# Editing B cells at the IGHA2 gene position provides alternative route to therapeutic IgA production

Marine Cahen, Jenny Leonard, Ophélie Dézé, Laurent Deleurme, Maiwenn Pineau, Anne-Laure Tanguy, Stéphane Paul, Jérôme Moreaux, Gregory Noël, Natsuko Ueda, et al.

► To cite this version:

Marine Cahen, Jenny Leonard, Ophélie Dézé, Laurent Deleurme, Maiwenn Pineau, et al. Editing B cells at the IGHA2 gene position provides alternative route to therapeutic IgA production. Mucosal Immunology, 2025, 18 (5), pp.1027-1035. 10.1016/j.mucimm.2025.06.001. hal-05163114

HAL Id: hal-05163114

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Submitted on 9 Oct 2025

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<figure><img src="image_1.png" /></figure>
Editing B cells at the IGHA2 gene position provides alternative route to therapeutic IgA production

Marine Cahen<sup>a</sup>, Jenny Léonard<sup>a</sup>, Ophélie Dézé<sup>a</sup>, Laurent Deleurme<sup>a</sup>, Maiwenn Pineau<sup>a</sup>,
Anne-Laure Tanguy<sup>a</sup>, Stéphane Paul<sup>b</sup>, Jérôme Moreaux<sup>c</sup>, Grégory Noël<sup>a</sup>, Natsuko Ueda<sup>a</sup>,
Yannic Danger<sup>a</sup>, Michel Cogné<sup>a,∗</sup>

ARTICLE INFO

Keywords:

IgA

CSR

Immunotherapy

A B S T R A C T

As professional and long-lived immunoglobulin (Ig) producers, B cells represent attractive candidates for adoptive immunotherapy and their highly expressed Ig heavy (IgH) chain locus is ideal for editing. Each of its constant genes, expressed after class switch recombination (CSR), affords an attractive platform where an adoptive Ig variable domain would acquire IgM, IgG, IgE or IgA class-specific functions. In particular, IgA plays a unique role in mucosal immunity but has remained excluded from therapeutic applicability due to unfavorable chemistry, manufacturing, and control (CMC) issues. To test whether these barriers could be overcome by producing IgA in vivo rather than in vitro, we edited the human B cell-specific IGHA2 gene and found it to be a suitable platform for inserting gene cassettes for expression in B cells. Targeted deletions can also induce CSR to IgA2, while, by combining IgA2 CSR with the insertion of a linked VH and a complete light chain, we have replaced the endogenous Ig chains with a customized full-size but single-chain IgA carrying an adoptive antigen specificity.

Taken together, we show that IGHA2-editing of B cells could provide a novel avenue to B-cell targeted delivery of therapeutic IgA, overcoming the problems that have so far excluded IgA from clinical use.

Introduction

Adoptive immunotherapy with ex vivo engineered autologous cells,
such as CAR T cells, has recently revolutionized the genre, including
with CRISPR/Cas9-mediated genome editing. ¹⁻³ Such strategies are
expanding and diversifying in terms of methods, receptors expressed,
and cell lineages edited. Recently, several reports have provided proof of
concept for the feasibility of B cell edition for adoptive immunotherapy
in mouse models. ⁴⁻¹⁰ B cells are clearly ideal candidates for the pro-
duction of membrane immunoglobulins (Ig) at the lymphocyte stage or
secreted Ig at the plasma cell stage. Their ability to survive as memory B
cells or terminally differentiated long-lived plasma cells (LLPCs), further
makes them attractive for therapeutic aims, to consistently provide an-
tibodies (Abs) both locally and in the circulation. Long-term provision of
therapeutic monoclonal Abs (mAbs) is desirable in many chronic

diseases such as cancer, autoimmunity, allergy or chronic infections. B
cell edition could therefore provide a new way to deliver specific ther-
apeutic mAbs to patients through their direct *in vivo* production,
whether these mAbs have classical or reengineered formats. This has
been used to introduce a cassette encoding an anti-PD1 antibody into the
mouse GAPDH gene.¹¹ In particular, this approach is relevant for short-
lived biotherapies, such as some bispecific Abs, which could be hereby
permanently infused into the body fluids. It could also enable the pro-
duction of complex biomolecules for which *in vivo* secretion by plasma
cells is possible while *in vitro* production and purification are not
mastered, as it is notably the case for IgA with its complex glycosylation
patterns.

IgA exported to mucosal surfaces in polymeric forms of secretory IgA (sIgA), play a key role in the protection against pathogens, as demonstrated for SARS-CoV-2.<sup>12-13</sup> It can not only control pathogens but also

<footnote><sup>a</sup> INSERM U1236, University of Rennes, Etablissement Français du Sang, 35000 Rennes, France</footnote>

<footnote><sup>b</sup> Centre International de Recherche en Infectiologie, University Claude Bernard Lyon 1, Inserm, U1111, CNRS UMR530, CIC1408 Vaccinology, University Hospital of Saint-Etienne F42055 Saint-Etienne, France</footnote>

<footnote><sup>c</sup> Institute of Human Genetics, University of Montpellier, CNRS-UMR 9002, Montpellier, France</footnote>

