Systems and methods to generate b cells genetically modified to express selected antibodies

Abstract

The present application relates to systems and methods to generate B cells genetically modified to express selected antibodies. The systems and methods can be used to: eliminate the need for classical vaccination; providing protection against infections available for current vaccination; providing protection against infections when the patient is additionally subjected to immunosuppression; and / or provides benefits provided by therapeutic antibodies, such as in the treatment of autoimmune disorders.

Description

[0001] This application is a divisional application of an invention patent application with an application date of October 19, 2018, application number "201880080021.5", and name "Systems and methods for producing B cells genetically modified to express selected antibodies".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 575,275 filed on October 20, 2017, U.S. Provisional Patent Application No. 62 / 580,303 filed on November 1, 2017, and U.S. Provisional Patent Application No. 62 / 623,371 filed on January 29, 2018, each of which is incorporated herein by reference in its entirety to the same extent as if fully set forth herein.

[0004] Statement regarding sequence listing

[0005] The Sequence Listing associated with this application is provided in XML format. Technical Field

[0006] The present disclosure provides systems and methods for genetically modifying B cells to express selected antibodies. The systems and methods can be used to: obviate the need for classical vaccination; provide protection against infectious agents for which no vaccination is currently available; provide protection against infectious agents when the patient is otherwise immunosuppressed; and / or provide the benefits provided by therapeutic antibodies, such as in the treatment of autoimmune disorders. Background Art

[0007] Vaccines are designed to increase the immunity of subjects to specific infections by stimulating B cells to produce antibodies to targeted infectious agents. Conventional pediatric vaccination is a long-established clinical intervention with relatively low risk and high efficacy. Unfortunately, however, vaccination is not available for all infectious agents. As an example, in the United States, millions of children visit doctors or emergency rooms every year due to infection with respiratory syncytial virus (RSV).

[0008] For decades, researchers have tried to develop vaccines that can induce B cells to produce antibodies that effectively protect against viruses such as RSV, human immunodeficiency virus (HIV), and Zika virus. But all attempts to induce protective antibodies have failed. The only RSV vaccine that has been widely tested actually makes the infection worse: the antibodies produced after vaccination do not disable the virus, but instead enhance its ability to infect cells. In addition to RSV, HIV, and Zika virus, there are many other infectious agents for which no effective vaccines are available.

[0009] In addition to fighting infections, antibodies may also be useful as therapeutic agents for other conditions such as autoimmune diseases. However, these antibody-based therapies typically require repeated injections of the antibody to maintain protection.

[0010] It is also worth noting that numerous patients undergo bone marrow or hematopoietic stem cell transplantation as treatment for hematological malignancies (e.g., leukemia, lymphoma, myeloma). Other patients receive infusions of genetically modified hematopoietic stem cells providing therapeutic genes that the patient lacks. All of these treatments require removing the patient's existing immune system before applying the transplant or genetically modified hematopoietic stem cells, thereby leaving a dangerous immunosuppression window before the patient's immune system reaches colony recovery after treatment. During this immunosuppression time, patients are extremely susceptible to infection such as RSV, influenza, parainfluenza and metapneumovirus (MPV). These infections are high risk factors and are associated with numerous deaths after these treatments. Summary of the invention

[0011] The present disclosure provides systems and methods for genetically modifying B cells to express selected antibodies. In specific embodiments, the selected antibodies reduce or eliminate the need for existing vaccinations. In specific embodiments, the selected antibodies protect against infections caused by viruses (e.g., RSV, HIV, Zika) that are currently unavailable for effective vaccination strategies. In specific embodiments, the selected antibodies reduce or eliminate the need for therapeutic antibody injections such as those that are administered to treat various autoimmune disorders. In specific embodiments, the selected antibodies protect immunosuppressed patients from infection. In specific embodiments, the disclosed methods can be used to reprogram B cells to protect against hundreds of different infections or pathogens, all of which are reprogrammed by single laboratory operations covering several days.

[0012] In a specific embodiment, the disclosure provides these benefits by inserting the genetic construct targeting into the specific region of the endogenous genome of the B cell.Importantly, because the genetic sequence in the B cell has the high variability required for antibody diversity, it is difficult to genetically modify these cells.This high genetic variability makes it impractical to directly target the antibody coding region to carry out genetic manipulation.In addition, it is also ineffective to remove and replace the coding portion of the genome of the B cell, because this method can negatively affect the B cell function.

[0013] An additional challenge with genetically modifying B cells to express an antibody of choice is that antibodies are produced from discrete protein units called heavy and light chains. The different chains are encoded by different parts of the B cell genome, yet must come together to form a functional antibody.

[0014] The present disclosure overcomes the indicated challenges and other challenges by identifying constant regions of the B cell genome that can be reliably targeted for genetic insertion and that, when modified, result in preferential expression of the inserted genetic construct over the corresponding portion of the native genome of the B cell. This strategy overcomes the sequence variability associated with the B cell genome and also overcomes the need to remove and replace portions of the endogenous B cell genome to achieve functional expression of the selected antibody. Overcoming the need to remove and replace portions of the endogenous B cell genome will maintain B cell function after genetic manipulation.

[0015] In a specific embodiment, the indicated region targeted for genetic insertion is an intron region upstream or downstream of the Eμ enhancer element of SEQ ID NO: 85 (human) or SEQ ID NO: 86 (mouse). In a specific embodiment, the region targeted for genetic insertion is a constant intron region selected from SEQ ID NO: 1 or 2 (human) or SEQ ID NO: 3 or 4 (mouse). In a specific embodiment, the human DNA sequence within SEQ ID NO: 1 for targeting for genetic insertion includes SEQ ID NO: 5-24. In a specific embodiment, the human DNA sequence within SEQ ID NO: 2 for targeting for genetic insertion includes SEQ ID NO: 25-44. In a specific embodiment, the mouse DNA sequence within SEQ ID NO: 3 for targeting for genetic insertion includes SEQ ID NO: 45-64. In a specific embodiment, the mouse DNA sequence within SEQ ID NO: 4 for targeting for genetic insertion includes SEQ ID NO: 65-84. Genetic sequences that are particularly capable of targeting these sites for genetic modification are described within the present disclosure as guide RNA (gRNA) SEQ ID NOs: 87-89 and 290-366.

[0016] In certain embodiments, the placement and composition of the inserted genetic construct results in preferential expression of the inserted genetic construct over the corresponding portion of the endogenous genome of the B cell. These embodiments also include elements that overcome the challenges associated with portions of the antibody being encoded by different regions of the endogenous B cell genome.

[0017] In a specific embodiment, the genetic construct is inserted into one of SEQ ID NOs: 1, 2, 3, and 4, and includes (i) a promoter; (ii) a signal peptide; (iii) a transgene encoding the entire light chain of the selected antibody; (iv) a flexible linker or jumping element; (v) a variable portion of the heavy chain of the selected antibody; and (vi) a splice junction that results in the expression of the endogenous heavy chain constant region of the B cell. In these embodiments, expressing the selected antibody in a single construct overcomes the challenges associated with portions of the antibody being encoded by different regions of the endogenous B cell genome. The inclusion of a flexible linker allows the light chain portion and the heavy chain portion of the expressed selected antibody to be physically connected in a manner that allows them to form a functional unit, while at the same time reducing the risk of the antibody portion binding to other potential expressed antibody chains from the endogenous genome of the B cell. The use of jumping elements does not physically connect the light chain portion and the heavy chain portion, but their expression in a close proximity also results in association to form a functional unit, while reducing the risk of the antibody portion binding to other potential expressed antibody chains from the endogenous genome of the B cell. The inclusion of the splice junction results in the selected antibody including a heavy chain constant region that is appropriate for the current activation and / or maturation state of the B cell. In other words, the selected expressed antibody can be expressed with any of the endogenous heavy chain constant regions of the B cell, and the heavy chain constant region expressed with the selected antibody can naturally change over time.

[0018] The present disclosure also provides methods for ensuring that only B cells that have been effectively genetically modified to express the selected antibody are collected for formulation and administration to a patient. For example, prior to genetic modification, the B cell will naturally express an antibody comprising a κ light chain or a λ light chain. The B cell can be modified to express a light chain different from the κ or λ chain that it naturally expresses, and only those B cells that express the replacement chain are selected for formulation and administration.

[0019] The present disclosure also provides numerous additional strategies for effectively modifying B cells to provide the benefits described herein. These and other strategies are more fully described in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . Schematic representation of the B cell response to classical vaccination strategies.

[0021] Figure 2A-2C .Schematic diagram of prior art vaccination strategies against respiratory syncytial virus (RSV): ( Figure 2A )Formalin-inactivated RSV vaccine; Figure 2B ) “Improved” RSV vaccines; and ( Figure 2C )RSV epitope backbone vaccine.

[0022] Figure 3A-3C Overview of previous attempts to bypass vaccination and directly provide protective antibodies against RSV: Figure 3A )palivizumab injection; Figure 3B ) adenovirus-mediated palivizumab expression; and ( Figure 3C ) Stem cell genetic modification and differentiation.

[0023] Figure 4A , Figure 4B . Schematic diagram of a specific embodiment of the protection strategy disclosed herein: ( Figure 4A ) B cell modification for protection against RSV; and ( Figure 4B )Strategies for simultaneous protection.

[0024] Figure 5 . Hypothetical secretion of palivizumab by B cell subsets in the presence and absence of infection.

[0025] Fig. 6A , Figure 6B .( Fig. 6A ) Antibody genes and ( Figure 6B ) antibody protein structure, thereby emphasizing the technical challenge of the present disclosure: antibodies are proteins made from two separate gene products. In certain embodiments, the synthetic genetic constructs encoding selected antibodies disclosed herein utilize jumping elements (e.g., self-cleaving peptides) to address this challenge.

[0026] Figure 7 . Schematic diagram depicting the endogenous heavy chain locus, including the V, D and J segments that undergo recombination, enhancer elements shown as circles and ovals, and 6 potential constant regions that can be expressed. B cells are initiated by expressing the μ / δ constant region, but can be switched to use the γ, α or ε constant region by deleting the intervening DNA. It should also be noted that each V segment is associated with a heavy chain promoter represented by an arrow that drives the expression of the heavy chain after recombination.

[0027] Fig. 8A , Figure 8B .Because two proteins come together to form an antibody, it may be desirable to target or inactivate the endogenous antibody heavy chains (IgH) and / or endogenous antibody light chains (IgL) of the B cell. In the absence of such targeting or inactivation, undesirable hybrid antibodies may be formed (i.e., endogenous light chains pair with selected antibody heavy chains, or vice versa). ( Fig. 8A ) A method for achieving endogenous IgH targeting, and the resulting chimera includes a portion of a selected antibody (e.g., palivizumab). ( Figure 8B) A method for achieving IgL inactivation. In the depicted method, a stop codon can be placed upstream of (or as part of) the inserted genetic construct.

[0028] Fig. 9 . Schematic diagram depicting the interaction of the heavy chain enhancer with (upper) endogenous VDJ or (lower) synthetic VDJ encoded by the inserted genetic construct. Promoters are depicted as arrows. Nucleic acids are depicted as boxes. B cells naturally express nucleic acids downstream of the promoter closest to the Eμ enhancer. Fig. 9 In the endogenous B cell genome depicted in , the first upstream promoter closest to the Eμ enhancer then drives the expression of the endogenous heavy chain VDJ segment. Inserting a genetic construct including a promoter between the Eμ enhancer and the first endogenous promoter causes the B cell to express the inserted genetic construct rather than the endogenous heavy chain VDJ segment. This inserted gene can be the VDJ variable region of the heavy chain, a paired complete antibody light chain together with the heavy chain variable VDJ, or another synthetic gene that can be expressed as a fusion with the B cell heavy chain constant region. Fig. 9 In the Figures, the constant region is represented alone to emphasize that the inserted genetic construct can be expressed with any of the potential heavy chain constant regions.

[0029] Fig.10 . Target region for insertion (human). Regions used to achieve insertion of genetic constructs into the genome: No. 1: from the terminal J region (IGHJ6 in humans, IGHJ4 in mice) to the Eμ enhancer, or No. 2: from the Eμ enhancer to the repeat sequence of the constant domain switch region.

[0030] Fig.11A , Fig. 11B .( Fig.11A ) human Eμ intronic enhancer (SEQ ID NO: 85) and a human DNA sequence including from IGHJ6 to Eμ intronic enhancer for targeting for genetic insertion (SEQ ID NO: 1); ( Fig. 11B ) Exemplary related gRNA (e.g., sgRNA) target sites (SEQ ID NO. 5-24) and gRNA sequences (SEQ ID NO: 88, 89, and 290-307).

[0031] Fig. 12A , Fig. 12B .( Fig. 12A ) a human DNA sequence (SEQ ID NO: 2) for targeting for genetic insertion, including from the Eμ intronic enhancer to the switch region; ( Fig. 12B) Exemplary related gRNA (e.g., sgRNA) target sites (SEQ ID NO.25-44) and gRNA sequences (SEQ ID NO:308-327).

[0032] Fig.13A , Fig. 13B .( Fig.13A ) mouse Eμ intronic enhancer (SEQ ID NO: 86) and a mouse DNA sequence including from IGHJ4 to Eμ intronic enhancer for targeting for genetic insertion (SEQ ID NO: 3); ( Fig. 13B ) Exemplary related gRNA (e.g., sgRNA) target sites (SEQ ID NOs. 45-64) and gRNA sequences (SEQ ID NOs: 87 and 328-346).

[0033] Fig.14A , Fig. 14B .( Fig.14A ) a mouse DNA sequence (SEQ ID NO: 4) for targeting for genetic insertion, including from the Eμ intronic enhancer to the switch region; ( Fig. 14B ) Exemplary related gRNA (e.g., sgRNA) target sites (SEQ ID NO.65-84) and gRNA sequences (SEQ ID NO:347-366).

[0034] Fig.15A , Fig. 15B . Fig.15A Schematic diagram depicting the insertion of a genetic construct encoding an anti-RSV antibody into an endogenous heavy chain locus using the CRISPR / Cas9 gene editing system. The genetic construct may include homology arms or homology stitches that are nucleotide overhangs homologous to the genomic DNA at the insertion site. Fig. 15B Additional examples of DNA repair templates are depicted, including sequences flanked by sgRNA target sites for generating double-stranded DNA breaks flanking the inserted sequence in response to cleavage of the genome by Cas9 / sgRNA (top), single-stranded DNA containing long homology arms flanking the inserted sequence (middle), and short homology arms flanking the inserted sequence generated by annealing of DNA oligos (bottom).

[0035] Fig.16.Depicts: (upper) a modified heavy chain locus modified with a genetic construct encoding the heavy chain variable region of an anti-RSV antibody; (middle) a modified light chain locus modified with a genetic construct encoding the light chain variable region of an anti-RSV antibody; and (lower) a schematic diagram of a modified heavy chain locus modified with a genetic construct encoding the light chain (i.e., IgL) of an anti-RSV antibody and the heavy chain variable region of an anti-RSV antibody, wherein a linker (including a Strep-Tag) is provided between the light chain and the heavy chain variable region.

[0036] Fig.17 . Schematic diagram of simultaneous protection against multiple pathogens by modified memory B cells and modified antibody-secreting B cells as disclosed herein.

[0037] Figures 18A-18C . Efficient Cas9 cleavage of targeted intronic regions in mouse and human B cells. Electroporation with Cas9 / sgRNA ribonucleoprotein complexes mediates efficient cleavage in mouse and human B cells. Cells were electroporated with Cas9 / sgRNA complexes. Tracking of indels by decomposition (TIDE) was used to assess ( Fig.18A ) Mouse B cell line (A20); ( Fig.18B ) primary B cells; and ( Fig. 18C ) Editing efficiency in a human B cell line (Ramos).

[0038] Fig.19 .Insertion of genetic constructs encoding RSV-specific antibodies into mouse B cells.

[0039] Fig. 20 .Enrichment and analysis of genetically modified B cells.

[0040] Fig.21 .Genetically modified B cells secrete RSV-binding antibodies.

[0041] Fig.22A , Fig. 22B Nonhomologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) methods offer alternatives to long homology-directed repair (HDR) for genome engineering of primary mouse B cells. Fig.22A ) Primary B cells were primed for 24 hours, incubated with adeno-associated virus (AAV) for 12 hours, washed, and electroporated or directly transferred to secondary culture for 3 days and then analyzed for mCherry expression. Fig. 22B ) Primary B cells were sensitized for 24 h, mock electroporated, or electroporated with template+Cas9 / sgRNA, and transferred to secondary culture for 5 days before analysis of mCherry expression.

[0042] Figure 23A-23E Insertion of novel genetic constructs encoding functional antibodies into mouse and human B-cell lines enables expression of both surface-bound and secreted antibodies. Fig.23A ) Schematic of the IgH locus showing the insertion site of a partial antibody construct, and depictions of surface-bound and secreted antibodies. emAb = synthetic antibody, used interchangeably with synAb in this article. ( Fig. 23B ) Staining of the mouse A20 B cell line, unmodified or modified with anti-RSV synthetic antibody (αRSV synAb), with RSV prefusion F protein tetramer (RSV-tetramer) and anti-Streptag II tetramer (αTagAb tetramer). ( Fig.23C ) Staining of unmodified or anti-RSV synAb-modified human RAMOS B cell lines with RSV prefusion F protein tetramers and anti-Streptag II antibody tetramers. Fig.23D ) ELISA for binding of antibodies obtained from culture medium of A20 cells unmodified or modified with anti-RSV synAb to RSV prefusion F protein. Palivizumab was used as a positive control. ( Fig.23E ) ELISA for the binding of antibodies obtained from culture medium of RAMOS cells unmodified or modified with anti-RSV synAb to RSV prefusion F protein. Palivizumab was used as a positive control.

[0043] Fig.24A – Fig.24C .Generation of primary mouse B cells with novel specificities. Fig.24A ) Surface staining of a mock-treated mouse B cell line (upper) or an anti-RSV synAb-modified mouse B cell line (lower) with anti-Streptag II tetramer before enrichment (left panel) and with anti-Streptag II tetramer (middle panel) and RSV pro-fusion protein tetramer (right panel) after enrichment and amplification. Fig. 24B ) ELISA for binding of antibodies obtained from culture media of unmodified or anti-RSV synAb modified mouse B cell cultures to RSV prefusion F protein. Palivizumab was used as a positive control. Antibody binding was detected with a 1:1 mixture of polyclonal anti-human Ig antibody and anti-mouse Ig antibody conjugated to HRP. (24C) Rapid expansion of B synAb enriched cells cultured with 3T3-CD40L feeder cells and IL-21.

[0044] Figure 25A-Figure 25I .Exemplary sequences. ( Fig.25A) exemplary sgRNA sequences (SEQ ID NOs: 87, 88, 89), genomic homology regions (SEQ ID NOs: 90-95), and splicing oligonucleotides (SEQ ID NOs: 96-101); ( Fig.25B ) human anti-RSVemAb AAV (2531bp (SEQ ID NO: 102) and related nucleotide and protein sequences (SEQ ID NO: 110-126, 280, 285)); ( Fig.25C ) Mouse anti-RSV emAb AAV (3134 bp (SEQ ID NO: 103) and related nucleotide and protein sequences (SEQ ID NO: 127-141, 281, 286)); ( Fig.25D ) mouse emAb-RSV-dsDNA (1736 bp (SEQ ID NO: 104) and related nucleotide and protein sequences (SEQ ID NO: 142-144)); ( Fig.25E ) human emAb-VRC01-AAV (2551 bp (SEQ ID NO: 105) and related nucleotide and protein sequences (SEQ ID NO: 145-152, 282, 287)); ( Fig.25F )human-emAb-Medi8852-AAV (2544 bp (SEQ ID NO: 106) and related nucleotide and protein sequences (SEQ ID NO: 153-160, 283, 288)); ( Figure 25G )human-emAb-AMM01-AAV (2555 bp (SEQ ID NO: 107) and related nucleotide and protein sequences (SEQ ID NO: 161-169, 284, 289)); ( Fig.25H )Balb / CmRSV splicing integration sequence (2261bp (SEQ ID NO:108) and related nucleotide and protein sequences (SEQ ID NO:170-172)); and ( Fig.25I )TT-hRSV-T7 integration sequence (1707 bp (SEQ ID NO: 109) and related nucleotide and protein sequences (SEQ ID NO: 173-175)).

[0045] Figure 26A-26D .( Fig.26A) A targeted region upstream of the Eμ enhancer for inserting a new antibody cassette; by targeting this region, the inserted emAb gene can be driven by the natural (but inserted) IgH promoter, thereby maximizing natural control over immunoglobulin expression. To enable one-shot insertion and minimize off-target interactions, the emAb construct is expressed as a single-chain fusion. This fusion consists of a complete light chain sequence connected to the variable region of the heavy chain with a glycine-serine linker having 57 amino acids. Physically connecting the light chain and the heavy chain minimizes the possibility of mispairing between the inserted emAb and the endogenous light chain. Optimizing the splicing junction allows emAb to be spliced ​​to the downstream endogenous IgH constant region. This allows emAb to be expressed in any of the heavy chain isotype categories. ( Fig.26B ) Burkitt's lymphoma-derived B cell lines naturally express surface and secreted forms of IgM paired with lambda light chains. Expression of engineered αRSV-emAb derived from palivizumab was detected using monomeric RSV-F protein and streptactin, a modified streptavidin with high affinity for the Streptag II motif in the linker. RAMOS cells modified with αRSV-emAb express engineered RSV-specific antibodies detectable on the surface of the cells. ( Fig.26C ) Engineered RSV-specific antibodies were also detected in the supernatant in a secreted form. Fig.26D ) αRSV-emAb-modified cells, but not control cells, exhibit rapid and sustained calcium signaling in response to protein antigens.

[0046] Figure 27A-27G Human B cells were efficiently genetically modified to express single-chain emAbs via paired cas9-sgRNA and AAV template delivery. Fig.27A ) Schematic representation of the human cell engineering process. Day 0: B cells were isolated from PBMCs and sensitized with CD40L, IL2, IL10, IL15, and CpG oligonucleotides. Day 2: Cells were electroporated with cas9 / sgRNA RNPs and treated with AAV encoding emAb HR template 1 hour after electroporation and then cultured as described for Day 0. Day 4: Cells were selected for antigen binding or tag expression. Days 4-15L: Selected cells were expanded on irradiated feeder cells expressing CD40L, IL2, and IL21, supplemented with IL15. Days 15-18: Cells were switched to feeder-free differentiation culture with IL6, IL15, and IFNγ. ( Fig.27B) Indel frequencies in B cells treated with emAb targeting Cas9 / sgRNA RNP from 6 independent PBMC donors. ( Fig.27C ) All human SNPs with a certain reported frequency across the targeted sgRNA site. Fig.27D ) Representative FACS for the binding of RSV-F prefusion monomers to cultured control or RSV-emAb genetically modified human B cells at day 4 of culture. ( Fig.27E )Frequency of RSV-emAb B cells after engineering of B cells from 6 independent donors. ( Fig.27F ) FACS for plasma cell markers (CD19, CD27, CD38 and CD138) in the case of primed cells (day 2) and in vitro differentiated cells (day 18). Figure 27G ) ELISA for secreted anti-HA stem antibodies in the culture medium of control B cells or influenza-targeted MEDI8852-emAb B cells on day 18 of culture.

