Methods and compositions comprising antigen specific b cells

Abstract

Some embodiments provided herein relate to antigen specific B cells lacking a capacity to differentiate. Some embodiments include the use of such cells to reduce an antibody response in a subject to an antigen.

Description

SCRI.539WO PATENT METHODS AND COMPOSITIONS COMPRISING ANTIGEN SPECIFIC B CELLS RELATED APPLICATIONS

[0001] This application claims priority to U.S. Prov. App. No. 63 / 656,736 filed June 6, 2024 entitled “METHODS AND COMPOSITIONS COMPRISING ANTIGEN SPECIFIC B CELLS” and U.S. Prov. App. No. 63 / 584,432 filed September 21, 2023 entitled “METHODS AND COMPOSITIONS COMPRISING ANTIGEN SPECIFIC B CELLS” which are each expressly incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] Some embodiments provided herein relate to antigen specific B cells lacking a capacity to differentiate. Some embodiments include the use of such cells to reduce an antibody response in a subject to an antigen. BACKGROUND OF THE INVENTION

[0003] Cell therapy provides great promise for the treatment of a variety of diseases and conditions. In cell therapy, autologous or allogeneic cells are transplanted into a patient to replace or repair defective or damaged tissue or cells, target certain tumor cells, or express certain proteins. Many different types of cells may be used, such as pluripotent stem cells; multipotent stem cells, such as hematopoietic stem cells and mesenchymal stem cells; or differentiated cells, such as B cells, plasma cells, dopaminergic neurons, lymphocytes, cardiomyocytes, and pancreatic islet cells. Potential applications of cell therapy include treatment of cancers, autoimmune diseases, and regeneration of damaged tissues in, for example, joints, the heart, and the central and / or peripheral nervous system. SUMMARY OF THE INVENTION

[0004] Some embodiments of the methods and compositions provided herein include a method for preparing an antigen specific B cell, comprising: (a) obtaining a modified B cell, wherein the modified B cell is derived from a B cell and: (i) lacks a capacity to differentiate, and (ii) comprises a B cell receptor (BCR) capable of specifically binding anantigen; and (b) contacting the modified B cell with the antigen, thereby preparing the antigen specific B cell.

[0005] In some embodiments, step (a) comprises modifying the B cell by inactivating a PRDM1 gene in the genome of the B cell. In some embodiments, the inactivating comprises introducing a nuclease capable of specifically cleaving the PRDM1 gene. In some embodiments, the nuclease is selected from a Cas nuclease, a modified or mutant Cas nuclease, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease or a modified or mutant Cas9 nuclease. Some embodiments also include introducing a genomic RNA (gRNA) into a B cell. In some embodiments, the modified B cell lacks the capacity to differentiate into a plasma cell.

[0006] In some embodiments, step (a) comprises modifying the B cell by introducing a polynucleotide encoding the BCR into the B cell. In some embodiments, a vector comprises the polynucleotide. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, or a lentivirus vector.

[0007] In some embodiments, step (a) comprises selecting for a B cell comprising the BCR. In some embodiments, the selecting comprises contacting a population of B cells with the antigen.

[0008] Some embodiments also include stimulating the modified B cell prior to step (b). In some embodiments, the stimulating comprises contacting the modified B cell with multimeric CD40 ligand (MCD40L), anti-CD180 antibody, IL-4, IL-2, IL-6, IL-10, IL-15, lipopolysaccharide (LPS), and / or CpG oligonucleotide (ODN) 2006. In some embodiments, the stimulating comprises contacting the modified B cell with MCD40L, anti-CD180 antibody, and IL-4.

[0009] In some embodiments, step (b) comprises contacting the modified B cell with the antigen for a period less than or equal to 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or 1 hour but not zero. In some embodiments, the period is in a range from or from about 1 hour to 6 hours or about 6 hours, from or from about 1 hour to 4 hours or about 4 hours, or from or from about 2 hours to 4 hours or about 4 hours.

[0010] In some embodiments, step (b) comprises contacting the modified B cell with a cytokine; optionally, wherein the cytokine comprises IL-4.

[0011] In some embodiments, the B cell is selected from the group consisting of a B cell, a hematopoietic stem cell, an early pro-B cell, a late pro-B cells, a large pre-B cell, a small pre-B cell, an immature B cell, a T1 B cell, a T2 B cell, a marginal zone B cell, a mature B cell, and a naïve B cell,

[0012] In some embodiments, the antigen is multimeric. In some embodiments, the antigen further comprises a non-cognate antigen. In some embodiments, the BCR lacks a capacity to specifically bind the non-cognate antigen. In some embodiments, the antigen and the non-cognate antigen are covalently bound to each other. In some embodiments, the antigen or non-cognate antigen comprises a polyethylene glycol (PEG), a viral protein, a non-human protein, or a recombinant protein. In some embodiments, the antigen or the non-cognate antigen comprises an AAV protein or a therapeutic protein, such as Factor VIII. In some embodiments, the AAV protein is selected from viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3). In some embodiments, the AAV protein is from AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVD-J, or Ark313.

[0013] In some embodiments, the antigen specific B cell is mammalian; optionally, wherein the antigen specific B cell is human. In some embodiments, the antigen specific B cell is ex vivo.

[0014] In some embodiments, the antigen specific B cell expresses a therapeutic protein. Some embodiments also include introducing into the antigen specific B cell a polynucleotide encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a cytokine; optionally, wherein the cytokine comprises IL-10. In some embodiments, the therapeutic protein is selected from TGF-ȕ1, IL-35, granzyme B, indoleamine-pyrrole 2,3-dioxygenase (IDO), T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), CD25, CTLA4, T- cell immunoglobulin and mucin domain 1 (TIM-1), FasL, lymphocyte-activation gene 3 (LAG-3), Galectin-9, or CD39.

[0015] Some embodiments of the methods and compositions provided herein include a cell prepared by any one of the methods provided herein.

[0016] Some embodiments of the methods and compositions provided herein include a pharmaceutical composition comprising any one of the cells provided herein and pharmaceutically acceptable excipient.

[0017] Some embodiments of the methods and compositions provided herein include a method for treating, inhibiting or ameliorating a disorder or disease, such as a protein deficiency disorder (e.g., a Factor VIII deficiency), a disease or disorder characterized as having an antibody-antigen based inhibition, antibody inhibitors, or an autoimmune disease (e.g., hemophilia A, multiple sclerosis or systemic lupus erythematosus), in a subject comprising administering any one of the cells provided herein to the subject. In some embodiments, the administration reduces an antibody response to the antigen or a cognate antigen in the subject compared to a subject, which has not been administered the cell. Thus, some embodiments concern methods of reducing an antigen specific antibody response in a subject without the administration of an immunosuppression therapy by administering the antigen specific B cells prepared as described herein to the subject. In some embodiments, the cell is autologous to the subject. In some embodiments, the cell is allogeneic to the subject. Additional embodiments include the use of any one or more of the antigen specific B cells set forth herein as a medicament, for example, a medicament for the treatment or inhibition of a disease or disorder characterized as having an antibody-antigen based inhibition, antibody inhibitors, or an autoimmune disease (e.g., a Factor VIII deficiency, multiple sclerosis or systemic lupus erythematosus).

[0018] Some embodiments of the methods and compositions provided herein include a system comprising: an antigen specific B cell, wherein the antigen specific B cell (i) lacks a capacity to differentiate, and (ii) comprises a B cell receptor (BCR) capable of specifically binding an antigen; and some embodiments also include the antigen.

[0019] In some embodiments, a PRDM1 gene in the genome of the antigen specific B cell is inactivated. Some embodiments also include a nuclease capable of specifically cleaving the PRDM1 gene; optionally, wherein the nuclease is selected from a Cas nuclease, a modified or mutant Cas nuclease, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease; optionally, wherein the Cas nuclease is a Cas9 nuclease or a modified or mutant Cas9 nuclease. Some embodiments also include a genomic RNA (gRNA) capableof specifically binding a PRDM1 gene in the genome of the antigen specific B cell. In some embodiments, the modified B cell lacks the capacity to differentiate into a plasma cell.

[0020] Some embodiments also include a polynucleotide encoding the BCR. In some embodiments, a vector comprises the polynucleotide. In some embodiments, the vector is a viral vector; optionally, wherein the viral vector is an adeno-associated virus (AAV) vector, or a lentivirus vector.

[0021] Some embodiments also include MCD40L and / or anti-CD180. Some embodiments also include a cytokine; optionally wherein the cytokine comprises IL-4.

[0022] In some embodiments, the antigen specific B cell is derived from a B cell selected from the group consisting of a B cell, a hematopoietic stem cell, an early pro-B cell, a late pro-B cells, a large pre-B cell, a small pre-B cell, an immature B cell, a T1 B cell, a T2 B cell, a marginal zone B cell, a mature B cell, and a naïve B cell.

[0023] In some embodiments, the antigen is multimeric. In some embodiments, the antigen further comprises a non-cognate antigen. In some embodiments, the BCR lacks a capacity to specifically bind the non-cognate antigen. In some embodiments, the antigen and the non-cognate antigen are covalently bound to each other. In some embodiments, the antigen or non-cognate antigen comprises a polyethylene glycol (PEG), a viral protein, a non-human protein, or a recombinant protein. In some embodiments, the antigen or the non-cognate antigen comprises an AAV protein or a therapeutic protein, such as Factor VIII. In some embodiments, the AAV protein is selected from viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3). In some embodiments, the AAV protein is from AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVD-J, or Ark313.

[0024] In some embodiments, the antigen specific B cell is mammalian; optionally, wherein the antigen specific B cell is human. In some embodiments, the antigen specific B cell is ex vivo.

[0025] In some embodiments, the antigen specific B cell expresses a therapeutic protein. Some embodiments also include introducing into the antigen specific B cell a polynucleotide encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a cytokine; optionally, wherein the cytokine comprises IL-10. In some embodiments, the therapeutic protein is selected from TGF-ȕ1, IL-35, granzyme B,indoleamine-pyrrole 2,3-dioxygenase (IDO), T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), CD25, CTLA4, T- cell immunoglobulin and mucin domain 1 (TIM-1), FasL, lymphocyte-activation gene 3 (LAG-3), Galectin-9, or CD39. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG.1A depicts an experimental workflow.

[0027] FIG.1B depicts bar graphs of Į-HEL and Į-OV IgM and IgG production in immunized mice following treatment, as measured by antigen specific ELISA.

[0028] FIG.2A depicts an experimental workflow.

[0029] FIG.2B depicts a bar graph with an ICE score for Prdm1 sgRNA.

[0030] FIG. 2C depicts a FACS analysis (left panel) and a graph (right panel) for B cell differentiation in vitro following gene editing.

[0031] FIG. 2D depicts a bar graph of total Ig secretion in vitro following gene editing.

[0032] FIG.3A depicts an experimental workflow.

[0033] FIG. 3B depicts a FACS analysis (left panel) and a graph (right panel) for splenic engraftment of B cells at different doses.

[0034] FIG. 3C depicts graphs for Į-HEL IgM, and Į-HEL IgG, and Į-OVA IgG production in immunized mice following treatment, as measured by antigen specific ELISA.

[0035] FIG.4A depicts an experimental workflow.

[0036] FIG. 4B depicts graphs for Į-HEL, Į-OVA, and Į-NP IgG production in immunized mice following treatment, as measured by antigen specific ELISA.

[0037] FIG.5A depicts an experimental workflow.

[0038] FIG. 5B depicts confocal microscopy of splenic germinal centers and their penetration by transferred CD45.2 B cells.

[0039] FIG. 5C depicts graphs for HEL- and OVA-specific IgG as measured by ELISA and HEL- and OVA-specific GC B cells and ASCs as measured by flow cytometry.

[0040] FIG.6 depicts a schematic for interactions between certain cells in a T cell zone, T-B cell border zone, and B cell follicle / germinal center.

[0041] FIG. 7 depicts a schematic for an embodiment in which antigen specific decoy B cells may greatly reduce undesired humoral responses detrimental to certain therapies.

[0042] FIG.8A depicts a schematic for Cas 9-mediated NHEJ of PRDM1 prevents plasma cell differentiation.

[0043] FIG. 8B depicts a FACS analysis for cells treated with an sgRNA targeting PRDM1.

[0044] FIG.8C depicts a primary B cell editing work flow.

[0045] FIG.8D depicts a FACS analysis.

[0046] FIG.8E depicts a bar graph for IL-10 secretion.