<footnote>* Corresponding author at: INSERM U1236, University of Rennes, Etablissement Français du Sang, 35000 Rennes, France.
E-mail address: michel.cogne@inserm.fr (M. Cogné).</footnote>
help master the composition of the microbiota.<sup>14–16</sup> IgA deficiency, the most common form of immunodeficiency, although asymptomatic in over 70 % of cases, may also be associated with increased incidence or severity of sinopulmonary and gastrointestinal infections.<sup>17–18</sup> As a therapeutic agent for immunodeficient patients, IgA is then only available within donor-derived IgA-enriched preparations of intravenous immunoglobulins.<sup>19–20</sup>

Targeted therapy with IgA mAbs has shown promise in several pre-clinical infection and tumor models.<sup>21–25</sup> However, this has not yet been translated into human applications, where systemic delivery of antigen-specific IgA (and its mucosal export as specific sIgA) can only be achieved by its endogenous production after immunization *in vivo*. Indeed, human IgA are highly N-glycosylated, and O-glycosylated for IgA1,

heterogeneously polymerized and poorly stable. Despite recent advances with IgA2 re-tailored for glycosylation,<sup>26–27</sup> the manufacturing of IgA is incompletely mastered and its systemic administration to patients remains an unmet need.<sup>21</sup>

In this context, B cell engineering may offer alternative strategies to IgA-based therapy. The proof of concept that B cells can be switched using CRISPR/Cas9 tools has been established *in vitro*.<sup>28</sup> Whether this could be used to purposefully modify the class of Abs produced *in vivo* against a given Ag, or whether the human IGHA genes could be engineered in order to graft V regions from a therapeutic mAb remains to be determined. In this study, we investigated various aspects of IGHA gene edition, focusing on IgA2 expression which is more efficient than IgA1 at neutralizing pathogens and activating effector cells.<sup>29–30</sup> We therefore

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Validation of IGHA2 for B cell edition. A. IGHA2 gene targeting strategy inserting an anti-HER2 single-chain IgG cassette flanked with homology arms. B. PCR validation of the anti-HER2 cassette insertion into the IGHA2 gene in LP1 and primary B cells, 2 days after transfection. C. RT-PCR detection of anti-HER2 scFull-IgG RNA transcripts by RT-PCR after integration in LP1 and primary B cells. D. ELISA evaluation of secreted anti-HER2 scFull-IgG1 in supernatant from LP1 cells and primary B cells on days 4–7–10 post-transfection.</figcaption></figure>
evaluated IGHA2 gene edition either for on purpose CSR or the use of the IGHA2 gene as an efficient site for the expression of transgenic Abs (either from the IgA class or not). As this gene is in an open configuration in B cells and proximal to the strong 3' IgH super-enhancer, it does indeed appear to be a potentially valuable "platform" for the B cell-specific expression of transgenes and in particular for chimeric IgA mAbs.

## Results

### The human IGHA2 gene is accessible to CRISPR/Cas9-mediated gene edition

We targeted the intron between Sα2 and the actively transcribed IGHA2 gene, which we observed in a previous study³¹ to harbor the H3K9me3 chromatin mark in plasmablasts, and which is close to the IgH 3'RR2 superenhancer, itself carrying the H3K27Ac chromatin mark abundantly (Suppl. Fig. 1A). We designed an efficient single-guide RNA (sgRNA) fulfilling the requirement to be unique and not present in the IGHA1 gene. Its cleavage efficiency reached 19% in primary B cells (Suppl. Fig. 1B). To assess whether this site could support insertion in B cells and efficient expression of an inserted gene, a cassette combining a pVH B cell specific promoter and the reporter gene tdTomato was flanked by 0.5 kb long arms to support homology-directed recombination (HDR) on both sides of the IGHA2 site (Suppl. Fig. 1C). The pVH-tdTomato cassette was then co-transfected with RNP into either the DG75 cell line or human primary B cells. This resulted in efficient expression of tdTomato in up to 24% of DG75 cells and 5% of primary B cells by flow cytometry (Suppl. Fig. 1D).

The lower efficiency of insertion in primary B cells compared to a cell line is not specific to IGHA2, but reflects the less efficient HDR in primary cells, as previously observed.³²

Beyond expressing a reporter gene, we investigated whether a therapeutic protein such as a single-chain IgG could be produced by B cells after non-deleterial insertion at the IGHA2 position, without disrupting the production of the endogenous IgH chain encoded by upstream constant genes. A cassette encoding a previously described anti-HER2 single-chain (scFull) IgG1³³ was thus flanked by the IGHA2 specific homology arms, to be inserted upstream of the CH1α2 exon (Fig. 1A). The homology arms were additionally flanked by truncated CRISPR target sites (tCTS) to improve HDR.³⁴ The scFull IgG format combines all IgH and IgL domains in a single chain, together with linkers.³³ The corresponding construct was electroporated together with appropriate RNP into the LP1 cell line, which normally secretes human IgG, or into primary B cells. Specific PCR of the recombinant band, with one primer in the inserted cassette and the other within the targeted sequence, validated the correct insertion at the expected position (Fig. 1B). Expression of the knock-in gene in both LP1 and primary B cells was assessed by RT-PCR, specifically amplifying the expected transcripts (Fig. 1C). Secretion of anti-HER2 IgG in the culture supernatant was validated by ELISA 7 days after electroporation for LP1 and at days 4, 7 and 10 for primary cells (Fig. 1D).