[0047] Fig.28 Single-chain emAbs are a flexible platform for expressing antiviral antibodies. Human B cells were mock electroporated (control B, upper row) or genetically modified with emAb constructs derived from the variable regions of the indicated broadly neutralizing antibody constructs and human kappa (palivizumab, VRC01, and MEDI8852) or lambda (AMM01) light chains (lower row). Control and emAb-engineered cells were stained with matching antigens derived from the indicated pathogens: RSV-F monomers, or tetramers of HIV-ENV, EBV GH / GL, or HA backbones.

[0048] Figure 29A-Figure 29D Insertion of .emAb into the effective IgH allele can block endogenous IgH production. Fig.29A ) Illustration of RAMOS IgH alleles: one effective allele contains the emAb target site and one allele has a c-myc translocation that eliminates the emAb target site. ( Fig.29B ) shows flow cytometry of surface expression of lambda light chain and RSV-F antigen binding in input RAMOS cells (gated on CD79b+) and αRSV-emAb engineered RAMOS cells (gated on CD79b+ / RSV-F+). ( Fig.29C ) Illustration of primary IgH alleles: one effective allele, and one ineffective allele without functional recombinant VDJ, both of which contain emAb target sites. ( Fig.29D) shows flow cytometry of surface expression of λ light chain and RSV-F antigen binding on input sorted λ light chain+ B cells (gated on CD79b+) and αRSV-emAb engineered B cells (gated on CD79b+ / RSV-F+).

[0049] Figure 30A-Figure 30E . Engineering of primary mouse B cells with the αRSV-emAb cassette. ( Fig. 30A ) Schematic representation of the mouse B cell engineering process. Day 0: B cells were isolated from spleen and peripheral lymph nodes (PLN) by negative selection and sensitized with CD40L-HA, anti-HA mAb and IL4. Day 1: Cells were electroporated with cas9 / sgRNA RNPs together with dsDNA (dsDNA conditions) or with cas9 / sgRNA RNPs alone, followed by treatment with AAV containing emAbHR template 1 hour after electroporation (AAV conditions). Cells were then maintained in culture as described for Day 0. Day 3: Cells were selected for antigen binding or tag expression. Days 4-8L: Selected cells were expanded on irradiated feeder cells expressing CD40L, supplemented with IL-21. ( Fig. 30B ) Percentage of indels in B cells treated with IgH-targeted cas9 / sgRNA RNP. ( Fig. 30C ) Representative FACS for binding to monomeric prefusion RSV-F protein in control B cells or emAb B cells engineered with dsDNA or AAV templates. ( Fig.30D ) Frequency of emAb cells in B cells engineered with dsDNA or AAV templates. Fig.30E ) Anti-RSV-specific secreted antibodies in the supernatants of control B cells or B cells engineered with dsDNA or AAV templates.

[0050] Figures 31A-31D .Protection from viral infection achieved by engineered αRSV-emAb B cells. ( Fig.31A ) Graph showing the antiviral protection achieved by transferred emAb cells. Day 0: 1.5x10 7 Day 5: Palivizumab was injected intraperitoneally at 15 mg / kg. Day 6: Blood was drawn to measure the antiviral Ab titer. Day 7: 10 6 pfu RSV virus was challenged intranasally. Day 12: Virus titers in the lungs were measured. ( Fig.31B) Surface expression of RSV-emAb receptor measured by RSV-F monomer and streptactin tag binding before and 24 hours after RSV-emAb cell transfer. ( Fig.31C ) Plasma titers of αRSV-F antibodies in mice on day 6. ( Fig.31D ) RSV virus titers in the lungs of mice without transferred cells, with αRSV-emAb B cells, with control B cells, or with 15 mg / kg palivizumab delivered intraperitoneally 48 hours prior to infection.

[0051] Fig.32A , Fig.32B .Multiplex transfer of human antibody-secreting cells into NSG mice. Fig.32A ) Schematic diagram of human emAb B cell transfer into NSG mice. Day 0: 5×10 B cells generated as described in FIG. 27 were transferred by intraperitoneal injection. 6 anti-influenza emAb B cells and 5×10 6 Anti-RSV emAb B. Day 7: Blood was drawn to measure antibody production in serum. Fig.32B ) ELISA for serum titers of anti-RSV-F antibodies and anti-HA stem antibodies (double transfer) in mice receiving emAb cells relative to control serum (no transfer).

[0052] Fig.33A , Fig.33B .For mice ( Fig.33A ) and people( Fig.33B ) Template for long insertion of emAb cassette in IgH locus. Indicated on the upper horizontal line of each genome is the position of elements in germline IgH locus, including the final J region, Eμ intron enhancer elements and the beginning of μ constant domain. Indicated is the position (cleavage site) of cas9 / sgRNA target site. Shown below is included in mouse AAV and dsDNA constructs ( Fig.33A ) and human AAV constructs ( Fig.33B ) The positions of the targeting homology arms in ). Also shown is the emAb cassette as inserted in the genome. DETAILED DESCRIPTION

[0053] Vaccines are designed to increase the immunity of a subject against a specific infection by stimulating B cells to produce antibodies against the targeted infectious agent. Antibodies are proteins that can provide protection against pathogens. Antibodies can bind to pathogens and are protective when this binding interferes with the normal function of the pathogen. For example, many protective antibodies bind to parts of pathogens that block the pathogen from entering cells. Antibodies can be attached to the surface of B cells (called B cell receptors), but most of their protective functions are exerted when they are secreted into the blood.

[0054] Pathogens may refer to any substance that can cause disease, and pathogenicity may refer to the ability of a substance to cause disease. Examples of pathogens include viruses, bacteria, and fungi that can infect a host and cause disease. Other examples of pathogens include host-derived proteins or other host-derived substances that cause disease, such as tumor necrosis factor α (TNFα), an inflammatory molecule associated with numerous autoimmune diseases (e.g., arthritis), and amyloid beta plaques, a fibrinous protein that accumulates in the brain during Alzheimer's disease. In certain embodiments, cancer cells and / or tumors may also be referred to as pathogens or pathogenic substances based on their ability to cause disease.

[0055] After exposure to a vaccine or natural pathogen, epitopes provided in the vaccine and / or present on the pathogen can bind to B cell receptors present on naive B cells. This binding can lead to activation of the B cells and the production of protective antibodies.

[0056] A naive B cell refers to a B cell before it has come into contact with its epitope. Each naive B cell expresses a unique antibody with a unique epitope specificity. The unique antibody expressed by each naive B cell is randomly generated by genetic recombination. Naive B cells express membrane-bound antibodies (i.e., B cell receptors), and after epitope binding, they can proliferate rapidly. During proliferation and maturation, antibody genes are mutated by receptor cells, which is used to increase the affinity for epitope binding. The increase in epitope binding affinity that occurs during B cell maturation is required for effective protection against pathogens. A single naive B cell can undergo dozens of cell divisions to produce thousands of antibody-secreting B cells and memory B cells ( Figure 1 ).

[0057] In addition to active antibody-secreting B cells, memory B cells are also important for protection against pathogens. Memory B cells usually do not actively secrete antibodies, but can quickly differentiate into antibody-secreting cells. The rapid differentiation of memory B cells into antibody-secreting cells can help the immune system launch a rapid response to secondary infections or pathogens previously encountered by vaccination (McHeyzer-Williams et al., Nat Rev Immunol. 2011; 12 (1): 24-34; Taylor et al., Trends Immunol. 2012; 33 (12): 590-7). For example, when the level of antibodies produced by antibody-secreting B cells has weakened, memory B cells maintain protection against hepatitis B virus (Williams et al., Vaccine. 2001; 19 (28-29): 4081-5; Bauer et al., Vaccine. 2006; 24 (5): 572-7). Thus, a successful vaccine stimulates the production of antibody-secreting B cells and long-lived memory B cells, all of which are capable of expressing antibodies that bind epitopes on pathogens with high affinity.

[0058] Unfortunately, there are many infectious agents for which no vaccine is available. Examples of infectious agents for which no effective vaccine strategies are available include RSV, HIV, and Zika virus.

[0059] With regard to RSV, the catastrophic failure of the formalin-inactivated RSV vaccine in the 1960s could not be attributed to a failure to induce antibody-secreting B cells and memory B cells targeting RSV. It is possible that the vaccine induced the production of antibodies that did not neutralize RSV but instead enhanced RSV infection ( Figure 2A ) (Blanco et al., Hum Vaccin. 2010; 6(6):482-92; Broadbent et al., Influenza Other Respir Viruses. 2015; 9(4):169-78). This emphasizes the delicate balance that vaccines must achieve: inducing the production of "protective" antibodies that target certain epitopes while avoiding stimulating the production of "pathogenic" antibodies that target incorrect epitopes ( Figure 2A ).

[0060] An analysis of the World Health Organization International Clinical Trials Registry Platform in 2015 identified nine candidate RSV vaccines that have been clinically evaluated since 2008, none of which have progressed beyond Phase 2 testing (Broadbent et al., Influenza Other Respir Viruses. 2015; 9(4): 169-78). Of these trials, only three have been completed and only one has reported results. The vaccine, called MEDI-559, appears to reduce RSV infection, but respiratory symptoms are too high for further testing (Malkin et al., PLoS One. 2013; 8(10): e77104). These data suggest that although MEDI-559 induces the production of protective antibodies, it may also induce the production of pathogenic antibodies ( Figure 2B ).

[0061] Other "improved" vaccination strategies involve changing the formulation administered to the patient. These include alternative approaches to inactivate / attenuate the virus, and changes to adjuvants designed to increase inflammatory responses (Broadbent et al., Influenza Other Respir Viruses. 2015; 9(4): 169-78; Garg et al., The Journal of general virology. 2014; 95(Pt5): 1043-54; Swanson et al., J Virol. 2014; 88(20): 11802-10; Widjaja et al., PLoS One. 2015; 10(6): e0130829; Stewart-Jones et al., PLoS One. 2015; 10(6): e0128779). Some of these approaches have produced increases in protective antibodies in animal models, but the potential for inducing pathogenic antibodies makes it possible that these "improved" RSV vaccines will suffer the same fate as MEDI-559.

[0062] In an attempt to focus the immune response to the epitope targeted by the protective antibodies, a recent approach has been to graft single RSV epitopes onto non-RSV backbones ( Figure 2C). This approach eliminates the possibility of pathogenic antibodies specific for other RSV epitopes because they are not present in the framework. Vaccination of rhesus macaques with RSV epitope frameworks resulted in the production of neutralizing antibodies by some animals, but only after 3-5 injections (Correia et al., Nature. 2014; 507(7491): 201-6).

[0063] Methods have also been developed to bypass vaccination and provide protective antibodies directly. The only clinically approved preventive treatment for RSV is injection of the high-affinity RSV-specific protective antibody palivizumab ( Figure 3A )(The PREVENT Study Group. Pediatrics. 1997; 99(1):93-9; The IMpact-RSV Study Group. Pediatrics. 1998; 102(3Pt 1):531-7). Unfortunately, the $10,000 cost of a 5-month series of palivizumab has limited its use to children at high risk for severe RSV infection (Meissner and Kimberlin, Pediatrics. 2013; 132(5):915-8). Other RSV-specific antibodies designed to last up to 1 year are currently undergoing clinical evaluation (Influenza Other Respir Viruses. 2015; 9(4):169-78). However, annual antibody reinjections are not practical for achieving lifelong protection.

[0064] To eliminate the need for lifelong injections, a method has been developed in which adenoviral vectors are used to transfer genes encoding protective antibodies into muscle cells ( Figure 3B) (Schnepp and Johnson, Curr Opin HIV AIDS. 2014; 9(3): 250-6). Promisingly, adenovirus-mediated expression of palivizumab partially protected mice from RSV infection (Skaricic et al., Virology. 2008; 378(1): 79-85). However, a limitation of this approach is the high cost of producing the high doses of virus necessary to achieve protective levels of antibody (24). High doses are necessary because the expression of antibodies by muscle cells is low compared to the estimated 10,000 antibodies secreted per second by a single B cell (Helmreich et al., J Biol Chem. 1961; 236: 464-73; Hibi and Dosch, Eur J Immunol. 1986; 16(2): 139-45). B cells achieve this high secretion rate by completely reprogramming their protein production machinery to focus on antibody secretion. Without major changes in manufacturing capabilities, adenovirus-mediated antibody gene transfer into muscle cells is not a practical option for RSV prevention.

[0065] Another approach uses lentiviral vectors to incorporate genes encoding protective antibodies into the genome of hematopoietic stem cells, which will then be induced to differentiate into antibody-secreting B cells ( Figure 3C ). One limitation of this approach is that the insertion of antibody genes is random, which introduces the risk of off-target genetic effects that cause disease. A second limitation of this approach is that the differentiation of antibody-secreting cells from hematopoietic stem cells requires long in vitro culture conditions of two months (Luo et al., Blood. 2009; 113(7): 1422-31). The final limitation is that this strategy will not produce a source of antibodies that can be enhanced after infection. Therefore, if antibody-secreting cells are not present in higher numbers or are not long-lived, protection against infection will be insufficient.

[0066] In certain embodiments, the present disclosure provides a method for expressing a selected antibody (e.g., an antibody to an infectious agent (e.g., palivizumab; Figure 4A)) to provide bypassing vaccination and / or eliminating the need for repeated therapeutic antibody injections. The types of B cells that are particularly useful for genetic engineering include existing antibody secreting B cells, memory B cells, naive B cells, B1 B cells and marginal zone B cells. Naive B cells have the greatest proliferation and functional potential, and can enter the germinal center response and improve their binding ability. B1 B cells express BCR and migrate to different locations such as the peritoneal cavity. B1 B cells rapidly differentiate into antibody secreting cells after being stimulated by BCR, and do not require signals for T cells to achieve optimal function. Marginal zone B cells are mainly located in the marginal zone of the spleen, and rapidly differentiate into antibody secreting cells after being stimulated by BCR. Marginal zone B cells also do not require signals from T cells to achieve optimal function. Genetically engineering one or more of these B cell subsets can produce antibodies at baseline levels for treating progressive or immediate infections, as well as long-lived inducible antibody sources in the event of future reinfection. Figure 4B A relevant strategy for simultaneous protection against numerous pathogens utilizing the teachings of the present disclosure is shown, while Figure 5 Depicted is the hypothetical secretion of palivizumab by exemplary B cell subsets in the presence and absence of infection.

[0067] The present disclosure provides genetic engineering of B cells by inserting a genetic construct including a transgene into an endogenous antibody locus, the locus being specifically selected to utilize the structure and function of the endogenous B cell genome. For example, inserting a transgene encoding at least a portion of a selected antibody into an endogenous antibody locus can allow robust production of the selected antibody by utilizing endogenous antibody expression regulatory mechanisms. A transgene can refer to a section of DNA encoding an external (i.e., exogenous) protein. A genetic construct can refer to an artificially constructed nucleic acid segment intended for introduction into a cell to allow expression of an external protein.

[0068] In certain embodiments, the disclosure provides B cells modified to express a selected antibody. The antibody is produced by two genes, a heavy chain gene and a light chain gene. Typically, the antibody includes two identical copies of the heavy chain and two identical copies of the light chain (see, e.g., Figure 6B The heavy chain is the larger of the two subunits, and each heavy chain includes a VDJ segment and a constant region (in Figure 6B) VDJ segment (or VDJ) refers to a unique pairing of V, D, and J gene segments that encode a unique portion of an antibody heavy chain that binds an epitope on a pathogen. Thus, V refers to one of the gene segments that randomly pairs with the D and J segments to encode a unique portion of an antibody heavy chain that binds an epitope on a pathogen. Similarly, D refers to one of the gene segments that randomly pairs with the V and J segments to encode a unique portion of an antibody heavy chain that binds an epitope on a pathogen. Finally, J refers to one of the gene segments that randomly pairs with the V and D segments to encode a unique portion of an antibody heavy chain that binds an epitope on a pathogen. There are several V segments, D segments, and J segments that can be assembled in a variety of different combinations to form a specific VDJ segment of a specific heavy chain (see, e.g., Figure 7 ).

[0069] Each B cell pairs only a single VDJ combination with a conserved constant C region to form a full-length heavy chain. The heavy chain C region can interact with other immune proteins such as Fc receptors to activate other immune cells. All naive B cells express the same C region segment, but can become to express different C region segments after activation, and different C regions confer different functions to antibodies. For example, one C gene segment encodes ε, and an antibody expressing ε is "IgE". IgE-type antibodies bind to the body's cells and often mediate allergic reactions. Antibodies expressing αC regions are IgA antibodies; antibodies expressing γC regions are IgG antibodies, and antibodies expressing μC regions are IgM antibodies. The human genome includes a single heavy chain locus present on chromosome 14.

[0070] Refer again Figure 6B , the light chain (IgL) of an antibody includes a variable region and a constant region. The light chain variable region includes V and J gene segments, and the light chain constant region may include a single immunoglobulin constant domain. Humans express two different light chains: Igκ, which is encoded by the immunoglobulin kappa locus on chromosome 2; and Igλ, which is encoded by the immunoglobulin λ locus on chromosome 22.

[0071] Fig. 6A Schematic diagram depicting the endogenous B cell genome encoding the IgH and IgL chains. Fig. 8A and Figure 8B An initial schematic depicting where an exogenous genetic construct of the present disclosure may be inserted to achieve expression of an antibody of choice. Fig. 8ADescribe the insertion of a genetic construct including [termination signal, IgL chain of the selected antibody (PV here), jumping element (2A here), and VDJ segment of the heavy chain] into the endogenous IgH genome between the endogenous VDJ segment and the endogenous C region coding segment. This method results in the expression of the entire exogenous IgL chain, the exogenous VDJ segment of the heavy chain, and the endogenous C region of the heavy chain. It can be useful to express antibodies including endogenous C regions, for example because this allows modified B cells to regulate C region expression based on natural B cell activation and maturation state. For example, alternative splicing in the constant region of the heavy chain locus allows modified B cells to switch between expressing membrane-bound antibodies and expressing secretory antibodies. This method also allows the expression of exogenous VDJ without the need to remove endogenous VDJ. This feature is beneficial because VDJ is a relatively large DNA segment, and its removal can negatively affect cell function.

[0072] Fig. 9 A similar schematic diagram is depicted with more details about the structure and function of the endogenous B cell genome, and how the present disclosure exploits this structure and function to achieve expression of selected antibodies. The promoter region is necessary to achieve transcription of the gene segment. The heavy chain variable region (V H ) promoter is selectively active in the B-cell lineage and includes a TATA box, an Inr element, and an octamer element within 100 base pairs (bp) of the transcription start site. H Promoter activity is under proximity-dependent regulation by the Eμ enhancer element (grey oval) of the endogenous B cell genome and an enhancer element (grey circle) located at the 3' end of the heavy chain locus, adjacent to the heavy chain alpha constant gene. The Eμ enhancer element is a DNA intron region (40 to 1500 bp in length) within the 700 bp intron between the J heavy chain segment and the Cμ (mu) segment of the immunoglobulin heavy chain locus. It can bind to activator proteins to increase or activate transcription of the heavy chain gene. The sequence of the human Eμ enhancer element is provided as SEQ ID NO: 85. Fig.11A The sequence of the mouse Eμ enhancer element is provided as SEQ ID NO:86 in Fig.13A middle.

[0073] Will include V H Insertion of a genetic construct that expresses a promoter between the endogenous heavy chain variable region and the endogenous Eμ enhancer can reduce or block the expression of endogenous V H The transcriptional activation caused by the Eμ promoter is inhibited because the Eμ enhancer will initiate transcription at the nearest upstream promoter. In this way, the expression of endogenous VDJ can be blocked without the need to remove this large DNA segment (which, as indicated, is problematic for cell function and survival). In a specific embodiment, VDJ recombination removes VH In certain embodiments, the endogenous genetic material is not removed. In certain embodiments, less than 50 base pairs are removed. In certain embodiments, the V in the exogenous genetic construct is located at an appropriate distance from the exogenous promoter of the genetic construct disclosed herein to activate transcription from the promoter inserted into the genetic construct. H Promoters include the native light chain promoter of IgK or IgL, the native human IgH promoter, the spleen focus forming virus promoter SFFV, the J558 h10 promoter, or the IgVH1-69 promoter.

[0074] Fig.10 Another schematic diagram of target regions for insertion of genetic constructs is provided. These target regions cover two conserved regions present in all B cells: from the terminal J gene segment (IGHJ6 in humans and IGHJ4 in mice) to the heavy chain intronic enhancer (Eμ), and from Eμ to the repetitive sequences associated with DNA switch recombination.

[0075] In specific embodiments, the region of the endogenous B cell genome targeted for insertion of the genetic construct is upstream of the Eμ enhancer of SEQ ID NO: 85 or 86. Figures 11A-14B Specific sequences are provided that can be targeted for insertion of genetic constructs to achieve expression of selected antibodies as disclosed herein.

[0076] Fig.11A The human DNA sequence of the IGHJ6 to Eμ intronic enhancer is provided (SEQ ID NO: 1; >hg38_dna range = chr14: 105862523-105863244 5' additive = 0 3' additive = 0 strand = - repeat shield = none). Fig. 11B Exemplary ranges (e.g., gRNA sites) for targeting within this sequence include SEQ ID NOs: 5-24 and related gRNA sequences (SEQ ID NOs: 88, 89, and 290-307). As an example, in a specific embodiment, the sgRNA of SEQ ID NO: 88 (see also Fig.25A ) can be used to target the gRNA site of SEQ ID NO: 7. In a specific embodiment, the sgRNA of SEQ ID NO: 89 (see also Fig.25A ) can be used to target the gRNA site of SEQ ID NO:10.

[0077] Fig. 12AThe human DNA sequence for region 2 is provided: Eμ intronic enhancer to switch region (SEQ ID NO:2; >hg38_dna range = chr14: 105860383-105861690 5' addition = 0 3' addition = 0 strand = -). Fig. 12B Exemplary ranges (e.g., gRNA sites) for targeting within this sequence are provided, including SEQ ID NOs: 25-44, as well as related gRNA sequences (SEQ ID NOs: 308-327).

[0078] Fig.13A The mouse DNA sequence for region 1 is provided: from IGHJ4 to the Eμ intronic enhancer (SEQ ID NO:3; >mm10_dna range=chr12:113427973-113428554 5' addition=0 3' addition=0 strand=-repeat shield=none). Fig. 13B Exemplary ranges (e.g., gRNA sites) for targeting within this sequence include SEQ ID NOs: 45-64 and related gRNA sequences (SEQ ID NOs: 87 and 328-346). As an example, in a specific embodiment, the sgRNA of SEQ ID NO: 87 (see also Fig.25A ) can be used to target the gRNA site of SEQ ID NO:46.

[0079] Fig.14A The mouse DNA sequence for region 2 is provided: from the Eμ intronic enhancer to the switch region (SEQ ID NO:4; >mm10_dna range=chr12:113425446-113426973 5' addition=0 3' addition=0 strand=-repeat shield=none). Fig. 14B Exemplary ranges (e.g., gRNA sites) for targeting within this sequence are provided, including SEQ ID NOs. 65-84, as well as related gRNA sequences (SEQ ID NOs: 347-366).