[0047] FIGs 9A-9C depict antigen-primed MD4 B cells reduce endogenous humoral responses to both cognate and linked antigen. FIG. 9A depicts a workflow for experiment in which splenic B cells were enriched from a single-cell suspension and incubated ex vivo in the presence of multimeric CD40 ligand (MCD40L) and Į-CD180 antibody for 2 days before IL-4 was then added with or without HEL-OVA for an additional 2 hours. B cells were then washed and 1 x 107were transferred via tail vein into B6 mice. These mice in addition to “No Transfer” (NT) controls were immunized 3 days later with HEL-OVA antigen in Sigma Adjuvant (IP). Mice were bled thereafter for ELISA analysis. FIG. 9B depicts line graphs for a time course ELISA data for Į-HEL and Į-OVA IgM and total IgG. Data presented as mean ng / mL IgM or IgG. p values shown represent comparisons between the treatment condition and NT and were calculated using a Kruskal-Wallis test to analyze area under the curve values with Dunn’s test for multiple comparisons. FIG.9C depicts bar graphs for day 14 Į-HEL and Į-OVA IgM and total IgG. Data presented as measured antibody normalized to the NT population average. p values were calculated using Kruskal-Wallis with Dunn’s test for multiple comparisons. n=13 per condition from 2 experimental repeats. All error bars indicate 95% CI.

[0048] FIGs 10A-10F depict antigen-primed Blimp1-deficient MD4 decoy B cells blunt specific endogenous humoral responses without secreting antibody. FIG. 10A depicts a schematic for a experimental workflow for knocking out Blimp1 in which enriched B cells were electroporated with Cas9 ribonucleoproteins (RNP) targeting either Rosa26 or Prdm1 after 2 days of stimulation in vitro, then cultured for 8 days on 40LB feeder cells in the presence of IL-4. Feeders were depleted and genomic DNA collected from expanded B cells for ICEanalysis. B cells were analyzed for differentiation via FACS. Supernatant was collected and total Ig secretion was measured via ELISA. FIG. 10B depicts a bar graph for mean indel and knockout rates in the Prdm1 gene of edited B cells at Day 8, as measured via ICE. N=3 biological replicates. FIG. 10C depicts a bar graph for mean concentration of total Ig (combined IgM, IgG, IgA, and IgE) in supernatant of expanded B cells at Day 8, as measured by ELISA. n=3 biological replicates per condition. p values calculated via one-way ANOVA with a Tukey’s test for multiple comparisons. FIG.10D depicts representative FACS plots (left panels) and summary data (right panel) showing CD19loCD138+ ASC proportion within treated cell populations, gated as Live / Dead-H-2Kd- lymphocyte singlets. n=3 biological replicates per condition. p values calculated via one-way ANOVA with a Tukey’s test for multiple comparisons. FIG. 10E depicts a schematic for a workflow for treating mice with CRISPR-edited B cells in which enriched B cells were electroporated with Rosa26- or Prdm1- targeted RNP and primed with IL-4 and HEL-OVA for 2 hours before adoptive transfer of 6 x 106cells into B6 mice. Recipient and NT control mice were immunized 3 days later with HEL- OVA antigen in Sigma Adjuvant. Mice were bled thereafter for ELISA analysis. FIG. 10F depicts bar graphs for day 14 Į-HEL IgM, Į-HEL IgG, and Į-OVA IgG. Data presented as measured antibody normalized to the NT population average. p values were calculated using Kruskal-Wallis with Dunn’s test for multiple comparisons. Total n per condition from 2 experimental repeats indicated on graph. All error bars indicate 95% CI.

[0049] FIGs 11A-11E depict Blimp1KOMD4 B cells mediate dose-dependent suppression of antigen-specific IgG responses without impacting simultaneous responses to an unrelated antigenic challenge. FIG.11A depicts a schematic for a workflow for examining the impact of cell dose on decoy-mediated suppression of antigen-specific antibody. Created with BioRender. FIG. 11B depicts representative FACS plots (left) and summary data (right) showing mean engraftment of antigen-primed Blimp1KOMD4 B cells across a range of adoptive cell doses, gated as Live / Dead-Dump-CD19+ lymphocyte singlets. n=3 biological replicates per condition. p values calculated via one-way ANOVA with a Tukey’s test for multiple comparisons. FIG. 11C depicts bar graphs for day 14 Į-HEL IgG and Į-OVA IgG. Data presented as measured antibody normalized to the NT population average. p values were calculated using Kruskal-Wallis with Dunn’s test for multiple comparisons. n=13 per condition from 2 experimental repeats. FIG.11D depicts a schematic for a workflow for dual immunizingmice following adoptive transfer of Blimp1KO B cells in which Blimp1KO B6 or MD4 B cells were antigen-primed and 7 x 106cells were transferred into B6 mice. Recipient and NT controls were immunized 3 days later with both HEL-OVA and NP-CGG antigen in Sigma Adjuvant. Mice were bled thereafter for ELISA analysis. FIG.11E depicts bar graphs for day 14 Į-HEL, Į-OVA, and Į-NP IgG. Data presented as measured antibody normalized to the NT population average. p values were calculated using Kruskal-Wallis with Dunn’s test for multiple comparisons. Total n per condition from 2 experimental repeats indicated on graph. All error bars indicate 95% CI.

[0050] FIGs 12A-12D depict Blimp1KOMD4 decoy B cells dominate germinal centers. FIG. 12A depicts a schematic for a workflow for microscopy experiments in which CD45.2+B6 or CD45.2+MD4 B cells are enriched and stimulated, electroporated with either Rosa26- or Prdm1-targeted RNPs followed by antigen priming. 6 x 106Blimp1WTB6, Blimp1KOB6, Blimp1WTMD4, or Blimp1KOMD4 B cells were adoptively transferred into CD45.1+B6 recipient mice. Three days later recipient mice were immunized with HEL-OVA. Mice were sacrificed at Day 5 or Day 11 for GC analysis via confocal microscopy. FIG. 12B depicts representative micrographs of splenic GCs in mice 5 days after immunization. FIG. 12C depicts representative micrographs of splenic GCs in mice 11 days after immunization. FIG.12D depicts a graphs for CD45.2 mean fluorescence intensity of GCs on Day 5 (left) and Day 11 (right). p values were calculated using a one-way ANOVA with Šidák’s test for multiple comparisons. Number of mice per condition as well as total number of GCs analyzed are indicated for each group. Representative of two experimental repeats.

[0051] FIGs 13A-13F depict Blimp1KOMD4 decoy B cells outcompete endogenous B cells for germinal center entry and access to the light zone. FIG. 13A depicts a schematic for a workflow for GC analysis experiments in which CD45.2+B6 or CD45.2+MD4 B cells were enriched, stimulated, then electroporated with either Rosa26- or Prdm1-targeted RNPs prior to 2-hour antigen priming. 6 x 106Blimp1KOB6, Blimp1WTMD4, or Blimp1KOMD4 B cells were adoptively transferred into CD45.1+B6 recipient mice. Three days later recipient mice were immunized with HEL-OVA. Mice were bled for ELISA analysis 10 days later and sacrificed 1 day later for GC analysis. Created with BioRender. FIG. 13B depicts bar graphs day 10 Į-HEL and Į-OVA IgG. Data presented as measured antibody normalized to the NT population average. p values were calculated using Kruskal-Wallis withDunn’s test for multiple comparisons. Total n per condition from 2 experimental repeats indicated on graph. FIG. 13C depicts representative FACS plots showing adoptively transferred antigen-specific vs. endogenous GC B cells. Gated as Live / Dead-Dump- CD19+PNA+CD38- lymphocyte singlets. FIG.13D depicts bar graphs for absolute counts per spleen of endogenous HEL-specific (top) or OVA-specific (bottom) GC B cells across treatment conditions. p values were calculated using one-way ANOVA with Tukey’s test for multiple comparisons. FIG. 13E depicts bar graphs for absolute counts per spleen of endogenous HEL-specific (top) or OVA-specific (bottom) GC ASCs, defined as the CD138+ subset of (d), across treatment conditions. p values were calculated using one-way ANOVA with Tukey’s test for multiple comparisons. FIG. 13F depicts bar graphs for light zone residency (defined as GC B cells in the CXCR4loCD86hi subset) of transferred cells vs. endogenous HEL-specific (top) or OVA-specific (bottom) GC B cells. p values were calculated using a repeated measures one-way ANOVA with Šidák’s test for multiple comparisons. n=9 per condition from 2 experimental repeats for D-F. All error bars indicate 95% CI. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)

[0052] FIGs 14A-14D depict antigen-primed Blimp1KOMD4 decoy B cells suppress the generation of C2-specific IgG and reduce the incidence of functional FVIII inhibition. FIG.14A depicts a schematic for a workflow for experiment in which Blimp1KOB6 or Blimp1KOMD4 B cells were primed with HEL-C2 conjugate antigen and 107cells were transferred via tail vein into HemA mice. Recipient mice were immunized i.p. three days later with the HEL-C2 antigen. Mice were bled at days 7, 14, and 28 and their serum tested for Į- C2 IgG via ELISA and functional FVIII inhibitors via clotting assay. FIG. 14B depicts a line graph for a time course ELISA data for Į-C2 IgG. Data presented as mean fold difference in antibody relative to pooled serum from unimmunized HemA mice. p value shown represents comparison between the Blimp1KOMD4 and NT conditions and was calculated using a Kruskal-Wallis test to analyze area under the curve values with Dunn’s test for multiple comparisons. FIG. 14C depicts a bar graph for day 28 Į-C2 IgG. Data presented as measured antibody normalized to the NT population average. p values were calculated using Kruskal- Wallis with Dunn’s test for multiple comparisons. Total n per condition from 2 experimental repeats indicated on graph. FIG. 14D depicts a graph for day 28 FVIII inhibition as measured via Bethesda clotting assay. Data presented as % inhibition of FVIII-mediated clotting relativeto control. p values were calculated using Kruskal-Wallis with Dunn’s test for multiple comparisons. Total n per condition from 2 experimental repeats indicated on graph. All error bars indicate 95% CI.

[0053] FIG. 15 depicts a schematic for a proposed model: Decoy B cells exploit GC biology to negatively modulate antigen-specific humoral responses. Following adoptive transfer, HEL-OVA-primed Blimp1KOMD4 “decoy” B cells (1) interact with activated pre- GC HEL- and OVA-specific T cells (2), competing with relatively lower affinity host HEL- and OVA-specific B cells (3) for access to T cell help. Due to this competition, host B cells undergo reduced pre-GC plasmablast differentiation and transferred decoy B cells do not differentiate due to Blimp1 deficiency (4). Instead, decoy B cells are diverted en masse into GCs, also reducing the number of host B cells that enter those GCs (5). Inside GCs, decoy B cells outcompete the lower-affinity host B cells for access to antigen presented on FDCs (6), leading to a reduction in access by host B cells to help from antigen-specific Tfh (7). By dominating GC responses, some decoy B cells may exit the GC as memory B cells (8) but they do not differentiate into plasma cells (9) due to Blimp1 deficiency. Importantly, the number of host antigen-specific plasma cells exiting GCs is reduced (10). It is presently unknown how the host memory B cell compartment (11) or secondary responses are impacted by decoy- mediated suppression.

[0054] FIG.16A depicts an experimental workflow for dual-editing B cells.

[0055] FIG. 16B depicts FACS plots showing CD19loCD138+ ASC proportion and LNGFR+ Rosa26-edited proportions within treated cell populations, gated as Live / Dead- H-2Kd- lymphocyte singlets.

[0056] FIG.16C depicts a graph of mean NHEJ and HDR rates in the Rosa26 gene of edited B cells at Day 6, as measured via ddPCR.

[0057] FIG.16D depicts a graph of Mean concentration of IL-10 in supernatant of expanded B cells at Day 9, as measured by ELISA. n=3 technical replicates per condition. p values calculated via one-way ANOVA with a Tukey’s test for multiple comparisons. Data from one experiment but representative of greater than 10 experiments. All error bars indicate 95% CI.

[0058] FIG. 17 depicts FACS analyses of cells edited to express GFP, TIGIT, CD25, or CTLA4.DETAILED DESCRIPTION

[0059] Some embodiments provided herein relate to antigen specific B cells lacking a capacity to differentiate. Some embodiments include the use of such cells to reduce an antibody response in a subject to an antigen.

[0060] Despite being vital for immune defense against numerous pathogens, some antibody responses pose challenges to modern therapies. Of particular concern are monogenic diseases such as hemophilia A where the success of both recombinant factor VIII (FVIII) therapy and viral-mediated gene therapy are limited by the generation of antibody-derived inhibition of the foreign therapeutic protein and / or immune responses targeting components of the viral vector.1-4The only presently approved strategy to eliminate FVIII inhibitors is immune tolerance induction (ITI), the basic premise of which is long-term treatment with large quantities of FVIII – a process that is time-consuming and often incompletely effective.5,6In the case of gene therapy, repeat administration of potentially therapeutic adeno-associated viruses (AAV) is limited by the generation of antibody inhibitors to multiple components of the vector.7Strategies for preventing such unwanted immune responses might permit repeat vector administration, for example, in cases where a patient first receives gene therapy early in life or where repeat administration of a low dose of vector is needed to limit immunogenicity of the gene product or toxicity of the viral vector.8-10Thus, a prophylactic therapy for preventing or reducing the generation of undesired antibody inhibitors without requiring broader immunosuppression would be a valuable addition to existing strategies for treating genetic disorders.11,12

[0061] Engineered B cells and plasma cells are emerging as a potential therapy to secrete pathogen-specific antibodies or a broad range of other protein therapeutics.13–17These new therapy platforms leverage key properties of plasma cells including longevity and capacity for very high rates of protein secretion. Importantly, B cells also function as antigen-specific antigen-presenting cells (APCs) and can therefore shape specific immune responses without secreting antibodies. B cells alone are sufficient to prime T follicular helper (Tfh) responses to Plasmodium infection in mice and in one model were found to be the dominant antigen APC following immunization with virus-like particles.18,19Moreover, because germinal center (GC) entry is regulated by affinity-mediated access to limited CD4 T cell help, it should be possibleto influence the humoral output of specific T-dependent antibody responses with engineered high-affinity B cells.20Consistent with this concept, adoptively transferred, naïve mature B cells expressing a defined, high-affinity B cell antigen receptor (BCR) can enter GCs and compete with endogenous B cells recognizing the same antigen.20Importantly, in this competitive setting, high-affinity, antigen-specific B cells can out-compete lower-affinity B cells for access to antigen and receipt of activation, expansion and differentiation signals mediated by Tfh cells, thereby reducing the generation of antibody secreting cells21(ASCs) derived from the endogenous B cell populations.

