Hybridoma Technology: the Preferred Method for Monoclonal Antibody

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ReportsExpert Opinion 2019FIRST DRAFT SUBMITTED: 29 04 2019; Hybridoma technology: the preferred method for ACCEPTED FOR PUBLI- CATION: 5 monoclonal antibody generation for in vivo applications 0 7 2019; Samantha Zaroff*,1 & Grace Tan1 PUBLISHED ONLINE: 00 00 0000 Prior to the development of hybridoma mAbs. In brief, the first stage involves technology, researchers relied on polyclonal the development and optimization of an antibodies (pAbs) for experiments involving immunogenic antigen (Ag) [3]. Next, a the identification and quantification of host animal is immunized with the Ag to specific proteins of interest within complex elicit an immune response and initiate biological environments. While pAbs the process of B-cell maturation [2,4]. The offered their own set of benefits, they could third stage involves the isolation of these B not be used for in vivo experiments or thera- cells from the spleen of the host animal and peutics due to their batch-to-batch incon- their fusion with myeloma cells to generate sistency and high levels of background hybridomas [5]. During the fourth stage, reactivity. It was not until 1975 when Köhler the generated hybridomas are subject to and Milstein used hybridomas to generate multiple rounds of screening and selection monoclonal antibodies (mAbs), that the in order to identify the hybridomas that worlds of antibodies and in vivo scientific produce the best mAbs for the intended research could meet for the first time [1,2]. downstream application. The fifth and The introduction of mAbs to in vivo research final stage is the amplification of these With ADCs and gave rise to some of the greatest scientific specific hybridomas and subsequent mAb © 2019 SAMANTHA ZAROFF targeted“ biologics achievements of the 21st century, including purification [3]. 2019 on the rise, there in vivo diagnostics, mAb therapeutics and is no telling what antibody–drug conjugation. While other COMMON IN VIVO astounding discovery technologies such as display libraries have APPLICATION OF mAbS will revolutionize mAb since been developed for mAb discovery, In vivo diagnostics therapeutics next. hybridoma technology remains the primary In vivo diagnostics are a noninvasive way for ” discovery platform preferred by in vivo clinicians to diagnose disease progression scientists. This editorial will address the through analysis of biomarkers within the benefits of using hybridoma technology for body rather than through biologic samples in vivo applications and describe some of inside a laboratory. Most antibody-based the major contributions that hybridoma- in vivo diagnostics are used for highly derived mAbs have made in in vivo research specific imaging. Some common imaging and therapy. methods include positron emission tomography (PET), magnetic resonance imaging WHAT IS HYBRIDOMA (MRI), fluorescent molecular tomography 3 TECHNOLOGY? (FMT) and ultrasound [6,7]. The main Hybridomas are cells formed via fusion difference between immune imaging and 67 between a short-lived antibody-producing standard imaging is that, rather than imaging B cell and an immortal myeloma cell. Each a large, nonspecific section of the body, a hybridoma constitutively expresses a large tagged antibody targets a precise location amount of one specific mAb, and favored for diagnostic imaging instead. This idea of hybridoma cell lines can be cryopreserved conjugating a full-length antibody or an KEYWORDS for long-lasting mAb production. As a result, antibody fragment to a nanoparticle, be it a antibodies • cancer • immunology researchers usually prefer generating radioisotope, fluorophore or positron emitter, • immunotherapy • in vitro models hybridomas over other mAb production would have not been possible without • reagents methods in order to maintain a convenient, hybridoma-based antibody discovery. It was 1GenScript USA, Piscataway, NJ 08854, USA; never-ending supply of important mAbs. only through hybridoma technology that *Author for correspondence: samantha. Hybridoma generation is a five-step fully natural mAb variable domains that did [email protected] process that takes advantage of a host not adversely impact a patient’s own

BioTechniques 67: 00-00 (September 2019) animal’s natural ability to generate immune system during an examination were 10.2144/btn-2019-0054 functional, highly specific, high-affinity discovered.