[0080] Therefore, in a specific embodiment, the disclosure provides a targeted insertion of a genetic construct comprising (i) a promoter and (ii) a transgenic portion encoding a portion of a selected antibody at an intronic region, the intronic region being constant in all B cells (before and after recombination), and (i) being positioned relative to an enhancer element interacting with the promoter; and (ii) being in a configuration such that the endogenous heavy chain VDJ sequence of the B cell is not expressed. In a specific embodiment, the encoded portion of the selected antibody comprises the entire light chain of the antibody and the VDJ segment of the heavy chain. These portions of the selected antibody can be expressed together with the heavy chain constant region expressed by the modified B cell at any given time. Specific embodiments of the genetic construct may also include or encode a signal peptide, a flexible linker, a jumping element, and / or a splice junction.

[0081] One technical challenge of the present disclosure is that antibodies are proteins made from two separate gene products, a heavy chain (IgH) and a light chain (IgL). Fig. 6A , Figure 6B ). This means that, in certain embodiments, both genetic positions must be modified simultaneously to properly express the selected antibody. However, the present disclosure also provides strategies for producing functional selected antibodies without having to modify both genetic positions. One method of allowing this to be achieved is by using sequences that allow antibody expression by a single construct. In certain embodiments, this is achieved by including a jumping element within the genetic construct. An example of a jumping element is a self-cleaving peptide, such as a self-cleaving "2A" peptide. The 2A peptide works by causing the ribosome to skip the synthesis of the peptide bond at a determined position, resulting in the production of two proteins from one mRNA. The 2A sequence is short (e.g., 20 amino acids), which facilitates use in size-restricted constructs, and the protein is produced at a 1:1 ratio. Specific examples include T2A(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 176); P2A(GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 177); E2A(GSG)QCTNYALLKLAGDVESNPGPP (SEQ ID NO: 178); and F2A(GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 179).

[0082] In a specific embodiment, the genetic construct includes an internal ribosome entry site (IRES) sequence. IRES can be located upstream of the heavy chain VDJ of the genetic construct. IRES is a non-coding structured RNA sequence that allows ribosomes to initiate translation at the second internal site on the mRNA molecule, thereby resulting in the production of two proteins from one mRNA. However, compared to the translation driven by 2A, the translation driven by IRES has a lower efficiency, thereby resulting in a lower expression of the second protein in the transcript.

[0083] In a specific embodiment, the genetic construct encodes a flexible linker between the light chain portion of a selected antibody and the heavy chain portion of a selected antibody. A linker can be a series of amino acids that flexibly connects one protein domain to another protein domain in a manner that allows the connected sequences to interact to form a functional unit.

[0084] In a particular sequence, a flexible linker may include one or more series of glycine and serine combinations that provide flexibility to the linker sequence. Exemplary Gly-Ser linkers include (GGS)n(SEQ ID NO:180), (GGGS)n(SEQ ID NO:181) and (GGGGS)n(SEQ ID NO:182), wherein n=1–100 and each integer therebetween. In a particular embodiment, n=10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In a particular embodiment, the Gly-Ser linker includes 50-80 amino acids. In a particular embodiment, the Gly-Ser linker includes 54, 57 or 60 amino acids. In a particular embodiment, the Gly-Ser linker is encoded by SEQ ID NO:116. In certain embodiments, the Gly-Ser linker comprises SEQ ID NO:122.

[0085] Additional examples of flexible linkers include (KESGSVSSEQLAQFRSLD)n (SEQ ID NO: 183) and (EGKSSGSGSESKST)n (SEQ ID NO: 184). In these linkers, the Gly and Ser residues in the linker are designed to provide flexibility, while Glu and Lys are added to improve solubility. Bird, RE et al. Science, 1988; 242: 423–426. In specific embodiments, n=1–100 and every integer therebetween. In specific embodiments, n=10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In specific embodiments, these linkers include 50-80 amino acids.

[0086] Specific embodiments include splice junctions that allow splicing between RNA encoded by a genetic construct and RNA encoded by an endogenous heavy chain constant region. In specific embodiments, the genetic construct includes a splice junction sequence at the 3' end. Splicing may refer to removing introns and joining exons together by an RNA / protein complex known as a spliceosome. A splice junction refers to an intron sequence that is directly flanked by an exon. The splice junction at the 3' end of an exon may include a splice donor site. The splice donor site sequence typically starts with "GU". In specific embodiments, the splice junction may include 40-80bp of an intron after the last exon of VDJ. In specific embodiments, the splice junction includes 40-80bp of an intron flanked by the 3' end of a human IGHJ1 gene segment or a mouse IGHJ3 gene. In specific embodiments, the splice junction includes CAG / gtaagt, wherein cutting and splicing occur after a capital G (indicated by the "splicing" annotation). In a specific embodiment, the splice junction includes CAG / gtgagt. CA forms the end of the serine codon, and G starts the first codon from the constant region. In a specific embodiment, in a genetic construct for insertion into a human locus, the splice junction with the flanking sequence includes SEQ ID NO: 124 or 151. In a specific embodiment, in a genetic construct for insertion into a mouse locus, the splice junction with the flanking sequence includes SEQ ID NO: 139.

[0087] The genetic constructs disclosed herein may also encode a signal peptide. Exemplary signal peptides include signal peptides derived from human IgH heavy chains, such as MELGLSWIFLLAILKGVQC (SEQ ID NO: 185); MELGLRWVFLVAILEGVQC (SEQ ID NO: 186); MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 187); MDWTWRILFLVAAATGAHS (SEQ ID NO: 188); MDWTWRFLFVVAAATGVQS (SEQ ID NO: 189); MEFGLSWLFLVAILKGVQC (SEQ ID NO: 190); MEFGLSWVFLVALFRGVQC (SEQ ID NO: 191); and MDLLHKNM KHLWFFLLLVAAPRWVLS (SEQ ID NO: 192); and signal peptides derived from human IgL light chains, such as MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 193). NO:193); and MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO:194). In a specific embodiment, in a genetic construct for insertion into a human locus, the signal peptide is encoded by SEQ ID NO:112 and includes SEQ ID NO:118. In a specific embodiment, in a genetic construct for insertion into a mouse locus, the signal peptide is encoded by SEQ ID NO:129 and includes SEQ ID NO:134. See also Figure 25B-Figure 25I and Haryadi R et al., tPLoS One v.10(2);2015PMC4338144.

[0088] As indicated, specific embodiments of the present disclosure utilize insertion of exogenous genetic constructs at the targeted position within the endogenous B cell genome. In specific embodiments, the targeted insertion can be promoted by including a homology region on one or both ends of the genetic construct. The homology region (i.e., homology suture or homology arm) is homologous to the sequence at the desired insertion site. In specific embodiments, the homology arm refers to a DNA segment included in the genetic construct that is 100% identical to the DNA region being modified. In specific embodiments, 100% identity may not be required for achieving targeted insertion (e.g., at least 90% identity may be sufficient).

[0089] The homology region causes the genetic construct to be arranged next to the targeted genetic region, and the DNA portion from the genetic construct is swapped into the region cut by the gene editing technology. In a specific embodiment, the genetic construct may include an upstream genomic homology end having 20 to 1,500 bp of genomic homology, and a downstream genomic homology end having 20 to 1,500 bp of genomic homology. The homology region may, for example, provide Fig.15A In certain embodiments, the homology region may include a "homology stitch" shown in , which can mediate the insertion of the genetic construct into the targeted insertion site. In certain embodiments, the upstream genomic homology end and the downstream genomic homology end may include a sequence having homology to the genomic sequence between the heavy chain VDJ region and the heavy chain Eμ enhancer element. In certain embodiments, the homology region may specifically include 20-50 base pairs; 300-500 base pairs; 350-550 base pairs; 900-1,000 base pairs or 400-600 base pairs. In certain embodiments, the homology region may specifically include 30-40 base pairs (e.g., 36 base pairs); 445-455 base pairs (e.g., 450 base pairs); 495-510 base pairs (e.g., 503 base pairs); and / or 960-980 base pairs (e.g., 968 base pairs). In specific embodiments, homology regions for use in mouse genetic constructs include SEQ ID NOs: 90, 91, 96, 97, 127, 140, 142, 143, 170, and 171. In specific embodiments, homology regions for use in human genetic constructs include SEQ ID NOs: 92-95, 98-101, 110, 125, 153, 173, and 174.

[0090] In certain embodiments, the genetic construct also encodes a tag sequence. The tag sequence can be useful, for example, so that cells expressing the genetic construct can be identified and / or sorted during the genetic modification process, and / or so that they can be controlled after administration to a subject. For example, in certain embodiments, it can be used to track and / or terminate the genetically modified cells after administration to a subject. Exemplary tags include (GmbH, LLC, Gottingen, DE), Tag II (WSHPQFEK (SEQ ID NO: 195)) or any variant thereof; see, e.g., U.S. Pat. No. 7,981,632, His tag, Flag tag (DYKDDDDK (SEQ ID NO: 196)), Xpress tag (DLYDDDDK (SEQ ID NO: 197)), Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 198)), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 199)), Polyglutamate tag, HA tag (YPYDVPDYA (SEQ ID NO: 200)), Myc tag (EQKLISEEDL (SEQ ID NO: 201)), Nus tag, S tag, SBP tag, Softag 1 (SLAELLNAGLGGS (SEQ ID NO: 202)), Softag3 (TQDPSRVG (SEQ ID NO: 203)). NO:203)) and V5 tag (GKPIPNPLLGLDST (SEQ ID NO:204)).

[0091] In certain embodiments, the present disclosure provides a genetic construct for expression of a selected antibody, the genetic construct comprising or encoding (i) a heavy chain promoter, and / or (ii) an immunoglobulin light chain, and / or (iii) a heavy chain variable region, and / or (iv) a stop codon; and / or (v) a jumping element and / or (vi) a splice junction and / or (vii) a homology arm and / or (viii) a linker and / or (ix) a tag.

[0092] Particular embodiments comprise or encode: (i) a heavy chain promoter; (ii) a signal peptide; (iii) the entire light chain of a selected antibody; (iv) a flexible linker or skipping element; (v) the variable region of the selected antibody heavy chain; and (vi) a splice junction.

[0093] Specific embodiments comprise or encode: (i) a heavy chain promoter; (ii) a signal peptide; (iii) the entire light chain of a selected antibody; (iv) a flexible linker or jumping element; (v) the variable region of the selected antibody heavy chain; (vi) a splice junction, and (vii) homology arms.

[0094] Specific embodiments comprise or encode: (i) a heavy chain promoter; (ii) a signal peptide; (iii) the entire light chain of a selected antibody; (iv) a flexible linker or jumping element; (v) the variable region of the selected antibody heavy chain; (vi) a splice junction, (vii) homology arms; and (viii) a tag.

[0095] Fig. 15BDescribe the additional examples of DNA repair templates. The examples of DNA repair templates that can also be used include synthetic DNA templates and adeno-associated viruses. In a specific embodiment, the synthetic DNA template may include double-stranded DNA (dsDNA), which includes or encodes a promoter and a selected antibody portion flanked by 20-1,500 base pairs with homology to the target site in the genome. In a specific embodiment, the synthetic DNA template may include single-stranded DNA (ssDNA), which includes or encodes a promoter and a selected antibody portion flanked by 10-80 base pairs or 400-1000 base pairs with homology to the target site in the genome. In a specific embodiment, the synthetic DNA template may include both dsDNA and ssDNA modified by phosphorylation at the end to increase the DNA connection efficiency. In a specific embodiment, both dsDNA and ssDNA can be modified at the end with a phosphorothioate bond to increase stability and prevent endonuclease digestion.

[0096] In a specific embodiment, the adeno-associated virus may include a segment encoding a synthetic antibody portion flanked by 20-1,500 base pairs having homology to a target site in the genome. In a specific embodiment, the promoter and synthetic antibody portion coding sequence may be flanked by homologous sequences matching the target site in the genome.

[0097] In certain embodiments, genetic constructs including DNA repair machinery (e.g., homologous sutures, synthetic DNA templates) can be delivered using gene editing systems such as CRISPR, TALEN, megaTAL, zinc finger nucleases, and / or adeno-associated viruses as described in more detail below. For example, genome targeting elements, genome cleavage elements, and genetic constructs described herein can be administered to B cells.

[0098] As a specific example of the application of the present disclosure, B cells can be modified to express palivizumab antibodies. B cells can be modified with a genetic construct comprising an 80 bp homology arm flanking a heavy chain promoter, which is upstream of a complete light chain (IgLPV) and a VDJ heavy chain gene segment (VDJPV) separated by a 2A peptide from palivizumab. Here, a 2A peptide is included to induce a ribosome jump event (Donnelly et al., The Journal of general virology. 2001; 82 (Pt 5): 1013-25), which allows heavy and light chains to be produced in the form of separate subunits that will usually associate to form a selected antibody. In a specific embodiment, a stop codon may be included upstream of the inserted heavy chain promoter to stop any potential transcription of the endogenous heavy chain variable region.

[0099] Fig.16 Describes an example of an "as modified" B cell genome, and Fig.17 The resulting B cell populations expressing the selected antibodies are profiled.

[0100] The following paragraphs provide a more detailed description of (i) exemplary selected antibodies and sequences; (ii) gene editing techniques and cell sorting; (iii) preparation of modified B cells; and (iv) methods of use.

[0101] (i) Exemplary selected antibodies and sequences. In certain embodiments, the selected antibodies are antibodies that can provide protection against pathogens or diseases (e.g., autoimmune diseases). In certain embodiments, the selected antibodies are anti-RSV antibodies, anti-HIV antibodies, anti-dengue virus antibodies, anti-pertussis Bordetella antibodies, anti-hepatitis C antibodies, anti-influenza virus antibodies, anti-parainfluenza virus antibodies, anti-meta-pneumovirus (MPV) antibodies, anti-cytomegalovirus antibodies, anti-Epstein Barr virus antibodies; anti-herpes simplex virus antibodies, anti-Clostridium difficile bacterial toxin antibodies, or anti-tumor necrosis factor (TNF) antibodies.

[0102] In certain embodiments, the selected antibody is a chimeric antibody. In certain embodiments, a chimeric antibody refers to a synthetic antibody comprising: (i) at least one portion encoded by the endogenous genome of a B cell, and (ii) at least one portion encoded by an inserted genetic construct. In certain embodiments, a chimeric antibody comprises an endogenous heavy chain constant domain, an exogenous immunoglobulin variable and constant light chain, and an exogenous variable heavy chain.

[0103] The following antibodies and sequences can be used to provide selected antibodies with targeted binding to a pathogen or antigen of interest (Kabat numbering is intended unless otherwise indicated):

[0104] An exemplary anti-RSV antibody is palivizumab, which targets the RSV fusion protein and is used to prevent or reduce RSV infection.

[0105] In certain embodiments, the anti-RSV antibody is mouse palivizumab, which comprises a variable heavy chain sequence comprising:

[0106] QVELQESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSG EGLEWLADIWWDDKKDYNPSLKSRLTISKDTSSNQVFLKITGVDT ADTATYYCARSMITNWYFDVWGAGTTVTVSS (SEQ ID NO: 138); and a variable light chain sequence comprising:

[0107] DIQLTQSPAIMSASPGEKVTMTCSASSSVGYMHWYQQKLSTS PKLQIYDTSKLASGVPGRFSGSGSGNSYSLTISSIQAEDVATYYCFR GSGYPFTFGQGTKLEIK (SEQ ID NO: 205).

[0108] Another exemplary anti-RSV antibody is human palivizumab, and includes a variable light chain sequence comprising:

[0109] DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGK APKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDFATYYCFQ GSGYPFTFGGGTKLEIKR (SEQ ID NO: 206); and a variable heavy chain sequence comprising:

[0110] QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPP GKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAGTT (SEQ ID NO: 123).

[0111] Within the variable heavy and light chains, segments called complementarity determining regions (CDRs) determine epitope binding. Each heavy chain has three CDRs (i.e., CDRH1, CDRH2, and CDRH3), and each light chain has three CDRs (i.e., CDRL1, CDRL2, and CDRL3).

[0112] Another exemplary anti-RSV antibody is described in U.S. Pat. No. 9,403,900. This anti-RSV antibody includes a variable heavy chain containing a CDRH1 sequence including GASINSDNYYWT (SEQ ID NO: 207), a CDRH2 sequence including HISYTGNTYYTPSLKS (SEQ ID NO: 208), and a CDRH3 sequence including CGAYVLISNCGWFDS (SEQ ID NO: 209); and a variable light chain containing a CDRL1 sequence including QASQDISTYLN (SEQ ID NO: 210), a CDRL2 sequence including GASNLET (SEQ ID NO: 211), and a CDRL3 sequence including QQYQYLPYT (SEQ ID NO: 212).

[0113] Exemplary anti-RSV antibodies also include AB1128 (available from MILLIPORE) and ab20745 (available from ABCAM).

[0114] An example of an anti-HIV antibody is 10E8, which is a broadly neutralizing antibody that binds to gp41. The 10E8 anti-HIV antibody includes a variable heavy chain containing a CDRH1 sequence including GFDF DNAW (SEQ ID NO: 213), a CDRH2 sequence including ITGPGEGWSV (SEQ ID NO: 214), and a CDRH3 sequence including TGKYYDFWSGYPPGEEYF QD (SEQ ID NO: 215); and a variable light chain containing a CDRL1 sequence including TGDSLRSHYAS (SEQ ID NO: 216), a CDRL2 sequence including GKNNRPS (SEQ ID NO: 217), and a CDRL3 sequence including SSRDKS GSRLSV (SEQ ID NO: 218).

[0115] Another example of an anti-HIV antibody is VRC01, which is a broadly neutralizing antibody that binds to the CD4 binding site of gp120. The VRC01 antibody includes a variable heavy chain containing a CDRH1 sequence including GYEFIDCT (SEQ ID NO: 219), a CDRH2 sequence including KPRGGAVN (SEQ ID NO: 220), and a CDRH3 sequence including RGKNCDYNWDFEHW (SEQ ID NO: 221); and a variable light chain containing a CDRL1 sequence including QYGS, a CDRL2 sequence including SGS, and a CDRL3 sequence including QQYEF (SEQ ID NO: 222).

[0116] Exemplary anti-HIV antibodies also include ab18633 and 39 / 5.4A (available from ABCAM); and H81E (available from THERMOFISHER).

[0117] An example of an anti-dengue virus antibody is antibody 55 described in US20170233460, and includes a variable heavy chain containing a CDRH1 sequence including EVQLHQSGAELVKPGASVKLSCTVSGFNIK (SEQ ID NO: 223), a CDRH2 sequence including WVKQRPEQGLEWI (SEQ ID NO: 224), and a CDRH3 sequence including ATIKADTSSNTAYLQLISLTSEDTAVYYCAF (SEQ ID NO: 225); and a variable light chain containing a CDRL1 sequence including DIQMTQSPASLSVSVGETVTITC (SEQ ID NO: 226), a CDRL2 sequence including WYQQKQGKSPQLLVY (SEQ ID NO: 227), and a CDRL3 sequence including GVPSRFSGSGSGTQYSLKINSLQSEDFGTYYC (SEQ ID NO: 228).

[0118] Another example of an anti-dengue virus antibody is DB2-3 described in U.S. Pat. No. 8,637,035, and includes a variable heavy chain containing a CDRH1 sequence including YTFTDYAIT (SEQ ID NO: 229), a CDRH2 sequence including GLISTYYGDSFYNQKFKG (SEQ ID NO: 230), and a CDRH3 sequence including TIRDGKAMDY (SEQ ID NO: 231); and a variable light chain containing a CDRL1 sequence including RSSQSLVHSNGNTYLH (SEQ ID NO: 232), a CDRL2 sequence including KVSNRFS (SEQ ID NO: 233), and a CDRL3 sequence including SQSTHVPYT (SEQ ID NO: 234). Examples of anti-dengue virus antibodies also include ab155042 and ab80914 (both available from ABCAM).

[0119] An example of an anti-pertussis antibody is described in U.S. Pat. No. 9,512,204, and includes a variable heavy chain comprising QVQLQQPGSELVRPGASVKLSCKASGYKFTSYWMHWVKQRPGQGLEWIGNIFPGSGSTNYDEKFNSKATLTTVDTSSNTAYMQLSSLTSEDSAVYYCTRWLSGAYFDYWGQ GTTVTVSS (SEQ ID NO: 235), and a variable light chain comprising QIVLTQSPALMSASPGEKVTMTCSASSSVSFMYWYQQKPRSSP KPWIYLTSNLPSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQ WSSHPPTFGSGTKLEIK (SEQ ID NO: 236).

[0120] An example of an anti-hepatitis C antibody includes a variable heavy chain; the variable heavy chain contains a CDRH1 sequence including SYGMHW (SEQ ID NO: 237), a CDRH2 sequence including VIWLDGSNTYYADSVKGR (SEQ ID NO: 238), and a CDRH3 sequence including ARDIFTVARGVIIYFDY (SEQ ID NO: 239); and a variable light chain containing a CDRL1 sequence including RASQSVSSYLA (SEQ ID NO: 240), a CDRL2 sequence including DASNRAT (SEQ ID NO: 241), and a CDRL3 sequence including QQRSNWVT (SEQ ID NO: 242). Examples of anti-hepatitis C antibodies also include MAB8694 (available from MILLIPORE) and C7-50 (available from ABCAM).

[0121] An example of an anti-influenza virus antibody is described in U.S. Pat. No. 9,469,685 and includes a variable heavy chain containing a CDRH1 sequence including GMTSNSLA (SEQ ID NO: 243), a CDRH2 sequence including IIPVFETP (SEQ ID NO: 244), and a CDRH3 sequence including ATSAGGIVNYYLSFNI (SEQ ID NO: 245); and a variable light chain containing a CDRL1 sequence including QTITTW (SEQ ID NO: 246), a CDRL2 sequence including KTS, and a CDRL3 sequence including QQYSTYSGT (SEQ ID NO: 247). An example of an anti-influenza virus antibody also includes C102 (available from THERMOFISHER).

[0122] An exemplary anti-MPV antibody includes MPE8.

[0123] Exemplary anti-CMV antibodies include MCMV5322A, MCMV3068A, LJP538, and LJP539. RG7667 includes a mixture of MCMV5322A and MCMV3068A, while CSJ148 includes a mixture of LJP538 and LJP539. See also, for example, Deng et al., Antimicrobial Agents and Chemotherapy 62 (2) e01108-17 (February 2018); and Dole et al., Antimicrobial Agents and Chemotherapy 60 (5) 2881-2887 (May 2016).

[0124] An example of an anti-EBV antibody includes a variable heavy chain comprising an AMM01 CDRH1 sequence comprising YTFIHFGISW (SEQ ID NO: 248), an AMM01 CDRH2 sequence comprising IDTNNGNTNYAQSLQG (SEQ ID NO: 249), and an AMM01 CDRH3 sequence comprising RALEMGHRSGFPFDY (SEQ ID NO: 250); and a variable light chain comprising an AMM01 CDRL1 sequence comprising GGHNIGAKNVH (SEQ ID NO: 251), an AMM01 CDRL2 sequence comprising YDSDRPS (SEQ ID NO: 252), and an AMM01 CDRL3 sequence comprising CQVWDSGRGHPLYV (SEQ ID NO: 253).

[0125] Examples of anti-HSV antibodies include HSV8-N and MB66.

[0126] Exemplary anti-Clostridium difficile antibodies include actoxumab and bezlotoxumab. See also, e.g., Wilcox et al., N Engl J Med 376(4)305-317 (2017).

