Media was additionally supplemented with 50 M 2-mercaptoethanol (2-ME) (M6250, Sigma-Aldrich). Cells were kept at 37 C in a 5% CO2humidified incubator, and passaged three times per week. heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab molecule (DTFab), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab with two distinct cargos in a site-specific manner. This technology platform will be useful in the development of multimodal imaging brokers, theranostics, and next-generation ADCs. == Introduction == The use of antibodydrug conjugates (ADCs) has emerged as a potent strategy in the treatment of malignancies. As of late 2020, nine FDA-approved ADCs19are used in the clinic, and several hundred are currently under clinical inestigation.10First- and second-generation ADCs are classically produced by conjugation of drug molecules to the side chains of solvent-exposed lysines or interchain cysteines.11However, such approaches lead to highly heterogeneous end-products with variable molecular weights, drug coupling sites, and drug-to-antibody ratio (DAR), with the concomitant risk of influencing target binding affinity.12Indeed, monoclonal antibodies (mAbs) typically contain more SB269970 HCl than 60 accessible lysines, whereas the drug-to-antibody ratio (DAR) should remain low enough (34) to prevent aggregation.13,14Third-generation ADCs aim to address these challenges by using site-specific conjugation methods.11,12As opposed to random coupling, site-specific modification enables rigid control over payload conjugation to generate a homogeneous product. Antigen-binding fragments (Fab) are molecules derived from mAbs.15Their heavy chain (HC) is truncated to solely contain the variable domain VH and the constant domain CH1, enabling association with the light chain (LC), but lacks the CH2 and CH3 domains that dimerize to generate the Fc domain. While these Fab retain binding ability to their target, they do not exhibit Fc-mediated immune effector functions such as recruitment of effector cells, or fixation of complement.16Moreover, they have a shorter half-life in circulation,17,18and are more efficient at penetrating dense tissues in which conventional mAbs are excluded.17,19However, the probability of modifying the binding region of a Fab using classical stochastic labeling is higher than on full-size mAbs, due to the smaller size and reduced number of reaction sites.20Thus, Fab fragments represent attractive proof-of-concept candidates for third-generation ADCs, as well as for imaging and thera(g)nostic21applications. Functionalization SB269970 HCl of antibody fragments with distinct payloads is Rabbit Polyclonal to BRCA2 (phospho-Ser3291) an attractive strategy in for several applications. While combination therapies are gaining more attention in chemotherapeutic treatments, classical ADCs target only one drug to cancer cells. Similarly, multimodal imaging enables the visualization of targets of interest in different scales, from whole body imaging with radioisotopes down to the histological level with fluorescent tracer molecules. These applications would benefit from the development of a flexible plug-and-play antibody fragment engineering platform for dual site-specific labeling. Most site-specific conjugation strategies make use of a SB269970 HCl short peptide tag (e.g., a sortase A recognition motif22) or engineered residues11,23to introduce cargos. Thus, they only permit functionalization with multiple distinct payloads through the synthesis of orthogonal multivalent linker systems or multifunctional conjugates, with concomitant synthetic and potential solubility SB269970 HCl issues. Here, we report a widely applicable strategy to introduce two orthogonal site-selective labeling tags on a Fab fragment by capitalizing on our recently reported Clustered Regularly Interspaced Short Palindromic Repeats/Homology Directed Repair (CRISPR/HDR) hybridoma genomic engineering approach.24In this work, we expand the genomic engineering toolbox to enable modification of the HC and LC loci of the mouse IgG1 (mIgG1) hybridoma, available for a plethora of targets. SB269970 HCl With this, dual-tagged Fab (DTFab) are generated equipped with two distinct sortase A recognition motifs (sortags) on the HC and LC, each orthogonally recognized by a specific variant of the evolved sortase A (eSrtA) enzyme (eSrt2A-9 or eSrt4S-9).25These enzymes enable the ligation of virtually any payload bearing a synthetically easily accessible N-terminal polyglycine motif onto the target protein. To demonstrate feasibility, the DTFab were sequentially functionalized with two distinct cargos in a site-specific manner, and thoroughly characterized. We expect that this technology platform will be a valuable asset in the development of Fab conjugates for imaging, theranostics, and next-generation ADC applications. == Results == == Generation of a Genetically Engineered Cell Line Secreting Fab Fragments == To produce Fab fragments suitable for dual site-specific labeling, we sought to alter the immunoglobulin domain within the genome of an anti-hCD20-producing hybridoma cell line, which expresses a mAb of the mIgG1 isotype with a LC (C). We hypothesized this could be achieved by adapting our recently developed CRISPR/HDR approach, 24in which we genetically engineered the rat IgG2a IgH locus, for modification of the murine IgH and IgK loci. We started with the genetic modification of the HC (Figure1A and B). To this end, we selected a guide RNA (gRNA-H.m1) that directs the Cas9 protein to the CH1 region of the mIgG1 IgH locus.26The HDR template consists of 600 bp 5 and 3 homology arms (HA) flanking the intended modification site in the hinge region of the HC. It was designed to insert.