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.

Fig. 1. CRISPR/HDR engineering of hybridoma NLDC145 to obtain sortagable Fab′ fragments against DEC205.

(A) Schematic representation of the CRISPR/HDR approach to convert wild-type (WT) hybridomas to Fab′ fragment–producing cell lines. (B) The targeted IgH locus of NLDC-145 with the variable region (VH) and constant regions (CH1, Hinge, CH2, and CH3) annotated is shown. Cas9 is guided by gRNA-H to hinge region and creates a double-stranded break before the first cysteine. The double-stranded break is subsequently repaired by HDR via the donor construct consisting of a sortag and his-tag motif (srt-his), an internal ribosomal entry site (IRES), blasticidin-resistance gene (Bsr), polyA transcription termination signal (pA), and homology arms (5′ HA and 3′ HA). The first 10 amino acids of the hinge before and after successful CRISPR/HDR are indicated. (C) Three days after electroporation, PCR was performed on genomic DNA from WT and CRISPR/HDR-targeted population using primers 1 and 2 (B). Agarose gel shows amplicon of expected size exclusively for the CRISPR/HDR-targeted population, indicating the correct integration of the donor construct within the population. (D) After limiting dilution of CRISPR/HDR-targeted cells, a flow cytometry screen was performed on the supernatant of monoclonal cell suspensions. To this end, DEC205-expressing cells (JAWSII) were incubated with clonal supernatants in combination with secondary antibodies against his-tag (blue) or rIgG2a (red). Exclusive his-tag signal indicates production of Fab′ fragments. (E) Immunoblotting of the supernatant of clones 47 to 52 for his-tag (blue) and rat heavy and light chain (red) confirms flow cytometry results. (F) In competition assay, JAWSII were incubated with a serial dilution (150 to 20 ng/ml) of either purified Fab′DEC205srt (blue), αDEC205 mAb (red), or an isotype control (gray) in combination with fluorescently labeled αDEC205 mAb (1 μg/ml). Decrease in mean fluorescent intensity (MFI) relates to the increase in competition for DEC205 binding with fluorescent-labeled αDEC205 mAb. n = 3, mean ± SEM. (G) Fab′DEC205srt can be C-terminally functionalized with a fluorescently labeled probe [GGGCK(FAM)] by using sortase (3M srt)-mediated ligation. LC, light chain.

Fig. 2. CRISPR/HDR engineering of hybridoma MIH5 to obtain panel of isotype variants.

(A) CRISPR/HDR strategy to engineer rIgG2a hybridomas and obtain recombinant hybridomas secreting murine isotypes. (B) The targeted IgH locus of MIH5 with the variable region (VH), constant region 1 (CH1), and hinge annotated. Cas9 is guided by gRNA-ISO to the intronic region upstream of the CH1 is shown. The resulting double-stranded break is subsequently resolved via HDR through a donor construct, leading to an in-frame insertion of an splice acceptor (SA, gray), isotype of choice (yellow), a sortag and his-tag motif (srt-his, blue), an IRES, blasticidin-resistance gene (Bsr), and polyA transcription termination signal (pA) upstream of the native CH1. The insert is enclosed by homology arms (5′ HA and 3′ HA). (C) Three days after electroporation, DNA from CRISPR/HDR-targeted MIH5 populations is obtained for PCR with primer 3 and primer 2 (B). Agarose gel of the PCR product reveals amplicons of the correct size exclusively for the CRISPR/HDR-targeted populations. (D) After selecting monoclonal hybridoma for each isotype, the supernatant of each isotype modified clone was incubated with PD-L1–expressing target cells (CT26). Displayed plots demonstrate that supernatants exclusively contain the engineered isotype variant with a C-terminal his-tag, while the original rIgG2a mAbs is absent. (E) Purified MIH5 isotype variants were effectively labeled with the fluorescent probe GGGCK(FAM) using sortase-mediated ligation.

Fig. 3. Glycosylation profiling of MIH5 WT and Fc-silent variant using high-resolution native mass spectrometry.

Purified samples of WT MIH5 (A) and mIgG2a_silent (B) were treated overnight with PNGase F (orange) to remove existing glycans. Subsequently, these samples were compared to untreated samples (black) via high-resolution native mass spectrometry. The Pearson correlation coefficient between the two spectra over all ion signals is given in the top right corner. The molecule mass of untreated (black) and treated (orange) samples and the difference in leading mass (green) are given in the bottom right corner. m/z, mass/charge ratio.

Fig. 4. FcγR engagement of MIH5 isotype variants.

Representative sensograms (A) display interactions of MIH5 WT and MIH5-engineered mAbs (mIgG1, mIgG2a, mIgG2b, mIgA, and mIgG2a_silent) for immobilized mFcγRI, mFcγRIIb, and mFcγRIV at increasing concentrations. Binding to FcγR is expressed in resonance units (RU). SPR was performed on four different concentrations of immobilized FcγRs (1, 3, 10, and 30 nM) to determine affinity [K_d(M)] of the Fc variants for the different FcRs (B). n = 3, mean ± SEM. (C) Predicted antibody-dependent cellular cytotoxicity (ADCC) activity for each murine isotype variant on the basis of their differential affinity for FcγRs.

Fig. 5. Effector function of MIH5 isotype variants.

ADCC in vitro assay (A) to compare effector function of MIH5 isotype variants. MC38 cells were ⁵¹Cr-labeled, opsonized with different MIH5 isotype variants, and exposed to whole blood from C57BL/6 mice for 4 hours. Specific lysis was quantified by measuring ⁵¹Cr release. n = 3, mean ± SEM. *P < 0.05. (B) Experimental setup of in vivo depletion assay. Splenic B cells labeled with Violet and Red tracer dye were used as target cells for in vivo depletion. The violet B cells were opsonized with mIgG2a_silent variant, while the red B cells were opsonized with mIgG2a, mIgG2b, or mIgA variant. Subsequently, B cells were mixed 1:1 (top plot and diagram) and injected intravenously into C57BL/6 mice. Twenty-four hours later, the spleens were isolated, and the ratios between violet and red B cells were determined via flow cytometry (bottom plot and diagram). The ratios before injection and after spleen isolation were used to quantify the isotype-specific depletion of the target cells. (C) Representative experiment indicating mIgG2a, mIgG2b, and mIgA specific depletion of target cells with mIgG2a_silent opsonized cells as reference population. n = 3, mean ± SEM. *P < 0.05.