody bivalent, monospecific.
The Fc region binds to natural killer (NK) cells, macrophages, neutrophils, and Fc receptors on other cells in the immune system. This, in turn, is responsible for inducing phagocytosis, cytokine release, antibody-dependent cell-mediated cytotoxicity (ADCC), and other downstream effects. Similarly, binding of the Fc region to a particular Fc receptor (FcRn) protects the IgG from degradation, thereby extending the half-life of the antibody.
Humanized transformation
Typically, monoclonal antibodies for research are made by injecting mice with antigen. The antibody-producing cells are collected, fused with myeloma cells to form a hybridoma, and the extent to which the antibody recognizes the antigen is screened. However, such antibodies, if used for treatment, will be considered by the human immune system to be foreign proteins.
"We produce our own antibodies to drug antibodies, so the concentration of the drug must be reduced," explains David Bramhill, founder of Bramhill Bio Consulting. "Only three of the current market are pure mouse antibodies." Most therapeutic antibodies are "chimeric" or "human", meaning that the variable region (or CDR) of the mouse Fab is cloned into The backbone of a human antibody, or sequence, is altered so that it is not rejected by the human immune system.
However, the disadvantage of this approach is that most antibodies undergo a process of central tolerance, ie, the animal recognizes and removes autoreactive antibodies. "If you compare human and mouse proteins, you will find that important regions are often homologous. So you may lose a lot of therapeutically effective epitopes," Paul Kang, Chief Scientific Officer, Innovative Targeting Solutions (ITS) Point out.
ITS uses a system to produce antibodies in which all genetic elements are engineered by human embryonic kidney cells (HEK). "We amplify cells and induce recombination, so each cell undergoes a unique V(D)J recombination reaction, displaying antibodies on the surface, just as it happens in the body," Kang said. This produces billions of cells, each expressing a unique antibody without being centrally tolerated.
Today, there is another concept in antibody engineering: developability, meaning that they not only have the right combination of biological properties, but are also manufacturable. Residues that may oxidize or glycosylate antibodies during manufacture should be removed. These antibodies are designed to provide better stability, better folding, and higher yields. Using fed-batch culture, the yield is generally 2-5 g/L.
The part that can be engineered in the antibody is the Fc region. All four IgG subtypes have different interactions with different Fc gamma receptors, and have the ability to activate and inhibit the function of these receptors. Researchers are trying to exchange or mutate a part of these areas. Similarly, antibody re-use can be adjusted by engineering regions that interact with FcRn.
Some tumors and pathogens produce proteases that cleave the hinge structure of the antibody, making it less able to induce ADCC or complement cascades. Therefore, it is necessary to modify this region of anti-tumor or anti-bacterial antibodies so that it is not susceptible to proteolysis, Bramhill said.
Of course, the therapeutic affinity reagent does not have to be a standard IgG. Currently, at least three Fab segments are licensed. They block receptors but do not cause Fc-related effector functions. In one approach, a Fab2 fragment (two Fab regions are chemically linked, but no Fc) can be used to crosslink the target. The two Fab regions do not need to recognize the same epitope. This bispecific antibody recognizes both tumor-associated antigens and T cell receptors. In addition, an antigen recognition domain can be added to make it a trivalent antibody.
There are many goals that people need to target. “The key is to find the wonderful 'best combination,'” Kang said. The combination of drugs, mono- and bispecific antibodies, and effector functions is really a specific analysis of the specific problem, and the situation is different.
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