[0062] In this study, whether engineering competition for antigen could effectively regulate specific humoral responses was tested. It was conceived that by eliminating the capacity of antigen-specific B cells to differentiate into plasma cells, affinity-based selection could be exploited to flood antigen-specific GCs with non-differentiating “decoy” B cells. To achieve these goals, Prdm1-targeted CRISPR / Cas9 gene editing was utilized to inactivate or knock out (KO) Blimp122in the decoy B cell population, thereby blocking plasma cell development. In parallel, the APC activity of decoy B cells was leveraged so as to limit the host humoral response to a target protein antigen while leaving non-specific antibody responses intact. Strikingly, as disclosed herein, Prdm1-edited primary B cells were shown to enter and dominate GCs in an antigen-specific fashion, and these antigen-specific, Blimp1- knockout decoy B cells suppressed humoral responses to both cognate and cis-linked antigen. Finally, the therapeutic potential of this approach was demonstrated by preventing the generation of FVIII-specific clotting inhibitors in a murine model of hemophilia A.

[0063] Some engineered B cell therapies in development aim to leverage the secretory capacity of plasma cells to secrete protein biologics such as cancer-targeted antibodies or supplement deficiencies such as factor IX in hemophilia B. In contrast, certain embodiments provided herein inhibit the immune response in a specific fashion by leveraging germinal center biology to prevent endogenous antigen specific B cells from receiving sufficient help to mount a normal antibody response. Some such embodiments are useful in preventing undesired antibody responses.

[0064] Some embodiments provided herein include preventing or inhibiting the production of antibody inhibitors towards existing therapies. For example, ~30% of hemophilia A patients eventually generate inhibitors for therapeutic factor VIII, limiting the efficacy ofthe treatment. In addition, patients undergoing gene therapy cannot undergo more than one or two dosing regiments because their immune system begins to generate antibody inhibitors that prevent or inhibit gene therapy vectors (viruses) from introducing therapeutic genes upon future introduction. FVIII- or virus-specific Decoy B cells may prevent or inhibit the generation of these inhibitors by dominating any germinal centers that arise as well as the T cell help within them.

[0065] Since Decoys are proficient at dominating germinal centers and interacting with antigen specific CD4 T cells, Decoys that are also gene-edited to secrete regulatory cytokines, such as IL-10, TGF ȕ1, or IL-33, are thought to “tolerize” or otherwise suppress antigen specific CD4 T cells. If Decoys secreting regulatory cargo are found to suppress antigen specific CD4 T cell activity, then Decoy B cells are thought to treat, inhibit, or ameliorateCD4-driven autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus.

[0066] Engineered plasma cells are being investigated as a potential cell-based therapy that leverages the native capacities of plasma cells to secrete large quantities of therapeutic protein and engraft long-term. Importantly, B cells also manifest a unique combined ability for antigen specific recognition and subsequent antigen presentation. This latter capability suggests the potential to use engineered B cell therapies to directly manipulate endogenous humoral immune responses.

[0067] Consistent with this notion, adoptively transferred, naïve mature B cells expressing a defined high-affinity B cell antigen receptor are known to enter germinal centers (GCs) and compete with endogenous B cells recognizing the same antigen for access to antigen and subsequent differentiation signals from T follicular helper cells. B lymphocyte-induced maturation protein-1 (Blimp1), which is encoded by PR domain zinc finger protein 1 (PRDM1), is a master transcription factor for plasma cell differentiation. It was hypothesized that by utilizing CRISPR / Cas9 gene editing to knock out Blimp1, affinity-based selection could be exploited to flood antigen specific GCs with non-differentiating “decoy” B cells, thereby blunting the primary endogenous humoral response to cognate antigen while leaving non-specific responses intact. Using a combination of antigen specific ELISA, flow cytometric analysis, and confocal microscopy, it was found that: (1) gene edited primary B cells enteredand dominated GCs in an antigen specific fashion, and (2) antigen specific Blimp1-knockout decoy B cells suppressed the humoral response to both cognate and cis-linked antigen.

[0068] Taken together, these findings provided a novel approach that exploited GC biology to negatively modulate specific humoral immune responses including, for example, generation of antibody inhibitors to clinically important target antigens such as factor VIII or adeno-associated viruses. Further, decoy B cells paired with unique cytokine cargos may provide a robust platform for precise, antigen specific, engineered immune regulation.

[0069] As described herein, antigen specific primary B cells, which have been (1) activated with signals that direct them into T-dependent germinal center responses (i.e., multimeric CD40 ligand, Į-CD180 antibody, and IL-4); (2) gene-edited to knock out / inactivate the PRDM1 encoding Blimp1; and (3) pulsed with a sufficiently multimeric antigen, will enter and dominate germinal centers directing humoral responses against the multimeric antigen that the B cells were pulsed with. Such decoy B cells respond to antigen and participate in germinal center responses despite being unable to differentiate into antibody-secreting plasma cells after Blimp1 knockout. Without being bound to any one theory, this may be due to the decoy B cells outcompeting endogenous B cells for access to help from CD4 T cells since upon transfer they are both more numerous and highly specific for the antigen in question.

[0070] Some embodiments provided herein relate to generating immunoregulatory B cells by editing. In some embodiments, an antigen specific B cell cannot differentiate (Blimp KO / inactivated) into a plasma cell. In some embodiments, the cell carries an immunomodulatory payload. As described herein, it has been observed that pulsing an antigen specific, Blimp KO B cell with a dual antigen (cognate Ag recognized by the BCR and another chemically linked Ag recognized by other B cells) ex vivo and then adoptively transferring these cells to a subject leads to a significant reduction in the production of antibodies to both antigens. Some such embodiments, provide a way to turn off the antibody response to the non- cognate antigen. In some embodiments, this may be applied to limiting antibody responses to important clinical antigens, for example AAV envelope.

[0071] Certain embodiments provided herein include aspects of methods and compositions disclosed in U.S. 2018 / 0282692 and U.S. 2023 / 0272431, which are expressly incorporated by reference herein in their entireties.Definitions

[0072] B lymphocyte-induced maturation protein-1 (Blimp1) is encoded by a PR domain zinc finger protein 1 (PRDM1) gene. In some embodiments provided herein, a Blimp1 knockout (KO) can refer to inactivation of the PRDM1 gene in a cell. In some embodiments, inactivation of the PRDM1 can result in reduced expression of the PRDM1 gene such as expression of Blimp1 in a cell, for example where a single allele of PRDM1 is inactivated. In some embodiments, inactivation of the PRDM1 can result in absence of expression of the PRDM1 gene such as expression of Blimp1 in a cell, for example where all alleles of PRDM1 in a cell are inactivated.

[0073] “B cells” or “B lymphocytes” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a white blood cell type of the lymphocyte subtype. B cells are unlike lymphocytes such as T cells and natural killer cells, as B cells express B cell receptors on their cell membrane. The B cell receptors allow the B cell to bind a specific antigen, which will initiate an antibody response. B cells develop from hematopoietic stem cells. As described herein, B cells can include B cell precursors, stem cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, T1 B cells, T2 B cells, marginal zone B cells, mature B cells, naïve B cells, activated B cells derived from any starting B cell population, plasmablasts (short-lived) cells, GC B cells, memory B cells, and / or long- or short-lived plasma cells and / or any mixtures or combinations thereof depending on the context.

[0074] B cell precursors include the cells from which the B cells are derived. Like T cells, B cells are lymphatic cells that are originated from the bone marrow, where they can reside until they are mature. The B cells, as described in the alternatives herein, include stem cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, T1 B cells, T2 B cells, marginal zone B cells, mature B cells, naïve B cells, plasmablasts (short- lived) cells, GC B cells, memory B cells, plasmablast cells and / or long lived plasma cells. In some alternatives of the plasma cell for expressing a molecule such as a macromolecule, protein or a peptide, the plasma cell is derived from a B cell. In some alternatives, the B cell is a memory B cell. In some alternatives, the B cell is a stem cell, early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, T1 B cell, T2 B cell, marginal zone B cell, mature B cell, naïve B cell, plasmablasts (short-lived) cell, GC B cell, memory B cell,plasmablast cell or a long-lived plasma cell. In some alternatives, the B cells comprise B cell precursors such as hematopoietic stem cells (HSCs), multipotent progenitor (MPP) cells, lymphoid progenitor (CLP) cells, naïve B cells, GC B cell, plasmablasts, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, T1 B cells, T2 B cells, marginal zone B cells, mature B cells and / or memory B cells. In some alternatives, the macromolecule is a prodrug.

[0075] “Memory B cells” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, the B cell sub-types that are formed within germinal centers following primary infection and are important in generating an accelerated and more robust antibody-mediated immune response in the case of re-infection. The B lymphocytes form the memory cells that can remember the same pathogen for future antibody production during future infections. In some alternatives of the plasma cell for expressing a molecule such as a macromolecule, protein or a peptide, the plasma cell is derived from a B cell. In some alternatives, the B cell is a memory B cell. In some alternatives, the macromolecule is a prodrug.

[0076] “Naïve B cell” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a B cell that has not yet been exposed to an antigen. Once exposed to an antigen, the naïve B cell becomes a memory B cell. In some alternatives of the plasma cell for expressing a molecule such as a macromolecule, protein or a peptide, the plasma cell is derived from a B cell. In some alternatives, the B cell is a memory B cell. In some alternatives, the macromolecule is a prodrug. “Peripheral blood mononuclear cells” (PBNC) as described herein are peripheral blood cells having a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nuclei, and neutrophils, basophils, and eosinophils have multi-lobed nuclei. In the alternatives described herein, the B cells are subject derived or are allogeneic peripheral blood mononuclear cells. In some alternatives, the B cells are blood- derived human B cells.

[0077] “Plasma cells” as described herein, are also called plasma B cells, plasmocytes, plasmacytes, or effector B cells. Plasma cells have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, forexample, white blood cells that secrete antibodies and are transported by the blood plasma and the lymphatic system.

[0078] “Plasma cell precursor” can begin as an immature plasma cell. The most immature blood cell of the plasma cell lineage is called the plasmablast which can differentiate into a mature fully differentiated plasma cells. Plasmablasts can secrete more antibodies than a B cell, but less than a plasma cell. In some alternatives, a method of making a plasma cell that expresses a molecule is provided. In some alternatives, the plasma cell is a plasma cell precursor. In some alternatives, the plasma cell precursor is a plasmablast.

[0079] “Genome editing” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a process that include methods for genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism. Editing a gene is also known as gene editing. In some alternatives described herein, a method of making plasma cells or plasma cell precursors that express a molecule, such as a macromolecule is provided, in which B cells or B cell precursors are subjected to at least one round of genome editing. Methods of genome editing can include, but is not limited to nucleic acid being inserted, deleted or replaced in the genome of a cell. In some alternatives, a nuclease is used to achieve this process. In some alternatives, the nuclease is engineered. In some alternatives, the methods include inducing double strand breaks that are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR). In some alternatives, the step of genome editing is performed by introduction of a single stranded nucleic acid. In some alternatives, the at least one round of genome editing further comprises cycling the B-cells for homologous recombination of the single stranded DNA oligonucleotides or recombinant adeno-associated virus into the candidate genetic loci. In some alternatives, the genome editing of the B cells for protein expression is performed in the absence of viral integration. In some alternatives, a second round of genome editing is performed to excise a region. In some alternatives, a third round of genome editing is performed to result in expression of a drug activatable growth enhancer. In some alternatives herein, the genome editing is performed by nonpathogenic AAV mediated editing by direct homologous recombination.

[0080] Genome editing can employ the use of RNA and protein-based transfection. For example the CRISPR / Cas system can be modified to edit genomes. This technique requiresthe delivery of the Cas nuclease complexed with a synthetic guide RNA (gRNA) into a cell, thus the cell’s genome can be cut at a specific location and allow existing genes to be removed and / or add new ones. Thus, CRISPR / Cas and related programmable endonuclease systems are rapidly becoming significant genome editing tools of the biomedical research laboratory, with their application for gene disruption and / or gene targeting as demonstrated in a variety of cultured cell and model organism systems. In some alternatives, of the CRISPR / Cas system described herein, the Cas nuclease comprises a native Cas molecule, a modified Cas molecule, or a mutant Cas molecule, such as a Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease or a modified or mutant Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease.

