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In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice

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AbstractTransplantation of B cells engineered ex vivo to secrete broadly neutralizing antibodies (bNAbs) has shown efficacy in disease models. However, clinical translation of this approach would require specialized medical centers, technically demanding protocols and major histocompatibility complex compatibility of donor cells and recipients. Here we report in vivo B cell engineering using two adeno-associated viral vectors, with one coding for Staphylococcus aureus Cas9 (saCas9) and the other for 3BNC117, an anti-HIV bNAb. After intravenously injecting the vectors into mice, we observe successful editing of B cells leading to memory retention and bNAb secretion at neutralizing titers of up to 6.8 µg ml−1. We observed minimal clustered regularly interspaced palindromic repeats (CRISPR)–Cas9 off-target cleavage as detected by unbiased CHANGE-sequencing analysis, whereas on-target cleavage in undesired tissues is reduced by expressing saCas9 from a B cell-specific promoter. In vivo B cell engineering to express therapeutic antibodies is a safe, potent and scalable method, which may be applicable not only to infectious diseases but also in the treatment of noncommunicable conditions, such as cancer and autoimmune disease.

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Data availabilityData are available in the main text, in the Extended Data figures and Supplementary Data and materials. Illumina sequencing data can be accessed in the SRA database under accession code PRJNA706552. Source data are provided with this paper.

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AcknowledgementsWe thank the Veterinary Service Center, Tel Aviv University for animal husbandry. The IDRFU, Genomic Research Unit and SICF units, Tel Aviv University for logistic support and council. We also thank L. Vardi, M. Gelbart, H. Kobo, D. Burstein, I. Benhar, N. Freund, M. Kay, T. Akriv and N. Gritsenko for reagents and feedback. This research was funded by the Varda and Boaz Dotan donation (A.B.), the H2020 European Research Council grant no. 759296 570 (A.B.) and the Israel Science Foundation grant nos. 1632/16, 2157/16 and 2876/21 (A.B.), The Bill and Melinda Gates Foundation grant no. OPP1183956 (J.E.V.), National Institutes of Health grant nos. R01 AI167003-01 (A.B.) AI128836 and R01 AI073148 (D.N.), Edmond J. Safra Center for Bioinformatics fellowship (T.K. and A.S.), St. Jude Children’s Research Hospital and ALSAC, National Institutes of Health Office Of The Director, Somatic Cell Genome Editing initiative grant no. U01AI157189 (S.Q.T.), the Gertner Institute Scholarship, the Yoran Institute Scholarship and the SAIA Foundation (A.D.N.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author informationAuthor notes

Alessio D. Nahmad

Present address: Tabby Therapeutics Ltd, Ness Ziona, Israel

Authors and AffiliationsThe School of Neurobiology, Biochemistry and Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

Alessio D. Nahmad, Inbal Reuveni, Daniel Nataf, Yuval Raviv, Miriam Horovitz-Fried, Iris Dotan & Adi Barzel

The Varda and Boaz Dotan Center for Advanced Therapies, The Sourasky Medical Center and Tel Aviv University, Tel Aviv, Israel

Alessio D. Nahmad, Inbal Reuveni, Daniel Nataf, Yuval Raviv, Miriam Horovitz-Fried, Iris Dotan & Adi Barzel

Department of Hematology, St Jude Children’s Research Hospital, Memphis, TN, USA

Cicera R. Lazzarotto & Shengdar Q. Tsai

Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Natalie Zelikson & Rina Rosin-Arbesfeld

The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

Talia Kustin & Adi Stern

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA

Mary Tenuta, Deli Huang, David Nemazee & James E. Voss

Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Yaron Carmi

ContributionsA.D.N. designed, performed and analyzed the study. C.R.L. performed CHANGE-seq; S.Q.T. supervised CHANGE-seq experiments. N.Z. and T.K. performed bioinformatical analyses. A.S. and R.R.-A. supervised the bioinformatical analyses. N.Z., M.H.-F. and I.R. helped with sample processing. Y.R. helped with vector design and cloning. D. Nataf and I.D. designed the B cell progenitor enrichment. M.T. and D.H. performed neutralization assays. D. Nemazee and J.E.V. supervised neutralization assays. I.D. contributed to supervising the study. Y.C. helped with experimental design. A.D.N. and A.B. drafted and revised the manuscript. A.B. conceptualized and supervised the study.