Overall, both tdTomato expression in B cells and scFull IgG secretion in a plasma cell line and in activated primary B cells, show adequacy of the IGHA2 site for transgene expression, with the advantage of a highly transcribed and B cell specific locus. Furthermore, in cells previously expressing any Cμ, Cγ or Cα1 gene upstream of IGHA2, this position (which is at the extreme 3' end of the IgH locus) does not disrupt the upstream [VDJ-Eμ-C] transcription unit encoding the endogenous Ig / BCR. Therefore, such an IGHA2 edition may support the production of therapeutic molecules of interest encoded by a passenger transgene in B cells, irrespective of their previous Ag specificity.

### CRISPR/Cas9-mediated CSR to IgA2 in human B-cells

CSR normally occurs in B cells under cytokine stimulation but can

also be mimicked by CRISPR-induced DNA cleavage.²⁸ This strategy could open up the possibility of switching any non-IgA producing B cell of desired specificity towards IgA2 production, with all the prospects associated with the effector properties of IgA2 in terms of mucosal immunity and myeloid cell activation. We verified that IGHA2 expression indeed follows CRISPR-mediated CSR by deriving IgA2-switched variants from human B cell lines and primary B cells. This was achieved by simultaneously targeting both the above described CRISPR/Cas9 site downstream of Sα2, and an accessible upstream region between the JH cluster and Sμ³³ (Fig. 2A). Co-transfection of the sgRNAs that target these distant sites (as RNP complexes with the Cas9 enzyme) efficiently mimicked IgA2-CSR and readily resulted in 8–12% of DG75 cells with IgM replaced by IgA2 membrane expression, while it increased the number of IgA2-expressing cells by 2-fold in primary B cells from healthy donors (Fig. 2B, Suppl. Fig. 2), validating the strategy.

To assess whether off-target events could affect B cell-expressed genes in such CRISPR-switched IgA2⁺ with edits at 2 distant positions of the IgH locus, RNAseq comparison using the CRISPRRoots³⁵ algorithm was performed between data from CRISPR-switched IgA2⁺ sorted DG75 cells and parental IgM⁺ DG75 cells. CRISPRRoots did not identify any alterations in genes carrying potential off-target sites with less than 5 mismatches with the sgRNAs used in the experiment.

To evaluate not only BCR expression in lymphocytes but also IgA2 secretion and functionality, we targeted the hybridoma SpLT70, which secreted an IgG-class anti-SARS-CoV-2 broadly neutralizing antibody (bnAb). The resulting variant hybridoma secreted an equivalent bnAb switched to the IgA2 class (Fig. 2C), which was retained neutralizing activity against the Wuhan and delta variant strains of SARS-CoV-2, similar to the parental hybridoma (Fig. 2D).

Overall, CRISPR/Cas9-induced CSR to IGHA2 can thus promote membrane expression and/or secretion of IgA2. This could be of particular interest in situations where B cells of a given Ag-specificity can be enriched from blood, opening up the possibility of converting them into IgA2-switched variants, to promote the secretion of IgA2 antibodies, support myeloid cell activation and improve immunity against mucosal pathogens.²⁹

### Coupling CRIPSR/Cas9-mediated CSR to IGHA2 and V-region replacement

IgA mAbs are attractive tools for immunotherapy due to the unique properties of this Ig class. However, neither their *in vitro* production nor their systemic administration is currently mastered. Alternatively, IgA mAbs could be delivered to patients via the endogenous route of B cell edition. Based on the above observation that the IGHA2 gene is accessible to CRISPR-induced CSR and CRISPR-induced insertion, we combined both strategies by inducing coordinated CSR breaks upstream of Sμ and downstream of Sα2, and simultaneously inserting an upstream cassette.

To assess whether insertions between such distant breaks could be efficiently achieved, we first inserted a pVH-tdTomato reporter cassette, flanked by *ad hoc* HDR-promoting arms on either side of the junction (Suppl. Fig. 3A). The same IGHA2 site was targeted as for the CRISPR/Cas9-induced CSR described above. The inserted gene got efficiently expressed in 10% of DG75 cells and in primary B cells (although in a lower percentage of cells, around 1.5%) (Suppl. Fig. 3B). We then coupled IGHA2-CSR with a targeted upstream insertion to provide an antigen-specific [VDJ-VκJκ-Cκ] module (Fig. 3A). An upstream pVH promoter additionally supported transcription of this previously described single-chain scFull-Ig Upstream Switchable Module (USM), while it ended with a linker and a donor splice site to ensure in-frame fusion with the CH1 exon of an acceptor C gene.³² Overall, this USM can thus be inserted at the junction of the disrupted μ/α regions and spliced to the endogenous CH1-α2 gene for subsequent production of IgA2 under a scFull format (Fig. 3A). This strategy was designed to both disrupt endogenous IgH production and replace it with recombinant
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** CRISPR/Cas9-induced α2 CSR. **A.** Schematic of CRISPR-induced α2-CSR with RNPs creating DNA breaks downstream of JH and upstream of IGHA2 B. Cell cytometry validation of IgA2 membrane expression after CRISPR-induced α2-CSR in DG75 (n = 10) and primary B cells (n = 7). **C.** ELISA evaluation of IgA2 secretion after CRISPR-induced α2-CSR of an anti-SARS-CoV-2 hybridoma (left). Virus neutralisation by supernatants from the original IgG hybridoma and its IgA2 variant. **D.** Comparison of the neutralization capacity of anti-SARS CoV-2 IgG versus IgA2 antibodies from a hybridoma. Neutralisation assay performed on SARS CoV-2 Wuhan and Delta strains.</figcaption></figure>