[0081] The basic components of CRISPR / Cas system comprise a target gene, a protospacer adjacent motif (PAM), a guide RNA, Cas endonuclease. An important aspect of applying CRISPR / Cas for genome editing is the need for a system to deliver the guide RNAs efficiently to a wide variety of cell types. This could, for example, involve delivery of an in vitro generated guide RNA as a nucleic acid (the guide RNA generated by in vitro transcription or chemical synthesis). In some alternatives, the nucleic acid could be rendered nuclease resistant by incorporation of modified bases.

[0082] The CRISPR-Cas system falls into two classes. The Class 1 system has a complex of multiple Cas proteins for the degradation of foreign nucleic acids. The Class 2 system has a single large Cas protein for a same purpose for the degradation of foreign nucleic acids. There are a 93 cas genes that are grouped into 35 families. 11 of the 35 families from a cas core, which includes the protein families CAS1 to CAS9. As described herein, Cas can comprise Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 or a modified or mutant Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease.

[0083] AAV mediated editing by direct homologous recombination is initiated by design of homology sequence “arms” that are specific to a region of the genome and results in a permanent correction in the DNA when administered to cells. In some alternatives herein, the gene editing is performed by nonpathogenic AAV mediated editing by direct homologous recombination. The identification of novel AAV genomes are described in Smith et al. (Mol Ther.2014 Sep; 22(9): 1625–1634; incorporated by reference in its entirety herein). The novel AAVs described by Smith et al., represents a new class of genetic vector for the manipulationof HSC genomes. Furthermore, these vectors may greatly expand the ability to deliver genes to targeted tissues and cells including cells that are refractory to gene transfer which circumventing prevalent preexisting immunity to AAV2. In some alternatives, the gene editing is performed by nonpathogenic AAVs naturally present in hematopoietic cells, wherein the editing is performed by AAV mediated editing by direct homologous recombination using the nonpathogenic AAVs as described in Smith et al.

[0084] “Engineered nucleases” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, enzymes that are engineered to be hybrid enzymes which can be used to specifically recognize a DNA sequence and efficiently edit the genome by the introduction of double-strand breaks. Without being limiting, there are four families of engineered nucleases are meganucleases, zinc finger nucleases (ZFN), transcription activator like effector-based nucleases (TALEN), and the CRISPR-Cas system or a modified or mutant CRISPR-Cas system.

[0085] “Meganucleases” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). In some alternative methods for making a plasma cell or plasma cell precursor that expresses a molecule such as a macromolecule, the method comprises: (a) isolating B cells, (b) developing the B cells, (c) performing a first round of genome editing of the B cells for protein expression in absence of viral integration, (d) expanding the B cells, and (e) differentiating the B cells, optionally, after step (c) or (d), thereby producing plasma cells that express a protein. In some alternatives, the first round of genome editing is performed by an RNA and protein-based transfection. In some alternatives, the nuclease is a meganuclease.

[0086] “Zinc finger nucleases (ZFN)” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, engineered restriction enzymes that are generated by fusing a zinc finger DNA-binding domain to a DNA- cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. In some alternative methods for making a plasma cell that expresses a molecule, such as a macromolecule, the method comprises: (a) isolating B cells, (b) developing the Bcells, (c) performing a first round of genome editing of the B cells for protein expression in absence of viral integration, (d) expanding the B cells, and (e) differentiating the B cells, optionally, after step (c) or (d), thereby producing plasma cells that express a protein. In some alternatives, the first round of genome editing is performed by an RNA and protein-based transfection. In some alternatives, the nuclease is a zinc finger nuclease. Certain methods for preparing cells

[0087] Some embodiments of the methods and compositions provided herein include methods for preparing an antigen specific B cell. Some such methods include (a) obtaining a modified B cell, wherein the modified B cell is derived from a B cell and: (i) lacks a capacity to differentiate, and (ii) comprises a B cell receptor (BCR) capable of specifically binding an antigen; and (b) contacting the modified B cell with the antigen, thereby preparing the antigen specific B cell.

[0088] In some embodiments, step (a) comprises modifying the B cell by inactivating a gene in the genome of the B cell required for the differentiation of the B cell, such as a PRDM1 gene which encodes B lymphocyte-induced maturation protein-1 (Blimp1). In some embodiments, the inactivating comprises introducing a nuclease capable of specifically cleaving the PRDM1 gene. In some embodiments, the nuclease is selected from a Cas nuclease, or a modified or mutant Cas nuclease, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease or a modified or mutant Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease. Some methods also include introducing a genomic RNA (gRNA) into a B cell, wherein the gRNA is capable of specifically binding to the PRDM1 gene. In some embodiments, the modified B cell lacks the capacity to differentiate into a plasma cell.

[0089] In some embodiments, step (a) comprises modifying the B cell by introducing a polynucleotide encoding the BCR into the B cell. In some embodiments, a vector comprises the polynucleotide. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, or a lentivirus vector.

[0090] In some embodiments, step (a) comprises selecting for a B cell comprising the BCR. In some embodiments, the selecting comprises contacting a population of B cells with the antigen.

[0091] Some embodiments also include stimulating the modified B cell prior to step (b). In some embodiments, the stimulating comprises contacting the modified B cell with multimeric CD40 ligand (MCD40L), anti-CD180 antibody, IL-4, IL-2, IL-6, IL-10, IL-15, lipopolysaccharide (LPS), and / or CpG oligonucleotide (ODN) 2006. In some embodiments, the stimulating comprises contacting the modified B cell with MCD40L, anti-CD180 antibody, and IL-4.

[0092] In some embodiments, step (b) comprises contacting the modified B cell with the antigen for a period less than or equal to 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or 1 hour but not zero. In some embodiments, the period is in a range from or from about 1 hour to 6 hours, from or from about 1 hour to 4 hours, or from or from about 2 hours to 4 hours.

[0093] In some embodiments, step (b) comprises contacting the modified B cell with a cytokine; optionally, wherein the cytokine comprises IL-4.

[0094] In some embodiments, the B cell is selected from the group consisting of a B cell, a hematopoietic stem cell, an early pro-B cell, a late pro-B cells, a large pre-B cell, a small pre-B cell, an immature B cell, a T1 B cell, a T2 B cell, a marginal zone B cell, a mature B cell, and a naïve B cell,

[0095] In some embodiments, the antigen is multimeric. In some embodiments, the antigen further comprises a non-cognate antigen. In some embodiments, the BCR lacks a capacity to specifically bind the non-cognate antigen. In some embodiments, the antigen and the non-cognate antigen are covalently bound to each other. In some embodiments, the antigen or non-cognate antigen comprises a polyethylene glycol (PEG), a viral protein, a non-human protein, or a recombinant protein. In some embodiments, the antigen or the non-cognate antigen comprises an AAV protein or a therapeutic protein, such as Factor VIII. In some embodiments, the AAV protein is selected from viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3). In some embodiments, the AAV protein is from AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVD-J, or Ark313.

[0096] In some embodiments, the antigen specific B cell is mammalian; optionally, wherein the antigen specific B cell is human. In some embodiments, the antigen specific B cell is ex vivo.

[0097] In some embodiments, the antigen specific B cell expresses a therapeutic protein. Some embodiments also include introducing into the antigen specific B cell a polynucleotide encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a cytokine; optionally, wherein the cytokine comprises IL-10. In some embodiments, the therapeutic protein is selected from TGF-ȕ1, IL-35, granzyme B, indoleamine-pyrrole 2,3-dioxygenase (IDO), T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), CD25, CTLA4, T- cell immunoglobulin and mucin domain 1 (TIM-1), FasL, lymphocyte-activation gene 3 (LAG-3), Galectin-9, or CD39.

[0098] Some embodiments include a cell prepared by any one of the methods provided herein. Therapies and therapeutic methods

[0099] Some embodiments of the methods and compositions provided herein include methods for treating, inhibiting or ameliorating a disorder in a subject comprising administering any one of the cells provided herein to the subject. In some embodiments, the administration reduces an antibody response to the antigen or a cognate antigen in the subject compared to a subject, which has not been administered the cell. In some embodiments, the cell is autologous to the subject. In some embodiments, the cell is allogeneic to the subject.

[0100] Some embodiments include a pharmaceutical composition comprising any one of the cells provided herein and a pharmaceutically acceptable excipient. Certain systems and kits

[0101] Some embodiments of the methods and compositions provided herein include systems. Some such systems include an antigen specific B cell, wherein the antigen specific B cell (i) lacks a capacity to differentiate, and (ii) comprises a B cell receptor (BCR) capable of specifically binding an antigen. Some embodiments also include the antigen.

[0102] In some embodiments, a PRDM1 gene in the genome of the antigen specific B cell is inactivated. Some embodiments also include a nuclease capable of specifically cleaving the PRDM1 gene; optionally, wherein the nuclease is selected from a Cas nuclease, or a modified or mutant Cas nuclease, such as Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8or Cas9 nuclease, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease; optionally, wherein the Cas nuclease is a Cas9 nuclease or a mutant or modified Cas9 nuclease. Some embodiments also include a genomic RNA (gRNA) capable of specifically binding a PRDM1 gene in the genome of the antigen specific B cell. In some embodiments, the modified B cell lacks the capacity to differentiate into a plasma cell.

[0103] Some embodiments also include a polynucleotide encoding the BCR. In some embodiments, a vector comprises the polynucleotide. In some embodiments, the vector is a viral vector; optionally, wherein the viral vector is an adeno-associated virus (AAV) vector, or a lentivirus vector.

[0104] Some embodiments also include multimeric CD40 ligand (MCD40L), anti- CD180 antibody, IL-4, IL-2, IL-6, IL-10, IL-15, lipopolysaccharide (LPS), and / or CpG oligonucleotide (ODN) 2006.

[0105] In some embodiments, the antigen specific B cell is derived from a B cell selected from the group consisting of a B cell, a hematopoietic stem cell, an early pro-B cell, a late pro-B cells, a large pre-B cell, a small pre-B cell, an immature B cell, a T1 B cell, a T2 B cell, a marginal zone B cell, a mature B cell, and a naïve B cell,

[0106] In some embodiments, the antigen is multimeric. Some embodiments also include a non-cognate antigen. In some embodiments, the BCR lacks a capacity to specifically bind the non-cognate antigen. In some embodiments, the antigen and the non-cognate antigen are covalently bound to each other. In some embodiments, the antigen or non-cognate antigen comprises a polyethylene glycol (PEG), a viral protein, a non-human protein, or a recombinant protein. In some embodiments, the antigen or the non-cognate antigen comprises an AAV protein or a therapeutic protein, such as Factor VIII. In some embodiments, the AAV protein is selected from viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3). In some embodiments, the AAV protein is from AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVD-J, or Ark313.

[0107] In some embodiments, the antigen specific B cell is mammalian; optionally, wherein the antigen specific B cell is human. In some embodiments, the antigen specific B cell is ex vivo.

[0108] In some embodiments, the antigen specific B cell expresses a therapeutic protein. Some embodiments also include a polynucleotide encoding a therapeutic protein. Insome embodiments, the therapeutic protein comprises a cytokine; optionally, wherein the cytokine comprises IL-10. In some embodiments, the therapeutic protein is selected from TGF- ȕ1, IL-35, granzyme B, indoleamine-pyrrole 2,3-dioxygenase (IDO), T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), CD25, CTLA4, T-cell immunoglobulin and mucin domain 1 (TIM-1), FasL, lymphocyte-activation gene 3 (LAG-3), Galectin-9, or CD39. EXAMPLES Example 1—Blunting specific T-dependent antibody responses by exploiting affinity selection with engineered decoy exogenous B cells

[0109] Splenic B cells were isolated from transgenic CD45.2 MD4 mice with B cell receptor specificity for hen egg lysozyme (HEL). These were then stimulated in vitro, electroporated with ribonucleoprotein (RNP) targeted to the Prdm1 gene resulting in CRISPR- mediated knockout of Blimp1. Blimp1KO MD4 B cells were then pulsed in vitro with HEL- OVA conjugate antigen for 2-4 hours and transferred to congenic mice via tail vein injection. Host mice were immunized three days later with HEL-OVA antigen in Sigma Adjuvant and Į-HEL and Į-OVA IgM and IgG measured from their serum on days 7, 14, and 28. In some cases, the spleen was later harvested and analyzed via flow cytometry or immunofluorescence imaging.

[0110] As depicted in FIGs. 1A-1B, antigen pulsed MD4 B cells secreted large quantities of Į-HEL IgM but reduced secretion of endogenous IgG specific for cognate and cis-linked antigen. FIG.1A depicts an experimental workflow. FIG.1B depicts bar graphs of Į-HEL and Į-OV IgM and IgG production in immunized mice following treatment, as measured by antigen specific ELISA.

