Hybridoma Technology

Monoclonal custom antibodies were engineered for the first time by Kohler and Milstein in 1975 by employing ‘hybridoma technology’ (Singh et al., 2018). The revolutionary hybridoma technology involves injecting a particular antigen into a mouse and then isolating antigen-specific plasma cells from the animal’s spleen. The procured plasma cell is then immortalized by fusing it with a cancer cell. The resulting hybrid cell is then cloned to produce identical daughter cells, which produce the immunoglobulin of interest. Only murine antibodies, i.e., antibodies derived from mice, for example, tositumomab, tiuxetan, and ibritumomab were initially produced by this method. However, these murine antibodies can produce strong immune reactions in humans, especially on repeated administration. Therefore, bioengineered and recombinant antibodies were prepared for successful administration in humans. Chimeric antibodies prepared as a result comprised VL and VH from mice and CH1, CH2, and CH3 from the human. Some of the chimeric antibodies include rituximab and cetuximab.

Further reduction of murine content led to the formation of ‘humanized mAbs.’ A major part of humanized antibodies is human-derived, with only one CDR derived from mice. Eventually, entirely human custom antibodies were prepared by employing either two technologies, i.e., phage display or transgenic mice (Ruker and Knopp. 2021). 

Applications

Use of Monoclonal Antibodies in Cancer Therapy

Zahavi and Weiner (2020) reviewed that monoclonal custom antibodies against antigens unique to cancer cells can cause tumor cell death by numerous mechanisms. A direct mechanism is the induction of tumor cell death by directly blocking the growth factor receptor signaling. When monoclonal antibodies bind to their specific ‘growth factor receptor,’ the survival signals are interrupted, thus manipulating their activation state or blocking their binding to the ligand. For instance, in different types of cancers, the overexpression of EGFR (Epidermal Growth Factor Receptor) occurs, and this overexpression signals tumor cell proliferation. An anti-EGFR mAb, cetuximab, dimerizes the receptor and blocks ligand binding, ultimately resulting in tumor cell apoptosis.

Similarly, Human Epidermal Growth Factor Receptor 2 (HER2) is another type of tyrosine kinase receptor overexpressed in ovarian and breast cancers. This receptor does not bind to any ligand and rather hetero-dimerizes with the growth factor. Therefore, custom monoclonal antibodies, Trastuzumab, are used for the treatment of such cancers. It suppresses the overexpression of HER2 and ultimately causes tumor cell apoptosis. 

Indirect ELISA using Monoclonal antibodies

Mai et al. (2020) studied how custom-made monoclonal antibodies against Photorhabdus-related toxins PirAVp and PirBVp released from Vibrio species show immunoreactivity in indirect ELISA. PCR-based methods are used to detect PirA and PirB genes in AHPND (Acute hepatopancreatic necrosis disease) diagnosis. However, some bacterial strains containing these genes do not express the binary toxin. This lack of expression led to the flawed estimation of the virulence of PirAVp and PirBVp-containing strains. Therefore, immunoassay like ELISA is a promising approach in this regard. The researchers optimized the codons of recombinant PirAVp and PirBVp genes for protein expression in the E. coli expression system and then purified these recombinant proteins and used them for custom monoclonal antibody production. Followed by protein sequencing, they analyzed a total of forty custom antibodies, including twenty PirAVp mAbs and twenty PirBVp mAbs by Western blot. They concluded that four out of these forty show the strongest immunoreactivity in indirect ELISA.

Human Monoclonal Antibodies for Flow Cytometry

Nanamiya et al. (2021) developed an anti-Human CC Chemokine Receptor 9 monoclonal antibody for flow cytometry. The CCR9 receptor is related to the ‘beta chemokine receptors’ on the surface of immature T lymphocytes and enterocytes. The overexpression of this receptor occurs in various diseases like rheumatoid arthritis, diabetes type 2, colitis, etc. so, highly sensitive antibodies are required to forecast the onset of such diseases. Anti-CCR9 mAbs cannot be developed by a traditional method as CC chemokine receptor 9 is a highly unstable G-protein coupled receptor. Therefore, the researchers developed an anti-CCR9 mAbs by using a cell-based immunization and screening method. They obtained Chinese hamster ovary cell lines (CHO)-K1 and P3U1 cell lines, a leukemia T-lymphoblast cell line (MOLT-4), and purchased anti-hCCR9 mAb and conjugated anti-mouse IgG. The experimenters took six weeks old BALB/c female mice and housed them under pathogen-free conditions. Throughout the 28 days duration of the experiment, the mice were regularly monitored for health, and loss of up to 25% of total body weight was regarded as a humane endpoint. Mice were euthanized, and death was confirmed by respiratory and cardiac arrest. To develop anti-hCCR9 mAbs by cell-based immunization and screening method. Two mice were injected by intraperitoneal route with 1×108 CHO/hCCR9 cells. Three additional immunity boosters were provided through the immunization procedure, and then they injected a final dose two days before spleen cell harvesting. The procured spleen cells were fused with P3U1, and the resulting hybridomas were cultured in an RPMI medium supplemented with aminopterin, thymidine, and hypoxanthine. The cultures were centrifuged, and the supernatants were analyzed by flow cytometry. They concluded that the CBIS method could more rapidly generate anti-hCCR9 mAb as compared to the traditional method.

Strengths and Limitations

Custom monoclonal antibodies show high homogeneity, i.e., batch-to-batch reproducibility and high specificity to a single epitope of a particular/target antigen. They yield low background noise and are sensitive to protein quantification assays. Yet, a potential disadvantage of monoclonal antibody therapy is that a high dosage of mAbs can form circulating immune complexes between ADAs and therapeutic or custom antibodies that might be harmful. Monoclonal antibody production is expensive, and when labeled, they become more prone to binding alterations.