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

Abstract

To provide systems and methods to genetically modify B cells to express selected antibodies.SOLUTION: A method of genetically modifying B cells to express Palivizumab is provided, the method comprising genetically engineering the B cells to express Palivizumab by means of a genetic construct comprising a heavy chain promoter of Palivizumab and a nucleic acid sequence encoding a full-length light chain, a Gly-Ser linker, and a variable region of the heavy chain of Palivizumab.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 575,275, filed Oct. 20, 2017; U.S. Provisional Patent Application No. 62 / 580,303, filed Nov. 1, 2017; and U.S. Provisional Patent Application No. 62 / 623,371, filed Jan. 29, 2018, each of which is hereby incorporated by reference in its entirety as if fully set forth herein.

[0002] Statement Regarding the Sequence Listing The sequence listing associated with this application is provided in text format instead of a hard copy, and is hereby incorporated by reference in this specification. The text file name containing the sequence listing is 18 - 024 - WO - PCT_ST25.txt. The text file is 184 KB, was created on Oct. 19, 2018, and was electronically submitted via EFS - Web.

[0003] The present disclosure provides systems and methods for genetically modifying B cells to express selected antibodies. Using these systems and methods, it is possible to eliminate the need for classical vaccination, provide protection against infectious pathogens for which currently available vaccinations are not available, provide protection against infectious pathogens when the patient is immunosuppressed by other means, and / or provide the benefits provided by therapeutic antibodies, such as in the treatment of autoimmune disorders.

Background Art

[0004] Vaccines are designed to increase the immunity of a subject against a specific infection by stimulating B cells to produce antibodies against the target infectious pathogen. Routine childhood vaccination is a long - established clinical intervention that is relatively low - risk and highly effective. Unfortunately, vaccination is not available for every infectious pathogen. As an example, in the United States, millions of children are seen by doctors or in emergency rooms each year due to infection with respiratory syncytial virus (RSV).

[0005] For decades, researchers have been working to develop vaccines that can induce B cells to produce antibodies effective against viruses such as respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), and Zika virus. However, all efforts to induce protective antibodies have failed. The only RSV vaccine that has been widely tested actually worsened infections, and the antibodies produced after vaccination did not neutralize the virus; instead, they enhanced the virus's ability to infect cells. In addition to RSV, HIV, and Zika virus, there are several other infectious pathogens for which no effective vaccines are available.

[0006] In addition to combating infections, antibodies can also be useful as treatments for other conditions such as autoimmune diseases. However, these antibody-based therapies typically require repeated injections of the antibody to maintain protection.

[0007] Moreover, notably, many patients receive bone marrow or hematopoietic stem cell transplants as treatment for blood cancers (e.g., leukemia, lymphoma, myeloma). Other patients receive infusions of genetically modified hematopoietic stem cells that give the patient the missing therapeutic gene. All of these treatments require that the patient's existing immune system be removed before the transplant or infusion of genetically modified hematopoietic stem cells, leaving a dangerous period of immunosuppression before the patient's immune system re-colonizes following the treatment. During this time of immunosuppression, patients are incredibly susceptible to infections such as RSV, influenza, parainfluenza, and metapneumovirus (MPV). These infections are high risk factors and are associated with a significant number of deaths following such treatments. Summary of the Invention Means for Solving the Problems

[0008] (Summary of the Invention) The present disclosure provides systems and methods for genetically modifying B cells to express selected antibodies. In certain embodiments, the selected antibodies reduce or eliminate the need for existing vaccinations. In certain embodiments, the selected antibodies protect against infections from viruses for which there are no effective vaccination strategies currently available (e.g., RSV, HIV, Zika). In certain embodiments, the selected antibodies reduce or eliminate the need for therapeutic antibody injections, such as those administered to treat various autoimmune disorders. In certain embodiments, the selected antibodies protect immunosuppressed patients from infections. In certain embodiments, using the methods of the present disclosure, B cells can be reprogrammed to protect against hundreds of different infectious pathogens or pathogenic bacteria, all by a single laboratory procedure that takes only a few days.

[0009] In certain embodiments, the present disclosure provides these advantages through targeted insertion of genetic constructs into specific regions of the B cell's endogenous genome. Importantly, genetic modification of B cells is difficult due to the high variability of these intracellular gene sequences required for antibody diversity. This high gene variability makes it infeasible to directly target antibody coding regions for genetic manipulation. Furthermore, removing and replacing the coding portion of the B cell genome is also not effective in this approach as it has a negative impact on B cell function.

[0010] Additional challenges arise with respect to genetically modifying B cells to express selected antibodies because antibodies are formed from separate protein units called heavy and light chains. The different chains are encoded by different parts of the B cell genome but must assemble to form a functional antibody.

[0011] The present disclosure, among other things, overcomes the noted difficulties by identifying a constant region of the B cell genome that can be reliably targeted for gene insertion and that, when modified, preferentially expresses the inserted gene construct over the corresponding portion of the B cell's native genome. 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 preserves B cell function after genetic manipulation.