[0111] As shown in FIGs. 2A-2D, Blimp1 knockout (KO) using a Prdm1-specific RNP was highly efficient. FIG.2A depicts the experimental workflow. FIG.2B depicts a bar graph with an ICE score for Prdm1 sgRNA. FIG.2C depicts a FACS analysis (left panel) and a graph (right panel) for B cell differentiation in vitro following gene editing. FIG.2D depicts a bar graph of total Ig secretion in vitro following gene editing.

[0112] As depicted in FIGs. 3A-3C, antigen specific Blimp1 KO decoy B cells reduced endogenous IgG specific for both cognate and cis-linked antigen in a dose-dependentmanner. FIG. 3A depicts the experimental workflow. FIG. 3B depicts a FACS analysis (left panel) and a graph (right panel) for splenic engraftment of B cells at different doses. FIG.3C depicts graphs for Į-HEL IgM, and Į-HEL IgG, and Į-OVA IgG production in immunized mice following treatment, as measured by antigen specific ELISA.

[0113] As depicted in FIGs. 4A-4B, decoy B cells reduced IgG in an antigen specific manner. FIG. 4A depicts the experimental workflow. FIG. 4B depicts graphs for Į- HEL, Į-OVA, and Į-NP IgG production in immunized mice following treatment, as measured by antigen specific ELISA.

[0114] As depicted in FIGs. 5A-5C, decoy B cells entered and dominated GCs, reducing the number of endogenous antigen specific B cells that participated in GCs and differentiated into antibody-secreting cells (ASCs). FIG. 5A depicts the experimental workflow. FIG. 5B depicts confocal microscopy of splenic germinal centers and their penetration by transferred CD45.2 B cells. FIG.5C depicts graphs for HEL- and OVA-specific IgG as measured by ELISA and HEL- and OVA-specific GC B cells and ASCs as measured by flow cytometry.

[0115] FIG.6 depicts a schematic for interactions between certain cells in a T cell zone, T-B cell border zone, and B cell follicle / germinal center. Without being bound to any one theory it was hypothesized that (1) HEL- and OVA-specific CD4 T cells were activated by antigen presenting DCs and Blimp1KOMD4 B cells, then (2) HEL- and OVA-specific pre- Tfh activated the Blimp1KOMD4 B cells. (3) Unable to differentiate into PBs, (4) the Blimp1KOMD4 favored entry into GCs where (5) they outcompeted endogenous HEL- and OVA-specific B cells in the LZ, (6) returned to the DZ (7) where they proliferated (8) and continued to dominate selection. (9) Some Blimp1KOMD4 exited the GC as memory B cells, although they were unable to differentiate into LLPCs. (10) Endogenous HEL-and OVA- specific B cells contributed limited T-dependent antibody due to inadequate help.

[0116] Antigen specific decoy B cells may greatly reduce undesired humoral responses detrimental to certain therapies (FIG. 7). For example, a significant problem in the treatment of hemophilia A and other protein deficiency disorders is that replacement therapy frequently (30% of the time in the case of hemophilia A) results in the generation of antibody inhibitors to the therapy itself since these patients have no central tolerance for the missing factor. Decoy B cells may be an option for preventing such undesired humoral responses.Example 2—Precise gene editing of primary B cells to secrete cytokines and regulate the immune response

[0117] Regulatory B cells have been implicated as important mediators of peripheral tolerance. Their promise as a cellular therapy for autoimmune disorders is limited by the transient nature of the regulatory B cell phenotype. We are attempting to address this limitation by engineering antigen specific B cells that constitutively secrete IL-10 (eBRegs).

[0118] B cells were harvested from mouse spleen and stimulated. NHEJ and HDR- mediated gene editing were performed using Cas9 RNP complexes and AAV-delivered recombinant repair templates. Products were expanded post-editing on feeder cells and enriched for edited cells based on cis-linked LNGFR surface tag. Products were further assessed editing via flow cytometry and IL-10 secretion via ELISA.

[0119] Primary murine B cells were dual edited to prevent differentiation and constitutively secrete regulatory cytokines. FIG. 8A depicts a schematic for Cas 9-mediated NHEJ of PRDM1 prevents plasma cell differentiation. FIG. 8B depicts a FACS analysis for cells treated with an sgRNA targeting PRDM1. FIG.8C depicts a primary B cell editing work flow. FIG. 8D and 8E depict day 4 post-dual editing, in which FIG. 8D depicts a FACS analysis and FIG.8E depicts a bar graph for IL-10 secretion. Example 3— Blunting specific T-dependent antibody responses with engineered 'decoy' B cells Adoptive transfer of antigen-primed, antigen-specific B cells reduces the humoral response to both cognate and cis-linked antigen

[0120] It was hypothesized that adoptively transferred, high-affinity B cells specific for one protein antigen might be able to outcompete lower-affinity host (endogenous) B cells specific for the same and / or linked antigens. Transferred B cells might achieve this dominance by competing more effectively for access to antigen, subsequent peptide presentation on MHCII, and / or access to T cell help sufficient to mount a robust antibody response. Further, a competitive advantage conferred by high affinity B cells could be amplified by exposing activated, antigen-specific B cells to cognate antigen ex vivo prior to adoptive transfer and subsequent immunization.

[0121] To begin to examine these ideas, adoptive transfer studies were performed (FIG. 9A). To obtain primary B cells with a defined antigen-specificity, B cells were utilized from MD4 transgenic mice, in which ~95% of splenic B cells express a high-affinity BCR (HyHEL10) which recognizes hen egg lysozyme (HEL) and is expressed on the B cell surface as non-switchable IgM and IgD.23,24Primary B cells were first enriched from the spleens of MD4 or control C57Bl6 (B6) mice. To drive B cell activation, these respective bulk B cell populations were cultured for 2 days in media supplemented with multimeric CD40 ligand (MCD40L) to mimic CD4 T-cell help and Į-CD180 antibody to mimic Toll-like receptor 4 engagement without driving cytokine secretion.25-28In some embodiments, B cells can be stimulated / activated in conditions that drive B cell activation and cell cycling without driving differentiation. This can be accomplished with a combination of one or more of MCD40L, Į- CD180 antibody, LPS, IL-4, IL-2, IL-6, IL-10, IL-15, and / or a TLR9 agonist, such as CpG oligonucleotide (ODN) 2006.

[0122] Next, to initiate cis-linked antigen presentation, activated B cell populations were exposed to HEL conjugated to ovalbumin (HEL-OVA conjugate) in the presence of soluble IL-4 for 2 hours. IL-4 was utilized to promote B cell survival.29,30Activated HEL- OVA-pulsed B cells were then adoptively transferred into cohorts of antigen-naïve B6 mice and three days later these mice were immunized intraperitoneally (i.p.) with HEL-OVA antigen in Sigma Adjuvant.

[0123] To track antigen-specific humoral responses, serum was collected at days 7, 14, and 28. It was found that Į-HEL IgM was increased and that Į-HEL IgG was decreased in mice receiving MD4 cells regardless of whether they had been primed in vitro (FIGs 9B, FIG. 9C). Strikingly, it was also found that MD4 cells primed with HEL-OVA significantly depressed OVA-specific IgG responses. This along with the increased inhibition of Į-HEL IgG suggested that ex vivo antigen priming prior to adoptive transfer may confer an additional competitive advantage to the transferred B cells in T-dependent antibody responses. Collectively, these data showed that transfer of B cells primed with cis-linked antigens can regulate subsequent humoral responses to both cognate and cis-linked antigen.Blimp1-deficient, antigen-specific B cells blunt the host humoral response without secreting antibody

[0124] While antigen-primed MD4 B cells markedly reduced the production of both Į-HEL and Į-OVA IgG, the recipient mice exhibited significantly increased levels of serum Į-HEL IgM. Differentiation of adoptively transferred MD4 B cells, rather than endogenous B cells, was likely responsible for the rise in serum HEL-specific IgM. The transcription factor Blimp1 plays an essential role in terminal differentiation of B cells into ASCs.22,31Therefore, to eliminate differentiation of adoptively transferred B cell populations a strategy was developed to inactivate and knock out Blimp1 expression using CRISPR / Cas9 to target the Prdm1 gene.32

[0125] The efficiency of Prdm1 disruption was assessed. Enriched splenic B cells were cultured for 2 days (FIG.9A) and then electroporated with ribonucleoprotein complexes (RNPs) consisting of Cas9 bound to single guide RNA (sgRNA) targeting either a control locus (Rosa26) or Prdm1. Electroporated cells were expanded in IL-4 on a fibroblast line (40LB feeder cells) expressing surface murine CD40L and secreting murine B cell-activating factor (BAFF)33 for 8 days (FIG. 10A). The Prdm1 gene was PCR-amplified from genomic DNA (gDNA) purified from feeder-depleted, expanded B cells and submitted for Sanger sequencing. Trace files were analyzed via inference of CRISPR edits (ICE) to estimate Prdm1 gene indel and knockout rates34 of 98% and 93%, respectively (FIG.10B). Total antibody levels and the size of the CD19loCD138+ ASC population were greatly reduced following Prdm1 targeting relative to mock or control edited populations (FIG 10C, FIG. 10D), findings consistent with near complete loss of Blimp1-mediated ASC differentiation and antibody secretion.35,36

[0126] To determine if Blimp1-deficient MD4 B cells lacking the ability to generate antibodies retain the ability to reduce antigen-specific IgG following immunization, HEL-OVA antigen-primed Prdm1-targeted B cells were adoptively transferred into B6 mice (FIG. 10E). Relative to Rosa26-targeted B cells, transfer of Blimp1-deficient B cells led to a marked reduction in Į-HEL IgM (FIG. 10F). Together with in vitro data, these findings were strong evidence that the increase in Į-HEL IgM in recipients of Blimp1WTMD4 B cells was derived from the transferred B cells. Consistent with data depicted in FIGs 9A-9C, Į-HEL IgG and Į-OVA IgG responses were also markedly reduced in recipients of antigen-primed Prdm1- targeted MD4 B cells (FIG.10F).

[0127] MD4 B cells compete for binding to HEL-OVA during ongoing humoral responses via their specificity for HEL; they should not be able to compete for binding to unconjugated OVA. No significant reduction was observed in Į-OVA IgG if mice were immunized with unconjugated OVA following the transfer of HEL-OVA-primed Blimp1KO MD4 B cells, suggesting that the reduction in antigen-specific antibody required the ongoing ability to compete for antigen. Because antigen-primed, Blimp1KO MD4 B cells effectively blunted both cognate and cis-linked humoral responses without contributing to the humoral response, they are referred to hereafter as “decoy” B cells. Decoy B cells exhibit cell dose-dependent antigen-specific suppression

[0128] The relationship between cell dose and decoy-mediated suppression was examined by transferring a range of doses of CD45.2+ antigen-pulsed MD4 decoy B cells into CD45.1+B6 mice. Spleens were collected from a subset of host mice at Day 3 post-transfer to assess B cell engraftment and the remaining animals were immunized as above (FIG. 11A). Flow cytometry-based cell enumeration showed a linear increase in B cell engraftment with an average splenic engraftment rate of ~1-2% of the total transferred decoy cell population (FIG. 11B). The reduction in Į-HEL IgG following immunization was dose-dependent, with reductions observed at each decoy B cell dose. In contrast, a significant reduction in serum Į- OVA IgG was present only at the highest dose of decoy B cells (Fig.3C).

[0129] As GC-dependent responses may be restricted by a finite number of follicular dendritic cell (FDC) niches, adoptive transfer of large numbers of antigen-specific B cells may limit the response to concurrent antigenic challenge.37To examine this possibility, mice were treated with either polyclonal or MD4 decoy B cells and then immunized simultaneously with HEL-OVA and NP-CGG, another T-dependent antigen (FIG. 11D). While Į-HEL and Į-OVA IgG were reduced with antigen-specific decoy treatment, Į-NP IgG was unaffected across conditions (FIG.11E). This suggested that antigen-primed decoy B cells outcompeted endogenous antigen-specific B cells without limiting the humoral output from B cells specific for other antigens.Blimp1KOMD4 decoy B cells outcompete endogenous B cells in germinal centers

[0130] To directly assess the impact of decoy B cells on GC responses in vivo, a series of imaging experiments and parallel flow cytometry studies evaluating the kinetics of GC formation and cellular distribution were performed. Antigen-primed Blimp1WT or Blimp1KO B6 or HEL-specific MD4 B cells expressing the CD45.2 congenic marker were adoptively transferred into cohorts of CD45.1+B6 recipient mice (FIG. 12A). Following immunization with HEL-OVA, spleens were harvested and analyzed via confocal microscopy at multiple timepoints to examine the impact of antigen-specificity and Blimp1 expression on decoy B cell localization across the GC response. Immediately prior to immunization, transferred B cells appeared to be distributed throughout the splenic B cell follicles regardless of specificity or Blimp1 expression. Strikingly, five days after immunization, Blimp1KOMD4 B cells dominated most GCs (FIG. 12B, FIG. 12D). In contrast, while GCs were readily detected in immunized mice that received Blimp1KOB6 or Blimp1WTMD4 B cells, those GCs exhibited significantly reduced penetrance of transferred CD45.2+ B cells and were largely composed of endogenous B cells (FIG. 12B, FIG. 12D). By day 11, GC penetrance by both Blimp1WTMD4 and Blimp1KOMD4 was equivalent, and both conditions exhibited increased penetrance over Blimp1WTB6 or Blimp1KOB6 transfer conditions (FIG. 12C, FIG. 12D). Adoptive transfer of Blimp1KO B cells appeared to increase the variance in GC size but did not appear to disrupt germinal center formation per se, as average GC size across mice across conditions was found to be comparable.