[0127] Commercially available anti-TNF antibodies include infliximab ( Centocor, Inc., Malvern, PA, and biosimilars Pfizer, Kent, UK and Pfizer, New York, NY), adalimumab ( Abbott Laboratories, Abbott Park, IL, and biosimilars Amgen, Thousand Oaks, CA, and Boehringer Ingelheim Int'l, Ingelheim, DE), golimumab ( Johnson & Johnson Corp., New Brunswick, NJ), etanercept (Enbrel ImmunexCorp, Thousand Oaks, CA, and biosimilars Novartis AG, Basel, CH) and certolizumab-pegol ( UCB Pharma, Brussels, BE).

[0128] In a specific embodiment, the CDRs of infliximab include: heavy chain residues 26-37, 52-70 and 103-116 and light chain residues 24-39, 55-61 and 94-102. In a specific embodiment, the heavy chain of infliximab begins with EVKLEESGGGLVQPGGSMK (SEQ ID NO: 254) and the light chain begins with DILLTQSPAILSVSPGER (SEQ ID NO: 255).

[0129] In a specific embodiment, infliximab comprises a variable heavy chain comprising a CDRH1 sequence comprising IFSNHW (SEQ ID NO:256), a CDRH2 sequence comprising RSKSINSATH (SEQ ID NO:257), and a CDRH3 sequence comprising NYYGSTY (SEQ ID NO:258); and a variable light chain comprising a CDRL1 sequence comprising FVGSSIH (SEQ ID NO:259), a CDRL2 sequence comprising KYASESM (SEQ ID NO:260), and a CDRL3 sequence comprising QSHSW (SEQ ID NO:261).

[0130] In a specific embodiment, adalimumab comprises a variable heavy chain comprising a CDRH1 sequence comprising TFDDYA (SEQ ID NO:262), a CDRH2 sequence comprising TWNSGHID (SEQ ID NO:263), and a CDRH3 sequence comprising VSYLSTASSL (SEQ ID NO:264); and a variable light chain comprising a CDRL1 sequence comprising GIRNYLA (SEQ ID NO:265), a CDRL2 sequence comprising YAASTLQ (SEQ ID NO:266), and a CDRL3 sequence comprising RYNRA (SEQ ID NO:267).

[0131] In a specific embodiment, selezizumab comprises a variable heavy chain comprising a CDRH1 sequence comprising VFTDYG (SEQ ID NO:268), a CDRH2 sequence comprising NTYIGEPI (SEQ ID NO:269), and a CDRH3 sequence comprising GYRSYAM (SEQ ID NO:270); and a variable light chain comprising a CDRL1 sequence comprising NVGTNVA (SEQ ID NO:271), a CDRL2 sequence comprising YSASFLY (SEQ ID NO:272), and a CDRL3 sequence comprising QYNIY (SEQ ID NO:273).

[0132] Numerous additional antibody sequences that can be used within the teachings of the present disclosure are available and known to those of ordinary skill in the art. Sequence information for commercially available antibodies can be found in the Drug Bank database, the CAS Registry, and / or the RSCB Protein Data Bank. In addition, nucleic acid sequences encoding portions of the selected antibodies described herein can be readily obtained by those of ordinary skill in the art.

[0133] (ii) Gene editing technology and cell sorting. Gene editing systems allow control of target sites for genetic therapy. Within the teachings of the present disclosure, any gene editing system capable of precise sequence targeting and modification can be used. These systems typically include a targeting element for precise targeting and a cutting element for cutting the targeted genetic site. Guide RNA is an example of a targeting element, and various nucleases provide examples of cutting elements. The targeting element and the cutting element can be separate molecules or connected, for example, by nanoparticles. Alternatively, the targeting element and the cutting element can be connected together to become a dual-purpose molecule. When it is intended to insert a therapeutic nucleic acid sequence, the system may also include a homology-guided repair template (i.e., a homology arm as described above) associated with the genetic construct. However, as further described below, different gene editing systems can use different components and configurations while maintaining the ability to precisely target, cut and modify selected genomic sites.

[0134] Specific embodiments utilize zinc finger nuclease (ZFN) as gene editing agent. ZFN is a class of site-specific nucleases that are engineered to bind and cleave DNA at specific locations. ZFN is used to introduce double-strand breaks (DSBs) at specific sites in DNA sequences, which enables ZFN to target unique sequences in the genome in a variety of different cells. In addition, after double-strand breaks, homology-guided repair (HDR) or non-homologous end joining (NHEJ) occurs to repair DSBs, thereby enabling genome editing.

[0135] ZFNs are synthesized by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The DNA binding domain includes three to six zinc finger proteins that are transcription factors. The DNA cleavage domain includes, for example, the catalytic domain of the FokI endonuclease. The FokI domain acts as a dimer, requiring two constructs with unique DNA binding domains for sites on the target sequence. The FokI cleavage domain cleaves within a spacer sequence of five or six base pairs that separates the two inversion half sites.

[0136] For additional information about ZFNs, see Kim et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); Wolfe et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Bibikova et al. Science 300, 764 (2003); Bibikova et al. Genetics 161, 1169-1175 (2002); Miller et al. The EMBO journal 4, 1609-1614 (1985); and Miller et al. Nature biotechnology 25, 778-785 (2007).

[0137] Specific embodiments can use transcription activator-like effector nuclease (TALEN) as a gene editing agent. TALEN refers to a fusion protein including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALEN is used to edit genes and genomes by inducing DSB in DNA, and the DSB induces the repair mechanism in cells. Generally, two TALENs must be combined, and each side of the target DNA site flanked by the DNA cleavage domain is dimerized and induced DSB. In cells, DSB is repaired by NHEJ, or if there is an exogenous double-stranded donor DNA fragment, it is repaired by HDR.

[0138] As indicated, TALEN has been engineered to bind to, for example, an endogenous genomic target sequence and cut DNA at the location of the target sequence. TALEN's TALE is a DNA binding protein secreted by Xanthomonas bacteria. The DNA binding domain of TALE includes a highly conserved repeat sequence with 33 or 34 amino acids, with different residues at the 12th and 13th positions of each repeat sequence. These two positions, referred to as repeat variable diresidues (RVDs), show a strong association with specific nucleotide recognition. Therefore, targeting specificity can be improved by changing the amino acids in the RVD and incorporating unconventional RVD amino acids.

[0139] Examples of DNA cleavage domains that can be used in TALEN fusions are wild-type and variant FokI endonucleases. For additional information on TALENs, see Boch et al. Science 326, 1509-1512 (2009); Moscou and Bogdanove, Science 326, 1501 (2009); Christian et al. Genetics 186, 757-761 (2010); and Miller et al. Nature biotechnology 29, 143-148 (2011).

[0140] Specific embodiments utilize MegaTAL as a gene editing agent. MegaTAL has a single-chain rare-cutting nuclease structure in which a TALE is fused to the DNA cleavage domain of a megabase meganuclease. Megabase meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease functions in the same domain. Compared to TALENs, megaTALs only require the delivery of a single peptide chain to achieve functional activity.

[0141] In a specific embodiment, the endogenous B cell genome can be targeted using the CRISPR gene editing system. The CRISPR nuclease system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages, and provides a form of acquired immunity. CRISPR is a DNA locus containing short repetitions of base sequences. In the case of the prokaryotic immune system, each repetition is followed by a short segment of spacer DNA, which belongs to the foreign genetic elements to which the prokaryotic organism is exposed. This CRISPR repeat array interspersed with spacers can be transcribed into RNA. RNA can be processed into a mature form and associated with nucleases such as cas (CRISPR-associated) nucleases. The CRISPR-Cas system including RNA and Cas nucleases with sequences that can hybridize to foreign genetic elements can then identify and cut these exogenous genetic elements in the genome.

[0142] The CRISPR-Cas system does not require the production of custom proteins to target specific sequences, but a single Cas enzyme can be programmed by a short guide RNA molecule (crRNA) to recognize a specific DNA target. The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show great diversity in protein composition and genomic locus structure. The CRISPR-Cas system loci have more than 50 gene families, and there are no absolutely universal genes, indicating the rapid evolution and great diversity of locus structure. So far, a comprehensive cas gene identification of 395 profiles of 93 Cas proteins has been conducted using a multi-pronged approach. The classification includes the characteristics of the characteristic gene profile plus the characteristics of the locus structure. A new classification of CRISPR-Cas systems is proposed, in which these systems are broadly divided into two categories, Category 1 has a multi-subunit effector complex, and Category 2 has a single subunit effector module exemplified by the Cas9 protein.

[0143] At least three different Cas9 nucleases have been developed for genome editing. The first is wild-type Cas9, which introduces double-strand breaks (DSBs) at specific DNA sites, resulting in activation of DSB repair mechanisms. DSBs can be repaired by non-homologous end joining (NHEJ), homology-guided repair (HDR), or microhomology-mediated repair (MMEJ). NHEJ may involve repairing DSBs when there is no homology (<5bp) between the two ends joined during repair; HDR may involve repairing DSBs when there is a large homology region (100 or more nucleotides) between the ends joined during repair; and MMEJ may involve repairing DSBs when there is a small (5 to 50bp) homology region between the ends joined during repair. Another type of Cas9 includes a mutant Cas9 referred to as Cas9D10A, which has only nickase activity, which means that it only cleaves one DNA chain and does not activate NHEJ. Therefore, DNA repair is only performed by the HDR path. The third is nuclease-deficient Cas9 (dCas9), which has no cleavage activity but can bind to DNA. Therefore, dCas9 can target specific sequences of the genome without cleavage. By fusing dCas9 with various effector domains, dCas9 can be used as a gene silencing or activation tool.

[0144] In addition to class 1 and class 2 CRISPR-Cas systems, more recently, a putative class 2, type V CRISPR-Cas class exemplified by Cpf1 has been identified, Zetsche et al. (2015) Cell 163(3):759-771. The Cpf1 nuclease can provide additional flexibility in target site selection by virtue of a short three-base pair recognition sequence (TTN), referred to as a protospacer adjacent motif or PAM. The cleavage site of Cpf1 is at least 18 bp from the PAM sequence, so the enzyme can repeatedly cleave a given locus after indels (insertions and deletions) have been formed, thereby increasing the efficiency of HDR. In addition, staggered DSBs with sticky ends allow for directional specific donor template insertion.

[0145] Additional information about CRISPR-Cas systems and components thereof is described in US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US 8993233, and US8999641, and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO 2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 20 4724、WO2014 / 204725、WO2014 / 204726、WO2014 / 204727、WO2014 / 204728、WO2014 / 204729、WO 2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711, WO 2017 / 106657, WO2017 / 127807 and applications related thereto.

[0146] Specific embodiments combine tracrRNA and crRNA into a single synthetic single guide RNA (sgRNA, using, for example, SEQ ID NO: 87-89 or 290-366). In specific embodiments, sgRNA may include a sequence having twenty nucleotides similar to crRNA and a tracrRNA sequence. For certain gene editing systems, the target sequence may be adjacent to PAM (e.g., 5'-20nt target -NGG-3'). In specific embodiments, the target sequence may include PAM (SEQ ID NO: 5-84). In specific embodiments, the guide RNA (gRNA) includes a target site targeted by a genome editing complex adjacent to PAM. The gRNA may include at least 16, 17, 18, 19, 20, 21, or 22 nucleotides adjacent to PAM.

[0147] In certain embodiments, the cleavage element is directed to the targeted DNA location ( Fig.25A (Sternberg et al., Mol Cell. 2015; 58(4):568-74). Genetic constructs with homology arms flanking the cleaved genomic region are efficiently inserted into this location by homology-directed DNA repair mechanisms (see e.g. Fig. 15B (Elliott et al., Mol Cell Biol. 1998; 18(1): 93-101). Using this method, the expression of endogenous antibodies will be eliminated, and the gene encoding the selected antibody will be inserted into the targeted genetic position. This targeted insertion eliminates or significantly reduces the possibility of off-target effects caused by random genetic insertion.

[0148] In a specific embodiment, the sgRNA targeting mouse or human IgH of each endogenous antibody targets a region 100 bp downstream of the J region ( Fig. 9 In experimental examples, this region was targeted to express a version of the selected antibody, palivizumab, containing the C region from the endogenous genome ( Figure 7 and Fig. 9). The crispr.mit.edu algorithm (Hsu et al., Nat Biotechnol. 2013; 31(9): 827-32) identified 22 targeting sequences for this region that were predicted to have little, if any, off-target binding. A single targeting sequence can be inserted into a full-length sgRNA and mixed with a nuclease such as Cas9 immediately before incubation as described (Schumann et al., Proc Natl Acad Sci USA. 2015; 112(33): 10437-42) and electroporated into B cells (Kim et al., J Immunol. 1979; 122(2): 549-54). Because cellular repair of DNA cuts made by Cas9 often results in loss of gene function (Symington and Gautier, Annu Rev Genet. 2011; 45: 247-71), efficient sgRNAs targeting antibody coding regions are expected to result in the appearance of some antibody-deficient B cells, which can be easily assessed by flow cytometry. The activity of sgRNAs targeting intronic sequences can be assessed by sequencing or by enzymatic assays such as the T7 endonuclease assay.

[0149] In certain embodiments, the genome targeting and cleavage elements can be administered by electroporation, nanoparticle-mediated delivery, and / or viral vector delivery. Electroporation can be used, for example, to deliver targeting elements and / or cleavage elements because the cell membrane typically does not allow the foreign molecule to enter the cell. Electroporation delivers an electric shock to the cell that temporarily allows the foreign molecule to pass through the cell membrane.

[0150] In certain embodiments, the genetic construct for insertion can be administered by electroporation, nanoparticle-mediated delivery, and / or viral vector delivery. Adeno-associated viral vectors include those derived from, for example, adenovirus 5 (Ad5), adenovirus 35 (Ad35), adenovirus 11 (Ad11), adenovirus 26 (Ad26), adenovirus 48 (Ad48), or adenovirus 50 (Ad50) and adeno-associated virus (AAV; see, for example, U.S. Patent No. 5,604,090; Kay et al., Nat. Genet. 24: 257 (2000); Nakai et al., Blood 91: 4600 (1998)).

[0151] In certain embodiments, the genomic targeting and cleavage elements may be administered by electroporation, and the genetic construct for insertion may be administered by AAV-mediated delivery. In certain embodiments, the genomic targeting and cleavage elements may be administered by nanoparticle-mediated delivery, and the genetic construct for insertion may be administered by AAV-mediated delivery.

[0152] In a particular embodiment, a genetic construct including a transgene can be mixed with a targeting element (e.g., sgRNA) and a cutting element (e.g., Cas9 or cpf1) immediately or shortly before electroporation. The selected antibody expression can be confirmed later (e.g., 3 days later) by measuring the binding of cells to fluorescently labeled target proteins by flow cytometry. Enrichment and analysis methods for detecting and analyzing epitope-specific B cells can be used. Pape et al., Science. 2011; 331(6021): 1203-7; Taylor et al., J Exp Med. 2012; 209(3): 597-606; Taylor et al., J Exp Med. 2012; 209(11): 2065-77; Haasken et al., J Immunol. 2013; 191(3): 1055-62; Taylor et al., J Immunol Methods. 2014; 405: 74-86; Nanton et al., Eur J Immunol. 2015; 45(2): 428-41; Hamilton et al., J Immunol. 2015; 194(10): 5022-34; Taylor et al., Science. 2015; 347(6223): 784-7). These methods allow detection of selected antibody-expressing B cells at frequencies exceptionally low as 0.00002% of the total B cell population (Taylor et al., Science. 2015; 347(6223):784-7).

[0153] In a particular embodiment, cells can be identified and / or sorted based on marker expression before or after the delivery of the genetic construct. For example, it may be useful to separate specific types of B cells (e.g., memory B cells, antibody secreting B cells, primary B cells, B1 B cells, marginal zone B cells) from a sample before the delivery of the genetic construct. As another example, it may be useful to separate B cells from other cells present in a blood sample. CD19 is an example of a protein expressed by B cells, with only a few other cells of the body expressing it. By labeling CD19 with a fluorescent molecule, B cells can be specifically identified. B220 is a marker that can be used to identify mouse B cells.

[0154] CD27 is an example of a protein expressed by memory but not naive human B cells. By labeling CD27 with a fluorescent molecule, memory B cells can be identified.

[0155] CD21 is an example of a protein that is not expressed (or expressed to a low degree) by some memory human B cells that are able to rapidly secrete antibodies after infection. Low CD21 expression can be used to identify B cells that are sensitized to achieve plasma cell differentiation. By labeling CD21 with a fluorescent molecule, these B cells can be specifically identified by, for example, negative selection.

[0156] Human naive B cells can be identified by the marker profile IgM+IgD+CD27-. Mouse naive B cells can be identified by the marker profile CD38+GL7-IgM+IgD+. Human B1 B cells can be identified by the marker profile CD5+CD43+. Mouse B1 B cells can be identified by the marker profile CD43+low B220. Human marginal zone B cells can be identified by the marker profile CD21+++IgM++IgD-CD27+. Mouse marginal zone B cells can be identified by the marker profile CD21+++IgM++IgD-.

[0157] Certain embodiments may utilize CD19 + CD27 + CD21 lo Overview of markers.

[0158] CD45 is a marker used to identify and / or isolate cell types for use in the experiments described herein. Different mouse strains express different forms of a protein called CD45, referred to as CD45.1 and CD45.2. In the experiments disclosed herein, B cells from mice expressing CD45.2 will be harvested and transferred into mice expressing CD45.1. By labeling CD45.1 and CD45.2 with different fluorescent molecules, cells from donor animals can be identified because they express CD45.2 but not CD45.1.

[0159] Specific embodiments include sorting B cells based on the expression of exogenous light chains after genetic modification. For example, B cells that naturally express kappa light chains can be modified to express selected antibodies including lambda light chains. B cells that naturally express lambda light chains can be modified to express selected antibodies including kappa light chains. Sorting based on the expression of exogenous light chains will allow only those B cells that express the selected antibodies to be separated. In specific embodiments, only those B cells that are completely lacking in the surface expression of their endogenous light chains are separated for preparation and administration to a subject.

[0160] In certain embodiments, cells can be identified and / or separated using flow cytometry. Flow cytometry is a sensitive and powerful analytical method that uses lasers to individually analyze fluorescent molecules that label millions of individual cells. By analyzing the combination of fluorescent molecules that label each cell, different B cell subtypes can be identified. Flow cytometry can be used to identify B cell subsets and analyze the expression of selected antibodies (e.g., palivizumab) within these cells.

[0161] In certain embodiments, methods of modifying B cells may include obtaining hematopoietic stem cells (HSCs), and / or delivering a genetic construct to HSCs. HSCs may refer to the type of stem cells that naturally produce B cells and all other cells of the immune system. HSCs may be obtained, for example, from umbilical cord blood.

[0162] The specific experimental results described herein utilized A20 cells, an immortalized cell line derived from mouse B cells, to develop genetic modification methods that were subsequently transferred to freshly isolated B cells.

[0163] In certain embodiments, B cells can be obtained from a human subject, and the resulting B cells, or a subset thereof, can be modified ex vivo.

[0164] Preparation of modified B cells. Once modified, the cells can be collected from the culture medium, washed and concentrated into a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0165] In certain embodiments, the carrier can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In certain embodiments, the infusion carrier comprises buffered saline with 5% hyaluronic acid sodium salt (HAS) or dextrose. Additional isotonic agents include polyhydroxy sugar alcohols, including trihydroxy or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol or mannitol.

[0166] The carrier may include a buffer such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer, and / or a trimethylamine salt.

[0167] Stabilizers refer to a broad class of excipients that can range in function from bulking agents to additives that help prevent cells from adhering to the vessel wall. Typical stabilizers may include polyhydroxy sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol and cyclic alcohols such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose and polysaccharides such as dextran.

[0168] Where necessary or helpful, the formulation may include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0169] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexahydroxyquaternium chloride, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0170] The formulation may include, for example, more than 10 2 Modified B cells, more than 10 3 Modified B cells, more than 10 4 Modified B cells, more than 10 5 Modified B cells, more than 10 6 Modified B cells, more than 10 7 Modified B cells, more than 10 8 Modified B cells, more than 10 9 Modified B cells, more than 10 10 Modified B cells or more than 10 11 modified B cells.

[0171] Methods of use. The methods disclosed herein include treating a subject (e.g., a human, veterinary animal (dog, cat, reptile, bird), livestock (e.g., horse, cattle, goat, pig, chicken), and research animal (e.g., monkey, rat, mouse, fish)) with a formulation disclosed herein. Treating a subject includes delivering a therapeutically effective amount. A therapeutically effective amount includes those that provide an effective amount, preventive treatment, and / or therapeutic treatment.

[0172] An "effective amount" is the amount of a composition necessary to produce a desired physiological change in a subject. An effective amount is often administered for research purposes. An effective amount disclosed herein can produce a statistically significant effect in an animal model or in vitro assay associated with evaluating the manifestation, progression and / or regression of a disorder.

[0173] "Preventive and therapeutic treatment" includes treatment administered to a subject who does not show signs or symptoms of a disease or only shows early signs or symptoms of a disease, so that the treatment is administered for the purpose of weakening or reducing the risk of developing the disease. Therefore, preventive and therapeutic treatment serves as a preventive treatment for the disease. In a specific embodiment, the preventive and therapeutic treatment alleviates, delays or prevents the deterioration of the disease. Specific embodiments include administering a formulation as described herein as a preventive and therapeutic protective measure in the absence of a currently effective vaccine. Specific embodiments include administering a formulation as described herein as a preventive and therapeutic protective measure as a substitute for conventional vaccination strategies. Specific embodiments include administering a formulation as described herein as a preventive and therapeutic protective measure as a supplement to conventional vaccination strategies.

[0174] "Therapeutic treatment" includes treatment administered to a subject who exhibits symptoms or signs of a disorder, and is administered to the subject for the purpose of reducing or eliminating those signs or symptoms of the disorder. Therapeutic treatment can reduce, control or eliminate the presence or activity of the disorder, and / or reduce, control or eliminate the side effects of the disorder.

[0175] In certain embodiments, the disorder is an infection.

[0176] Acting as an effective amount, a prophylactic treatment, or a therapeutic treatment are not mutually exclusive, and in certain embodiments, the administered dose may achieve more than one type of treatment.

[0177] In certain embodiments, the therapeutically effective amount provides an anti-pathogen effect. The anti-pathogen effect may include an anti-infective effect. The anti-infective effect may include reducing the occurrence of infection, reducing the severity of infection, reducing the duration of infection, reducing the number of infected cells, reducing the volume of infected tissue, increasing life expectancy, inducing sensitivity of infected cells to immune clearance, reducing pain associated with infection, and / or reducing or eliminating symptoms associated with the treated infection.

[0178] In certain embodiments, the therapeutically effective amount provides an anti-inflammatory effect. The anti-inflammatory effect may include reducing pain, heat, redness, swelling, and / or loss of function associated with inflammation.

[0179] In certain embodiments, the therapeutically effective amount provides an anti-Crohn's disease effect or an anti-ulcerative colitis effect. The anti-Crohn's disease effect or the anti-ulcerative colitis effect may include reducing diarrhea, reducing rectal bleeding, reducing unexplained weight loss, reducing fever, reducing abdominal pain and cramping, reducing fatigue and low energy feelings, and / or restoring appetite.

[0180] In certain embodiments, the therapeutically effective amount provides an anti-arthritic effect. The anti-arthritic effect may include reducing joint pain, stiffness, swelling, redness, and / or restoring range of motion. Types of arthritis include rheumatoid arthritis (RA), ankylosing spondylitis, and psoriatic arthritis.

