Molecular engineering of antibodies for site-specific covalent conjugation using CRISPR/Cas9

Abstract

Site-specific modification of antibodies has become a critical aspect in the development of next-generation immunoconjugates meeting criteria of clinically acceptable homogeneity, reproducibility, efficacy, ease of manufacturability, and cost-effectiveness. Using CRISPR/Cas9 genomic editing, we developed a simple and novel approach to produce site-specifically modified antibodies. A sortase tag was genetically incorporated into the C-terminal end of the third immunoglobulin heavy chain constant region (CH3) within a hybridoma cell line to manufacture antibodies capable of site-specific conjugation. This enabled an effective enzymatic site-controlled conjugation of fluorescent and radioactive cargoes to a genetically tagged mAb without impairment of antigen binding activity. After injection in mice, these immunoconjugates showed almost doubled specific targeting in the lung vs. chemically conjugated maternal mAb, and concomitant reduction in uptake in the liver and spleen. The approach outlined in this work provides a facile method for the development of more homogeneous, reproducible, effective, and scalable antibody conjugates for use as therapeutic and diagnostic tools.

Figure 1

Illustration of CRISPR/Cas9 genome editing approach of hybridoma cells for site-specific modification of antibodies. Hybridoma cells were modified by co-transfection with plasmid expressing sgRNA and Cas9, and linearized HDR repair plasmid. After 48 hrs, GFP-positive cells were isolated using fluorescence-activated cell sorting (FACS) into 96 well plates and cultured. Clones were analyzed for incorporation if inserted tag by gel and sequence analysis.

Figure 2

Schematic diagram and characterization of CRISPR/Cas9 genome editing of hybridoma cells for site-specific modification of antibodies. C-terminal end of Ig gamma-2B gene from (a) rat chromosome 13q24, (b) Fcgr2b locus, was modified by (c) homology directed repair to incorporate sortase and flag tags. sgRNAs with best On-Target/Off-Target scores were selected in the region near the IgG2b stop codon. (d) Anti-ICAMIgG2b antibodies were generated incorporating Sortase and FLAG tags at their C-terminal end. Confirmation of insert was performed by (e) Agarose gel analysis of PCR fragments from positive and negative clones. (f) Verification of integration in positive clones by sanger sequencing. (g) Coomassie blue stained gel of three positive clones. (h) Western blot of a positive clone under reducing and non-reducing conditions, stained with anti-Flag-HRP.

Figure 3

Conjugation efficiency and biodistribution analysis of CRISPR-modified antibody. (a) Coomassie blue stained gel of sortase-mediated conjugation of modified antibody to GGG-fluorophore. (b) Fluorescence scan of the antibody-fluorophore, stained with anti-Flag-HRP, showing the antibody conjugated fluorophore (red tab) compared to free fluorophore (black tab). (c) HPLC fluorescence trace antibody-fluorophore conjugates at different molar ratios. (d) Quantitative analysis of the percent of antibody modification. (e) Binding of ¹²⁵I-labeled CRISPR-modified antibody to REN-ICAM and REN-WT cells. (f) Biodistribution of ¹²⁵I-labeled CRISPR-modified anti-ICAM mAb in mice at 30 min. Tissue uptake is indicated as mean ± SEM (n = 3). Biodistribution analysis was performed comparing ¹¹¹In-labeled site-specific CRISPR/Cas9- to chemically-modified mAb. (g) Biodistribution of ¹¹¹In-labeled anti-ICAM mAb modified in mice at 30 min. Tissue uptake is indicated as mean ± SEM. (h) Localization ratio of selected organs. Significant differences were determined by t-test with Bonferroni correction to account for multiple comparisons.

Figure 4

Comparison of the pulmonary targeting of various ICAM-1 directed agents. Lung Biodistribution, calculated as percent injected dose per gram (%ID/g) of tissue. Control Isotype – purified mouse IgG2b antibody, PVP - 100-nm polyvinylphenol particles, liposomes - 150-nm PEGylated immunoliposomes, and FNP - ferritin nanoparticles^,–.