Corresponding authorCorrespondence to
Adi Barzel.

Ethics declarations

Competing interests
A.D.N., D. Nataf, M.H.-F., I.D. and A.B. are listed as inventors on patent applications covering B cell engineering. A.D.N. and A.B. have an equity stake in and receive monetary compensation from Tabby Therapeutics Ltd, a B cell engineering company. S.Q.T. is a coinventor on patents covering the CHANGE-seq method. S.Q.T. is a member of the scientific advisory boards of Kromatid, Inc. and Twelve Bio. The other authors declare no competing interests.

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Peer review information
Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended dataExtended Data Fig. 1 Multiple isotypes of the 3BNC117 antibody are expressed by engineered B cells.a–d. ELISA for each isotype. a. IgM, b. IgG1, c. IgG2c and d. IgA. All samples come from the CMV-Cas9gRNA + donor injected mice at different time points, as indicated in each legend. Mean and SD are indicated. n = 3 biologically independent animals. e,f. Area under the curve (AUC) for A-D. e. IgM, ns; pv=0.1288, ***; pv=0.0008, **; pv=0.0062, f. IgG1, ns; pv=0.131 and from top to bottom as presented in the graph, **; pv=0.0044 and pv=0.0013, g. IgG2c, ***; pv=0.0007, *; pv=0.0195, **; pv=0.0098, h. IgA, ns; pv=0.0587, **;=pv=0.0013, *; pv=0.0403, for two-sided unpaired t-test. n = 3 biologically independent animals. For A-H, sample collection day is indicated. Mean values are indicated. i. Fraction of 3BNC117 IgG titers as quantified by ELISA using purified gp120 binding sera from donor injected mice immunized with gp120, at day 37. ***; pv=0.0007 for two-sided unpaired t-test. n = 3 biologically independent samples. Mean values are indicated.

Source data

Extended Data Fig. 2 bNAb genomic integration, sera titers and neutralization as a function of immunizations and co-injection of the CRISPR-Cas9 vector.a. Area under the Curve (AUC) of Fig. 2d for YU2.DG (left) and JRFL (right) **; pv=0.0036 (YU2.DG) and pv=0.005 (JRFL) for unpaired t-test for CMV-Cas9gRNA + donor to PBS comparison and ##; pv=0.0072 (YU2.DG) and pv=0.0063 (JRFL) for one-sample t-test for Naïve to PBS comparison. n = 3 for CMV-Cas9gRNA + donor and PBS. Naïve sample is from a single, non-immunized, non-AAV-injected mouse. Mean values ± SD are indicated. b. Area under the curve (AUC) of Fig. 2c. From top to bottom, *; pv=0.0185 and pv=0.0103, **; pv=0.0036 for two-sided unpaired t-test. n = 3 biologically independent animals. Mean values ± SEM are indicated. c. RT-PCR on RNA from sorted, 3BNC117+, CD19+, CD4− blood lymphocytes from day 37. Here, we used a reverse primer in a membranal exon of either IgHCμ or IgHCγ (all subtypes) and a forward primer on the VH of the coded 3BNC117. Numbers indicate different mice, injected with either a) PBS, b) the donor vector and the CMV-Cas9gRNA vector, or c) the donor vector only, as indicated above the gels. Control sample (C+) comes from in-vitro engineered primary mouse splenic lymphocytes, as described previously7. Ladder sizes are indicated on the left. Arrow indicates the expected amplicon size. For each group, experiment was reproduced 3 times with independent samples, as indicated by the numbers. Molecular weight markers (M) and their respective size in base pairs (MW) are indicated. d. Total DNA from the previous reaction as in (C) was purified and a semi-nested PCR with the same forward primer and a reverse primer on the CH1 of the respective constant domains. Ladder sizes are indicated on the left. Arrow indicates the expected amplicon size. For each group, experiments were reproduced 3 times with independent samples. Molecular weight markers (M) and their respective size in base pairs (MW) are indicated. e. Sanger sequencing alignment and chromatogram of the purified amplicon from the previous step. Reference sequences are indicated above. For the IgHCγ, each subtype reference is indicated. Sequencing of the IgHCμ amplicon of donor 3 has failed. f. Experimental design for (G-I). Splenic lymphocytes were activated with LPS and IL-4 and engineered, ex vivo, by AAV transduction and Cas9 electroporation with or without a gRNA. h. Flow cytometry of engineered splenic lymphocytes two days following treatment. Pregated on live, singlets. FcR block was used in the staining. Engineering parameters are indicated above each plot. h. EtBr gel electrophoresis showing products of an RT-PCR reaction with RNA from cells two days following treatment as in (F). For each sample, a control (C) reaction was performed amplifying the endogenous IgHG1 cDNA. Ladder sizes are indicated on the left. Arrow indicates the expected amplicon size. The experiment was reproduced once, with similar results. Molecular weight markers (M) and their respective size in base pairs (MW) are indicated. i. Sanger sequencing of the previous amplicons, confirming the integration.