[0181] In certain embodiments, the therapeutically effective amount provides an anti-plaque psoriasis effect.Anti-plaque psoriasis effects may include reduction of erythema, scaling spots, itching, burning, soreness, nail bed abnormalities, and / or joint swelling or stiffness.

[0182] In a specific embodiment, B cells can be obtained from a subject, a subset of B cells can be modified in vitro, and then the modified B cells can be formulated and administered to the subject. In a specific embodiment, a first subset of the subject's B cells can be modified with a first genetic construct to produce selected antibodies for a first pathogen, and a second subset of the subject's B cells can be modified with a second genetic construct to produce selected antibodies for a second pathogen, thereby providing protective antibodies for two pathogens. As indicated, B cells for any number of pathogens can be formed and administered to the subject. In a specific embodiment, the selected antibodies can be anti-RSV antibodies, anti-HIV antibodies, anti-dengue virus antibodies, anti-pertussis Bordetella antibodies, anti-hepatitis C antibodies, anti-influenza virus antibodies, anti-parainfluenza virus antibodies, anti-MPV antibodies, anti-cytomegalovirus antibodies, anti-Epstein-Barr virus antibodies; anti-herpes simplex virus antibodies, anti-Clostridium difficile bacterial toxin antibodies and / or anti-TNF antibodies. In a specific embodiment, the selected antibodies can be one or more of anti-RSV antibodies, anti-influenza virus antibodies, anti-parainfluenza virus antibodies and / or anti-MPV antibodies. In certain embodiments, the selected antibody may be an anti-RSV antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, and an anti-MPV antibody. In certain embodiments, the selected antibody is palivizumab.

[0183] In a specific embodiment, B cells can be obtained from a bone marrow donor or hematopoietic stem cell donor that has been immunologically matched to the recipient. In a specific embodiment, a first subset of the donor's B cells can be modified with a first genetic construct to produce selected antibodies for a first pathogen, and a second subset of the donor's B cells can be modified with a second genetic construct to produce selected antibodies for a second pathogen, thereby providing protective antibodies for two pathogens. As indicated, B cells for any number of pathogens can be formed and administered to the subject. In a specific embodiment, the selected antibodies can be anti-RSV antibodies, anti-HIV antibodies, anti-dengue virus antibodies, anti-pertussis Bordetella antibodies, anti-hepatitis C antibodies, anti-influenza virus antibodies, anti-parainfluenza virus antibodies, anti-MPV antibodies, anti-cytomegalovirus antibodies, anti-Epstein-Barr virus antibodies; anti-herpes simplex virus antibodies, anti-clostridium difficile bacterial toxin antibodies and / or anti-TNF antibodies. In a specific embodiment, the selected antibodies can be one or more of anti-RSV antibodies, anti-influenza virus antibodies, anti-parainfluenza virus antibodies and / or anti-MPV antibodies. In certain embodiments, the selected antibodies may be anti-RSV antibodies, anti-influenza virus antibodies, anti-parainfluenza virus antibodies, and anti-MPV antibodies. In certain embodiments, the selected antibodies are palivizumab. Genetically modified B cells may be administered to recipients to provide protection (e.g., anti-infective effects) against infection until the transplanted cells achieve population recovery of the recipient's own immune system.

[0184] In certain embodiments, the recipient is receiving a bone marrow or hematopoietic stem cell transplant from a donor as a treatment for a hematological malignancy. Examples of hematological malignancies include acute lymphocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's lymphoma / leukemia, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, follicular lymphoma (grade I, II, III, or IV), Hodgkin's lymphoma, intravascular large B-cell lymphoma, lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, marginal zone lymphoma (extranodal and nodal), mediastinal (thymic) large B-cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, POEMS syndrome / osteosclerotic myeloma, primary effusion lymphoma, splenic marginal zone lymphoma, small lymphocytic lymphoma, smoldering multiple myeloma (SMM), and Waldenstrom's macroglobulinemia.

[0185] In a specific embodiment, the recipient is receiving genetically modified hematopoietic stem cells that provide the gene that the recipient is lacking. These recipients may have primary or secondary immunodeficiencies that can be treated by providing therapeutic genes through hematopoietic stem cells. The World Health Organization has identified more than 80 primary immunodeficiency diseases. These diseases are characterized by an intrinsic defect in the immune system, in which in some cases, the body cannot produce any or enough antibodies to infection. In other cases, the cellular defenses used to combat infection fail to function properly. Typically, primary immunodeficiency is a hereditary disorder. X-chromosome-linked severe combined immunodeficiency (SCID-X1) is another example of primary immunodeficiency. X-chromosome-linked SCID causes both cellular immunity depletion and humoral immunity depletion caused by mutations in the common γ chain gene (γC), which results in the absence of T lymphocytes and natural killer (NK) lymphocytes.

[0186] Secondary or acquired immunodeficiency is not the result of an inherited genetic abnormality, but occurs in individuals in which the immune system is compromised by factors external to the immune system. Examples include trauma, viruses, chemotherapy, toxins, and pollution. Acquired immunodeficiency syndrome (AIDS) is an example of a secondary immunodeficiency disorder caused by a virus, human immunodeficiency virus (HIV), in which depletion of T lymphocytes renders the body unable to fight infection.

[0187] In certain embodiments, B cells can be obtained from a subject, a subset of B cells can be modified ex vivo, and then the modified B cells can be formulated and administered to the subject. In certain embodiments, a first subset of B cells of a subject can be modified with a first genetic construct to produce selected antibodies against inflammatory molecules such as inflammatory cytokines, thereby providing antibodies that protect against inflammation. In certain embodiments, the selected antibodies can be anti-TNF antibodies and / or anti-IL-1 antibodies. In certain embodiments, the selected antibodies are infliximab, adalimumab and / or golimumab and / or their approved biosimilars.

[0188] For administration, the therapeutically effective amount (also referred to herein as dosage) can be initially estimated based on the results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine dosages suitable for use in the target subject. The actual dosage administered to a particular subject can be determined by a physician, veterinarian, or researcher taking into account parameters such as physical and physiological factors, including age, previous vaccinations (if any), target, body weight, severity of the disorder, type of disorder, stage of the disorder, previous or concurrent therapeutic interventions, idiopathic conditions of the subject, and route of administration.

[0189] As indicated, in certain embodiments, the modified B cells express a tag that allows for tracking and / or elimination, for example, following administration to a subject.

[0190] Exemplary dosages may include greater than 10 2 Modified B cells, more than 10 3 Modified B cells, more than 10 4 Modified B cells, more than 10 5 Modified B cells, more than 10 6 Modified B cells, more than 10 7 Modified B cells, more than 10 8 Modified B cells, more than 10 9 Modified B cells, more than 10 10 Modified B cells or more than 10 11 modified B cells.

[0191] In certain embodiments, the effect of the selected antibodies can be measured using viral titer. Viral titer refers to the amount of virus that can be detected. High viral titer means high infection level. The best protective response is observed when the titer drops to zero.

[0192] As will be appreciated by one of ordinary skill in the art, although specific embodiments have been described, additional embodiments may be utilized within the scope of the present disclosure. The following description provides a description and implementation of representative additional embodiments.

[0193] Exemplary embodiments.

[0194] 1. A method for genetically engineering a B cell to express a selected antibody, the method comprising targeted insertion of a genetic construct comprising (i) a promoter and (ii) a transgene encoding a portion of a selected antibody at an intronic region that is constant in all B cells and (i) positioned relative to an enhancer element that interacts with the promoter to drive expression of the transgene; and (ii) in a configuration such that a portion of the endogenous antibody encoding genome of the B cell is not expressed.

[0195] 2. A method for genetically engineering a B cell to express a selected antibody, the method comprising inserting into SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 a genetic construct comprising or encoding (i) a heavy chain promoter, (ii) a signal peptide, (iii) a full-length light chain of the selected antibody; (iv) a flexible linker or a jumping element; (v) a variable region of the heavy chain of the selected antibody; and (vi) a splice junction, thereby genetically engineering the B cell to express the selected antibody.

[0196] 3. A method as described in embodiment 1 or 2, wherein the endogenous variable heavy chain encoding genome of the B cell is not removed during genetic modification.

[0197] 4. A method as described in any one of embodiments 1-3, wherein the selected antibody is an anti-respiratory syncytial virus (RSV) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-dengue virus antibody, an anti-Bordetella pertussis antibody, an anti-hepatitis C antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, an anti-metapulmonary virus (MPV) antibody, an anti-cytomegalovirus antibody, an anti-Epstein-Barr virus antibody; an anti-herpes simplex virus antibody, an anti-Clostridium difficile bacterial toxin antibody, or an anti-tumor necrosis factor (TNF) antibody.

[0198] 5. The method of any one of embodiments 1-4, wherein the genetic construct comprises SEQ ID NO: 102-175, 278, 279 or 280-289.

[0199] 6. A method as described in any one of embodiments 2-5, wherein the flexible linker is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody.

[0200] 7. A method as described in any one of embodiments 2-6, wherein the flexible linker is selected from SEQ ID NO: 180-184.

[0201] 8. A method as described in any one of embodiments 2-7, wherein the flexible linker is a Gly-Ser linker comprising 50-80 amino acids.

[0202] 9. A method as described in any one of embodiments 2-8, wherein the flexible linker is a Gly-Ser linker comprising 57 amino acids.

[0203] 10. A method as described in any one of embodiments 2-6, 8 or 9, wherein the flexible linker is SEQ ID NO: 122.

[0204] 11. The method of any one of embodiments 2-10, wherein the jumping element is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody.

[0205] 12. The method of any one of embodiments 2-11, wherein the jumping element is a self-cleaving peptide.

[0206] 13. The method of embodiment 12, wherein the self-cleaving peptide is selected from SEQ ID NOs: 176-179.

[0207] 14. The method of any one of embodiments 2-13, wherein the jumping element is an internal ribosome entry site (IRES).

[0208] 15. A method as described in any one of embodiments 2-14, wherein the heavy chain promoter is selected from SEQ ID NO: 111 and 128.

[0209] 16. A method as described in any one of embodiments 2-15, wherein the heavy chain promoter is IgVH1-69 or J558H10.

[0210] 17. The method of any one of embodiments 2-16, wherein the signal peptide is selected from SEQ ID NOs: 118, 134, and 185-194.

[0211] 18. The method of any one of embodiments 2-17, wherein the signal peptide is derived from a human IgH heavy chain or a human IgL light chain.

[0212] 19. The method of any one of embodiments 1-18, wherein the genetic construct comprises homology arms.

[0213] 20. A method as described in embodiment 19, wherein the homology arms include SEQ ID NO:90-101, 110, 125, 127, 140, 142, 143, 153, 170, 171, 173, 174, 278 or 279.

[0214] 21. The method of any one of embodiments 1-20, wherein the genetic construct encodes a tag.

[0215] 22. The method of embodiment 21, wherein the tag comprises Tag II, His tag, Flag tag, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Softag 1, Softag 3, or V5 tag.

[0216] 23. A method as described in embodiment 21 or 22, wherein the tag comprises SEQ ID NO: 122 or 195-204.

[0217] 24. A method as described in any one of embodiments 1-23, further comprising delivering a guide RNA (gRNA) sequence and a nuclease selected from one or more of SEQ ID NOs: 87-89 and 290-366 to the B cell.

[0218] 25. The method of embodiment 24, wherein the delivery is achieved by electroporation, nanoparticles, or virus-mediated delivery.

[0219] 26. The method of any one of embodiments 1-25, wherein the genetic construct is part of an adeno-associated viral vector.

[0220] 27. A method as described in any one of embodiments 24-26, wherein the gRNA and the nuclease are delivered by electroporation and the genetic construct is delivered as part of an adeno-associated viral vector.

[0221] 28. The method of any one of embodiments 24-27, wherein the nuclease is Cas9 or Cpf1.

[0222] 29. A method as described in any one of embodiments 24-28, wherein the target sequence targeted by one or more of the gRNA sequences is selected from one or more of SEQ ID NOs: 5-84, and the gRNA is selected from one or more of SEQ ID NOs: 87-89 and 290-366.

[0223] 30. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-RSV antibody, including palivizumab, AB1128, or ab20745.

[0224] 31. A method as described in any one of embodiments 1-30, wherein the selected antibody is: palivizumab containing a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 136; palivizumab containing a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 205; an anti-RSV antibody containing a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 120; or an anti-RSV antibody containing a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 206.

[0225] 32. A method as described in any one of embodiments 1-30, wherein the selected antibody is an anti-RSV antibody comprising CDRH1 comprising SEQ ID NO: 207, CDRH2 comprising SEQ ID NO: 208, CDRH3 comprising SEQ ID NO: 209; CDRL1 comprising SEQ ID NO: 210, CDRL2 comprising SEQ ID NO: 211, and CDRL3 comprising SEQ ID NO: 212.

[0226] 33. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-HIV antibody including 10E8, VRC01, ab18633, or 39 / 5.4A.

[0227] 34. The method of any one of embodiments 1-29 or 33, wherein the selected antibody is an anti-HIV antibody comprising a heavy chain comprising SEQ ID NO: 150 and a light chain comprising SEQ ID NO: 149.

[0228] 35. A method as described in any one of embodiments 1-29 or 33, wherein the selected antibody is an anti-HIV antibody comprising CDRH1 comprising SEQ ID NO: 213, CDRH2 comprising SEQ ID NO: 214, CDRH3 comprising SEQ ID NO: 215, CDRL1 comprising SEQ ID NO: 216, CDRL2 comprising SEQ ID NO: 217, and CDRL3 comprising SEQ ID NO: 218, or CDRH1 comprising SEQ ID NO: 219, CDRH2 comprising SEQ ID NO: 220, CDRH3 comprising SEQ ID NO: 221, CDRL1 comprising QYGS, CDRL2 comprising SGS, and CDRL3 comprising SEQ ID NO: 222.

[0229] 36. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-dengue virus antibody, including antibody 55, DB2-3, ab155042, or ab80914.

[0230] 37. A method as described in any one of embodiments 1-29 or 36, wherein the selected antibody is an anti-dengue virus antibody comprising CDRH1 comprising SEQ ID NO: 223, CDRH2 comprising SEQ ID NO: 224, CDRH3 comprising SEQ ID NO: 225; CDRL1 comprising SEQ ID NO: 226, CDRL2 comprising SEQ ID NO: 227, and CDRKL3 comprising SEQ ID NO: 228, or CDRH1 comprising SEQ ID NO: 229, CDRH2 comprising SEQ ID NO: 230, CDRH3 comprising SEQ ID NO: 231, CDRL1 comprising SEQ ID NO: 232, CDRL2 comprising SEQ ID NO: 233, and CDRL3 comprising SEQ ID NO: 234.

[0231] 38. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-pertussis antibody comprising a heavy chain comprising SEQ ID NO: 235 and a light chain comprising SEQ ID NO: 236.

[0232] 39. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-hepatitis C antibody, including MAB8694 or C7-50.

[0233] 40. A method as described in any one of embodiments 1-29 or 39, wherein the selected antibody is an anti-hepatitis C antibody comprising CDRH1 comprising SEQ ID NO:237, CDRH2 comprising SEQ ID NO:238, CDRH3 comprising SEQ ID NO:239, CDRL1 comprising SEQ ID NO:240, CDRL2 comprising SEQ ID NO:241, and CDRL3 comprising SEQ ID NO:242.

[0234] 41. The method of any one of embodiments 1-29, wherein the selected antibodies are anti-influenza virus antibodies, including C102.

[0235] 42. The method of any one of embodiments 1-29 or 41, wherein the selected antibody is an anti-influenza virus antibody comprising a heavy chain comprising SEQ ID NO: 159 and a light chain comprising SEQ ID NO: 158.

[0236] 43. The method of any one of embodiments 1-29 or 41, wherein the selected antibody is an anti-influenza virus antibody comprising a CDRH1 comprising SEQ ID NO: 243, a CDRH2 comprising SEQ ID NO: 244, a CDRH3 comprising SEQ ID NO: 245, a CDRL1 comprising SEQ ID NO: 246, a CDRL2 comprising KTS, and a CDRL3 comprising SEQ ID NO: 247.

[0237] 44. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-MPV antibody, including MPE8.

[0238] 45. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-CMV antibody, including MCMV5322A, MCMV3068A, LJP538, or LJP539.

[0239] 46. ​​A method as described in any one of embodiments 1-29, wherein the selected antibody is an anti-EBV antibody containing a heavy chain comprising SEQ ID NO: 168 and a light chain comprising SEQ ID NO: 166.

[0240] 47. A method as described in any one of embodiments 1-29, wherein the selected antibody is an anti-EBV antibody comprising CDRH1 comprising SEQ ID NO:248, CDRH2 comprising SEQ ID NO:249, CDRH3 comprising SEQ ID NO:250, CDRL1 comprising SEQ ID NO:251, CDRL2 comprising SEQ ID NO:252, and CDRL3 comprising SEQ ID NO:253.

[0241] 48. A method as described in any one of embodiments 1-29, wherein the selected antibodies are anti-HSV antibodies, including HSV8-N and MB66.

[0242] 49. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-Clostridium difficile antibody, comprising actomyosine or bezentuzumab.

[0243] 50. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-TNF antibody, including infliximab, adalimumab, etanercept, celizumab, or accepted biosimilars thereof.

[0244] 51. The method of any one of embodiments 1-29 or 50, wherein the selected antibody is a heavy chain comprising SEQ ID NO: 254 and a light chain comprising SEQ ID NO: 255; a CDRH1 comprising SEQ ID NO: 256, a CDRH2 comprising SEQ ID NO: 257, and a CDRH3 comprising SEQ ID NO: 258; a CDRL1 comprising SEQ ID NO: 259, a CDRL2 comprising SEQ ID NO: 260, and a CDRL3 comprising SEQ ID NO: 261; a CDRH1 comprising SEQ ID NO: 262, a CDRH2 comprising SEQ ID NO: 263, and a CDRH3 comprising SEQ ID NO: 264; a CDRL1 comprising SEQ ID NO: 265, a CDRL2 comprising SEQ ID NO: 266, and a CDRL3 comprising SEQ ID NO: 267; a CDRH1 comprising SEQ ID NO: 268, a CDRH2 comprising SEQ ID NO: 269, and a CDRH3 comprising SEQ ID NO: 270; or a CDRH1 comprising SEQ ID NO: 271. An anti-TNF antibody comprising CDRL1 of SEQ ID NO:271, CDRL2 comprising SEQ ID NO:272, and CDRL3 comprising SEQ ID NO:273.

[0245] 52. A method as described in any one of embodiments 1-51, wherein the genetic modification utilizes a sequence comprising any one of SEQ ID NOs: 87, 88, 89, 90-175, 278-366.

[0246] 53. The method of any one of embodiments 1-52, wherein the B cell is an antibody-producing B cell, a memory B cell, a naive B cell, a B1 B cell, or a marginal zone B cell.

[0247] 54. A B cell modified according to the method of any one of embodiments 1-53.

[0248] 55. The B cell of embodiment 54, wherein the B cell is an antibody-secreting B cell, a memory B cell, a naive B cell, a B1 B cell, or a marginal zone B cell.

[0249] 56. A method of providing an anti-infective effect in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the B cells of embodiment 54 or 55, thereby providing an anti-infective effect.

[0250] 57. The method of embodiment 56, wherein the providing obviates the need for vaccination.

[0251] 58. The method of embodiment 56 or 57, wherein the administration is an alternative vaccination regimen.

[0252] 59. The method of any one of embodiments 56-58, wherein the subject is immunosuppressed.

[0253] 60. The method of any one of embodiments 56-59, wherein the subject is subjected to immunosuppression as part of a treatment regimen comprising bone marrow transplantation, hematopoietic stem cell transplantation, or administration of genetically modified hematopoietic stem cells.

[0254] 61. A method of providing an anti-inflammatory effect in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the B cells of embodiment 54 or 55, thereby providing an anti-inflammatory effect.

[0255] 62. A genetic construct for modifying B cells to express a selected antibody, the genetic construct comprising or encoding (i) a heavy chain promoter, (ii) a signal peptide, (iii) a full-length light chain of the selected antibody; (iv) a flexible linker or jumping element; (v) a variable region of the heavy chain of the selected antibody; and (vi) a splice junction.

[0256] 63. The genetic construct of embodiment 62, comprising SEQ ID NO: 102-175 or 280-289.

[0257] 64. A genetic construct as described in embodiment 62 or 63, wherein the flexible linker is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody.

[0258] 65. The genetic construct of any one of embodiments 62-64, wherein the flexible linker is selected from SEQ ID NOs: 180-184.

[0259] 66. The genetic construct of any one of embodiments 62-65, wherein the flexible linker is a Gly-Ser linker comprising 50-80 amino acids.

[0260] 67. The genetic construct of any one of embodiments 62-66, wherein the flexible linker is a Gly-Ser linker comprising 57 amino acids.

[0261] 68. The genetic construct of any one of embodiments 62-64, 66 or 67, wherein the flexible linker is SEQ ID NO: 122.

[0262] 69. The genetic construct of any one of embodiments 62-68, wherein the jumping element is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody.

[0263] 70. The genetic construct of any one of embodiments 62-69, wherein the jumping element is a self-cleaving peptide.

[0264] 71. The genetic construct of embodiment 70, wherein the self-cleaving peptide is selected from SEQ ID NOs: 176-179.

[0265] 72. The genetic construct of any one of embodiments 62-69, wherein the jumping element is an internal ribosome entry site (IRES).

[0266] 73. The genetic construct of any one of embodiments 62-72, wherein the heavy chain promoter is selected from SEQ ID NOs: 111 and 128.

[0267] 74. The genetic construct of any one of embodiments 62-73, wherein the heavy chain promoter is IgVH1-69 or J558H10.

[0268] 75. The genetic construct of any one of embodiments 62-74, wherein the signal peptide is selected from SEQ ID NOs: 118, 134, and 185-194.

[0269] 76. The genetic construct of any one of embodiments 62-75, wherein the signal peptide is derived from a human IgH heavy chain or a human IgL light chain.

[0270] 77. The genetic construct of any one of embodiments 62-76, wherein the genetic construct comprises homology arms.

[0271] 78. The genetic construct of embodiment 77, wherein the homology arms comprise SEQ ID NO: 90-101, 110, 125, 127, 140, 142, 143, 153, 170, 171, 173, 174, 278, or 279.

[0272] 79. The genetic construct of any one of embodiments 62-78, wherein the genetic construct encodes a tag.

[0273] 80. The genetic construct of embodiment 79, wherein the tag comprises Tag II, His tag, Flag tag, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Softag 1, Softag 3, or V5 tag.

[0274] 81. The genetic construct of embodiment 79 or 80, wherein the tag comprises SEQ ID NO: 122 or 195-204.

[0275] 82. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-RSV antibody, including palivizumab, AB1128, or ab20745.

[0276] 83. A genetic construct as described in any one of embodiments 62-82, wherein the selected antibody is: palivizumab containing a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 136; palivizumab containing a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 205; an anti-RSV antibody containing a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 120; or an anti-RSV antibody containing a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 206.

[0277] 84. The genetic construct of any one of embodiments 62-82, wherein the selected antibody is an anti-RSV antibody comprising CDRH1 comprising SEQ ID NO:207, CDRH2 comprising SEQ ID NO:208, CDRH3 comprising SEQ ID NO:209; CDRL1 comprising SEQ ID NO:210, CDRL2 comprising SEQ ID NO:211, and CDRL3 comprising SEQ ID NO:212.