[0131] Flow cytometry was used to further define the impact of Blimp1KO antigen-specific decoy B cells on the host GC response, utilizing congenic markers and HEL and OVA tetramers to track and quantify endogenous and decoy B cells following immunization. The experimental approach is shown schematically in FIG.13A. Ten days after immunization, Į-HEL and Į-OVA serum IgG were similarly reduced in mice treated with Blimp1WT or Blimp1KOMD4 (FIG. 13B). Importantly, the reduction in serum antibody correlated with a marked decrease in the proportion of endogenous antigen-specific GC B cell populations (FIG. 13C, shows representative data for one Blimp1KOMD4 recipient). Specifically, in comparison to the no cell transfer control, at day 11, Blimp1WT or Blimp1KOMD4 recipient mice exhibited a significant reduction in both HEL- and OVA- specific GC B cells (FIG. 13D) and HEL- and OVA-specific GC ASCs (FIG. 13E).Surprisingly, despite not impacting day 14 serum concentrations of IgG in previous experiments, Į-HEL and Į-OVA serum IgG (FIG.13B), Į-HEL and Į-OVA GC B cells (FIG. 13D), and Į-HEL and Į-OVA GC ASCs (FIG.13E) were also partially reduced in mice treated with polyclonal Blimp1KO B6 B cells, though to a lesser extent than the reduction upon treatment with Blimp1WT or Blimp1KO MD4 B cells. Notably, in recipients of adoptively transferred Blimp1WT and Blimp1KO MD4 B cells, but not Blimp1KOB6 B cells, transferred B cell populations present within the GC expressed increased light zone (LZ) markers (CXCR4loCD86hi) relative to endogenous HEL- or OVA-antigen-specific GC B cells (FIG. 13F). Together, these results demonstrated that high-affinity antigen-specific B cells primed with antigen conjugate not only invade GCs but also inhibited participation of endogenous B cells specific for either antigen in GCs initiated following immunization. Interestingly, the increased dominance of early GCs suggested that this effect was augmented by Blimp1 deficiency during the early GC response. HEL-specific decoy B cells prevent the generation of factor VIII inhibitors in a murine model of hemophilia A

[0132] To explore whether decoy B cells could be used to suppress an undesired antibody response to a clinically relevant therapeutic protein, studies assessing inhibitory responses to human factor VIII (FVIII) were performed. The C2 domain of FVIII contains binding sites for von Willebrand factor and phosphatidylserine and may serve as a docking module to promote FVIII proteolytic activation by thrombin or activated factor X.38-41The C2 domain’s necessity for the proper function of FVIII paired with its immunogenicity makes the C2 domain a frequent target of inhibitory antibodies following FVIII therapy.42,43Therefore the C2 domain of human FVIII was chemically conjugated to HEL in a 1:1 ratio and Blimp1KOMD4 B cells were primed with the conjugate. These antigen-primed decoy B cells, or alternatively, HEL-C2 primed, polyclonal B cells, were then injected into the FVIII exon 16-mutated HemA mouse strain prior to immunization with the same HEL-C2 conjugate to model the generation of FVIII inhibitors with or without decoy B cell treatment (FIG.14A).44,45Strikingly, transfer of HEL-C2 primed, HEL-specific, Blimp1-deficient decoy B cells significantly reduced the generation of Į-C2 IgG following immunization relative to the antibody produced in untreated mice (FIG.14B, FIG.14C). In contrast, mice treated with HEL-C2 primed, polyclonal B cells exhibited marginally increased Į-C2 IgG. Importantly, the reduction in Į-C2 IgG in Blimp1KOMD4 B cell-treated mice translated to a stark reduction in functional inhibition of FVIII-mediated clotting 28 days after immunization (FIG. 14D). Collectively, these results showed that antigen-specific decoy B cells reduced not only host humoral responses to relatively weak cis-linked antigens such as OVA, but also functional inhibitors of immunogenic and clinically relevant antigens.

[0133] Antibody inhibitors are a frequent complication in the treatment of patients with inherited protein deficiency disorders;1-12the lack of targeted prophylactics that do not require broad immunosuppression limits the efficacy of the care available to these patients. In the studies disclosed herein, a cell-based approach is described for reducing antigen-specific antibody responses without the use of immunosuppression. Adoptive transfer of exogenously antigen-primed, high-affinity B cells were shown to inhibit the generation of host ASCs and antibody responses specific for both cognate and cis-linked antigens. Rather than hindering this effect, Blimp1 deficiency appeared to convey an early advantage to antigen-primed “decoy” B cells for GC dominance and accomplished the same reduction in systemic antibody without undermining that reduction by differentiating and secreting decoy-encoded antibody. Decoy B cells outcompeted endogenous B cells for participation in GCs, which likely explains the observation of significant decreases in endogenous antigen-specific ASCs emerging from those GCs – all while leaving concurrent unrelated antibody responses intact. Finally, the linked decoy effect was leveraged to prevent the generation of FVIII-specific clotting inhibitors in a murine model of hemophilia A, demonstrating the therapeutic potential of this platform.

[0134] Previous studies have demonstrated that relatively lower-affinity B cells are outcompeted by high-affinity B cells for germinal center entry20and in adoptive transfer models, this correlates with a decrease in antigen-specific antibody produced by the lower- affinity endogenous B cells when high-affinity transgenic B cells are present.21However, it was unknown whether this effect could be leveraged to reduce antibody responses specific for a cis-linked antigen. Studies disclosed herein, demonstrated that when transgenic high-affinity B cells were primed with conjugate antigen ex vivo prior to adoptive transfer, not only was this effect increased, but it extended to endogenous antibody responses specific for the linked antigen (FIG. 9B, FIG. 9C). This critical observation, and the subsequent use in the FVIIImodel, suggested this cis-linked antigen approach was likely leverageable for B cell-based therapies designed to limit the humoral response to a broad range of candidate antigens.

[0135] Notably, Blimp1WT MD4 B cells retained the capacity for secretion of antigen-specific antibodies following adoptive transfer. Prdm1 was chosen as a target for gene ablation to curtail antibody secretion by transferred cells and, in parallel via limiting differentiation, to potentially enhance decoy cell antigen-presenting function.46,47A CRISPR / Cas9 protocol was developed to disrupt the Prdm1 gene in activated primary B cells before adoptive transfer (FIG. 10A). Mice treated with antigen-primed, Blimp1-deficient, high-affinity decoy B cells mounted significantly reduced antigen-specific antibody responses to both cognate and cis-linked antigen upon immunization (FIG. 10D, FIG. 10E, FIG. 10F). The ability of Blimp1-deficent decoy cells to retain their suppressive activity supports the concept that the inhibitory impact of decoy B cells is driven primarily by competition for access to finite T cell help rather than by antibody feedback limiting the availability of soluble antigen potentially required for maintaining the GC response. This latter inference was supported by the observation that the suppression of antibody specific for either cognate (HEL) or cis-linked (OVA) antigen was dose-dependent – cognate responses exquisitely so, with stepped reductions at every dose (FIG. 11C). Finally, Blimp1 deficiency appeared to improve rather than handicap decoy access to antigen-specific T cells by driving early dominance of GCs (FIG.12B, FIG.12D).

[0136] FIG.15 discloses a working model for decoy B cell function based upon the above detailed observations. Following adoptive transfer and immunization, both Blimp1+ and Blimp1-deficient MD4 decoy B cells compete with endogenous B cells for help from pre-GC T cells and receive signals to either differentiate into short-lived plasmablasts or enter a GC. A large proportion of Blimp1WTMD4 B cells likely become short-lived plasmablasts instead of entering a GC (supported by the observation of high serum levels of Į-HEL IgM at Day 7 in HEL-OVA-immunized mice receiving Blimp1WTMD4 B cells (FIG. 9B, FIG. 9C)), whereas the day 5 confocal microscopy suggests that Blimp1KOMD4 B cells were diverted into GCs en masse to a greater degree than are Blimp1WTMD4 (FIG. 12B, FIG. 12D). The microscopy findings disclosed herein further suggested that between days 5 and 11, the relatively fewer Blimp1WTMD4 B cells that did enter GCs were also able to outcompete endogenous B cells and proliferated to the extent that domination by Blimp1KO vs. Blimp1WTMD4 B cells was indistinguishable at day 11. Not only did both Blimp1-sufficient and Blimp1- deficient MD4 B cells enter and seemingly dominate GCs, but their presence also correlated with a steep reduction in the number of endogenous HEL- and OVA-specific B cells and ASCs present in peak GCs (FIG.13C, FIG.13D, FIG.13E). Importantly, both Blimp1-sufficient and Blimp1-deficient MD4 B cells exhibited phenotypic features consistent with increased access to the GC light zone relative to endogenous antigen-specific B cells (FIG. 13F). This likely translates to superior access to Tfh help relative to endogenous B cells and therefore decoy B cells may suppress endogenous affinity maturation. Overall, the reduction in endogenous antibody specific for both cognate and cis-linked antigen is likely due to a reduction in both pre- and post-GC endogenous antigen-specific ASCs, resulting from reduced competition for finite T cell help in the presence of decoy B cells.

[0137] The combined data disclosed herein, supports future therapeutic application. First, decoy B cells limited responses specific for cognate and cis-linked antigen but did not impact concurrent GC responses to an unrelated antigenic challenge (FIG. 11E), suggesting this approach is unlikely to impact a simultaneous, but independent, infectious challenge. The therapeutic potential for decoy-mediated suppression was further demonstrated in a clinically relevant setting, as decoy B cells limited the generation of pathogenic clotting inhibitors in a murine model of hemophilia A. Upon immunization of FVIIIKO HemA mice with an antigen comprised of HEL chemically conjugated to the immunogenic C2 domain of human FVIII (FIG. 14A), nearly half of immunized control animals exhibited functional inhibition of FVIII-mediated clotting (FIG. 14D). Pretreatment with HEL-C2-primed MD4 decoy B cells resulted in a profound reduction of serum Į-C2 IgG (FIG. 14B, FIG. 14C) and the proportion of mice that developed FVIII inhibitors dropped six-fold (FIG. 14D). A prophylactic therapy that can reduce or mitigate the generation of inhibitors to biologic therapies may be less time-consuming and more effective than ITI and might provide a powerful means to extend the therapeutic window of existing treatments. The utility of the decoy B cell platform described herein is applicable to current challenges in gene therapy and, in parallel, is likely to be augmented by additional cell engineering..

[0138] In summary, pretreatment with antigen-primed, antigen-specific, Blimp1- deficient B cells reduced serum antibody specific for both cognate and cis-linked antigen following immunization without impacting concurrent unrelated humoral responses. These“decoy” B cells readily entered germinal centers, reducing the number of endogenous antigen- specific B cells that emerged from these GCs as functional antibody-secreting cells. This approach has clinical implications, as decoy prophylaxis profoundly reduced the emergence of FVIII-targeted clotting inhibitors in a murine model of hemophilia A. Taken together, the studies disclosed herein suggest that Blimp1-knockout decoy B cells can provide a robust platform for precise, antigen-specific, engineered immune regulation. Methods

[0139] B6 (C57Bl / 6), CD45.1-B6 (Ptprca Pepcb / BoyJ), MD4, and CD45.1-HemA mice (Factor VIII exon 16-knockout) on a CD45.1-B6 background44,45were maintained in a specific pathogen-free environment.

[0140] Antigen preparation. HEL and OVA were purchased from ThermoFisher Scientific. NP-CGG, ratio >40 was purchased from Biosearch Technologies. The gene for human FVIII C2 domain (residues 2171–2332) was subcloned into a pET28a-TEV(+) plasmid with NdeI and XhoI restriction sites, and an N-terminal His6 tag by Genscript. C2 was then expressed in SHuffle T7 E.coli B cells (New England Biolabs) and grown in 2XYT media containing 20 mM MgCl2and 50 μg / mL Kanamycin at 30°C until OD600 reached 0.6-0.8. C2 was purified from whole cell lysate via nickel column purification. The His-tag of the purified protein was cleaved with TEV protease and the His-tag was removed via nickel column purification. Purity was analyzed via SDS-PAGE.