Source data

Extended Data Fig. 3 Detection of engineered B cells in the spleen, the blood and the bone marrow.a. Flow cytometry plots demonstrating 3BNC117 expression among CD19+ CD11b− cells in the spleen at day 82 of 2CC immunized mice. Pregated on live, singlets. FcR block was used in the staining. b. Quantification of B. ***; pv=0.0006 for two-sided unpaired t-test. c. Flow cytometry plots demonstrating 3BNC117 expression among blood B cells (CD19+, CD4−). d. Quantification of blood 3BNC117-expressing cells over time. The black arrows indicate immunizations and the blue arrow indicates AAV injection. ####; pv 0.9999, *; pv=0.0387 and pv=0.0372 (H) for One-Way ANOVA with Tukey’s multiple comparison.

Source data

Extended Data Fig. 4 Assessing expression of the transgene in different subsets of cells.a. Flow cytometry examples for Fig. 5c-g and Extended Data Fig. 3b-e, j-k. b. Quantification of 3BNC117+ cells in bone marrow. #; pv=0.0129 for Two-Way ANOVA and *; pv=0.0255 for Two-Way ANOVA with Tukey’s multiple comparison. c. Quantification of the indicated populations from 3BNC117+ B220− cells in the bone marrow. *; pv=0.0335, ****=pv 0.9999 for Two-Way ANOVA with Tukey’s multiple comparison. f-g. Quantification of D. for the rate of 3BNC117-expressing cells for the spleen (F) or the bone marrow (G). From top to bottom: ns; pv=0.9994 and pv=0.0668 (F) and pv=0.2371 and pv=0.0560 for Two-Way ANOVA with Tukey’s multiple comparison. h. Serum 3BNC117 IgG titers at the indicated time points. The scale of the Y-axis was chosen to correspond to the other 3BNC117 titer plots in this manuscript. ns; pv=0.1726t for Two-Way ANOVA. i. Representative ELISPOT assay of a day 140 bone marrow from CD45.1 recipient mice. j. Quantification I. ns; pv=0.1756 for two-sided unpaired t-test. For B-D, FcR block was used in staining.

Source data

Extended Data Fig. 10 Assessment of the specificity and sensitivity of the anti-idiotype for 3BNC117.a. Experimental scheme. Cells are engineered a day following extraction from spleen and activation. We used three different donor AAVs, each expressing a different antibody: either 3BNC117, VRC01 or 10−1074. b. Flow cytometry of gp120 or anti-idiotype binding of engineered cells, two days following treatment. Staining procedure is indicated above the plots. FcR block was used in staining. Each row indicates a different AAV used. Untransduced cells serve as the negative control. c. Quantification of B. ****; pv Read More

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