mAb therapeutics T-cell surface. Once the ScFv interacts with those CDRs causing an immune response. Compared with other biologics, mAbs are able the TAA, the T cell becomes activated and In vitro methods, such as display libraries, to maintain an extremely high affinity towards initiates an immune response directly can generate unnatural pairings of VH and their target. Due to this high affinity and speci- targeting the tumor. Novel forms of targeted VL and cause the resulting mAb to have high ficity, researchers began investigating the biologics are being discovered every day, immunogenicity, especially after unnatural therapeutic potential of mAbs as metabolic such as bispecific CARs and T-cell receptor AM and recombinant pairing with a standard activators, inhibitors and immunotherapy [13]. With ADCs and targeted IgG backbone [17]. modulators. While the first few US biologics on the rise, there is no telling what FDA-approved mAb therapeutics, such as astounding discovery will revolutionize mAb Constant domain activity muromonab-CD3, were generated solely in therapeutics next. SHM and GCV improve antibody affinity mice, it became evident that in order to avoid within the variable region; however, the immune rejection, future mAb-based thera- BENEFITS OF HYBRIDOMA constant domain of the antibody also has to peutics needed to undergo human- TECHNOLOGY FOR IN VIVO go through its own maturation process. ization [8–10]. Since the approval of APPLICATIONS Newly generated antibodies will generally muromonab-CD3 in 1986, the FDA has Natural affinity maturation be of the IgM isotype. In order to change their approved approximately 80 more mAb thera- Since hybridoma technology relies on isotype, antibodies will go through class peutics for diseases ranging from mature B cells taken from secondary switch recombination (CSR), a process that autoimmune disorders, to inflammatory lymphatic organs, purified mAbs from these facilitates isotype switching [18]. IgG is the diseases, HIV and cancer. Interestingly, cells will have already gone through multiple most common isotype used in biological despite the discovery of combinatorial display stages of stringent genetic modifications research and drug development; however, libraries in 1984 as an alternative mAb within the host animal. Natural affinity CSR can generate antibodies from all five discovery platform, the majority of these mAb maturation (AM) is a complex antibody isotypes in order to ensure that the antibody therapeutics were originally discovered using refinement process that occurs within the is equipped with the necessary effector hybridoma technology in either fully murine germinal centers of the host animal [14]. functions and different bio-distribution. or humanized mice. The reason for this During AM, DNA encoding the variable preference is likely attributed to the natural regions of antibody chains are diversified SUMMARY ability of the murine immune system to via somatic hypermutation (SHM) [15,16]. This editorial reviews the basics of generate highly specific mAbs that elicit Through SHM, mutations accumulate in the hybridoma technology, the common uses strong constant domain functionality with complementary-determining regions (CDRs) of mAbs in in vivo applications, and the limited immunoreactivity after humanization. of the variable genes, and the tightest technical benefits of using hybridomas to binders to the Ag are actively selected for, generate mAbs for these applications. Alter- Antibody–drug conjugates & targeted resulting in the production of antibodies with native methods for mAb discovery and biologics high affinity. In addition to SHM, several development, such as in vitro combinatorial Like in vivo diagnostics, mAbs can be conju- mammalian species (rabbits) and birds also display libraries and in vivo B-cell sorting, gated to almost any small molecule, be it a undergo gene conversion (GCV). GCV takes have also been successfully developed fluorophore or a drug, in order to deliver place when a dsDNA break occurs within the [17,19,20]. Display libraries rely on screening cargo to a specific target. Targeted thera- already rearranged V(D)J segments of an large libraries of randomized, synthetic peutics come in two flavors, antibody–drug antibody’s variable region and the break is variable VH and VL antibody fragments in conjugates (ADCs) and targeted repaired via a homologous recombination- order to select the tightest Ag binders, while biologics [11]. ADCs are formed when a mAb like mechanism, introducing affinity- B-cell sorting relies on identifying and or mAb fragment is conjugated to a small- increasing mutations in the process. sorting mature B cells from peripheral molecule drug for targeted delivery to a blood. Unlike hybridoma technology, these specific organ, tissue or tumor. These “magic Low level of immunogenicity methods rely heavily on recombinant mAb bullets” have revolutionized the field of Since hybridoma technology depends on the expression as the basis of mAb production. targeted drug delivery, especially for immunization of a host animal, the generated While each method certainly has their oncology and immunology. After the high-affinity antibodies will possess natural respective merits and drawbacks, success of ADCs, researchers took the idea pairing of variable light (VL) and heavy (VH) hybridoma technology, despite being a one step further and discovered a way of gene segments with a class-matched “senior” antibody generation methodology expressing antibody variable chains on the constant domain. During the process of in 2019, still prevails as the most preferred surface of living drugs, or biologics. One of humanization, all regions surrounding the method for discovering mAbs for in vivo the most popular forms of targeted biologics CDRs are modified in order to reduce the applications. With new discoveries made is chimeric antigen receptor T-cell therapy chance of the antibody causing a negative every day to enhance antibody discovery, (CAR-T) [12]. Unlike ADCs, CAR-T targets a immune response once inside a patient. the future looks very bright for adapting T cell to a tumor-associated antigen (TAA) Since the process of hybridoma-based mAb hybridoma technology to support a wide through single-chain variable fragment generation takes advantage of natural CDR range of in vivo applications. (ScFvs) recombinantly expressed on the development, there is very limited risk of

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