[0141] Maleimide-activated OVA was conjugated to sulfhydrylated HEL to generate stable HEL-OVA conjugates. OVA was maleimide-activated with a 100-fold molar excess of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo- SMCC; ThermoFisher). HEL was incubated with a 7-10-fold molar excess of N-succinimidyl S-acetylthioacetate (SATA; ThermoFisher), and HEL-SATA was deacetylated in 0.5 M hydroxylamine / 25 mM EDTA in PBS, pH 7.2-7.5. Activated proteins were then conjugated with a 4-fold molar excess of HEL to OVA before removal of unconjugated HEL and OVA monomers via size exclusion chromatography.

[0142] Maleimide-activated C2 was conjugated to sulfhydrylated HEL similarly, except C2 was activated with PEGylated SMCC (SM(PEG)6; ThermoFisher) and HEL wasactivated with PEGylated SATA (SAT(PEG)4; ThermoFisher) to maintain antigen solubility. HEL and C2 were conjugated at a 1:1 molar ratio.

[0143] CRISPR / Cas9 editing and culturing of B cells. Spleens were harvested from 8-14 week-old B6 or MD4 donor mice and B cells were enriched using the EasySep™ Mouse B Cell Isolation Kit (StemCell) according to the manufacturer’s instructions. Enriched B cells were activated for 2 days in IMDM supplemented with 55 μM 2-mercaptoethanol, 10% FBS, 100 ng / mL murine multimeric CD40L (AdipoGen), 1 μg / mL rat Į-mouse CD180 (BD Pharmingen), and 100 IU ml–1 of penicillin and 100 μg ml–1 of streptomycin (Gibco) at a density of 3-4 x 106cells / mL. After 2 days in media at 37°C, B cells were harvested from plates, washed with MACS buffer (0.5% BSA / 2mM EDTA in PBS), and counted via hemocytometer.

[0144] For Rosa26 or Prdm1 gene targeting, Rosa26 or Prdm1 sgRNA (Synthego) was complexed with Alt-R™ S.p. Cas9 Nuclease V3 (IDT) at a molar ratio of 3:1 sgRNA:Cas9 in electroporation buffer (MaxCyte) for 10-20 minutes at room temperature (RT). Electroporation reactions consisted of 1E8 cells / mL in electroporation buffer with a final Cas9 concentration of 1.25 μM. All electroporations were performed on a MaxCyte ExPERT GTx with the “B Cell 2” protocol.

[0145] Following electroporation, cells were immediately diluted into IMDM supplemented with 55 μM 2-mercaptoethanol, 10% FBS, 100 ng / mL murine multimeric CD40L, 1 μg / mL rat Į-mouse CD180, 10 ng / mL murine IL-4 (PeproTech), 100 IU ml–1 of penicillin and 100 μg ml–1 of streptomycin, and 1 μg / mL HEL-OVA or HEL-C2 (where indicated) at a cell density of 3-5 x 106cells / mL. Cells recovered in media at 37°C for 2 hours before adoptive transfer.

[0146] For characterization of gene knockout efficiency, 5 x 104electroporated cells were added to each of 3 wells of a 12-well plate coated with 2 x 105irradiated 40LB feeder cells33in 4 mL B cell media supplemented with 1 ng / mL IL-4 and Pen / Strep. After 4 days, all cells were harvested from each well, washed with MACS buffer, and transferred to a fresh 10 cm plate coated with 3 x 106irradiated 40LB feeder cells in 40 mL B cell media supplemented with 1 ng / mL IL-4 and Pen / Strep. After 4 more days, all cells were harvested from the 10 cm plates, incubated with biotinylated Į-H-2Kd antibody (BioLegend), and 40LB feeder cells were depleted with Į-biotin microbeads (Miltenyi) and LD columns (Miltenyi)according to the manufacturer’s directions. Half of the remaining B cells were phenotypically characterized for expression of surface CD138 via flow cytometry. Genomic DNA was isolated from the other half of the B cells using the Zymo Research Quick-DNA Microprep Kit and gene regions surrounding Cas9 cut sites were amplified via PCR with primers manufactured by IDT and PrimeSTAR GXL DNA Polymerase (Takara Bio). Gene amplicons were cleaned with the QIAquick PCR Purification Kit, submitted to Genewiz for Sanger sequencing, and Inference of CRISPR Edits (ICE) analysis was performed to obtain indel and knockout scores by submitting .ab1 files to Synthego’s online tool.

[0147] Adoptive transfer and immunization. B cells were harvested from plates, washed with MACS buffer, and counted via hemocytometer before exchanging buffer into sterile PBS at an appropriate cell density. Cells were transferred intravenously (i.v.) in 200 μL via tail vein. Pooled B cells from 8-14 week-old mice of both sexes were transferred into age- matched male B6 and CD45.1-B6 mice in HEL-OVA immunization experiments. Pooled B cells from 8-14 week-old female mice were transferred into age-matched CD45.1-HemA mice of both sexes in HEL-C2 immunization experiments.

[0148] Where indicated, mice were immunized intraperitoneally (i.p.) with 25 μg HEL-OVA, 25 μg HEL-C2, or 100 μg NP-CGG prepared in Sigma Adjuvant System (Sigma- Aldrich) according to the manufacturer’s directions.

[0149] ELISAs and Bethesda assays. For all mice except those of the HemA phenotype, serum was collected via bleeds from the submandibular or submental vein or terminally via cardiac puncture. In these experiments, blood was collected in BD Microtainer Blood Collection Tubes with clot activator and SST gel, spun at maximum speed on a benchtop microcentrifuge for 5 minutes. For experiments using HemA mice, serum was collected via retroorbital bleed with plain capillary tubes and dispensed into polypropylene microcentrifuge tubes, followed by centrifugation for 5 min at 5,000 x g. Supernatant was either used for ELISAs immediately or stored at -80°C until assayed. For measurement of total antibody secreted from cultured cells, culture supernatant was collected from centrifuged cell cultures and stored at -80°C until assayed.96-well polystyrene plates were coated overnight at 4°C with 2 μg / mL goat Į-mouse Ig (Southern Biotech), HEL, OVA, C2, or NP-BSA (ratio 1-9, Biosearch Technologies) in PBS. Wells were washed three times with 300 μL 0.05% Tween- 20 in PBS (PBST) and blocked overnight with 200 μL 1% BSA, 0.05% Tween-20 in PBS at4°C. Wells were washed three times with PBST and sevenfold serial dilutions of sera in duplicate were added to the wells with sevenfold dilutions of appropriate standards (mouse IgM, IgG, IgE, or IgA (Southern Biotech) and pooled B6 serum) in blocking buffer and the plates were incubated at RT for 90 mins. Wells were then washed six times with PBST and HRP goat Į-mouse IgM, IgG, IgE, or IgA (Southern Biotech), diluted 1:2000 in blocking buffer, was added to wells and incubated at RT for 60 mins. Wells were then washed six times with PBST and treated with 100 μL TMB substrate (BD Bioscience) for 3-5 minutes before the addition of 100 μL stop solution (2N H2SO4). Absorbance at 450 nm was measured with a SpectraMax microplate reader (Molecular Devices). Concentrations of antibodies were calculated from standard curves generated from diluted mouse IgM and IgG then corrected for plate-to-plate variation by normalization with the pooled B6 serum on every plate. Statistical differences in antibody in all experiments were calculated using a Kruskal-Willis test with a Dunn’s test for multiple comparisons.

[0150] Bethesda assays were performed to examine FVIII inhibition54using a modified clotting assay with aPTT reagent and FVIII-deficient plasma. Residual activity numbers below 100 were subtracted from 100 and reported as percent inhibition of normal FVIII activity while residual activity calculated above 100 was reported as 0 percent inhibition. Statistical significance was calculated via Kruskal-Wallis test with a Dunn’s multiple comparisons test.

[0151] Flow cytometry. Single-cell suspensions from spleens or cell cultures were incubated with fluorochrome-conjugated antibodies or antigen tetramer55for 20 min at RT in PBS, washed with 5 ml PBS supplemented with 2% FBS (FACS buffer), and resuspended in FACS buffer for acquisition. For experiments requiring cell counting, 50 μL of AccuCount Fluorescent Particles (Spherotech) was added to samples after final wash and used to quantify total cell numbers. Data were acquired on a LSR II or LSRFortessa and analysed using FlowJo (TreeStar).

[0152] Confocal microscopy and analysis. For confocal imaging, PFA-fixed and sectioned spleens were imaged as previously described using a Leica SP8 microscope56. Briefly, isolated spleens were fixed using BD Cytofix (BD Biosciences) diluted 1:3 in PBS for 24 hours at 4°C, and were then dehydrated with 30% sucrose solution for 1-3 days at 4°C. Spleens were cut longitudinally and then embedded in OCT compound (Tissue-Tek) and storedat -20°C. Spleens were sectioned on a Thermo Scientific Micron HM550 cryostat into 20 μm sections and stained.56A Leica SP8 tiling confocal microscope equipped with a 20X 0.8NA oil objective was used for confocal image acquisition. Multiparameter confocal images were corrected for fluorophore spillover using the built-in Leica Channel Dye Separation module. Single stained controls were acquired using UltraComp eBeads (Invitrogen) that were incubated with fluorescently conjugated antibodies, mounted on slides with Fluormount-G slide mounting media (ThermoFisher), and imaged. All raw imaging data was processed, visualized, and analyzed using Imaris (Bitplane).

[0153] For analysis of germinal centers, surfaces were created on GL7 signal using the surface object creation wizard in Imaris. Surfaces were then filtered by volume and positive Bcl6 signal. In some experiments, another filter was applied for positive CD21 / 35 signal. Volume and CD45.2 mean intensity signal, representing relative abundance of transferred B cells, was then exported for each germinal center surface and plotted. Example 4—Dual-edited primary murine B cells constitutively secreted IL-10 without differentiating

[0154] As depicted in FIG. 16A, enriched B cells were electroporated with RNPs targeting both Rosa26 and Prdm1 after 2 days of stimulation in vitro. The cells were then cultured for 6 days on 40LB feeder cells in the presence of IL-4. B cells were analyzed for differentiation via FACS. Genomic DNA was collected from cells and HDR was calculated via ddPCR. Supernatant was collected and Il-10 secretion was measured via ELISA. Generated with BioRender.

[0155] FIG.16B depicts representative FACS plots showing CD19loCD138+ ASC proportion and LNGFR+ Rosa26-edited proportions within treated cell populations, gated as Live / Dead-H-2Kd- lymphocyte singlets. FIG. 16C depicts a graph of mean NHEJ and HDR rates in the Rosa26 gene of edited B cells at Day 6, as measured via ddPCR. FIG.16D depicts a graph of mean concentration of IL-10 in supernatant of expanded B cells at Day 9, as measured by ELISA. n=3 technical replicates per condition. p values calculated via one-way ANOVA with a Tukey’s test for multiple comparisons. Data from one experiment but representative of greater than 10 experiments. All error bars indicate 95% CI.

[0156] The expression of LNGFR on the flow plot, paired with the measurable increase in IL-10 expression, for dual-edited cells, indicated that the HDR worked and that the B cells were secreting IL-10. Simultaneously, the dual-edited B cells also did not have significant CD138 expression. This indicated that they were not differentiating. Example 5—Primary murine B cells edited to express a broad range of regulatory receptors

[0157] Primary murine B cells were edited to express a broad range of regulatory receptors. Murine B cells were edited in a manner similar to the workflow depicted in FIG. 16A. Briefly, cells were edited using Rosa26-targeted Cas9 cutting via transduction using rAAV HDR donors encoding either GFP, TIGIT, CD25, or CTLA-4 along with cis-linked LNGFR.

