Immune checkpoint inhibitors in the form of monoclonal antibody drugs have emerged as efficacious anti-cancer immunotherapies. The first immune checkpoint blocker, ipilimumab, a monoclonal antibody targeting CTLA-4, was FDA-approved in 2011. Since then, several other antibody drugs have entered the clinic targeting other checkpoint molecules, such as PD-1 (e.g., Pembrolizumab, Nivolumab, and Cemiplimab) and PD-L1 (e.g., Atezolizumab, Avelumab, and Durvalumab).
CTLA-4 and PD-1 are T cell-expressed surface molecules that become upregulated upon T cell activation. As inhibitory checkpoints, CTLA-4 and PD-1 binding to their respective ligands, B7 and PD-L1, results in the activation of signaling pathways that serve to refrain T cell activity (Marin-Acevedo et al. 2021). Conversely, antibodies that specifically bind and block such inhibitory checkpoints help boost T cell activity.
“Mechanism of action of immune checkpoint inhibitors (ICIs). The binding together of MHC-peptide (peptide = tumor antigen) and TCR activates the anti-cancer pathway of T-cell–mediated immune response. The binding of CTLA-4 to its ligand and PD-1 to PD-L1, on the contrary, inhibit the anti-cancer pathway. The binding of monoclonal antibodies, such as ICIs, to CTLA-4, PD-1 or PD-L1 prevent the deactivation of the anti-cancer pathway.” Retrieved without modifications from Vani et al. 2020). https://creativecommons.org/licenses/by/4.0/
While highly effective in boosting anti-cancer immunity, not all patients respond to checkpoint inhibitor monotherapy (Marin-Acevedo et al. 2021). This observation has prompted investigators to use checkpoint blockers in combined immunotherapy modalities. For instance, multi-specific checkpoint blockers in the format of bispecific antibodies are providing new opportunities to improve the efficacy of these immunotherapies further.
There is continued interest and much effort being devoted to developing new antibody blockers targeting immune checkpoints. Although several antibody blockers for PD-1 are already approved and used clinically, new antibody leads may potentially offer improved properties to more effectively target this checkpoint alone or in combined formats. For example, a recent study by investigators at the Center of Excellence in Systems Biology, Chulalongkorn University, Thailand, led by Dr. Trairak Pisitkun, leveraged hybridoma technology, and duplex high-throughput screening to identify new PD-1 checkpoint blockers (Phakham et al. 2022). Their optimized approach could be easily extended to developing blocking antibodies to novel checkpoint targets.
Hybridoma technology was first developed in the 1970s, and it is a well-established approach that enables the development of highly specific monoclonal antibodies at high yields (Köhler and Milstein, 1975). Nevertheless, recognizing some factors limiting hybridoma-based antibody discovery success rate, scientists have implemented improved processes over decades. Such is the optimized approach followed by Phakham and colleagues, where specific improvements to immunization, fusion , and hybridoma selection and cloning enabled the identification of several blocking anti-PD-1 antibody leads.
First, Phakham et al. hyperimmunized mice with human PD-1 (hPD-1) either utilizing the purified protein (5 times) or CHO-K1 cells expressing hPD-1 (8 times), which were obtained from GenScript Biotech. Each approach was optimized to induce the best immune response. For instance, immunization with PD-1 protein through various routes (i.e., subcutaneous, intramuscular, and intraperitoneal) aimed at inducing humoral and cell-mediated responses. In contrast, the use of cells expressing hPD-1 enabled stimulating immune responses with the most native form of the PD-1 protein, that is, embedded on the cell surface and having appropriate conformation and glycosylation.
Next, the team opted to use the electrofusion technique because the fusion of B and myeloma cells through the standard polyethylene glycol (PEG) method has low efficiency. Electrofusion not only yields more hybrid cells but also favors their growth rate (Rems et al. 2013). Lastly, for hybridoma selection and cloning, Phakham and colleagues used a methylcellulose-based semisolid medium, improving cloning efficiency.
Screening is a labor-intensive phase in antibody discovery workflows. In order to identify the maximum number of clones producing blocking anti-PD-1 antibodies, the team opted for a combined approach. First, over 10,000 clone mini-pools were screened by ELISA, identifying over 50 producing high binders. Next, they implemented an efficient duplex high-throughput flow cytometry screen using cells expressing hPD-1 to identify high-binders having anti-PD-1 blocking activity. Overall, this approach enabled Phakham and colleagues to streamline the identification of five mini-pools producing blocking anti-PD1 antibodies. Subsequent subcloning and expansion enriched monoclones and supported antibody generation for functional characterization.
Four anti-PD1 monoclonal antibodies were shown to block the PD1-PD-L1 interaction and to have high binding affinity even in the subnanomolar range. The team went on to develop chimeras from the identified antibody leads by relying on GenScript’s services for grafting mouse variable region sequences to human IgG4/kappa constant regions and cloning the resulting chimeras into pcDNA3.4 expression vectors. Significantly, most of these chimeric antibodies demonstrated to have anti-PD-1 binding and blocking activities on par with Keytruda and Opdivo, two clinically used anti-PD-1 antibodies.
The therapeutic efficacy of checkpoint inhibitors depends on their ability to induce T cell activation. Therefore, to properly evaluate the newly identified anti-PD1 leads, GenScript conducted a Mixed Lymphocyte Reaction (MLR) assay. This in vitro functional assay relies on the co-culture of antigen-presenting cells and T cells to evaluate the effects of PD-1 blocking antibodies on T cell cytokine secretion. These studies successfully identified an anti-PD1 lead having T cell activating properties similar to Keytruda and Opdivo.
Ultimately, Phakham and colleagues have successfully developed an efficient workflow based on optimized hybridoma, cloning, and screening methods to streamline the discovery of new antibody drug leads. Some of the anti-PD-1 blocking antibody leads already discovered may provide opportunities for further development as candidates for clinical use.
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