Abstract
The immunoglobulin locus of B cells can be reprogrammed by genome editing to produce custom or non-natural antibodies that are not induced by immunization. However, current strategies for antibody reprogramming require complex expression cassettes and do not allow for customization of the constant region of the antibody. Here we show that human B cells can be edited at the immunoglobulin heavy-chain locus to express heavy-chain-only antibodies that support alterations to both the fragment crystallizable domain and the antigen-binding domain, which can be based on both antibody and non-antibody components. Using the envelope protein (Env) from the human immunodeficiency virus as a model antigen, we show that B cells edited to express heavy-chain antibodies to Env support the regulated expression of B cell receptors and antibodies through alternative splicing and that the cells respond to the Env antigen in a tonsil organoid model of immunization. This strategy allows for the reprogramming of human B cells to retain the potential for in vivo amplification while producing molecules with flexibility of composition beyond that of standard antibodies.
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Data availability
Sequences of all guide RNAs, expression plasmids, homology-donor plasmids, AAV vectors and primers are provided with this paper and can be found in Supplementary Tables 1 and 3–6, respectively. Sequences of all insert plasmid features are also described in Supplementary Tables 3 and 4. The antibodies used are listed in Supplementary Table 7. Uncropped gel images for Extended Data Figs. 1c, 2b, 5b and Extended Data Fig. 6d can be found in Supplementary Fig. 3. Representative gating schemes for the flow cytometry analysis of peripheral blood B cells in Figs. 3a,b, 6a,d,i and Extended Data Figs. 4f, 5c are in Supplementary Fig. 8. Gating schemes for Fig. 3g–j are in Supplementary Fig. 10. Gating schemes for Fig. 4a, Extended Data Fig. 6b–d and Supplementary Fig. 12 are in Supplementary Fig. 11. The gating scheme for Fig. 5c–e is in Supplementary Fig. 14. Deep sequencing data have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive database: sequencing data of primary B cells treated with Cas9-sg05 RNPs in Fig. 1d and Extended Data Fig. 1d,e can be found under accession code PRJNA994591 and sequencing data of SHM in edited Raji cells in Supplementary Figs. 4 and 5 can be found under accession code PRJNA1095559. Source data are provided with this paper.
Code availability
Core functions of NGS analysis were performed using publicly available open-source software, as described in the Methods section. R scripts used for Cas9-sg05 RNP activity and SHM in Raji cells are available on reasonable request.
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Acknowledgements
We thank G.N. Llewellyn and C. Diadhiou for critical discussions and assistance with reviewing the manuscript. P.M.C. discloses support for the research described in this study from National Institutes of Health grants HL156274, AI164561, AI164556 and MH130178. G.L.R. discloses support for the research described in this study from a Career Development Award from the American Society of Gene & Cell Therapy. H.-Y.C. discloses support for the research described in this study from a Taiwan USC scholarship. Tonsil material was provided by the Norris Comprehensive Cancer Center Translational Pathology Core, supported in part by National Institutes of Health grant CA014089 from the National Cancer Institute. The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the American Society of Gene & Cell Therapy, National Cancer Institute or National Institutes of Health.
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G.L.R., C.H., K.S., A.M., H.M. and H.-Y.C. developed editing reagents and performed cell-editing experiments. G.L.R., C.H., A.M., X.H., K.S. and C.-H.C. established assays and performed antibody and sample analyses. G.L.R. performed NGS experiments, analysed data and visualized results. E.J.K. supervised the collection of tonsil tissue. G.L.R. and P.M.C. conceived the study, designed experiments, interpreted data and wrote the manuscript with input from other authors. P.M.C. supervised the study.
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Extended data
Extended Data Fig. 1 Extended analyses of genome editing at the constant region of the IgH locus.
(a) K562 cells were electroporated with Cas9 RNPs containing indicated gRNAs and matched ssODN homology donors to insert an XhoI restriction site (n = 3). HDR editing was measured by Sanger sequencing and ICE analysis. (b) K562 cells were electroporated with Cas9 RNPs for indicated gRNAs and a matched plasmid homology donor containing a GFP expression cassette (n = 3). HDR editing was measured by flow cytometry for GFP expression after 3 weeks. (c) Site-specific insertion of GFP expression cassettes in AAV6-edited K562 cells was confirmed by in-out PCR for each tested gRNA. Uncropped gel is available in Supplementary Fig. 3a. Created with Biorender.com. (d) On- and off-target activity of sg05 was measured at indicated IGHG genes in primary human B cells, 5 days after editing, by targeted amplicon deep sequencing. Aggregate mutations at each base in a 50 bp window surrounding the sg05 cut site (0; orange dotted line) are shown for each gene. (e) Percentage mutated reads at each IGHG gene calculated as for all changes (≥1 bp changed), which gives a higher background than when a cutoff of ≥2 bp is selected, as shown in Fig. 1d (n = 2). Error bars show mean ± SEM. Statistics were calculated by 2-way ANOVA with Fisher’s LSD test.
Extended Data Fig. 2 Extended analyses of engineered B cell lines expressing anti-HIV HCAbs.
(a) Raji B cells were edited with sg05 Cas9 RNPs plus a plasmid homology donor to insert a GFP expression cassette and editing rates were measured by flow cytometry. (b) Ramos B cells were edited with sg05 Cas9 RNPs and plasmid homology donors for J3, A6 or a control GFP expression cassette. Editing rates were determined by flow cytometry. (c-d) Raji (c) and Ramos (d) cells edited by sg05 Cas9 RNPs and J3, A6, or GFP plasmid homology donors, post sorting by FACS. (e) GFP-edited Raji cells were sorted by FACS for GFP expression, and the enriched population was subjected to in-out PCR and Sanger sequencing of PCR bands to confirm precise insertion. The dotted line indicates the predicted sg05 cut site. Uncropped gel is available in Supplementary Fig. 3b. (f) Secretion of HCAbs was detected by total IgG ELISA from J3 or A6-edited Raji and Ramos cells, but not from GFP-edited control cells.