[0278] 85. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-HIV antibody including 10E8, VRC01, ab18633, or 39 / 5.4A.

[0279] 86. The genetic construct of any one of embodiments 62-81 or 85, wherein the selected antibody is an anti-HIV antibody comprising a heavy chain comprising SEQ ID NO: 150 and a light chain comprising SEQ ID NO: 149.

[0280] 87. The genetic construct of any one of embodiments 62-81 or 85, wherein the selected antibody is an anti-HIV antibody comprising CDRH1 comprising SEQ ID NO: 213, CDRH2 comprising SEQ ID NO: 214, CDRH3 comprising SEQ ID NO: 215, CDRL1 comprising SEQ ID NO: 216, CDRL2 comprising SEQ ID NO: 217, and CDRL3 comprising SEQ ID NO: 218, or CDRH1 comprising SEQ ID NO: 219, CDRH2 comprising SEQ ID NO: 220, CDRH3 comprising SEQ ID NO: 221, CDRL1 comprising QYGS, CDRL2 comprising SGS, and CDRL3 comprising SEQ ID NO: 222.

[0281] 88. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-dengue virus antibody, including antibody 55, DB2-3, ab155042, or ab80914.

[0282] 89. The genetic construct of any one of embodiments 62-81 or 88, wherein the selected antibody is an anti-dengue virus antibody comprising CDRH1 comprising SEQ ID NO: 223, CDRH2 comprising SEQ ID NO: 224, CDRH3 comprising SEQ ID NO: 225; CDRL1 comprising SEQ ID NO: 226, CDRL2 comprising SEQ ID NO: 227, and CDRKL3 comprising SEQ ID NO: 228, or CDRH1 comprising SEQ ID NO: 229, CDRH2 comprising SEQ ID NO: 230, CDRH3 comprising SEQ ID NO: 231, CDRL1 comprising SEQ ID NO: 232, CDRL2 comprising SEQ ID NO: 233, and CDRL3 comprising SEQ ID NO: 234.

[0283] 90. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-pertussis antibody comprising a heavy chain comprising SEQ ID NO: 235 and a light chain comprising SEQ ID NO: 236.

[0284] 91. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-hepatitis C antibody, including MAB8694 or C7-50.

[0285] 92. The genetic construct of any one of embodiments 62-81 or 91, wherein the selected antibody is an anti-hepatitis C antibody comprising CDRH1 comprising SEQ ID NO:237, CDRH2 comprising SEQ ID NO:238, CDRH3 comprising SEQ ID NO:239, CDRL1 comprising SEQ ID NO:240, CDRL2 comprising SEQ ID NO:241, and CDRL3 comprising SEQ ID NO:242.

[0286] 93. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-influenza virus antibody, including C102.

[0287] 94. The genetic construct of any one of embodiments 62-81 or 93, wherein the selected antibody is an anti-influenza virus antibody comprising a heavy chain comprising SEQ ID NO: 159 and a light chain comprising SEQ ID NO: 158.

[0288] 95. The genetic construct of any one of embodiments 62-81 or 93, wherein the selected antibody is an anti-influenza virus antibody comprising CDRH1 comprising SEQ ID NO: 243, CDRH2 comprising SEQ ID NO: 244, CDRH3 comprising SEQ ID NO: 245, CDRL1 comprising SEQ ID NO: 246, CDRL2 comprising KTS, and CDRL3 comprising SEQ ID NO: 247.

[0289] 96. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-MPV antibody, including MPE8.

[0290] 97. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-CMV antibody, including MCMV5322A, MCMV3068A, LJP538 or LJP539.

[0291] 98. A genetic construct as described in any one of embodiments 62-81, wherein the selected antibody is an anti-EBV antibody comprising a heavy chain comprising SEQ ID NO: 168 and a light chain comprising SEQ ID NO: 166.

[0292] 99. A genetic construct as described in any one of embodiments 62-81, wherein the selected antibody is an anti-EBV antibody comprising CDRH1 comprising SEQ ID NO:248, CDRH2 comprising SEQ ID NO:249, CDRH3 comprising SEQ ID NO:250, CDRL1 comprising SEQ ID NO:251, CDRL2 comprising SEQ ID NO:252, and CDRL3 comprising SEQ ID NO:253.

[0293] 100. The genetic construct of any one of embodiments 62-81, wherein the selected antibodies are anti-HSV antibodies, including HSV8-N and MB66.

[0294] 101. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-Clostridium difficile antibody, comprising actomyosine or bezentuzumab.

[0295] 102. The genetic construct of any one of embodiments 62-81, wherein the selected antibody is an anti-TNF antibody, including infliximab, adalimumab, etanercept, celizumab, or accepted biosimilars thereof.

[0296] 103. The genetic construct of any one of embodiments 62-81 or 102, wherein the selected antibody is a heavy chain comprising SEQ ID NO: 254 and a light chain comprising SEQ ID NO: 255; a CDRH1 comprising SEQ ID NO: 256, a CDRH2 comprising SEQ ID NO: 257, and a CDRH3 comprising SEQ ID NO: 258; a CDRL1 comprising SEQ ID NO: 259, a CDRL2 comprising SEQ ID NO: 260, and a CDRL3 comprising SEQ ID NO: 261; a CDRH1 comprising SEQ ID NO: 262, a CDRH2 comprising SEQ ID NO: 263, and a CDRH3 comprising SEQ ID NO: 264; a CDRL1 comprising SEQ ID NO: 265, a CDRL2 comprising SEQ ID NO: 266, and a CDRL3 comprising SEQ ID NO: 267; a CDRH1 comprising SEQ ID NO: 268, a CDRH2 comprising SEQ ID NO: 269, and a CDRH3 comprising SEQ ID NO: 270. or an anti-TNF antibody comprising CDRL1 comprising SEQ ID NO:271, CDRL2 comprising SEQ ID NO:272, and CDRL3 comprising SEQ ID NO:273.

[0297] 104. A kit for genetically modifying B cells, the kit comprising a genetic construct as described in any one of embodiments 62-103 and a gRNA targeting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0298] 105. The kit of embodiment 104, wherein the gRNA is selected from one or more of SEQ ID NOs: 87, 88, 89, and 290-366.

[0299] 106. The kit of embodiment 104 or 105, further comprising a nuclease.

[0300] 107. The kit of embodiment 106, wherein the nuclease is Cas9 or Cpf1.

[0301] 108. The kit of any one of embodiments 104-107, further comprising nanoparticles or adeno-associated viral vectors.

[0302] 109. The kit of any one of embodiments 104-108, wherein the gRNA and the nuclease are associated with nanoparticles.

[0303] 110. The kit of any one of embodiments 104-109, wherein the genetic construct is part of an adeno-associated viral vector.

[0304] Example 1. Providing lifelong protection against respiratory syncytial virus infection without a vaccine. Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in young children, especially infants, with chronic lung disease, congenital heart disease or premature birth. Humoral immunity can mediate effective protection against RSV, and recombinant antibodies (MedImmune, Inc.) / palivizumab. However, both natural infection and previous vaccine trials have failed to induce adequate protective immune responses against RSV.

[0305] For RSV and other diseases that are difficult to be resisted by vaccination, it is extremely attractive to bypass vaccination by engineering primary B cells to elicit the expression of desired therapeutic antibodies. The immunoglobulin (Ig) locus is extremely large, diverse, and is subject to extensive genome reorganization and editing. In addition, the transcription of immunoglobulin genes depends on the regulation of mRNA splicing and polyadenylation by regulatory DNA elements to produce both membrane and secretory forms. This complexity has made viral transduction, which is a traditional method for cell engineering of lymphocytes, technically impractical for producing therapeutic B cells.

[0306] Specific embodiments include a platform for rapid and selective reprogramming of primary B cell antibody specificity by single-click immunogenetic engineering. This platform uses the high activity of microhomology-mediated end-joining DNA repair pathways in primary B cells to insert fully synthetic hybrid double-stranded / single-stranded DNA templates after DNA breaks are created by Cas9 / sgRNA riboproteins. The key to this method is to retain endogenous regulatory elements, which allows natural control of surface-bound and secreted antibody expression. In addition, the strategy is not limited to RSV. It may be possible to express protective antibodies against virtually any pathogen using only a single blood draw and subsequent cell infusions a few days later.

[0307] Materials and Methods. Design of sgRNA. sgRNAs targeting intronic sequences in the mouse and human IgH loci were designed using CrispRGold (crisprgold.mdc-berlin.de) and were produced in a synthetic form with 2'-O-methyl analogs and 3' phosphorothioate internucleotide linkages at the first three 5' and 3' terminal RNA residues (Synthego). The genomic targeting sequence is as follows:

[0308] Mouse: UUAUACAGUAUCCGAUGCAU (SEQ ID NO: 87)

[0309] Person:GUCUCAGGAGCGGUGUCUGU(SEQ ID NO:89)

[0310] Design and assembly of template sequences.

[0311] Human: The antibody construct included the IgVH1-69 heavy chain promoter region (SEQ ID NO: 111), the full-length codon-optimized light chain of palivizumab (SEQ ID NO: 113 (nucleotides) and SEQ ID NO: 120 (amino acids)), a glycine-serine linker with 57 amino acids containing 3 tandem copies of the StreptagII motif (SEQ ID NO: 116 (nucleotides) and SEQ ID NO: 122 (amino acids)), the codon-optimized variable region of the palivizumab heavy chain (SEQ ID NO: 117 (nucleotides) and SEQ ID NO: 123 (amino acids)), and a splice junction with 60 bp of flanking sequences derived from the human IGHJ1 gene segment (SEQ ID NO: 124).

[0312] Mouse: mCherry template includes J5558H10 heavy chain promoter, complete codon optimized mCherry open reading frame and sv40 polyadenylation site. Antibody construct includes J5558H10 heavy chain promoter (SEQ ID NO: 128, V. A Love et al. Molecular Immunology 2000), full length codon optimized antibody light chain (SEQ ID NO: 130 (nucleotide) and SEQ ID NO: 135 (amino acid)), a 57 amino acid glycine-serine linker containing two tandem copies of Streptag II sequence (SEQ ID NO: 116 (nucleotide) and SEQ ID NO: 122 (amino acid)), codon optimized variable region of heavy antibody chain (SEQ ID NO: 133 (nucleotide) and SEQ ID NO: 138 (amino acid)), and a splice junction with 60 bp of flanking sequence derived from mouse IGHJ3 gene segment (SEQ ID NO: 139).

[0313] Annealing of stitching oligonucleotides (i.e., splicing oligonucleotides). Stitching oligonucleotides with 36 bp complementarity to the pam distal and pam proximal non-target DNA strands, and 50-100 bp complementarity to the inserted template were synthetically generated and pre-annealed to the DNA template (e.g., SEQ ID No: 96-101) prior to use. The splicing oligonucleotides can provide a "homologous stitch" as shown in Figure 15.

[0314] Adeno-associated virus (AAV) viral vector template delivery. The AAV viral vector contains the MND promoter, a complete codon-optimized mCherry open reading frame, and an sv40 polyadenylation site, flanked by two mouse sgRNA recognition sites or 400 bp of homologous sequences. AAV virions pseudotyped with AAV6 viral capsids were produced in 293T cells and purified by sucrose gradient centrifugation and stored at -80 ° C. For viral delivery template DNA, concentrated AAV virus was added 12 hours before electroporation to a final volume of 10% of the total culture volume.

[0315] Mouse B cell culture and electroporation. Basal B cell culture medium included RPMI medium with 10% fetal bovine serum (Hyclone), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 55 μM β-mercaptoethanol (Sigma), and 100 U / ml penicillin plus 100 μg / mL streptomycin (Gibco), except in the antibiotic-free steps as indicated.

[0316] B cells were isolated from spleen and lymph nodes by negative selection with magnetic beads (Miltenyi) and cultured at 2 × 10 cells / mL in B cell culture medium supplemented with 100 ng / ml recombinant vector-free HA-tagged mouse CD40L (R&D Systems), 100 ng / ml anti-HA antibody (clone 543851, R&D Systems), and 4 ng / ml mouse IL-4 (R&D Systems). 6 / ml for 24 hours. Next, B cells were electroporated using the Neon transfection system and a 10 μl pipette as follows. Cas9 protein (Invitrogen) and synthetic sgRNA (Synthego) were mixed at a ratio of 3 μg Cas9 / 900 ng sgRNA and incubated at room temperature for at least 10 minutes. B cells were washed with PBS and incubated at 2.5×10 7 Cells were suspended with Cas9 / sgRNA and pre-assembled DNA templates at a final density of 10 cells / ml in Neon buffer T. Cells were electroporated (1675 V, 10 ms, 3 pulses) and immediately dispensed into pre-warmed antibiotic-free medium.

[0317] For cell expansion, B cells were co-cultured with irradiated (80 Gy) 3T3-CD40L feeder cells in the presence of 20 ng / ml mouse IL-21.

[0318] sgRNA activity was assessed using Tracking of Indels by Decomposition (TIDE). Total genomic DNA was isolated from mock-treated and Cas9-treated cells 3-5 days after electroporation. A 500-600 bp region flanking the cleavage site was amplified by PCR using the following oligos:

[0319] Mouse: Forward: GGCTCCACCAGACCTCTCTA (SEQ ID NO: 274) Reverse: AACCTCAGTCACCGTCTCCT (SEQ ID NO: 275)

[0320] Human: Forward: ACAGTAAGCATGCCTCCTAAG (SEQ ID NO: 276)

[0321] Reverse: GCCACTCTAGGGCCTTTGTT (SEQ ID NO: 277)

[0322] Results. Results are shown in Figures 18A-18C , Fig.22A and Fig. 22B , Figure 23B-Figure 23E ,as well as Figure 24A-Figure 24C middle. Fig.18A , Fig.18B and Fig. 18C Shown are successful cleavage of mouse B cell line A20, primary mouse B cells, and human B cell line RAMOS at the target IgH locus following electroporation of cells with Cas9 / sgRNA ribonucleoprotein, respectively. Fig.22A and Fig. 22B Depicts the insertion of the mCherry fluorescent protein reporter into the IgH locus in primary mouse B cells. Fig. 23B and Fig.23C Shown in part are antibody cassette insertions in the A20 mouse B cell line ( Fig. 23B ) and RAMOS human B cell lines ( Fig.23C ) followed by surface expression of anti-RSV antibodies. Fig.23D and Fig.23E Shown in part are antibody cassette insertions in the A20 mouse B cell line ( Fig.23D ) and RAMOS human B cell lines ( Fig.23E ) followed by secretion of anti-RSV antibodies. Fig.24A Shown are surface expression of anti-RSV antibodies following insertion of a portion of the antibody cassette into primary mouse B cells, initially (left panel) and after in vitro enrichment and expansion (right panel). Fig. 24B Shown is the secretion of anti-RSV antibodies following insertion of a partial antibody cassette into a primary mouse B cell line. Fig.24C Demonstrating the in vitro proliferation potential of engineered B cells.

[0323] Embodiment 2. The goal of this embodiment is to produce genetically modified B cells with determined specificity that maintain natural control of secretory and surface Ig expression by genome engineering of IgH loci. Due to the highly variable sequences present in B cells, the IgH loci in B cells are difficult to target for genome engineering regions. B cell development leads to the recombination of V, D and J elements over 1 trillion DNA bases to produce VDJ variable regions important for antibody diversity. Later in the development of B cell individuals, class switching between different constant regions leads to the loss of DNA over a similar sequence range (reviewed in Watson et al., (2017). Trends Immunol 38 (7): 459-470).

[0324] This sequence variability makes direct targeting of antibody coding regions impractical. However, a small DNA region between the last J gene segment and the switch region involved in class switching is present in all B cells. This universal target contains the key intron Eμ enhancer, which is one of several powerful enhancer elements that collaborate to drive high-level expression of IgH genes despite their presence in a weak promoter. The activity of these enhancers is partly regulated by the proximity of the promoter to the Eμ enhancer, and inserting a transgene between the recombinant VDJ segment and the Eμ enhancer can completely block VDJ transcription (Delpy et al., (2002). J Immunol 169 (12): 6875-6882). For this reason, the method used in this embodiment targets the region upstream of the Eμ enhancer to insert a new antibody cassette ( Fig.26A ). By targeting this region, the inserted emAb genes can be driven by the native (but inserted) IgH promoter, thereby maximizing natural control over immunoglobulin expression.

[0325] To enable one-shot insertion and minimize off-target interactions, the emAb construct was expressed as a single-chain fusion. This fusion consisted of the complete light chain sequence connected to the variable region of the heavy chain with a 57-amino acid glycine-serine linker, as described for single-chain F(ab) fragments. (Koerber et al., (2015). J Mol Biol 427(2):576-586) Fig.26A). This linker contains 3 tandem repeats of the StreptagII motif to facilitate detection and enrichment of genetically modified cells (Schmidt and Skerra (2007). Nat Protoc 2 (6): 1528-1535). Physically connecting the light and heavy chains minimizes the possibility of mispairing between the inserted emAb and the endogenous light chain. Optimizing the splice junction allows the emAb to be spliced ​​to the downstream endogenous IgH constant region. This allows the emAb to be expressed in any of the heavy chain isotype classes.

[0326] The strategy was tested in the RAMOS human B cell line. This Burkitt lymphoma-derived B cell line naturally expresses surface and secreted forms of IgM paired with lambda light chains. In these experiments, the expression of engineered αRSV-emAb derived from palivizumab was detected using monomeric RSV-F protein and streptactin, a modified streptavidin with high affinity for the Streptag II motif in the linker. RAMOS cells were genetically modified with αRSV-emAb to express engineered RSV-specific antibodies that can be expressed on the surface of the cells ( Fig.26B ) and in secreted form in the supernatant ( Fig.26C ) detected. To confirm that the emAb BCR is assembled with a secondary protein complex that is critical for BCR signaling, RAMOS cells were exposed to stimulation with multimeric RSV-F antigen. αRSV-emAb engineered cells, but not control cells, exhibited rapid and sustained calcium signaling in response to protein antigen ( Fig.26D ). These data were used to confirm the feasibility of the emAb engineering approach.

[0327] Next, a multistep expansion and differentiation process was used to genetically modify primary human B cells ( Fig.27A Electroporation with pre-complexed guide RNA and Cas9 resulted in highly efficient cleavage of genomic DNA, leading to defective repair of this region in 70% of the target alleles analyzed across multiple independent donors ( Fig.27B ). The sgRNA target site is strongly conserved in humans, with no single nucleotide polymorphisms reported at frequencies above 1% ( Fig.27C ). Addition of AAV-delivered αRSV-emAb cassette efficiently reprograms human B cells to bind RSV-F protein ( Fig.27D Notably, emAb B cells were successfully generated from every human donor tested, with an average engineering rate of 24% ( Fig.27E). In vitro culture and differentiation during emAb production increased the antibody secretion potential. Sensitized cells expressed high levels of CD19 and low amounts of plasma cell markers CD138, CD27, and CD138 at day 2, which was different from cells with lower levels of CD19 and increased CD38, CD27, and CD138 at day 18 ( Fig.27F Corresponding to these changes in cell surface markers, differentiated emAb-engineered B cells secrete large amounts of targeted antibodies ( Figure 27G ). Taken together, these data demonstrate that primary B cells can be rapidly and efficiently engineered to produce specific protective antibodies.

[0328] To demonstrate the flexible aspect of the platform, emAb cassettes derived from 3 additional broadly neutralizing antiviral antibodies were tested, including anti-HIV targeting VRC01, EBV targeting AMM01, and influenza HA backbone targeting MEDI8852. Primary B cells were efficiently reprogrammed by all 4 constructs, including antibodies with both kappa (palivizumab, VRC01, Medi8852) and lambda (AMM01) light chains ( Fig.28 ). These data demonstrate the flexible and widely applicable nature of the emAb platform.

[0329] Blocking the production of endogenous Ig heavy chains is important for maximizing the production of emAbs and minimizing the potential for the production of unknown endogenous antibodies from genetically modified cells. The RAMOS B cell strain endogenously expresses IgH paired with λ light chains. Engineering these cells with αRSV-emAbs connected to κ light chains enables the use of surface λ light chain expression as an effective measure of IgH expression. In addition, RAMOS cells have been subjected to c-myc translocations, thereby destroying one IgH allele, so that any emAb insertion will necessarily be in the effective allele ( Fig.29A ). The injected RAMOS cells expressed high levels of λ light chain on their surface, whereas cells expressing αRSV-emAb had almost completely lost λ expression ( Fig.29B ). These data indicate that the insertion of emAb on the effective allele can effectively block the expression of endogenous IgH. In almost all primary B cells, one IgH allele has an effective VDJ rearrangement, while the other allele does not undergo VDJ recombination, or undergoes ineffective recombination. However, these alleles all have potential sites for emAb insertion ( Fig.29C ). To test the impact of emAb insertion, purified primary B cells expressing λ light chain were genetically modified with αRSV-emAb. The input cells continued to express endogenous antibodies paired with λ light chain on their surface. In contrast, half of the αRSV-emAb-engineered B cells had lost λ light chain expression ( Fig.29D). The differential expression patterns seen in the case of RAMOS and primary B cells indicate that emAb insertion can block endogenous IgH expression if inserted into the effective allele. Differential expression of surface light chains is a way to purify cells that only express emAb constructs. Alternatively, the potential for insertion at either allele provides the possibility of generating dual antibody-expressing emAb cells by selecting an initial pool of antiviral memory B cells for engineering, or by inserting different boxes on each allele.

[0330] It has been shown that B cells can be engineered and then the protective capacity of the cells in a murine viral infection model is confirmed. A process of sensitization, electroporation + emAb cassette delivery and expansion similar to that used in the case of human primary B cells was used ( Fig. 30A ), producing murine emAb B cells. Using electroporation in combination with pre-complexed guide RNA and Cas9, cleavage was highly efficient, resulting in defective repair of this region in 80% of the DNA analyzed ( Fig. 30B AAV delivery of the murine αRSV-emAb cassette reproducibly modified mouse B cells, with 8-24% of the murine B cells binding RSV-F ( Fig. 30C , Fig.30D Using double-stranded DNA (dsDNA) containing short homology regions instead of AAV also achieved insertion in 1-7% of cells ( Fig. 30C , Fig.30D , see also Example 1), thus providing the potential for emAb engineering of B cells using purely synthetic components. High titers of secreted engineered antibodies were also detected in culture supernatants produced by both methods ( Fig.30E ).

[0331] To test the potential for antiviral protection, 1.5 x 10 7 Genetically modified mouse B cells were infused into wild-type Balbc / byJ mice, followed by blood draw and RSV challenge ( Fig.31A RSV-specific antibodies and genetically modified B cells were present in the blood 6 days after transfer of the genetically modified B cells ( Fig.31B , Fig.31C Importantly, mice that received the genetically modified B cells were almost completely protected against RSV infection ( Fig.31D This protection is close to that provided by palivizumab injected 2 days before infection ( Fig.31D ). Cell transfer of mixed human emAb targeting RSV and influenza into NOD-scid IL2Rγnull (NSG) mice generated serum titers of antibodies targeting both viruses ( Fig.32A , Fig.32B). These results show that the genetically modified B cells disclosed herein protect against viral infection.

