Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering

Abstract

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab′ fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering

“…In this study, we present for the first time a strategy to generate dual-tagged Fab′ fragments bearing two orthogonal site-specific modification tags (DTFab′), which can readily be utilized for the chemoenzymatic conjugation with two distinct cargos. Extending on our previous work, 24 we demonstrate the successful engineering of a hybridoma secreting mIgG1 antibodies (anti-CD20 WT) to a stable daughter cell line producing Fab′ fragments carrying an eSrt2A-9 (LAETGG) motif on its HC, and an eSrt4S-9 (LPESGG) motif on its κ LC (anti-CD20 DTFab′). This method enables robust biorthogonal engineering of virtually any antibody-secreting hybridoma, to reproducibly produce high yields of modified DTFab′ molecules.…”

“…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 κ LC (Cκ). We hypothesized this could be achieved by adapting our recently developed CRISPR/HDR approach, 24 in 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 ( Figure 1 A and B).…”

“…Nucleofection of the HDR template and CRISPR-Cas9 vectors was performed as described previously. 24 Briefly, hybridoma cells were assessed for viability, centrifuged (90 g, 5 min), resuspended in phosphate buffered saline (PBS)/1% FBS, and centrifuged again (90 g, 5 min). 1 × 10 6 cells were resuspended in 100 μL of SF medium with 1 μg of HDR template and 1 μg of Cas9 vector, or 2 μg of GFP vector (control) and transferred to cuvettes for nucleofection with the 4D-Nucleofection System (V4XC-2024, Lonza, CQ-104, Program SF).…”

“…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. 24 In 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. 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).…”

Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system

“…In the present study, we evaluated the feasibility of using the Cas9 RNP in combination with single strand donor DNA to obtain a stable hybridoma cell line able to express and secrete protein of diagnostic interest into the culture medium.The hybridoma cell line 3E1 was chosen for use in the present study because of the high production yield (more than 1 mg/mL) of the specific mAb when grown in a high-density stationary culture system (CELLine bioreactor flask) (Moreno et al, 2019). The specificity of the CRISPR/Cas9 system allowed us to insert a coding sequence of interest into a precise region of the hybridoma genome, thereby avoiding random integration (Smith et al, 2014). Targeted insertion of a coding sequence into the genome can considerably increase the protein expression level compared with random integration (Lee et al, 2015).…”