[0158] LNGFR+-edited subpopulations were compared across samples demonstrating that: LNGFR+ cells in populations edited with GFP_LNGFR AAV specifically expressed GFP: LNGFR+ cells in populations edited with TIGIT_LNGFR AAV specifically expressed TIGIT; LNGFR+ cells in populations edited with CD25_LNGFR AAV specifically expressed CD25; and LNGFR+ cells in populations edited with CTLA4_LNGFR AAV specifically expressed CTLA4 (FIG.17). References 1. Walsh, C. E., et al., Am. J. Hematol.90, 400–405 (2015). 2. Leissinger, C. A. Semin. Hematol. 53, 20–27 (2016). 3. Vandamme, C., et al., Hum. Gene Ther. 28, 1061–1074 (2017). 4. Verdera, H. C., et al., Mol. Ther.28, 723–746 (2020). 5. Schep, S. J., et al., Blood Rev. 32, 326–338 (2018). 6. Scott, D. W., et al., Blood 121, 4449–4456 (2013). 7. Shirley, J. L., et al., Mol. Ther. 28, 709–722 (2020). 8. Earley, J., et al., Trends Biotechnol.41, 836–845 (2023). 9. Kishimoto, T. K. et al., Expert Opin. Biol. Ther.22, 1067–1071 (2022). 10. Ertl, H. C. J. et al., Front. Immunol.13, 975803 (2022). 11. Mingozzi, F. et al., Mol. Ther.20, 1410–1416 (2012). 12. Yamada, K. et al., Mol. Ther.28, 1381–1382 (2020). 13. Moffett, H. F. et al., Sci. Immunol. (2019) doi:10.1126 / sciimmunol.aax0644. 14. Voss, J. E. et al., Elife 8, (2019). 15. Hung, K. L. et al., Mol. Ther.26, 456–467 (2018). 16. Cheng, R. Y. H. et al., Nat. Commun.13, (2022).David, M. et al., Mol. Ther. Methods Clin. Dev.31, 101111 (2023). Arroyo, E. N. et al., J. Exp. Med. 217, (2020). Hong, S. et al., Immunity 49, 695-708.e4 (2018). Schwickert, T. A. et al., J. Exp. Med.208, 1243–1252 (2011). Le, T. L., et al., J. Immunol. 181, 6027–6037 (2008). Shapiro-Shelef, M. et al., Immunity 19, 607–620 (2003). Goodnow, C. C. et al., Nature 334, 676–682 (1988). Mason, D. Y., et al., Int. Immunol. 4, 163–175 (1992). Kornbluth, R. S., et al., Int. Rev. Immunol.31, 279–288 (2012). Chan, V. W. F. et al., J. Exp. Med. 188, 93 (1998). Divanovic, S. et al., Nat. Immunol.6, 571–578 (2005). Chaplin, J. W., et al., J. Immunol.187, 4199 (2011). Wurster, A. L., et al., J. Biol. Chem.277, 27169–27175 (2002). Granato, A., et al., J. Immunol.192, 5761–5775 (2014). Klein, U. et al., Nat. Immunol. (2006) doi:10.1038 / ni1357. Chu, V. T. et al., Proc. Natl. Acad. Sci. U. S. A. (2016) doi:10.1073 / pnas.1613884113. Nojima, T. et al., Nat. Commun. (2011) doi:10.1038 / ncomms1475. Conant, D. et al., Cris. J.5, 123–130 (2022). Oracki, S. A., et al., Immunological Reviews at https: / / doi.org / 10.1111 / j.1600-X.2010.00940.x (2010). Brynjolfsson, S. F. et al., Front. Immunol. 9, 386243 (2018). Avancena, P. et al., Proc. Natl. Acad. Sci. U. S. A.118, e2100576118 (2021). Saenko, E. L., et al., J. Biol. Chem. 269, 11601–11605 (1994). Saenko, E. L. et al., J. Biol. Chem.272, 18007–18014 (1997). Nogami, K. et al., J. Biol. Chem.275, 25774–25780 (2000). Walter, J. D. et al., J. Biol. Chem.288, 9905–9914 (2013). Prescott, R. et al., Blood 89, 3663–3671 (1997). Arai, M., et al., J. Clin. Invest.83, 1978–1984 (1989). Bi, L. et al., Nat. Genet.10, 119–121 (1995). Miao, C. H. et al., Blood 114, 4034 (2009). Silacci, P., et al., J. Exp. Med.180, 1329–1336 (1994). Piskurich, J. F. et al., Nat. Immunol.1, 526–532 (2000). Viant, C. et al., Cell 183, 1298-1311.e11 (2020). Tedder, T. F. et al., J. Immunol.194, 1395–1401 (2015). Cerqueira, C., et al., Semin. Immunol.44, (2019). Wallace, C. H. et al., JCI Insight 3, (2018). Xiao, S. et al., Cell Rep.32, (2020). Rosser, E. C. et al., Immunity 42, 607–612 (2015). Chen, C. Y. et al., Mol. Ther. Nucleic Acids 20, 534–544 (2020). Taylor, J. J. et al., J. Exp. Med.209, 2065 (2012).56. Leal, J. M. et al., Sci. Immunol. 6, (2021).

[0159] The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0160] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

[0161] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing an antigen specific B cell, comprising: (a) obtaining a modified B cell, wherein the modified B cell is derived from a B cell and: (i) lacks a capacity to differentiate, and (ii) comprises a B cell receptor (BCR) capable of specifically binding an antigen; and (b) contacting the modified B cell with the antigen, thereby preparing the antigen specific B cell.

2. The method of claim 1, wherein step (a) comprises modifying the B cell by inactivating a PRDM1 gene in the genome of the B cell.

3. The method of claim 2, wherein the inactivating comprises introducing a nuclease capable of specifically cleaving the PRDM1 gene.

4. The method of claim 2, wherein the nuclease is selected from a Cas nuclease, such as Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease or a modified or mutant Cas nuclease, such as a modified or mutant Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease.

5. The method of claim 4, wherein the Cas nuclease is a Cas9 nuclease or a modified or mutant Cas9 nuclease.

6. The method of any one of claims 1-5, further comprising introducing a genomic RNA (gRNA) into a B cell.

7. The method of any one of claims 1-6, wherein the modified B cell lacks the capacity to differentiate into a plasma cell.

8. The method of any one of claims 1-7, wherein step (a) comprises modifying the B cell by introducing a polynucleotide encoding the BCR into the B cell.

9. The method of claim 8, wherein a vector comprises the polynucleotide.

10. The method of claim 9, wherein the vector is a viral vector.

11. The method of claim 10, wherein the viral vector is an adeno-associated virus (AAV) vector, or a lentivirus vector.

12. The method of any one 1-7, wherein step (a) comprises selecting for a B cell comprising the BCR.

13. The method of claim 12, wherein the selecting comprises contacting a population of B cells with the antigen.

14. The method of any one of claims 1-13, further comprising stimulating the modified B cell prior to step (b).

15. The method of claim 14, wherein the stimulating comprises contacting the modified B cell with multimeric CD40 ligand (MCD40L), anti-CD180 antibody, IL-4, IL-2, IL-6, IL-10, IL-15, lipopolysaccharide (LPS), and / or CpG oligonucleotide (ODN) 2006.

16. The method of any one of claims 1-15, wherein step (b) comprises contacting the modified B cell with the antigen for a period of less than 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or 1 hour but not zero.

17. The method of claim 16, wherein the period is in a range from about 1 hour to about 6 hours, from about 1 hour to about 4 hours, or from about 2 hours to about 4 hours.

18. The method of any one of claims 1-17, wherein step (b) comprises contacting the modified B cell with a cytokine; optionally, wherein the cytokine comprises IL-4.

19. The method of any one of claims 1-18, wherein the B cell is selected from the group consisting of a B cell, a hematopoietic stem cell, an early pro-B cell, a late pro-B cells, a large pre-B cell, a small pre-B cell, an immature B cell, a T1 B cell, a T2 B cell, a marginal zone B cell, a mature B cell, and a naïve B cell.

20. The method of any one of claims 1-19, wherein the antigen is multimeric.

21. The method of any one of claims 1-20, wherein the antigen further comprises a non-cognate antigen; optionally, wherein the BCR lacks a capacity to specifically bind the non- cognate antigen; optionally, wherein the antigen and the non-cognate antigen are covalently bound to each other.

22. The method of any one of claims 1-21, wherein the antigen or the non-cognate antigen comprise a polyethylene glycol (PEG), a viral protein, a non-human protein, or a recombinant protein; optionally, wherein the antigen or the non-cognate antigen comprises an AAV protein or a therapeutic protein, such as Factor VIII;optionally, wherein the AAV protein is selected from viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3); and optionally, wherein the AAV protein is from AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVD-J, or Ark313.

23. The method of any one of claims 1-22, wherein the antigen specific B cell is mammalian; optionally, wherein the antigen specific B cell is human.

24. The method of any one of claims 1-23, wherein the antigen specific B cell is ex vivo.

25. The method of any one of claims 1-24, wherein the antigen specific B cell expresses a therapeutic protein.

26. The method of any one of claims 1-25, further comprising introducing into the antigen specific B cell a polynucleotide encoding a therapeutic protein.

27. The method of claim 25 or 26, wherein the therapeutic protein is selected from TGF-ȕ1, granzyme B, indoleamine-pyrrole 2,3-dioxygenase (IDO), T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), CD25, CTLA4, T-cell immunoglobulin and mucin domain 1 (TIM-1), FasL, lymphocyte-activation gene 3 (LAG-3), Galectin-9, CD39, and a cytokine, such as IL-10 or IL-35.

28. A cell prepared by the method of any one of claim 1-27.

29. A pharmaceutical composition comprising the cell of claim 28 and pharmaceutically acceptable excipient.

30. A method for treating, inhibiting or ameliorating a disorder in a subject comprising administering the cell of claim 28 or the pharmaceutical composition of claim 29 to the subject.

31. The method of claim 30, wherein the administration reduces an antibody response to the antigen or a cognate antigen in the subject as compared to a subject which has not been administered the cell.

32. The method of claim 30 or 31, wherein the cell is autologous to the subject.

33. The method of claim 30 or 31, wherein the cell is allogeneic to the subject.

34. A system comprising:an antigen specific B cell, wherein the antigen specific B cell (i) lacks a capacity to differentiate, and (ii) comprises a B cell receptor (BCR) capable of specifically binding an antigen.

35. The system of claim 34, further comprising the antigen.

36. The system of claim 34 or 35, wherein a PRDM1 gene in the genome of the antigen specific B cell is inactivated.

37. The system of any one of claims 34-36, further comprising a nuclease capable of specifically cleaving the PRDM1 gene; optionally, wherein the nuclease is selected from a Cas nuclease, such as Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease or a modified or mutant Cas nuclease, such as a modified or mutant Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9 nuclease, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease; preferably, wherein the Cas nuclease is a Cas9 nuclease or a modified or mutant Cas9 nuclease.

38. The system of any one of claims 34-37, further comprising a genomic RNA (gRNA) capable of specifically binding a PRDM1 gene in the genome of the antigen specific B cell.

39. The system of any one of claims 34-38, wherein the modified B cell lacks the capacity to differentiate into a plasma cell.

40. The system of any one of claims 34-39, further comprising a polynucleotide encoding the BCR.

41. The system of claim 40, wherein a vector comprises the polynucleotide.

42. The system of claim 41, wherein the vector is a viral vector; optionally, wherein the viral vector is an adeno-associated virus (AAV) vector, or a lentivirus vector.

43. The system of any one of claims 34-42, further comprising multimeric CD40 ligand (MCD40L), anti-CD180 antibody, IL-4, IL-2, IL-6, IL-10, IL-15, lipopolysaccharide (LPS), and / or CpG oligonucleotide (ODN) 2006.

44. The system of any one of claims 34-43, further comprising a cytokine; optionally wherein the cytokine comprises IL-4.

45. The system of any one of claims 34-44, wherein the antigen specific B cell is derived from a B cell selected from the group consisting of a B cell, a hematopoietic stem cell,an early pro-B cell, a late pro-B cells, a large pre-B cell, a small pre-B cell, an immature B cell, a T1 B cell, a T2 B cell, a marginal zone B cell, a mature B cell, and a naïve B cell.

46. The system of any one of claims 34-45, wherein the antigen is multimeric.

47. The system of any one of claims 34-46, wherein the antigen further comprises a non-cognate antigen; optionally, wherein the BCR lacks a capacity to specifically bind the non- cognate antigen; optionally, wherein the antigen and the non-cognate antigen are covalently bound to each other; optionally, wherein the antigen or the non-cognate antigen comprise a polyethylene glycol (PEG), a viral protein, a non-human protein, or a recombinant protein; optionally, wherein the antigen or the non-cognate antigen comprises an AAV protein or a therapeutic protein, such as Factor VIII; optionally, wherein the AAV protein is selected from viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3); and optionally, wherein the AAV protein is from AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVD-J, or Ark313.

48. The system of any one of claims 34-47, wherein the antigen specific B cell is mammalian; optionally, wherein the antigen specific B cell is human.

49. The system of any one of claims 34-48, wherein the antigen specific B cell is ex vivo.

50. The system of any one of claims 34-49, wherein the antigen specific B cell expresses a therapeutic protein.

51. The system of any one of claims 34-50, further comprising introducing into the antigen specific B cell a polynucleotide encoding a therapeutic protein.

52. The system of claim 50 or 51, wherein the therapeutic protein is selected from TGF-ȕ1, granzyme B, indoleamine-pyrrole 2,3-dioxygenase (IDO), T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), CD25,CTLA4, T-cell immunoglobulin and mucin domain 1 (TIM-1), FasL, lymphocyte-activation gene 3 (LAG-3), Galectin-9, CD39, or a cytokine, such as IL-10 or IL-35.

53. Use of the cell of claim 28, the pharmaceutical composition of claim 29, or the system of anyone of claims 34-52 as a medicament.

54. The cell of claim 28, the pharmaceutical composition of claim 29, or the system of anyone of claims 34-52 for use in treating, inhibiting or ameliorating a disease or disorder in a subject, such as hemophilia A, multiple sclerosis or systemic lupus erythematosus or a protein deficiency disorder, such as a Factor VIII deficiency, or a disease or disorder characterized as having an antibody-antigen based inhibition, or antibody inhibitors.

55. A method of reducing an antigen-specific antibody response in a subject comprising administering the cell of claim 28, the pharmaceutical composition of claim 29, or the system of anyone of claims 34-52 to a subject in the absence of an immunosuppression therapy.

Images