[0332] Methods. Design of single-chain antibody template sequences. Human: Antibody constructs include the IgVH1-69 heavy chain promoter region (SEQ ID NO: 111), full-length antibody light chains (e.g., SEQ ID NOs: 113, 145, 154, and 161 (nucleotides) and SEQ ID NOs: 119, 148, 157, and 165 (amino acids)), a 57-amino acid glycine-serine linker containing three tandem copies of the StreptagII motif (SEQ ID NO: 116 (nucleotides) and SEQ ID NO: 122 (amino acids)), the variable region of the heavy chain (e.g., SEQ ID NOs: 117, 147, 156, and 164 (nucleotides) and SEQ ID NOs: 123, 150, 159, and 168 (amino acids)), and a 60-base pair splice junction derived from flanking sequences matching the IgHJ variable region (e.g., SEQ ID NOs: 124 and 151).

[0333] Mouse: The antibody construct included the J5558H10 heavy chain promoter (SEQ ID NO: 128, VA Love et al. Molecular Immunology 2000), a full-length codon-optimized antibody light chain (e.g., SEQ ID NO: 130 (nucleotides) and SEQ ID NO: 135 (amino acids)), a 57-amino acid glycine-serine linker containing three tandem copies of the Streptag II sequence (SEQ ID NO: 116 (nucleotides) and SEQ ID NO: 122 (amino acids)), a codon-optimized variable region of the heavy antibody chain (e.g., SEQ ID NO: 133 (nucleotides) and SEQ ID NO: 138 (amino acids)), and a splice junction with 60 base pairs of flanking sequences derived from the mouse IGHJ3 gene segment (e.g., SEQ ID NO: 139).

[0334] The complete sequences of exemplary antibody constructs can be found at Figure 25B-Figure 25I Obtained in.

[0335] Production of recombinant AAV vectors. AAV vectors were produced by triple transfection of AAV vectors, serotype 6 capsids, and adenovirus helper plasmids (pHelper) into HEK293T cells using PEI. 24 hours after transfection, the culture medium was changed to serum-free DMEM, and after 72 hours, the cells were collected, dissolved by freezing-thawing, treated with benzonase, purified by iodixanol gradient, and then concentrated into PBS using Amicon Ultra-15 columns (EMDMillipore) (Choi et al., (2007). Curr Protoc Mol Biol Chapter 16: Unit 16 25). The titer of the virus stock was determined by qPCR of the AAV genome, and the titer was 5×10 per microliter. 10 Up to 1×10 12 within the range of (Aurnhammer et al., (2012). Hum Gene Ther Methods 23(1):18-28).

[0336] Generation of mouse dsDNA emAb templates.

[0337] The αRSV-emAb template was amplified by modified DNA oligos as follows and short homology regions were added.

[0338] Forward primer: (contains 5' phosphate, mouse genome homologous region is in bold)

[0339] / 5Phos / ACCACCTCTGTGACAGCATTTATACAGTATCCGATGGACAAGTGAGTGTCTCAGGTTAGGATTCT(SEQ ID NO:278)

[0340] Reverse primer (containing a phosphorothioate-stabilized DNA bond (*), the mouse genome homologous region is in bold)

[0341] T*A*A*AGAAAGTGCCCCACTCCACTCTTTGTCCCTATG CTTGACCACAATGAATACTCCCACC(SEQID NO:279)

[0342] The dsDNA template was amplified by PCR, purified using minElute PCR cleanup columns (Qiagen), and concentrated.

[0343] Cell lines. 3T3-msCD40L was obtained from Dr. Mark Connors with the assistance of the NIH AIDS Reagent Program (Division of AIDS, NIAID, NIH: Catalog No. 12535). 3T3 cells were cultured in DMEM medium with 10% fetal bovine serum (Gibco), 100 U / ml penicillin plus 100 μg / mL streptomycin (Gibco), and G418 (350 μg / mL).

[0344] RAMOS cells were purchased from ATCC (CRL-1596 TM RAMOS cells were cultured in RPMI medium with 10% fetal bovine serum (Gibco) and 100 U / ml penicillin plus 100 μg / mL streptomycin (Gibco).

[0345] Mouse B cell culture and electroporation. Basal B cell culture medium included RPMI medium with 10% fetal bovine serum (Gemini Biosciences), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 55 μM β-mercaptoethanol (Sigma), and 100 U / ml penicillin plus 100 μg / mL streptomycin (Gibco), except in the antibiotic-free steps as indicated.

[0346] B cells were isolated from spleen and lymph nodes by negative selection with magnetic beads (Miltenyi) and cultured at 2 × 10 cells / mL in B cell culture medium supplemented with 100 ng / ml recombinant vector-free HA-tagged mouse CD40L (R&D Systems), 100 ng / ml anti-HA antibody (clone 543851, R&D Systems), and 4 ng / ml mouse IL-4 (R&D Systems). 6 / ml for 24 hours. Next, B cells were electroporated using the Neon transfection system as follows. Cas9 protein (Invitrogen) and synthetic sgRNA (Synthego) were mixed at a ratio of 1 μg Cas9 to 300 ng sgRNA and incubated at room temperature for at least 10 minutes. B cells were washed with PBS and incubated at 2.5×10 7 At a final density of 10 cells / mL, 12 μg Cas9 RNP / 10 6 cells were suspended together in Neon buffer T. For dsDNA conditions, 7.5 μg dsDNA template / 10 6Cells were electroporated (1675V, 10 milliseconds, 3 pulses) and immediately distributed to pre-warmed antibiotic-free culture medium. For AAV conditions, concentrated AAV in PBS was added until 15% of the final culture volume. After electroporation, B cells were expanded for another 48 hours with B cell culture medium supplemented with 100ng / ml recombinant vector-free HA-tagged mouse CD40L (R&D systems), 100ng / ml anti-HA antibody (clone 543851, R&D Systems), 4ng / ml mouse IL-4 (R&D Systems) and 20ng / ml mouse IL-21 (Biolegend). For secondary amplification, B cells were co-cultured with irradiated (80 gray) NIH 3T3-CD40L feeder cells in the presence of 20ng / ml mouse IL-21 (Biolegend).

[0347] Human B cell culture and electroporation. Basal medium (hBCM) for human B cell culture was 10% FBS (Gemini Biosciences), 100 U / ml penicillin and 100 μg / mL streptomycin (Gibco) in IMDM medium, except in antibiotic-free steps as indicated.

[0348] Human PBMC is obtained by Fred Hutchinson Cancer Research Center. Cells are thawed and separated using Militenyi B cell separation kit II (people) according to the manufacturer's protocol using negative selection. Separated cells are resuspended in hBCM supplemented with 100ng / mL MEGACD40L (Enzo Life Sciences), 50ng / mL recombinant IL-2 (Biolegend), 50ng / mL IL-10 (Shenendoah Biotech), 10ng / mL IL-15 (Shenandoah Biotech), 1μg / mL CpG ODN2006 (IDT) at 0.5-1.0*10^6 individual cells / ml.

[0349] After 48 hours of stimulation, cells were electroporated using the Neon transfection system. Cas9 protein (Invitrogen) and H7 sgRNA (Synthego) were pre-compounded in buffer T at a 2: 1 ratio for 20 minutes at room temperature. The cells were washed with PBS (Gibco) and resuspended in buffer T containing pre-compounded Cas9 RNP at a final concentration of 2.5*10^7 cells / ml in buffer T. The cell-RNP mixture was loaded into a 10uL Neon transfection tip and electroporated at 1750V, 20ms, and 1 pulse according to the manufacturer's protocol. Immediately after electroporation, the cells were plated in the stimulation medium as described above without antibiotics. After 30 minutes, AAV was added to a final concentration of 10-15% of the culture volume and mixed thoroughly. After 2-4 hours, the cells were transferred to a larger culture dish to allow further amplification.

[0350] Two days after electroporation, cells were stained with fluorescent dye-labeled antigen or streptactin, and genetically modified cells were selected. For secondary amplification, B cells were co-cultured with irradiated (80 gray) NIH 3T3-CD40L feeder cells in hBCM containing 5 μg / mL human recombinant insulin (Sigma), 50 μg / mL transferrins (Sigma), 50 ng / mL recombinant IL-2 (Biolegend), 20 ng / mL IL-21 (Biolegend) and 10 ng / mL IL-15 (Shenandoah Biotech).

[0351] To promote differentiation into plasma cells, cells were transferred from expansion conditions to fresh feeder-free culture conditions containing hBCM supplemented with 5 μg / mL human recombinant insulin (Sigma), 50 μg / mL transferrin (Sigma), 500 U / mL universal type I IFN protein (R&D Systems), 50 ng / mL IL-6 (Shenendoah Biotech), 10 ng / mL IL-15 (Shenendoah Biotech).

[0352] sgRNA activity was assessed by TIDE. Total genomic DNA was isolated from mock-treated and cas9 / sgRNA-treated cells 3-5 days after electroporation. A 500-600 base pair region flanking the sgRNA target site was amplified by PCR using the following oligos:

[0353] Mouse:

[0354] Forward: GGCTCCACCAGACCTCTCTA (SEQ.ID NO: 274)

[0355] Reverse: AACCTCAGTCACCGTCTCCT (SEQ ID NO: 275)

[0356] people:

[0357] Forward: ACAGTAAGCATGCCTCCTAAG (SEQ ID NO: 276)

[0358] Reverse: GCCACTCTAGGGCCTTTGTT (SEQ ID NO: 277)

[0359] Purified PCR products were subjected to Sanger sequencing, and the ICE algorithm (Hsiau et al., (2018). "Inference of CRISPR Edits from Sanger Trace Data." bioRxiv) was used to determine the frequency of indels in Cas9 / sgRNA electroporated cells relative to mock-electroporated cells.

[0360] Protein antigens. Prefusion RSV-F protein, EBV gh / gl complex, and modified HIV envelope antigen (426cTM4 d1-3) were produced as described (McLellan et al., (2013). Science 342(6158):592-598; McGuire et al., (2016). Nat Commun 7:10618; Snijder et al., (2018). Immunity 48(4):799-811e799). Stabilized influenza HA backbone was produced as described (Yassine et al., (2015). Nat Med 21(9):1065-1070) from VRC clone 3925 derived from strain H1 1999NC. Monomer prefusion RSV-F protein was labeled with Alexa-488 (Thermo Fisher). All other proteins were conjugated to biotin using a biotin:protein molar ratio between 0.8 and 2, followed by tetramerization with streptavidin-PE or streptavidin-APC (prozyme).

[0361] Flow cytometry. Flow cytometric analysis was performed on a FACSymphony machine (BD bioscience), cells were sorted on an Aria II (BD bioscience), and data were analyzed using FlowJo software (Tree Star).

[0362] EmAb Therapeutic Studies in Mice Animal studies were approved and performed in compliance with the requirements of the Fred Hutchinson Cancer Center Institutional Animal Care and Use Committee.

[0363] For RSV challenge, EmAb or control B cells were added at 1.5 × 10 7 A single intraperitoneal (IP) dose of 150 cells was administered. For passive transfer of palivizumab, mice received a single intraperitoneal dose of 15 mg / kg. GFP-expressing RSV (referred to herein as RSV for simplicity) was generously provided (Munir et al., (2008). J Virol 82(17):8780-8796). Age-matched BALB / cByJ mice (Jackson Labs) were inoculated with 10 μL of PBS. 6 pfu of sucrose-purified RSV was inoculated intranasally. Lungs were collected on the 5th day after infection, and titers were determined as previously described by plaque assay (Murphy et al., (1990). Vaccine 8 (5): 497-502). Briefly, lungs were homogenized in 2 ml culture medium in a GentleMACS dissociator, clarified by centrifugation at 400 × g for 10 minutes, then snap-frozen and stored at -80 ° C. Supernatants were diluted 1:10 and 1:20 in duplicate in DMEM culture medium. Each dilution of 100 μL was added to confluent Vero cells in a 24-well plate for 2 hours at 37 ° C. An overlay of 0.8% methylcellulose was then added, and the plate was incubated for 5 days, followed by imaging using a filter set for GFP on a Typhoon imager. The titer represented by pfu / lung was calculated by counting the number of plaques in the case of the highest positive dilution and correcting for the dilution factor.

[0364] For human cell engraftment, human emAb B cells were used at 5 × 10 6 cells / emAb specificity (total 1×10 7 A single intraperitoneal dose of NOD-scid IL2Rγ null (NSG) mice (generated by FHCRC breeding laboratory) were administered. 7 days after transfer, blood was drawn and the human emAb titers against RSV-F and HA backbone in the serum were determined by ELISA.

[0365] Statistical analysis. Statistical analysis was performed using GraphPad Prism 7. Pairwise comparisons were performed using unpaired t-test with Welch's correction.

[0366] The nucleic acid sequences described herein are shown using standard letter abbreviations for nucleotide bases as defined in 37 CFR § 1.822. In some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand should be understood to be included in the embodiments where it will be appropriate. For example, sequences complementary to target sites including SEQ ID NOs: 5-84 provide gRNA targeting sequences for targeting these sites.

[0367] Any nucleic acid encoding the selected antibody construct as described herein can be used. Variants of the nucleic acid sequences disclosed herein include various sequence polymorphisms, mutations and changes, wherein the sequence differences do not substantially affect the function of the encoded protein. The term nucleic acid or "gene" may include not only coding sequences, but also regulatory regions such as promoters, enhancers and termination regions. The term may also include all introns and other DNA sequences spliced ​​from mRNA transcripts, and variants caused by alternative splicing sites. The encoding nucleic acid may be a DNA or RNA that guides the expression of one or more selected antibody constructs. These nucleic acid sequences may be DNA chain sequences transcribed into RNA or RNA sequences translated into proteins. The nucleic acid sequence includes both full-length nucleic acid sequences and non-full-length sequences obtained by full-length proteins. The sequence may also include one or more natural sequences that may be introduced to provide degenerate codons that codon preference is provided in a specific cell type. The nucleic acid sequence encoding the selected antibody construct may be easily prepared according to the relevant amino acid sequence of the selected antibody construct.

[0368] "Variants" of protein sequences include those having one or more amino acid additions, deletions, end positions or substitutions compared to a protein sequence disclosed elsewhere herein.

[0369] Amino acid substitutions may be conservative or non-conservative substitutions. Variants of the protein sequences disclosed herein may include those with one or more conservative amino acid substitutions. "Conservative substitutions" or "conservative amino acid substitutions" refer to substitutions found in one of the following conservative substitution groups: Group 1: A, G, S, T; Group 2: D, E; Group 3: N, Q; Group 4: R, K, H; Group 5: I, L, M, V; and Group 6: F, Y, W.

[0370] In addition, amino acids can be grouped into conservative substitution groups (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing) based on similar function, chemical structure, or composition. For example, for substitution purposes, an aliphatic grouping may include G, A, V, L, and I. Other groups that include amino acids that are considered conservative substitutions of each other include: sulfur-containing: M and C; acidic: D, E, N, and Q; small aliphatic non-polar or slightly polar residues: A, S, T, P, and G; polar negatively charged residues and their amides: D, N, E, and Q; polar positively charged residues: H, R, and K; large aliphatic non-polar residues: M, L, I, V, and C; and large aromatic residues: F, Y, and W.

[0371] Non-conservative substitutions include those that significantly affect: the structure of the peptide backbone in the region with the change (e.g., alpha helical or beta sheet structure); the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. Non-conservative substitutions that are generally expected to produce the greatest changes in the properties of the protein are those in which (i) a hydrophilic residue (e.g., S or T) can be substituted for (or replaced by) a hydrophobic residue (e.g., L, I, F, V, or A); (ii) a C or P can be substituted for (or replaced by) any other residue; (iii) a residue with an electropositive side chain (e.g., K, R, or H) can be substituted for (or replaced by) an electronegative residue (e.g., Q or D); or (iv) a residue with a bulky side chain (e.g., F) can be substituted for (or replaced by) a residue without a bulky side chain (e.g., G). Additional information is found in Creighton (1984) Proteins, WH Freeman and Company.

[0372] Variants of the nucleic acid and protein sequences disclosed herein also include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a reference sequence disclosed herein.

[0373] "Percentage of sequence identity (%)" for sequences identified herein is defined as the percentage of nucleic acids or amino acid residues identical to those in a reference sequence in a candidate sequence after aligning the sequences and, if necessary, introducing spaces to achieve maximum percentage of sequence identity. Comparison for the purpose of determining nucleic acid or amino acid sequence identity percentage can be achieved in various ways belonging to the skills in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring comparisons, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. For example, sequence identity % values ​​are generated using WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology, 266:460-480 (1996)), which uses several search parameters, most of which are set to default values. Those not set to default values ​​(ie, adjustable parameters) were set with the following values: overlap spacing = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix BLOSUM62.

[0374] Variants will generally exhibit the same qualitative biological activity as the reference nucleic acid or peptide sequence, and elicit a substantially similar biological response, but variants may be selected to improve the characteristics of the reference nucleic acid or peptide as desired. Screening for variants can be performed using the experimental protocols described herein.

[0375] As will be appreciated by one of ordinary skill in the art, each embodiment disclosed herein may include, consist essentially of, or consist of its specific stated elements, steps, ingredients, or components. Therefore, the term "include / including" should be interpreted as a narrative: "comprise, consist of, or consist essentially of". The transitional term "comprise / comprises" means including, but not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in significant quantities. The transitional phrase "consisting of" excludes any unspecified elements, steps, ingredients, or components. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients, or components and those that do not substantially affect the embodiment. A substantial impact will result in a statistically significant reduction in the expression of the selected antibody by B cells.

[0376] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is needed, the term "about" when used in conjunction with a stated value or range has the meaning reasonably ascribed to it by one skilled in the art, i.e., to mean slightly more or slightly less than the stated value or range, within ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0377] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0378] Unless otherwise indicated herein or clearly contradicted by the context, the terms "a / an", "the" and similar indicators used in the context of describing the present invention (especially in the context of the following claims) should be interpreted as covering both the singular and the plural. The description of the numerical range herein is intended only to serve as a shorthand method for individually referring to each individual numerical value within the range. Unless otherwise indicated herein, each individual numerical value is incorporated into the specification as if it were individually described herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary wordings (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not limit the scope of the present invention. No wording in the specification should be interpreted as indicating that any non-required element is necessary for implementing the present invention.

[0379] The grouping of alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Each group member may be referred to and required individually or in any combination with other members of the group or other elements found herein. It is contemplated that one or more members of a group may be included in or deleted from a group for reasons of suitability and / or patentability. When any of the inclusions or deletions occur, the specification is deemed to contain the group as modified, thereby satisfying the written description of all Markush groups used in the appended claims.

[0380] Certain embodiments of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art after reading the previous description. The inventors expect that a skilled person will adopt the variations when appropriate, and the inventors intend that the present invention be implemented in a manner different from the manner specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter recited in the claims attached thereto as permitted by applicable law. In addition, unless otherwise indicated herein or otherwise clearly refuted by the context, any combination of the above elements with all possible variations thereof is encompassed by the present invention.

[0381] In addition, numerous references have been made throughout this specification to patents, printed publications, journal articles, and other written texts (herein referenced materials). Each of the referenced materials is individually incorporated herein by reference in its entirety with respect to their referenced teachings.

[0382] Finally, it should be understood that the embodiments of the present invention disclosed herein illustrate the principles of the present invention. Other modifications that may be adopted are within the scope of the present invention. Therefore, by way of example and not limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Therefore, the present invention is not limited to exactly as shown and described.

[0383] The details shown herein are by way of example and are presented only for the purpose of illustrative discussion of the preferred embodiments of the invention and in order to provide what is believed to be the most useful and understandable description of the principles and conceptual aspects of the various embodiments of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary to achieve a basic understanding of the invention, and the description together with the drawings and / or examples makes it apparent to those skilled in the art how several forms of the invention may be embodied in practice.

[0384] Unless explicitly and clearly modified in the following examples, or when the application of the meaning renders any interpretation meaningless or substantially meaningless, the definitions and explanations used in this disclosure are intended and intended to govern in any future interpretation. In cases where the interpretation of a term would render it meaningless or substantially meaningless, the definition should be obtained from Webster's Dictionary, 3rd Edition, or a dictionary known to those of ordinary skill in the art such as Oxford Dictionary of Biochemistry and Molecular Biology (ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. A method for genetically engineering B cells in vitro to express a selected antibody, the method comprising: Delivering gene editing agents to B cells, The gene editing agent cleaves a genomic region in a B cell that is targeted for insertion of a genetic construct; and The genetic construct comprises from 5' to 3': (i) the nucleotide sequence of the heavy chain promoter; (ii) a nucleotide sequence encoding a signal peptide; (iii) a nucleotide sequence encoding the light chain of the selected antibody; (iv) a nucleotide sequence encoding a flexible linker or a nucleotide sequence encoding a jumping element, wherein the jumping element is selected from a self-cleaving peptide and an internal ribosome entry site (IRES); (v) a nucleotide sequence encoding the heavy chain variable region of the selected antibody; and (vi) a nucleotide sequence comprising a splice junction that allows the antibody to be spliced ​​to an endogenous IgH constant region, The genomic region comprises the following sequence: (A) SEQ ID NO: 2; (B) SEQ ID NO: 3; or (C) SEQ ID NO: 4, And wherein, after cleavage by the gene editing agent, the genetic construct is inserted into the IgH constant region within SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, thereby genetically engineering the B cell to express the selected antibody.

2. The method of claim 1, wherein the endogenous variable heavy chain encoding genome of the B cell is not excised.

3. The method of claim 1, wherein the gene editing agent comprises a guide RNA (gRNA) and a nuclease, When the genomic region is (A), the sequence of the gRNA is as shown in SEQ ID NO: 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326 or 327; When the genomic region is (B), the sequence of the gRNA is as shown in SEQ ID NO: 87, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345 or 346; or When the genomic region is (C), the sequence of the gRNA is as shown in SEQ ID NO: 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365 or 366.

4. The method of claim 1, wherein the delivery is achieved by electroporation, nanoparticles, or virus-mediated delivery.

5. The method of claim 3, wherein the gRNA and the nuclease are delivered by electroporation and the genetic construct is delivered as part of an adeno-associated viral vector.

6. The method of claim 3, wherein the nuclease is Cas9 or Cpf1.

7. The method of claim 1, wherein: When the genomic region is (A), the heavy chain promoter consists of the sequence shown in SEQ ID NO: 111; or when the genomic region is (B) or (C), the heavy chain promoter consists of the sequence shown in SEQ ID NO:

128.

8. The method of claim 1, wherein: The signal peptide is derived from human IgH heavy chain and consists of the amino acid sequence shown in SEQ ID NO: 185, 186, 187, 188, 189, 190, 191 or 192; The signal peptide is derived from human IgL light chain and consists of the amino acid sequence shown in SEQ ID NO: 193 or 194; or When the genomic region is (A), the signal peptide consists of the amino acid sequence shown in SEQ ID NO: 118; or when the genomic region is (B) or (C), the signal peptide consists of the amino acid sequence shown in SEQ ID NO:

134.

9. The method of claim 1, wherein the flexible joint: Encoded by the nucleotide sequence shown in SEQ ID NO: 116; Composed of the amino acid sequence shown in SEQ ID NO: 122, 180, 181, 182, 183 or 184; or It is a Gly-Ser linker containing 50-80 amino acids.

10. The method of claim 1, wherein the self-cleaving peptide consists of the sequence shown in SEQ ID NO: 176, 177, 178 or 179.

