By Sharon Yang, Cell Biology, ‘20
Author’s Note: I first came across an article talking about this new innovation on Science X. Having worked with hybridomas and antibodies through various internships, I was deeply intrigued by this discovery and secured an original paper to learn more about its potential applications. Because of the revolutionizing usage of antibodies in the medical field, it is vital to understand how this finding will facilitate antibody-based therapies in clinical research.
Since the discovery of antibodies and their applications in therapeutics, many diseases once deemed incurable now have a treatment, if not a cure. Antibodies are proteins that recognize and bind to specific antigens (proteins that are considered “foreign” to the body). The immune system recognizes this antibody-antigen complex and removes the foreign substance from the body. Monoclonal antibodies (mAbs) are specific for one type of antigen and are produced using hybridomas, immortal cell lines that secrete only one type of antibody. The specificity of a mAb is determined by its antigen binding variable region. Though the variable region is of critical importance, the constant region (also known as the Fc region) is also essential to the therapeutic efficacy of mAbs. The Fc region has many different variants, called isotypes. Each isotype has its own unique function in making the immune system respond in different ways. After an antibody binds to an antigen by its variable region, the Fc region of the antibody elicits a response from the immune system, which serves as the basis for antibody-based therapeutics.
A recent study conducted in the summer of 2019 by Schoot and colleagues demonstrates how the use of genetic engineering on hybridomas can modify the Fc region of mAbs to that of a different species, isotype, or format. This new versatile platform grants ease of production of monoclonal antibodies that have different constant regions but retain the same variable regions.
The research team utilized a one-step clustered regularly interspaced short palindromic repeat (CRISPR)/homology-directed repair (HDR) technique to create a recombinant hybridoma that secretes a mAb in the Fc format of choice — a highly attractive alternative to the conventional recombinant production methods that were often time-consuming, challenging, and expensive.
As the team emphasizes, “[CRISPR/HDR] is a simple alternative approach requiring a single electroporation step to obtain an unlimited source of target antibody in the isotype format of choice” (1). Through using CRISPR/HDR, the team was able to seamlessly generate monovalent Fab’ fragments and a panel of different isotypes for the same monoclonal antibody.
CRISPR/Cas9 and Homology-Directed Repair
In their genetic engineering method, the researchers took advantage of an ancient bacterial immunity mechanism: the CRISPR/Cas9 system. When a bacteria is invaded by a virus, the bacteria stores snippets of viral DNA and creates segments of DNA called CRISPR arrays. When a virus with the same DNA segment attacks again, the bacteria creates RNA from the CRISPR arrays to target the virus; the RNA is called the guide RNA. The nuclease protein Cas9 is used to cut the DNA apart at a very specific site determined by the guide RNA, disabling the virus. CRISPR/Cas9 works in a similar fashion in the lab. Scientists create a guide RNA that binds to Cas9, which then targets a specific site on the DNA to be cut (2).
When CRISPR/Cas9 cuts DNA, it induces a double-strand break (DSB). Homology-directed repair (HDR) occurs when the intact donor strand contains high sequence homology to the damaged DNA strand. Through HDR, scientists can integrate a sequence or gene of their liking into the genome, which Schoot and colleagues perform in their study (3).
The Generation of Fab’ Fragments
The fragment antigen-binding (Fab’) is a region on the antibody that binds to the antigen. It consists of a single heavy chain and light chain. To create a Fab’ fragment-secreting hybridoma using CRISPR/HDR, the team selected NLDC-145, a hybridoma clone that secretes mAbs of rat IgG2a (rIgG2a) isotype. The antigen of rIgG2a is DEC205, an endocytic receptor found on immune cells. The team electroporated NLDC-145 cells with Cas9 and an appropriate guide RNA to induce double-strand breaks at the hinge region; to repair the double-strand break, they designed an HDR Fab’ donor construct for homology-directed repair. The HDR Fab’ donor construct also inserts specific tags onto the protein, allowing for easy purification of the Fab’ fragment.