IgA2. The inclusion of a L-chain in the scFull framework restricts the pairing of the recombinant H-chain to the covalently linked L-chain, precluding hybrid association with endogenous L-chains. Using an anti-HER2 scFull-USM cassette (based on pertuzumab sequences) in DG75 cells, this strategy effectively hijacked endogenous Ig expression and replaced it with that of a recombinant IgA2 BCR binding HER2 in approximately 1.5 % of cells. (Fig. 3B, top, Suppl. Fig. 4A and 4B). However, we observed lower membrane IgA staining for the edited anti-HER2 BCR than for the endogenous IgA BCRs, and a decrease in the

percentage of cells expressing the anti-HER2 BCR from day 4 to day 7 and day 10 (Suppl. Fig. 4B).

When used in primary B cells, such a scFull-USM insertion at the μα CRISPR/Cas9 junction strongly stimulated α2-CSR and resulted in the expression of an anti-HER2 IgA2 BCR in about 1.5 % of cells (Fig. 3B, bottom). As expected, cells having only underwent α2-CSR or having only integrated the anti-HER2 cassette without switching to IgA2 were also present. Validation of the on-target integration of the scFull-USM was performed using a multiplex PCR assay (with 2 primers flanking
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Coupled CRISPR/Cas9-induced α2 CSR and scFull-Ig-USM knock-in. A. Strategy for coupling CRISPR-induced α2 CSR and insertion of an anti-HER2 scFull-Ig-USM cassette flanked with homology arms. B. Flow cytometry detection of anti-HER2 scFull-IgA2 expressing B cells stained by anti-human IgA2 antibody and fluorescent antigen on DG75 cell line (n = 9) (top) and primary B cells (n = 9) (bottom). C. PCR validating the insertion of the anti-HER2 scFull-Ig-USM cassette in DG75 (top) and primary B cells (bottom), 2 days after transfection. D. RT-PCR detection of splicing of the anti-HER2 scFull-USM cassette on IGHA2 after insertion into DG75 (left) and primary B cells (right). E. ELISA detection of secreted anti-HER2 scFull-IgA in supernatants of transfected primary B cells at day 7 of stimulation (n = 8). F. Flow cytometric evaluation of the phosphorylation of PLCγ2, pERK1, pSYK and BLNK after cross-linking of the membrane-anchored anti-HER2 scFull BCR (n = 7).</figcaption></figure>
the IgH insertion site and 1 primer hybridizing to the inserted cassette) (Fig. 3C, Suppl. Fig. 4C). Correct splicing of the knocked-in scFull-USM fragment with to the acceptor Cα2 CH1 region was confirmed by RT-PCR amplification of the corresponding transcript (Fig. 3D). Secretion of anti-HER2 IgA by edited B cells was detected by ELISA (Fig. 3E).

To assess the functionality of this IgA BCR, a DG75 [IgA2⁺IgM⁻] anti-HER2 clone was selected (Suppl. Fig. 4D), stimulated *in vitro* with BCR ligands, permeabilized and monitored by cell cytometry for phosphorylation of members of the BCR signaling cascade (pERK1, PLCγ2, Syk, BLNK) (Fig. 3F).

The functionality of edited primary B cells stimulated *in vitro* was also assessed by simultaneously evaluating the anti-HER2 scFull-IgA2 BCR by flow cytometry (validating the expected gene editing), and the secreted anti-HER2 scFull-IgA2 antibody in their supernatant by a specific ELISA, confirming their correlation (Suppl. Fig. 5A and 5B). Analysis of membrane markers on these edited cells also confirmed that a significant part of HER2 binding cells were switched to IgA2 and were differentiated into CD20-CD38⁺ plasmablasts (Suppl. Fig. 5C and 5D), showing that such an edition does not compromise differentiation towards antibody secreting cells.

Collectively, these experiments validate the possibility to simultaneously replace both the Ag-binding region and the class of the expressed H chain in B cells.

## Discussion

After T cells and NK cells, B cells can now be considered for adoptive cell therapy approaches. As professional Ig producers, they appear ideally suited to deliver therapeutic Abs to patients after Ig gene edition. Although long thought to be fragile cells poorly suited to manipulation, several reports have now demonstrated the feasibility of editing B cells to modify Ig synthesis.4,6,7,32,36 We show that the IGHA2 gene, which carries chromatin marks of transcribed euchromatin, is an ideal site for edition, notably for expressing recombinant molecules to be secreted by plasma cells in parallel to their endogenous Ig. Plasma cells are indeed efficient “antibody factories”, capable of surviving for years as LLPCs.37 While the GAPDH and PRDM1 loci can support the transcription of inserted genes in plasma cells,7,38 the Ig loci represent the best possible sites for high expression, in particular for the IGHA2 gene, as the most downstream IgH gene, i.e. the closest to the locus 3'RR2 super-enhancer which is particularly active in activated B cells and plasma cells.39 Efficient insertion and expression of either a reporter cassette or a single-chain IgG at the IGHA2 position confirms that this is an interesting landing platform for transgenic cassettes in B cells. As few human B cells express IgA2, the endogenous Ig production and function is unlikely to be affected by such a distal insertion, either at the lymphocyte or the plasma cell stage.

