In 2019, the U.S. Environmental Protection Agency(EPA) announced that it would stop conducting or funding studies on mammals by 2035[1]. A year later, the European Commission also recommended developing non-animal-derived antibodies, urging government authorities, funding agencies, and publishers to endorse the use of these antibodies to improve scientific reproducibility[2]. There is no denying that the animal-friendly concept will soon become mainstream in the scientific community.
To reduce animal use in antibody production, non-animal-derived antibodies, which are mainly produced by display technologies, have been developed to replace animal-derived antibodies, which are primarily produced by hybridoma technology. However, display technologies have important limitations when it comes to generating antibodies against native structures like viruses, not to mention the demanding requirement of performing the technique[3]. On the other hand, animal-derived antibodies provide more information about how the body will react to a virus[4], and hybridoma technology is also relatively easier to be carried out. As a result, there is still a necessity for the existence of animal-derived antibodies.
Hybridoma technology is the primitive, most fundamental, and successful methodology in monoclonal antibody(mAb) isolation[5]. It is widely used compared with other methods because once hybridoma clones are well established, the following procedure to produce mAbs will be easy to start. Moreover, hybridoma technology depends on B cells that undergo a natural antibody maturation process, where somatic hypermutations(SHM) diversify variable regions, and the class switch recombination(CSR) matures constant regions of antibodies. These two natural processes are not possible in other mAbs isolation methods, making hybridoma a unique way to produce naturally developed in vivo antibodies in the laboratory.[6]
Hybridoma cells are generated via fusion between a short-lived antibody-producing B cell and an immortal myeloma cell.[7] The following is the process of mAb generation by the Hybridoma technology. First, we will induce myeloma and B cell fusion with Polyethylene glycol(PEG) and electrofusion. After that, we will conduct Hybridoma screening because even in the most efficient hybridoma fusions, only about 1% of the starting cells are fused, and only about 1 in 105 viable form hybrids. The cells will be cultured in HAT(hypoxanthine-aminopterin-thymidine) medium, and only the hybridoma cells can divide and increase in the HAT medium because the genome from the B-lymphocyte makes them HGPRT(hypoxanthine-guanine phosphoribosyl transferase) positive and cells containing a non-functional or lacking HGPRT protein will die in HAT medium. Finally, Hybridoma cells would be able to produce mAb in vitro and in vivo.
Monoclonal antibody(mAb) is well known for binding with specific domains of targeted antigens [7]. Monoclonal antibodies are monospecific and produced by identical B cells having high affinity and specificity towards a single epitope of an antigen. The difference between mAb and Polyclonal antibodies(pAbs) is that pAbs are a pool of immunoglobulin molecules secreted by different B cell lineages and react against multiple epitopes of a specific antigen[8].
Since mAb has higher affinity and specificity, mAb is widely used in treatment, diagnosis device development, and research techniques. First, mAb can be used to treat cancer, inflammatory and autoimmune disorders, nervous system disorders[4], and infections, including COVID-19. Regarding mAb application in research, enzyme-linked immunosorbent assay(ELISA) and western blot are ultimately required for the monoclonal reagent. IgG monoclonal antibodies are generally preferred because they are less prone to degradation and may be more helpful.[9]
During the original hybridoma production, we must inject the antigens into the mice's bodies to obtain the required B cell. Therefore, we started brainstorming about the process of generating B cells and combined synthetic biology into our project.
The generation of B cells includes two essential stages. The first one is SHM. In this stage, the gene on the variable region of B cells will be mutated to optimize the antibodies' affinity. The second one is CSR. In this stage, the gene in the constant region will be mutated, forming different antibodies with different functions. Regardless of the mechanism, the activation-induced cytidine deaminase(AID) plays a dominant role in these stages to induce random mutations in gene sequence from cytidine to uridine and help change the conformation of antibodies.
This is the docking result between the spike protein of SARS-CoV2(red) and the antibody variable region(blue). The antibody is generated by our original hybridoma, which has not been transfected with the AID gene. The CDR3 on the antibody variable region can interact with the RBD of the spike protein. If we transfect the AID gene into the hybridoma, the AID will mutate the variable region and change the conformation of the antibody, increasing the affinity.
As a result, we transfected AICDA, the gene of AID, into hybridoma cells with lentivirus and devise a method to shut the function of CSR down. Meanwhile, we added the Tet-On system into our construct to regulate the expression of AICDA to prevent hybridoma cells from dying of excessive mutation. We want to execute the SHM process in vitro to improve the affinity of monoclonal antibodies and eliminate the time-consuming step of inducing immunoreaction in mice's bodies. In the meantime, these changes can also offer scientists and biotechnology companies with hybridoma cells off the shelf a solution to respond to the trend of reducing laboratory animals.
Apart from improving the affinity of mAbs, we designed and made a bioreactor aiming to simplify the procedure of mAbs' mass production. In this bioreactor, we set Chinese hamster ovary cells(CHO cells) as target cells for culturing and revising the commonly used cell retention microfluidic device. Combining the microfluidic system with a biomimetic design to help sort CHO cells and antibodies, our bioreactor can hopefully increase the recovery rate of CHO cells and the collection rate of antibodies.
We hope our project can optimize the entire procedure of the production of monoclonal antibodies, from hybridoma technology to the latter stage of mass production, and contribute to the scientific research and biotechnology industry.