To test secretion of the Fab’ fragment, they stained JAWSII, a DEC205-expressing cell line, with the supernatants of NLDC-145 clones that had undergone CRISPR/HDR. Flow cytometry assays showed that a large portion of Fab’-secreting hybridomas were successfully created. Further assays showed that the secreted Fab’ fragments retained their binding capabilities. It is worth noting that the researchers also used the same strategy to convert other hybridoma lines to become recombinant, Fab’-producing lines, with similar success; this demonstrates that this engineering technique is flexible and not just limited to one cell line (1).
The Generation of Isotype Panels
In a similar manner to creating monoclonal Fab’-generating hybridomas, the team also used the one-step CRISPR/HDR technique to create hybridomas capable of producing a wide array of isotype variants for the same mAb. This time, the cell line subject was hybridoma MIH5, which secretes monoclonal rIgG2a that targets mouse PD-L1, an immune checkpoint protein. The goal was to make clones of MIH5 to each produce one isotype of the chimeric (having both rat and mouse-related parts) monoclonal antibodies: mIgG1, mIgG2a, mIgG2b, mIgG3, mIgA, and a mutant form of mIgG2a (mIgG2asilent).
MIH5 cells were cotransfected (introduced with DNA) with a Cas9 vector containing the appropriate guide RNA and a construct from a panel of isotype HDR donor constructs (each isotype had its own unique HDR donor construct). Following knock-in integration, flow cytometry analysis showed that the engineered chimeric mAbs were successfully secreted. Thus, the creation of recombinant hybridomas for a panel of isotypes was successfully engineered (1). This invention allows for the creation of monoclonal antibodies with different Fc regions, providing researchers an easy way to “customize” their antibodies to elicit a specific response from the immune system. Researchers may choose which isotype variant they want on their antibody, which is fully dependent on their target (antigen) of interest and how the immune system behaves towards it. This has vast potential in antibody-based therapeutics, in that this system can be used for the optimization of potential drugs to become more potent and dynamic.
To test the functional capability of isotype-switched mAbs, Schoot and colleagues tested the antibodies’ capability to induce an important immune mechanism: antibody-dependent cellular cytotoxicity (ADCC). In order to test ADCC in vitro, mouse colon adenocarcinoma cells were labeled with chromium-51, and then taken in by MIH5 Fc isotype variants. After adding whole blood, they measured chromium-51 release. On the other hand, B cell depletion by MIH5 Fc variants was used to measure ADCC in in vivo experiments. Analyses of these studies show that chimeric mAbs created by CRISPR/HDR hybridomas have the same biochemical and immune effector characteristics as their recombinant and naturally occurring counterparts (1). Something to highlight is that instead of treading through the laborious process of producing recombinant antibodies in the conventional way (often consisting of multiple rounds of optimized sequencing, cloning, transfection), this one-step mechanism grants smooth and rapid generation of recombinant antibodies that perform their expected functions (1).
The ability to create monoclonal antibodies with the freedom to choose what goes on their constant regions possess many applications in the vast field of medicine and engineering. Being able to construct a very specific monoclonal antibody (the engineering element) that stimulates the immune system in a certain, beneficial way (the medical component) intertwines the two fields together to propel us closer towards treating diseases more efficiently and effectively. This system also represents an optimized version of recombinant engineering, which saves valuable time and funds that can be used towards conducting further studies. A simple, yet powerful and flexible approach, this versatile CRISPR/HDR platform aims to facilitate antibody engineering and research for the scientific community, and is accelerating the rate at which new clinical trials can be performed.
- Schoot, J. M. V. D. et al. Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering. Science Advances 5, (2019).
- Ran, F Ann et al. “Genome engineering using the CRISPR-Cas9 system.” Nature protocols vol. 8,11 (2013): 2281-2308. doi:10.1038/nprot.2013.143
- Cortez, Chari. “CRISPR 101: Homology Directed Repair.” Addgene Blog, Addgene, 12 Mar. 2015, blog.addgene.org/crispr-101-homology-directed-repair.