Another attractive strategy is forced IgA-CSR. IgA Abs are abundant in biological fluids, activate the complement via the alternative and lectin pathways and are captured by several membrane receptors on immune cells, including DC-SIGN, transferrin receptor, Fcα/μ and FcαR1. Polymeric IgA is transported to mucosal surfaces after binding to the polymeric Ig receptor (pIgR) on the apical side of epithelial cells and is transported in the opposite direction by M cells after binding to dectin-1. Because of their unique ability to activate myeloid cells, trigger the complement cascade and act at mucosal sites, IgA Abs are essential in the elimination of pathogens from the mucosae. The induction of IgA after vaccination hereby correlates with protection against Rotavirus.40 While patients with IgA deficiency are susceptible to mucosal infections, they are also exposed to celiac disease and autoimmunity, indicating the dual role of IgA in immunity and tolerance.41 Regarding IgA mAbs, several reports have documented their unique properties against both pathogens and tumor antigens.21,23,25 Systemic administration of recombinant IgA however remains precluded by its short *in vivo* half-life (6 days), and the challenge of industrially mastering its instability and its complex glycosylation.42 It is therefore attractive to produce

therapeutic IgA *in vivo* in the best possible professional IgA factories: B cells.

Simple CRISPR-induced α2-CSR, which retains the previous Ag specificity, may also be valuable. In patients, IgA production is of interest for virus neutralization.43 The availability of IgA-switching procedures may open up strategies for IgA-based therapy, bypassing the need for *in vitro* production and purification of IgA, by using *ex vivo* CSR in selected B cells. This may be relevant for patients with IgA deficiency, as well as for promoting mucosal immunity following systemic vaccination.

B cell edition strategies have recently focused on editing the IgH locus, as this both disrupts endogenous H chain production and replaces it with a recombinant Ig. Both Ig chains can be simultaneously expressed using various types of gene cassettes, either encoding them as a long precursor further cleaved to generate H and L chains,⁶ or associating both Ig chains as a scAb.⁸,³² Here we used our previous design of a scFull-Ig USM cassette that combines the desired Ab VDJ and VJ regions within an upstream 1 kb transcribed domain that is most exposed to somatic hypermutation (SHM) in activated B cells. Accordingly edited B cells could undergo affinity maturation of the V regions, while the Ig constant exons are far from the VH promoter and away from deleterious hypermutation. This cassette gets associated with Ig μ chains when inserted upstream of Cμ. In the present study, we show that this scFull design can re-tailor B cell specificity for Ag with a single hit, while also forcing a switch to the IgA2 class. This strategy aims to combine two events, CRISPR-induced CSR and integration of a gene cassette between the distant IgH breaks, in cells that are then edited to express anti-HER2 IgA2. However, single edits can also occur in parallel, yielding IgA2-switched cells that don't bind HER2 and unswitched cells with only the anti-HER2 cassette integrated. In the future, recently described strategies to improve HDR¹⁴ will be valuable to further increase gene insertion after CSR-like DNA cleavage in the IgH locus. As the level of membrane expression was lower for the edited IgA BCR than for the endogenous one, optimizing transcription from the inserted promoter and the transport of the single-chain IgA to the membrane will also be important, while *in vivo* experiments will be necessary to evaluate the long-term fate of the edited B cells in terms of survival and terminal differentiation into LLPCs.

Overall, our experiments based on non-viral precise edition validate the efficient IGHA2 gene targeting and the expression of genes inserted at this position. This site appears to be competent for expressing therapeutic molecules, for purposely creating IgA-switched B cells of interest, and finally for the production by edited B cells of monoclonal IgA biotherapeutics, which are of great potential interest but currently not amenable to classical production methods.

The efficiency of primary B cell editing was found to be more variable than for cell lines, likely due to heterogeneous viability and proliferation of cells from different donors and as fragile cells, B cells will certainly benefit from future advances in gentle genome editing protocols.

Although no deleterious CRISPR off-target effects were observed in the present study, improvements of the edition strategy could be achieved by using a variant CRISPR strategy that requires only single-strand DNA breaks for gene integration⁴⁵ and/or by additionally inserting an inducible suicide gene to further control edited cells.⁴⁶ As with any cell therapy protocol requiring gene delivery but with a special concern about potential B cell malignancy, edition of human B cells will indeed require to maximize safety before any clinical application.

## Materials and methods

### Cell lines

DG75 human Burkitt's lymphoma cell lines and LP-1 human myeloma cell line were cultured in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% fetal bovine serum (FBS), 1 mM Sodium Pyruvate,
Minimal Essential Medium Non-Essential Amino Acids Solution, 1 unit/ml penicillin, 1 µg/mL –streptomycin at 37 °C with 5 % CO₂.