11. The method of claim 1, wherein: When the genomic region is (A), the nucleotide sequence comprising the splice junction is shown by SEQ ID NO: 124 or 151; or when the genomic region is (B) or (C), the nucleotide sequence comprising the splice junction is shown by SEQ ID NO:

139.

12. The method of claim 1, wherein the genetic construct further encodes a tag consisting of a sequence shown in SEQ ID NO: 195, 196, 197, 198, 199, 200, 201, 202, 203 or 204.

13. The method of claim 1, wherein the genetic construct is part of an adeno-associated viral vector.

14. The method of claim 13, wherein after insertion of the genetic construct, the genomic region comprises the sequence shown in SEQ ID NO:

109.

15. The method of claim 1, wherein the selected antibodies comprise anti-respiratory syncytial virus (RSV) antibodies, anti-human immunodeficiency virus (HIV) antibodies, anti-pertussis antibodies, anti-influenza virus antibodies, anti-Epstein-Barr virus (EBV) antibodies, or anti-tumor necrosis factor (TNF) antibodies, The anti-respiratory syncytial virus RSV antibody comprises: (a) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO: 138 and a variable light chain consisting of the amino acid sequence of SEQ ID NO: 136; (b) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO:138 and a variable light chain consisting of the amino acid sequence of SEQ ID NO:205; (c) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO: 123 and a variable light chain consisting of the amino acid sequence of SEQ ID NO: 120; or (d) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO:123 and a variable light chain consisting of the amino acid sequence of SEQ ID NO:206; The anti-human immunodeficiency virus HIV antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 150 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 149 The anti-pertussis antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 235 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 236; The anti-influenza virus antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 159 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 158; The anti-Epstein-Barr virus EBV antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 168 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 166; The anti-tumor necrosis factor (TNF) antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 254 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO:

255.

16. The method of claim 1, wherein the selected antibody comprises CDRH1 consisting of SEQ ID NO: 207, CDRH2 consisting of SEQ ID NO: 208, CDRH3 consisting of SEQ ID NO: 209; CDRL1 consisting of SEQ ID NO: 210, CDRL2 consisting of SEQ ID NO: 211, and CDRL3 consisting of SEQ ID NO: 212; CDRH1 consisting of SEQ ID NO:213, CDRH2 consisting of SEQ ID NO:214, CDRH3 consisting of SEQ ID NO:215, CDRL1 consisting of SEQ ID NO:216, CDRL2 consisting of SEQ ID NO:217, and CDRL3 consisting of SEQ ID NO:218; CDRH1 consisting of SEQ ID NO:219, CDRH2 consisting of SEQ ID NO:220, CDRH3 consisting of SEQ ID NO:221, CDRL1 consisting of QYGS, CDRL2 consisting of SGS, and CDRL3 consisting of SEQ ID NO:222; CDRH1 consisting of SEQ ID NO: 223, CDRH2 consisting of SEQ ID NO: 224, CDRH3 consisting of SEQ ID NO: 225; CDRL1 consisting of SEQ ID NO: 226, CDRL2 consisting of SEQ ID NO: 227, and CDRL3 consisting of SEQ ID NO: 228; CDRH1 consisting of SEQ ID NO:229, CDRH2 consisting of SEQ ID NO:230, CDRH3 consisting of SEQ ID NO:231, CDRL1 consisting of SEQ ID NO:232, CDRL2 consisting of SEQ ID NO:233, and CDRL3 consisting of SEQ ID NO:234; CDRH1 consisting of SEQ ID NO:237, CDRH2 consisting of SEQ ID NO:238, CDRH3 consisting of SEQ ID NO:239, CDRL1 consisting of SEQ ID NO:240, CDRL2 consisting of SEQ ID NO:241, and CDRL3 consisting of SEQ ID NO:242; CDRH1 consisting of SEQ ID NO:243, CDRH2 consisting of SEQ ID NO:244, CDRH3 consisting of SEQ ID NO:245, CDRL1 consisting of SEQ ID NO:246, CDRL2 consisting of KTS, and CDRL3 consisting of SEQ ID NO:247; CDRH1 consisting of SEQ ID NO:248, CDRH2 consisting of SEQ ID NO:249, CDRH3 consisting of SEQ ID NO:250, CDRL1 consisting of SEQ ID NO:251, CDRL2 consisting of SEQ ID NO:252, and CDRL3 consisting of SEQ ID NO:253; CDRH1 consisting of SEQ ID NO:256, CDRH2 consisting of SEQ ID NO:257, and CDRH3 consisting of SEQ ID NO:258; CDRL1 consisting of SEQ ID NO:259, CDRL2 consisting of SEQ ID NO:260, and CDRL3 consisting of SEQ ID NO:261; CDRH1 consisting of SEQ ID NO: 262, CDRH2 consisting of SEQ ID NO: 263, and CDRH3 consisting of SEQ ID NO: 264; CDRL1 consisting of SEQ ID NO: 265, CDRL2 consisting of SEQ ID NO: 266, and CDRL3 consisting of SEQ ID NO: 267; or CDRH1 consisting of SEQ ID NO:268, CDRH2 consisting of SEQ ID NO:269 and CDRH3 consisting of SEQ ID NO:270; or CDRL1 consisting of SEQ ID NO:271, CDRL2 consisting of SEQ ID NO:272 and CDRL3 consisting of SEQ ID NO:

273.

17. The method of claim 1, wherein the selected antibody is an anti-respiratory syncytial virus (RSV) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-dengue virus antibody, an anti-bordetella pertussis antibody, an anti-hepatitis C antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, an anti-metapulmonary virus (MPV) antibody, an anti-cytomegalovirus antibody, an anti-Epstein-Barr virus antibody; an anti-herpes simplex virus antibody, an anti-Clostridium difficile bacterial toxin antibody, or an anti-tumor necrosis factor (TNF) antibody.

18. The method of claim 1, wherein the selected antibodies comprise: An anti-RSV antibody selected from palivizumab, AB1128 or ab20745; An anti-HIV antibody selected from 10E8, VRC01, ab18633 or 39 / 5.4A; An anti-dengue virus antibody selected from antibody 55, DB2-3, ab155042 or ab80914; An anti-hepatitis C antibody selected from MAB8694 or C7-50; Anti-influenza virus antibodies including C102; Anti-MPV antibodies including MPE8; An anti-CMV antibody selected from MCMV5322A, MCMV3068A, LJP538 or LJP539; an anti-HSV antibody selected from HSV8-N or MB66; an anti-Clostridium difficile antibody selected from actoxumab or bezlotoxumab; or An anti-TNF antibody selected from infliximab, adalimumab, etanercept or certolizumab.

19. The method of claim 1, wherein the genetic construct comprises a homology arm 5' to the heavy chain promoter and a homology arm 3' to the splice junction.

20. The method of claim 19, wherein the genetic construct comprises: The nucleotide sequence shown in SEQ ID NO: 102 comprises a coding sequence encoding an anti-RSV antibody consisting of the amino acid sequence shown in SEQ ID NO: 126; The nucleotide sequence shown in SEQ ID NO: 105, which comprises a coding sequence encoding an anti-HIV antibody consisting of the amino acid sequence shown in SEQ ID NO: 152; The nucleotide sequence as shown in SEQ ID NO: 106, which comprises a coding sequence encoding an anti-influenza virus antibody consisting of the amino acid sequence shown in SEQ ID NO: 160; or The nucleotide sequence shown in SEQ ID NO: 107 comprises a coding sequence encoding an anti-EBV antibody consisting of the amino acid sequence shown in SEQ ID NO:

169.

21. The method of claim 19, wherein: When the genomic region is (B), the 5' homology arm is represented by SEQ ID NO: 127, and the 3' homology arm is represented by SEQ ID NO: 140; The 5' homology arm is represented by SEQ ID NO: 90, and the 3' homology arm is represented by SEQ ID NO: 91; or The 5' homology arm is represented by SEQ ID NO:142, and the 3' homology arm is represented by SEQ ID NO:

143.

22. The method of claim 19, wherein When the genomic region comprises (B), the genetic construct comprises: (i) a 5' homology arm sequence as shown in SEQ ID NO: 127; (ii) the heavy chain promoter having the sequence shown in SEQ ID NO: 128; (iii) a coding sequence encoding the signal peptide having the amino acid sequence shown in SEQ ID NO: 134; (iv) a coding sequence encoding the light chain of the selected antibody having the amino acid sequence shown in SEQ ID NO: 286; (v) a coding sequence encoding the flexible linker having the amino acid sequence shown in SEQ ID NO: 122; (vi) a coding sequence encoding the heavy chain variable region of the selected antibody having the amino acid sequence shown in SEQ ID NO: 138; (vii) the splice junction having the sequence shown in SEQ ID NO: 139; and (viii) a 3' homology arm sequence as shown in SEQ ID NO: 140; or When the genomic region comprises (B), the genetic construct comprises: (i) a 5' homology arm sequence as shown in SEQ ID NO: 142; (ii) the heavy chain promoter having the sequence shown in SEQ ID NO: 128; (iii) a coding sequence encoding the signal peptide having the amino acid sequence shown in SEQ ID NO: 134118; (iv) a coding sequence encoding the light chain of the selected antibody having the amino acid sequence shown in SEQ ID NO: 286; (v) a coding sequence encoding the flexible linker having the amino acid sequence shown in SEQ ID NO: 122; (vi) a coding sequence encoding the heavy chain variable region of the selected antibody having the amino acid sequence shown in SEQ ID NO: 138; (vii) the splice junction having the sequence shown in SEQ ID NO: 139; and (viii) the 3' homology arm sequence shown in SEQ ID NO:

143.

23. The method of claim 1, wherein the B cell is an antibody-producing B cell, a memory B cell, a naive B cell, a B1 B cell, or a marginal zone B cell.

24. A B cell modified according to the method of any one of claims 1-23.

25. The B cell of claim 24, wherein the B cell is an antibody-secreting B cell, a memory B cell, a naive B cell, a B1 B cell, or a marginal zone B cell.

26. A genetic construct for modifying a B cell to express a selected antibody by genetic insertion at the IgH constant region within SEQ ID NO: 2, 3 or 4, the genetic construct comprising from 5' to 3': (i) the nucleotide sequence of the heavy chain promoter; (ii) a nucleotide sequence encoding a signal peptide; (iii) a nucleotide sequence encoding the light chain of the selected antibody; (iv) a nucleotide sequence encoding a flexible linker or a nucleotide sequence encoding a jumping element, wherein the jumping element is selected from a self-cleaving peptide and an internal ribosome entry site (IRES); (v) a nucleotide sequence encoding the heavy chain variable region of the selected antibody; and (vi) a nucleotide sequence comprising a splice junction that allows the antibody to be spliced ​​to a downstream endogenous IgH constant region, Wherein the splice junction is used to express the endogenous heavy chain constant region of the B cell, wherein the heavy chain constant region is appropriate for the current activation and / or maturation state of the B cell.

27. The genetic construct of claim 26, wherein the genetic construct comprises a homology arm 5' to the heavy chain promoter and a homology arm 3' to the splice junction.

28. The genetic construct of claim 27, wherein the genetic construct comprises: The nucleotide sequence shown in SEQ ID NO: 102 comprises a coding sequence encoding an anti-RSV antibody consisting of the amino acid sequence shown in SEQ ID NO: 126; The nucleotide sequence shown in SEQ ID NO: 105, which comprises a coding sequence encoding an anti-HIV antibody consisting of the amino acid sequence shown in SEQ ID NO: 152; The nucleotide sequence shown in SEQ ID NO: 106, which comprises a coding sequence encoding an anti-influenza virus antibody consisting of the amino acid sequence shown in SEQ ID NO: 160; The nucleotide sequence shown in SEQ ID NO: 107 comprises a coding sequence encoding an anti-EBV antibody consisting of the amino acid sequence shown in SEQ ID NO:

169.

29. The genetic construct of claim 27, wherein The 5' homology arm is represented by SEQ ID NO: 110 or 153, and the 3' homology arm is represented by SEQ ID NO: 125; The 5' homology arm is represented by SEQ ID NO:92, and the 3' homology arm is represented by SEQ ID NO:93; The 5' homology arm is represented by SEQ ID NO:94, and the 3' homology arm is represented by SEQ ID NO:

95.

30. The genetic construct of claim 27, wherein The genetic construct comprises: (i) a 5' homology arm sequence as shown in SEQ ID NO: 110; (ii) the heavy chain promoter consisting of the sequence shown in SEQ ID NO: 111; (iii) a coding sequence encoding the signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 118; (iv) a coding sequence encoding the light chain of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 285; (v) a coding sequence encoding the flexible linker consisting of the amino acid sequence shown in SEQ ID NO: 122; (vi) a coding sequence encoding the heavy chain variable region of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 123; (vii) the splice junction consisting of the sequence shown in SEQ ID NO: 124; and (viii) a 3' homology arm sequence as shown in SEQ ID NO: 125; or The genetic construct comprises: (i) a 5' homology arm sequence as shown in SEQ ID NO: 110; (ii) the heavy chain promoter consisting of the sequence shown in SEQ ID NO: 111; (iii) a coding sequence encoding the signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 118; (iv) a coding sequence encoding the light chain of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 287; (v) a coding sequence encoding the flexible linker consisting of the amino acid sequence shown in SEQ ID NO: 122; (vi) a coding sequence encoding the heavy chain variable region of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 150; (vii) the splice junction consisting of the sequence shown in SEQ ID NO: 151; and (viii) a 3' homology arm sequence as shown in SEQ ID NO: 125; or The genetic construct comprises: (i) a 5' homology arm sequence as shown in SEQ ID NO: 153; (ii) the heavy chain promoter consisting of the sequence shown in SEQ ID NO: 111; (iii) a coding sequence encoding a signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 118; (iv) a coding sequence encoding the light chain of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 288; (v) a coding sequence encoding a flexible linker consisting of the amino acid sequence shown in SEQ ID NO: 122; (vi) a coding sequence encoding the heavy chain variable region of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 159; (vii) the splice junction consisting of the sequence shown in SEQ ID NO: 151; and (viii) a 3' homology arm sequence as shown in SEQ ID NO: 125; or The genetic construct comprises: (i) a 5' homology arm sequence as shown in SEQ ID NO: 153; (ii) the heavy chain promoter consisting of the sequence shown in SEQ ID NO: 111; (iii) a coding sequence encoding the signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 118; (iv) a coding sequence encoding the light chain of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 289; (v) a coding sequence encoding the flexible linker consisting of the amino acid sequence shown in SEQ ID NO: 122; (vi) a coding sequence encoding the heavy chain variable region of the selected antibody consisting of the amino acid sequence shown in SEQ ID NO: 168; (vii) the splice junction consisting of the sequence shown in SEQ ID NO: 151; and (viii) the 3' homology arm sequence shown in SEQ ID NO:

125.

31. The genetic construct of claim 26, wherein the heavy chain promoter consists of the sequence shown in SEQ ID NO:

111.

32. The genetic construct of claim 26, wherein the signal peptide: Derived from human IgH heavy chain, and consisting of the amino acid sequence shown in SEQ ID NO: 185, 186, 187, 188, 189, 190, 191 or 192; Derived from human IgL light chain, and consisting of the amino acid sequence shown in SEQ ID NO: 193 or 194; or It consists of the amino acid sequence shown in SEQ ID NO:

118.

33. The genetic construct of claim 26, wherein the flexible linker: Encoded by the nucleotide sequence shown in SEQ ID NO: 116; Composed of the amino acid sequence shown in SEQ ID NO: 122, 180, 181, 182, 183 or 184; or It is a Gly-Ser linker containing 50-80 amino acids.

34. The genetic construct of claim 26, wherein the self-cleaving peptide consists of the sequence shown in SEQ ID NO: 176, 177, 178 or 179.

35. The genetic construct of claim 26, wherein the nucleotide sequence comprising the splice junction is set forth in SEQ ID NO: 124 or 151.

36. The genetic construct of claim 26, wherein the genetic construct further encodes a tag consisting of a sequence as set forth in SEQ ID NO: 195, 196, 197, 198, 199, 200, 201, 202, 203 or 204.

37. The genetic construct of claim 26, wherein the genetic construct is part of an adeno-associated viral vector.

38. The genetic construct of claim 26, wherein the selected antibodies comprise anti-respiratory syncytial virus (RSV) antibodies, anti-human immunodeficiency virus (HIV) antibodies, anti-pertussis antibodies, anti-influenza virus antibodies, anti-Epstein-Barr virus (EBV) antibodies, or anti-tumor necrosis factor (TNF) antibodies, The anti-respiratory syncytial virus RSV antibody comprises: (a) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO: 138 and a variable light chain consisting of the amino acid sequence of SEQ ID NO: 136; (b) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO:138 and a variable light chain consisting of the amino acid sequence of SEQ ID NO:205; (c) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO: 123 and a variable light chain consisting of the amino acid sequence of SEQ ID NO: 120; or (d) a variable heavy chain consisting of the amino acid sequence of SEQ ID NO:123 and a variable light chain consisting of the amino acid sequence of SEQ ID NO:206; The anti-human immunodeficiency virus HIV antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 150 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 149; The anti-pertussis antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 235 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 236; The anti-influenza virus antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 159 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 158; The anti-Epstein-Barr virus EBV antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 168 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO: 166; The anti-tumor necrosis factor (TNF) antibody comprises a variable heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 254 and a variable light chain consisting of the amino acid sequence shown in SEQ ID NO:

255.

39. The genetic construct of claim 26, wherein the selected antibody comprises CDRH1 consisting of SEQ ID NO: 207, CDRH2 consisting of SEQ ID NO: 208, CDRH3 consisting of SEQ ID NO: 209; CDRL1 consisting of SEQ ID NO: 210, CDRL2 consisting of SEQ ID NO: 211, and CDRL3 consisting of SEQ ID NO: 212; CDRH1 consisting of SEQ ID NO:213, CDRH2 consisting of SEQ ID NO:214, CDRH3 consisting of SEQ ID NO:215, CDRL1 consisting of SEQ ID NO:216, CDRL2 consisting of SEQ ID NO:217, and CDRL3 consisting of SEQ ID NO:218; CDRH1 consisting of SEQ ID NO:219, CDRH2 consisting of SEQ ID NO:220, CDRH3 consisting of SEQ ID NO:221, CDRL1 consisting of QYGS, CDRL2 consisting of SGS, and CDRL3 consisting of SEQ ID NO:222; CDRH1 consisting of SEQ ID NO: 223, CDRH2 consisting of SEQ ID NO: 224, CDRH3 consisting of SEQ ID NO: 225; CDRL1 consisting of SEQ ID NO: 226, CDRL2 consisting of SEQ ID NO: 227, and CDRL3 consisting of SEQ ID NO: 228; CDRH1 consisting of SEQ ID NO:229, CDRH2 consisting of SEQ ID NO:230, CDRH3 consisting of SEQ ID NO:231, CDRL1 consisting of SEQ ID NO:232, CDRL2 consisting of SEQ ID NO:233, and CDRL3 consisting of SEQ ID NO:234; CDRH1 consisting of SEQ ID NO:237, CDRH2 consisting of SEQ ID NO:238, CDRH3 consisting of SEQ ID NO:239, CDRL1 consisting of SEQ ID NO:240, CDRL2 consisting of SEQ ID NO:241, and CDRL3 consisting of SEQ ID NO:242; CDRH1 consisting of SEQ ID NO:243, CDRH2 consisting of SEQ ID NO:244, CDRH3 consisting of SEQ ID NO:245, CDRL1 consisting of SEQ ID NO:246, CDRL2 consisting of KTS, and CDRL3 consisting of SEQ ID NO:247; CDRH1 consisting of SEQ ID NO:248, CDRH2 consisting of SEQ ID NO:249, CDRH3 consisting of SEQ ID NO:250, CDRL1 consisting of SEQ ID NO:251, CDRL2 consisting of SEQ ID NO:252, and CDRL3 consisting of SEQ ID NO:253; CDRH1 consisting of SEQ ID NO:256, CDRH2 consisting of SEQ ID NO:257, and CDRH3 consisting of SEQ ID NO:258; CDRL1 consisting of SEQ ID NO:259, CDRL2 consisting of SEQ ID NO:260, and CDRL3 consisting of SEQ ID NO:261; CDRH1 consisting of SEQ ID NO: 262, CDRH2 consisting of SEQ ID NO: 263, and CDRH3 consisting of SEQ ID NO: 264; CDRL1 consisting of SEQ ID NO: 265, CDRL2 consisting of SEQ ID NO: 266, and CDRL3 consisting of SEQ ID NO: 267; or CDRH1 consisting of SEQ ID NO:268, CDRH2 consisting of SEQ ID NO:269 and CDRH3 consisting of SEQ ID NO:270; or CDRL1 consisting of SEQ ID NO:271, CDRL2 consisting of SEQ ID NO:272 and CDRL3 consisting of SEQ ID NO:

273.

40. A genetic construct as described in claim 26, wherein the selected antibody is an anti-respiratory syncytial virus RSV antibody, an anti-human immunodeficiency virus HIV antibody, an anti-dengue virus antibody, an anti-bordetella pertussis antibody, an anti-hepatitis C antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, an anti-metapulmonary virus MPV antibody, an anti-cytomegalovirus antibody, an anti-Epstein-Barr virus antibody; an anti-herpes simplex virus antibody, an anti-Clostridium difficile bacterial toxin antibody or an anti-tumor necrosis factor TNF antibody.

41. The genetic construct of claim 26, wherein the selected antibodies comprise: An anti-RSV antibody selected from palivizumab, AB1128 or ab20745; An anti-HIV antibody selected from 10E8, VRC01, ab18633 or 39 / 5.4A; An anti-dengue virus antibody selected from antibody 55, DB2-3, ab155042 or ab80914; An anti-hepatitis C antibody selected from MAB8694 or C7-50; Anti-influenza virus antibodies including C102; Anti-MPV antibodies including MPE8; An anti-CMV antibody selected from MCMV5322A, MCMV3068A, LJP538 or LJP539; an anti-HSV antibody selected from HSV8-N or MB66; an anti-Clostridium difficile antibody selected from actumozumab or bezentuzumab; or An anti-TNF antibody selected from infliximab, adalimumab, etanercept or celizumab.

42. A kit for genetically modifying B cells, the kit comprising a genetic construct as described in any one of claims 26-41 and a gRNA targeting SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

43. A kit as described in claim 42, wherein the gRNA has a sequence shown in SEQ ID NO: 88, 89, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306 or 307.

44. The kit of claim 42, further comprising a nuclease.

45. The kit of claim 44, wherein the nuclease is Cas9 or Cpf1.

46. ​​The kit of claim 44, further comprising nanoparticles, and wherein the gRNA and the nuclease are connected by the nanoparticles.

47. The kit of claim 42, further comprising an adeno-associated viral vector, and wherein the genetic construct is part of an adeno-associated viral vector.

48. Use of the B cell of claim 24 for preparing a medicament for providing an anti-infective effect to a subject in need thereof.

49. The use of claim 48, wherein the medicament obviates the need for vaccination.

50. The use of claim 48, wherein the medicament replaces a vaccination regimen.

51. The use of claim 48, wherein the subject is immunosuppressed.

52. The use of claim 48, wherein the subject is subjected to immunosuppression as part of a treatment regimen comprising bone marrow transplantation, hematopoietic stem cell transplantation, or administration of genetically modified hematopoietic stem cells.

53. A preparation, characterized in that The preparation comprises the B cells of claim 24.

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