Extended Data Fig. 3 Lack of pairing between HCAb and co-expressed light chain.
(a) Assay schematic. 293T cells were transfected with expression plasmids for the J3 VHH HCAb, a full-length (FL) conventional human IgG1 derived from Ofatumumab, or its Igκ L chain only (LC), or combinations as indicated. Supernatants, including mixed supernatants as controls, were evaluated by ELISAs based on HIV gp120 binding by the J3 VHH and detection with an anti-VHH antibody (control), or anti-Igκ L chain antibodies to detect pairings between the J3 HCAb and either the FL or LC components. FabALACTICA digestion is expected to cleave FL antibodies into Fab and Fc fragments and may also cleave HCAbs. Created with Biorender.com. (b-c) Results of gp120-VHH (b) and gp120-Igκ (c) ELISAs, with or without FabALACTICA digestion (n = 4 J3 and J3/FL; n = 2 FL, LC, J3/LC, L3+LC, and J3 + FL). Cross-pairing was only observed after co-transfection of J3 HCAb and FL antibody, consistent with H chain interactions that were released by FabALACTICA digestion. In contrast, the L chain alone did not pair with the J3 HCAb. Error bars show mean ± SEM. Statistics were performed using 2-way ANOVA with Fisher’s LSD test.
Extended Data Fig. 4 Comparison of culture conditions for primary human B cells.
Primary human B cells were cultured with the indicated stimulation conditions (DP, BAC, RP105) and basal media (IMDM, XF, XF plus FBS) as described in Supplementary Fig. 7. Cultures were pre-activated for 3 days before measurements were started on day 0. (a) Fold expansion of cells over time (n = 3). (b) Viability of cells over time (n = 3). (c) Cell size, measured by flow cytometry as forward scatter (FSC) median fluorescence intensity (MFI) on day 0 (n = 3). (d) Total IgG secreted into supernatants was measured over time by ELISA (n = 4). (e) Total IgG amounts normalized for viable cell counts (n = 4). (f) B cell phenotypes were assessed at day 8 in indicated cultures by flow cytometry, as shown in Supplementary Fig. 8 (n = 3). For BAC + DP, the cells were started in BAC and treated for a total of 5 days, then switched to the DP protocol for the remaining 6 days, as shown in Supplementary Fig. 7. (g) Comparison of total IgG secretion and IgG/cell at day 8 for cells treated with indicated stimulation protocols (n = 3). DP was in IMDM, BAC and BAC + DP were in XF without FBS. Error bars show mean ± SEM. Statistics were calculated by 2-way ANOVA with Tukey’s test (c) or paired 1-way ANOVA with Tukey’s test (g).
Extended Data Fig. 5 Extended analyses of engineering with AAV6 homology donors.
(a) Editing rates achieved using sg05 Cas9 RNPs and AAV6-J3 homology donors on Raji and Ramos B cell lines. Representative plots are shown, together with summary data (n = 6 control Raji and AAV6 + Cas9 Ramos, n = 4 control Ramos and AAV6 + Cas9 Raji). (b) Site-specific insertion of the J3 cassette in primary B cells treated with sg05 RNPs and AAV6-J3 donor was confirmed by in-out PCR followed by Sanger sequencing of the band. The dotted line shows the presumed sg05 cleavage site. Uncropped gel image is provided in Supplementary Fig. 3c. (c-g) Primary human B cells were edited with sg05 Cas9 RNPs and AAV6-J3 at the indicated MOIs, with BAC activation in XF media (n = 5 5e5 MOI, n = 3 1e4 and 1e5 MOI). Data from the highest MOI, (5e5), is reproduced here from Fig. 3, for comparison. (c) Editing rates, measured at day 8 by flow cytometry for surface J3-BCR (n = 6 5e5 MOI, n = 3 1e4 and 1e5 MOI). (d) Editing quantified by in-out ddPCR at day 8 and normalized per cell against a control reaction. (e) The yield of edited cells at day 8 was calculated from 5 ×105 starting B cells. (f) Fold expansion of total cells at day 8. (g) J3 HCAb secretion, measured by gp120–IgG ELISA at day 8. Error bars show mean ± SEM. Statistics were calculated by 2-way ANVOA with Šídák method (a), or 1-way ANOVA with Tukey’s test (d-h).
Extended Data Fig. 6 J3-BCR surface expression is reduced with differentiation.
(a) Edited primary B cells were differentiated in DP plus IMDM media and RNA extracted at indicated time points. RT-PCR was performed using a J3-specific forward primer and IgG reverse primers specific for the secreted (S) or membrane (M) isoforms. Uncropped gel image is provided in Supplementary Fig. 3d. (b-d) Five x 105 primary human B cells were activated with BAC from day −3 and edited at day 0 with Cas9 RNPs and an AAV6-J3 homology donor (MOI = 5 × 105 vg/cell). Edited cells (intracellular IgG+ VHH+) were identified at day 8, as described in Supplementary Fig. 11, for analysis of surface J3-BCR expression (gp120 binding). (b) Representative plot of total (surface and intracellular) VHH vs. surface J3-BCR. c,d, Representative plot (c) and summary (d) of surface J3-BCR expression in edited cells of different populations for n = 3 independent donors. Summary MFIs were normalized to the median of the total population for that donor. Error bars show mean ± SEM.
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Rogers, G.L., Huang, C., Mathur, A. et al. Reprogramming human B cells with custom heavy-chain antibodies. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01240-4
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DOI: https://doi.org/10.1038/s41551-024-01240-4