## Purification, culture, and activation of human primary B lymphocytes

Human primary B cells were isolated from buffy coats collected from healthy volunteers at the Etablissement Français du Sang (Rennes, France). B cells were positively sorted with the StraightFromTM Buffy Coat CD19 Microbead Kit (Miltenyi Biotec) with a purity > 90 %.

The medium for human primary B cell culture was RPMI 1640 GlutaMAX medium (Gibco) supplemented with 10 % FBS (Gibco), 1 mM Sodium Pyruvate (Gibco), 1 unit/ml penicillin, 1 µg/mL –streptomycin (Gibco). For four days, B cells were activated in 24-well plates at 0.75·10⁶ cells/mL with 1 µg/ml CpG oligodeoxyribonucleotide 2006 (Miltenyi Biotec), 2.4 µg/ml F(ab')2 Fragment Goat Anti-Human IgA + IgG + IgM (H + L) (Jackson ImmunoResearch), 50 U/ml recombinant IL-2 (R&D Systems) and 100 ng/ml recombinant human soluble CD40L (Immunex). At Day 3, 5 ng/ml IL-10 (R&D Systems) was added in each well. At Day 4, medium was removed, B cells were washed twice and cultured again in 24-well plates at 0.5·10⁶ cells/mL in culture medium with 50 U/ml IL-2 (R&D Systems), 5 ng/ml IL-4 (R&D Systems) and 12 ng/ml IL-10 (R&D Systems) for 3 days, in order to promote differentiation into pre-plasmablasts. At day 7, B cells were washed and still cultured into 24 well plates at 0.5·10⁶ cells/mL in culture medium containing 50 U/ml IL-2 (R&D Systems), 250 U/ml IFN-α (R&D Systems), 20 ng/ml IL-6 (PeproTech) or 12 ng/ml IL-10 (R&D Systems), which promotes plasma cell differentiation.

## Plasmid constructions and donor DNA preparation

To obtain donor DNA for specific genomic integration after CRISPR/Cas9 cleavage at the IgH locus, we constructed *ad hoc* plasmid vectors containing cassettes that encoded either the tdTomato reporter gene, or the complete coding sequence of a previously studied anti-HER2 single-chain (sc) IgG, or a previously validated Upstream Switchable Module (USM) which could be spliced onto an IgH constant gene to encode an anti-HER2 scFull chimeric Ig.³³ In these plasmids, flanking arms were adapted to each homology-directed repair (HDR) strategy. All constructs were assembled from PCR-amplified fragments using the NEBuilder HIFI assembly kit (New England Biolabs) and validated by plasmid sequencing. PCR templates used for construction were either synthetic gene fragments, plasmids, or human genomic DNA.

Donor DNA used for transfection and HDR were produced by PCR amplification of above-mentioned plasmid constructions. All PCR products were purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). The purified DNA was then ethanol precipitated and resuspended in H₂O to a final target concentration of 2000–5000 ng/µl, as quantified by Qubit.

Donor DNA templates are listed in *supplementary Table 1*.

## Gene edition

The sgRNAs used in the study were designed as conserved sequences shared by the latest GRCh38.p14 and TtoT-CHM13v2.0 releases of the human genome. Using the CRISPOR tool (https://crispor.tefor.net/), on-target sequences were also selected to have at least 4 mismatches with any putative off-target site that shared a 12 bp homology adjacent to a PAM motif. An *ad hoc* cleavage site for CRISPR-Cas9 mediated insertion was thus selected upstream of the IGHA2 gene (CCAGAGCTCCCCTGGGTAAC; human genome GRCh38, CHr14, 105588997–105589016). For the strategies needing to mimic class switching, the IGHA2 targeting sgRNA was combined with an sgRNA that cleaved the intronic region following JH6 and preceding the IGHM gene (GGAAAGAGAACTGTCGGAGT; human genome GRCh38, CHr14, 105862762–105862781).

The cleavage efficiency provided by the IGHA2 sgRNA coupled with

Cas9 as an RNP complex was evaluated by PCR-amplifying the targeted site using flanking primers and sequencing PCR amplicons. Sequences obtained from edited vs non-edited cells were then compared through “Tracking of Indels by Decomposition” (TIDE) (https://shinyapps.datacurators.nl/tide/).

To edit B cell lines in an exponential growth phase, cells were seeded one day prior to the CRISPR experiments at 0.2 10⁶ cells/ml. RNPs were formed by incubating 100 pmol of Alt-RTM S.p. HiFi Cas9 Nuclease V3 (IDT) with 500 pmol of sgRNA (Synthego) for 15 min at 37 °C. The cells were electroporated using a Nucleofector Kit V (Lonza) as follows: 1.10⁶–3.10⁶ cells were centrifuged and resuspended in 100 µl of supplemented Nucleofector solution. For the third genome edition strategy, the cells were transfected with RNPs containing two sgRNAs (500 pmol each). Immediately after transfection, 900 µl of culture medium were added, and the cells allowed to rest for 5–10 min before transfer to 6-well plates in 5 ml of medium.

