The development of therapeutic monoclonal antibodies is continually expanding, and these immunotherapeutics find broad applications in the treatment of various diseases. In the field of oncology, their significance is underscored by the numerous antibodies available for treating various types of cancers, with many more antibodies currently undergoing different stages of clinical trials (Kaplon et al., 2022). Monoclonal antibodies also serve as potent agents for treating various other disorders, including chronic inflammation, cardiovascular diseases, transplantation-related issues, and infectious diseases (Carter, 2006). Radioconjugated antibodies prove effective not only for in vivo imaging but also for tumor therapy, and when combined with other therapeutics, they can enhance anti-tumor activity (Bethge and Sandmaier, 2005). Radioimmunoconjugates possess several characteristics that enable real-time detection of antigens, changes in antigen expression, and their heterogeneity. These features facilitate the precise dosing of the drug required for an effective anti-tumor response and provide pharmacokinetic information about the antibodies (Parakh et al., 2022). Furthermore, this class of immunotherapeutics has demonstrated its clinical effectiveness due to several advantages, including biochemical stability, high specificity, selectivity, and a lower incidence of side effects compared to other therapeutic classes.

Initially, monoclonal antibodies were produced using hybridoma technology, which involves the fusion of a myeloma cell line with spleen cells from an immunized animal (Köhler and Milstein, 1975). While antibodies generated through hybridoma technology have led to important tools in research, diagnosis, and immunotherapy, clinical applications of mouse-derived monoclonal antibodies are limited, primarily due to the human anti-mouse antibody (HAMA) response triggered by the human immune system (Renner and Stenner, 2018). To address this limitation, various humanization strategies were developed to reduce the HAMA response. In the humanization process, the murine constant domains are replaced with their corresponding human counterparts, and the complementarity-determining regions (CDRs) hypervariable loops from the VH and VL domains, responsible for antigen recognition and binding, are grafted onto a homologous human beta-sheet framework (Jones et al., 1986). In most cases, the CDR-grafting procedure leads to a significant reduction in antibody affinity to its antigen compared to the parental antibody, necessitating the use of affinity maturation to restore binding strength. As a result, these humanization strategies are time-consuming.

Another pivotal advancement in antibody technology was the development of antibody fragment phage display (McCafferty et al., 1990), which opened up the possibility of generating fully human antibodies (Marks et al., 1991) with therapeutic applications. Phage display is a powerful technique for displaying diverse molecules on the surface of a filamentous bacteriophage. Consequently, phage display is employed in studying protein-ligand interactions, antibody-receptor binding, monoclonal antibody epitope mapping, selecting enzyme substrates, and screening combinatorial peptide, protein, and antibody libraries (Bazan et al., 2012; Anand et al., 2021; Molek et al., 2011). In the realm of immunotherapy, phage display is utilized to select antibody fragments from a human combinatorial naive library with high selectivity and specificity against a given antigen. The most common antibody format in phage display selection is the single-chain Fv (scFv) fragment, in which the VH and VL domains are linked by a short peptide. Once the scFv fragment is selected for high affinity, selectivity, and specificity against an antigen, it must be reformatted. The scFv fragment should be reformatted into a larger structure, such as the complete IgG molecule, with a native-like conformation to be applicable in preclinical trials and disease treatments. While various expression systems exist for IgGs, clinical-grade full-length antibodies necessitate a mammalian expression system to ensure proper folding and glycosylation, preserving the effector functions of IgG.

The primary objective of this study was to create several mammalian expression vectors for producing high-quality, clinical-grade functional antibodies with applications in clinical trials, immunotherapy, and in vivo imaging. Specifically, we designed several generic expression vectors that can be utilized to reformat scFv fragments into complete IgG1 with a native-like structure. These cloning vectors for the expression of full-length human antibodies are based on the IgGγ1 structure, with options for lambda and kappa light chain isotypes for therapeutic antibody production. Additionally, two reformatting vectors were developed to produce antibodies for use in in vivo imaging. These constructs incorporate several mutations in the lower hinge region and CH2 domain to inhibit interaction with Fcγ receptors, effectively blocking engagement with immune cells, which is unnecessary for imaging. This expression system was tested with an anti-MT1-MMP IgG1/λ and anti-TACE IgG1/κ and evaluated for protein expression in transiently and stably transfected HEK-293 and CHO-K1 cells. In addition to extensive biochemical characterization, the antibodies were also assessed in functional assays.