Primary B cells were electroporated using an ATx device (MaxCyte). A total of 3.10⁶ cells were centrifuged, washed and resuspended in 25 µl of electroporation buffer (MaxCyte). The RNP complex was formed by incubating 25 pmol of Cas9 with 125 pmol of sgRNA for 15 min at 37 °C. When two sgRNAs were required, each was added at 125 pmol without changing the amount of Cas 9, thus limiting the final volume to be transfected.

RNP was then added to the cell suspension and cells were transfected with the OC-25 assembly using the program “B cell 2”. Briefly, 0.5 U of Pulmozyme (Roche, 2500 U/2.5 ml) was added immediately after transfection, the cells were allowed to rest for 30 min at 37 °C before being transferred to 24-well plates in 1.5 ml of medium, and Alt-R HDR Enhancer V2 (IDT) was added to the medium. After 24 h, the HDR enhancer was removed by renewing the medium and cells were incubated for 24 h at 32 °C. For KI experiments, 6–13 µg of donor DNA was added to the RNP complex before addition to cell lines or primary B cells, respectively.

## DNA and RNA analysis

For IGHA2 KI analysis, DNA was prepared from transfected cells using SDS, Proteinase K treatment and ethanol precipitation and analyzed by PCR experiments with primers specific for the inserted cassette and the targeted IGHA2 gene. PCR used Q5 Taq polymerase (New England Biolabs). Correct insertion was indicated by a specifically amplified 834 bp fragment.

For experiments coupling KI and IGHA2 CSR, a multiplex PCR was used, with Phusion Taq polymerase (ThermoFisher) and 3 primers competitively amplifying either a WT (897 bp) or a KI-specific band (772 bp).

For RNA analyses, RNA was reverse transcribed using RT Superscript II (Thermo) and random primers (Invitrogen). Transcripts of the inserted cassette were PCR-amplified using a forward primer included in the USM and reverse primers specific for the endogenous IgH C exons.

All primers used in the study are listed in *Supplementary Table 2*.

To assess off-target events that may affect B cell-expressed genes, an RNAseq comparison was performed between datasets from CRISPR-switched IgA2⁺ sorted cells of the DG75 cell line versus the original IgM⁺ DG75 cell line. This was done using the CRISPRroots package,³⁵ which monitors for potential off-target transcriptomic changes manifesting as either sequence variation or differential expression of predicted target genes.

## Flow cytometry

To monitor tdTomato expression, cells were washed and resuspended in 300 µL PBS, with addition of 1 µL of DAPI (Serva) to distinguish live and dead cells. Switched IgA cells were stained with mouse anti-IgA2 AF647-conjugated antibody (Southern Biotech).

To assess the expression of specific anti-HER2 BCR, we used
biotinylated human HER2/ErbB2 protein (Sino Biological) and PECF594 streptavidin (BD Horizon). For staining, 18 µl of 20 ng of HER2-Biot and 10 ng of streptavidin were premixed and incubated for 30 min at room temperature before being added to 0.5 10⁵ cells, after which the mixture was incubated for 30 min on ice in a final volume of 50 µl. To detect anti-HER2 BCR under the IgA2 form, we added mouse anti-IgA2 AF647-conjugated antibody (Southern Biotech) was added at the same time as the HER2/streptavidin pre-mix. The cells were washed again and resuspended in 300 µL of PBS, 1 µL of DAPI (Serva) was added just before cytometry analysis. Stained cells were analyzed on a Cytoflex flow cytometer (Beckman Coulter).

## Production of anti-SARS-CoV-2 hybridoma

Human monoclonal antibodies were obtained as already described [16]. Briefly, peripheral blood mononuclear cells (PMBC) were isolated from patient. PBMC were separated on MSL (Sigma-Aldrich) and incubated with EBV supernatant (1 ml per $10^7$ PBMC) from B95-8 cell line. Aliquots were plated at 0,1 – 0,5.10⁶ cells/ml in 200 µL wells in culture Iscove's medium + 15 % fetal calf serum and 0,5 µg/ml cyclosporin A (Novartis). Anti-SARS-COV-2-Spike protein secreting-cells were identified by a two-in-one ELISA assay developed to select neutralizing antibodies. After EBV transformation, selected positives cells were fused with Sp2/O-Ag14 murine myeloma. The hetero-hybridomas were cloned by two limiting dilutions before characterizing antibody production in supernatant. Hybridoma SpLT70, the supernatant of which provided the broadest neutralization of SARS-CoV variants was chosen for further experiments.

## Enzyme-linked immunosorbent assays

Detection of secreted IgA or IgG antibodies: For the anti-human IgA or IgG ELISA, MaxiSorp clear flat-bottom 96-well plates were coated overnight at 4 °C with 50 µL/well of unlabeled goat anti-human IgA antibody (Southern Biotech) at 1 µg/mL. After washings, coated wells were blocked with 200 µL/well PBS supplemented with 3 % bovine serum albumin (BSA) (one hour at 37 °C). Samples were added in duplicate at 50 µL/well and incubated for 2 h at 37 °C. After washing, 100 µL/well of alkaline phosphatase-conjugated goat anti-human IgA or IgG antibody (Southern Biotech) was added and incubated for 1 h at 37 °C. After washing, detection was performed with SIGMAFAST Nitrophenyl phosphate (SIGMA), the reaction was stopped with 30 µL/well of 3 N NaOH and the absorbance was read at 405 nm.

Detection of secreted anti-HER2 IgA antibodies: MaxiSorp 96-well plates (Thermo Scientific) were coated with 50/µL/well at 10 µg/ml streptavidin (Sigma) overnight at 4 °C and blocked with 200 µL/well of PBS containing 5 % BSA (Sigma) for one hour at room temperature. Then, 50 µL/well at 0.5 µg/ml biotinylated human HER2/ErbB2 protein (Sino Biological) was added, and the mixture was incubated at 37 °C for one hour. After washing the coated plates, cell supernatants (50 µL/well) were added and incubated for 1–2 h at 37 °C. Finally, presence of IgA was detected by adding 100 µL/well of alkaline phosphatase-coupled goat anti-human IgA (Southern Biotech) (1 h at 37 °C), and revealing bound antibodies with using SIGMAFAST p-nitrophenyl phosphate (Sigma). The reaction was stopped with 30 µL/well 3 N NaOH and the absorbance was read at 405 nm.

## Neutralization test of two strains of the SARS-CoV-2 virus

A plaque reduction neutralization test (PRNT) was used for the detection and titration of SARS-CoV-2 virus neutralizing antibodies. A tenfold dilution of each specimen in culture medium was first heated for 30 min at 56 °C to avoid complement-linked reduction of viral activity. Dilutions (in duplicate) of the specimens were mixed with the live SARS-CoV-2 virus. After a contact of 30 min, 150 µl of the mix was transferred into 96-well microplates covered with Vero E6 cells (American Type

Culture Collection, CRL-1586, tested for mycoplasma contamination). The plates were incubated at 37 °C and infection efficiency was evaluated by microscopy 5 days later when the cytopathic effect of the virus control reached 100–500 TCID50 (median culture infectious dose) per 150 µl. Neutralization was recorded if more than 50 % of the cells were preserved and the titre was expressed as the inverse of the higher serum dilution that exhibited neutralizing activity; a threshold of 20 was used (PRNT50 titre ≥ 20). Experiments were performed in a biosafety level 3 laboratory. The viral strains that were used were sequenced and deposited at GISAID (https://www.gisaid.org/) (accession numbers EPI_ISL_1707038 19A (B.38 lineage); EPI_ISL_1707039 Alpha (B.1.1.7 lineage); and EPI_ISL_1904989 Delta (B.1.617.2 lineage)).

## BCR signaling assays

The ability of the engineered BCR to trigger B cell activation was demonstrated using phospho-flow assays, which monitor the intracellular phosphorylation of elements of the BCR signalling cascade (SYK, BLNK PLCγ2 and pERK1). DG75 cells clonally expressing a scFull-IgA2 BCR were starved for 1 h with RPMI 1640 GlutaMAX medium (Gibco) supplemented with 1 % fetal bovine serum (FBS). A stimulation step was then performed by adding the F(ab')2 fragment goat anti-human Ig (anti-IgA + IgG + IgM, H + L) (Jackson ImmunoResearch) at 20 µg/ml. Cells were then permeabilized and stained with appropriate antibodies specific for the phosphorylated forms of SYK, BLNK, PLCγ2, and pERK1 (BD PhoSFL) according to the supplier's protocol. Stained cells were analyzed on a CytoFlex flow cytometer (Beckman Coulter).

## Statistical analysis

Data are presented as mean ± SD with 95 % confidence intervals (CIs). Statistical analyses were performed using GraphPad Prism 8 software and significance is indicated based on Mann-Whitney tests (ns = not significant; \* p < 0.05; \*\* p < 0.01; \*\*\* p < 0.001; \*\*\*\* p < 0.0001).

## Statement of compliance with research ethics and standards of research

All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee from INSERM and with the 1964 Helsinki Declaration and its subsequent amendments. Blood from healthy volunteers (with informed consent) was obtained from the Etablissement Français du Sang, Rennes, France (under agreement CODECOH #AC-2019-3853 from the French national ethics board). This article does not include any animal studies.

## Data availability statement

RNAseq and the associated CRISPRroots analysis were deposited on the European Nucleotide Archive (E.N.A.) database with the accession number PRJEB88673. Other datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

## CRediT authorship contribution statement

**Marine Cahen:** Writing – original draft, Investigation. **Jenny Léonard:** Investigation. **Ophélie Dézé:** Investigation. **Laurent Deleurme:** Methodology. **Maiwenn Pineau:** Visualization, Methodology. **Anne-Laure Tanguy:** Methodology. **Stéphane Paul:** Investigation. **Jérôme Moreaux:** Visualization, Methodology. **Grégory Noël:** Investigation. **Natsuko Ueda:** Investigation. **Yannic Danger:** Methodology, Investigation. **Michel Cogné:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Funding acquisition, Data curation, Conceptualization.
Declaration of interests

The authors declare no competing interest.

Acknowledgments

This work was supported by Agence Nationale de la Recherche (Grants PEPR THERA-B & ANR-23-CE17-0024-01) and by Leucémie Espoir 22. We thank Matthieu Angles for help with the transfer of ChIP-seq data.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mucimm.2025.06.001.

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