[0012] In certain embodiments, the region of interest targeted for gene 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 certain embodiments, the region targeted for gene insertion is a constant intron region selected from SEQ ID NO: 1 or 2 (human) or SEQ ID NO: 3 or 4 (mouse). In certain embodiments, the human DNA sequence within SEQ ID NO: 1 targeted for gene insertion comprises SEQ ID NOs: 5-24. In certain embodiments, the human DNA sequence within SEQ ID NO: 2 targeted for gene insertion comprises SEQ ID NOs: 25-44. In certain embodiments, the mouse DNA sequence within SEQ ID NO: 3 targeted for gene insertion comprises SEQ ID NOs: 45-64. In certain embodiments, the mouse DNA sequence within SEQ ID NO: 4 targeted for gene insertion comprises SEQ ID NOs: 65-84. Gene sequences that can specifically target these sites for gene modification are described within the present disclosure as guide RNA (gRNA) SEQ ID NOs: 87-89, and 290-366.

[0013] In certain embodiments, the placement and components of the inserted gene construct result in preferential expression of the inserted gene construct over the corresponding portion of the B cell's endogenous genome. These embodiments also include elements that overcome challenges associated with portions of antibodies encoded by different regions of the endogenous B cell genome.

[0014] In certain embodiments, the gene construct is inserted into one of SEQ ID NOs: 1, 2, 3, and 4 and comprises (i) a promoter; (ii) a signal peptide; (iii) a transgene encoding the entire light chain of a selected antibody; (iv) a flexible linker or skipping element; (v) the variable portion of the heavy chain of the selected antibody; and (vi) a splice junction that results in expression of the endogenous heavy chain constant region of a B cell. In these embodiments, if the selected antibody is expressed as a single construct, the challenges associated with portions of the antibody encoded by different regions of the endogenous B cell genome are overcome. Inclusion of a flexible linker physically links the expressed light and heavy chain portions of the selected antibody such that the light and heavy chains are able to form a functional unit while simultaneously reducing the risk that the antibody portion binds to other potentially expressed antibody chains from the endogenous genome of the B cell. Use of a skipping element does not physically link the light and heavy chain portions, but if those chains are expressed in proximity, they associate to form a functional unit while simultaneously reducing the risk that the antibody portion binds to other potentially expressed antibody chains from the endogenous genome of the B cell. Inclusion of a splice junction results in a selected antibody that includes a heavy chain constant region suitable for the current activation and / or maturation state of the B cell. In other words, it is possible to express a selected antibody having any of the endogenous heavy chain constant regions of a B cell, and the heavy chain constant region expressed with the selected antibody can naturally change over time.

[0015] The present disclosure also provides a method that ensures that only B cells that have been effectively genetically modified to express a selected antibody are collected for formulation and administration to a patient. For example, prior to genetic modification, the B cells naturally express an antibody that includes a kappa or lambda light chain. The B cells can be modified to express a light chain that is different from the kappa or lambda chain that it naturally expresses, and only the B cells that express the replaced chain are selected for formulation and administration.

[0016] The present disclosure also provides numerous additional strategies for effectively modifying B cells to provide the advantages described herein. These and other strategies are more fully described in the detailed description below.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0018] Detailed Description Vaccines are designed to increase a subject's immunity against a specific infection by stimulating B cells to produce antibodies against a target infectious pathogen. Antibodies are proteins that can provide defense against pathogens. Antibodies can bind to pathogens, and when this binding interferes with the normal function of the pathogen, the antibody is defensive. For example, many defensive antibodies bind to portions of the pathogen that prevent the pathogen from entering cells. Antibodies can be bound to the surface of B cells (known as B cell receptors), but most of their defensive function is exerted when secreted into the blood.

[0019] A pathogen can refer to any substance that can cause a disease, and pathogenicity can refer to the ability of a substance to cause a disease. Examples of pathogens include viruses, bacteria, and fungi that can infect a host and cause a disease. Other examples of pathogens include host-derived proteins or other host-derived substances such as tumor necrosis factor alpha (TNFα), inflammatory molecules associated with numerous autoimmune conditions (e.g., arthritis), and beta-amyloid plaques, a fibrous protein that accumulates in the diencephalon in Alzheimer's disease. In certain embodiments, cancer cells and / or tumors can also be referred to as pathogens or pathogenic substances based on their ability to cause disease.

[0020] When exposed to a vaccine or a natural pathogen, the epitopes provided by the vaccine and / or present on the pathogen can bind to B cell receptors present on naive B cells. This binding enables the B cells to be activated and produce defensive antibodies.

[0021] Naive B cells refer to B cells before they have finished contacting their epitopes. Each naive B cell expresses a unique antibody with a unique epitope specificity. The unique antibodies expressed by each naive B cell are randomly generated through genetic recombination. Naive B cells express membrane-bound antibodies (i.e., B cell receptors) and can rapidly proliferate when they bind to epitopes. During proliferation and maturation, antibody genes undergo somatic mutations, which help increase the affinity of epitope binding. The increase in epitope-binding affinity that occurs during B cell maturation is necessary for effective defense against pathogens. A single naive B cell can undergo cell division dozens of times to create thousands of antibody-secreting B cells and memory B cells that express the same antibody (Figure 1), or related antibodies that have been mutated to improve binding to the pathogen.

[0022] In addition to active antibody-secreting B cells, memory B cells are important for defense against pathogens. Memory B cells usually do not actively secrete antibodies but can rapidly differentiate into antibody-secreting cells. The rapid differentiation of memory B cells into antibody-secreting cells can help the immune system initiate a rapid response against pathogens encountered through secondary infection or previous 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, memory B cells maintain defense against hepatitis B virus when the levels of antibodies produced by antibody-secreting B cells decrease (Williams et al., Vaccine. 2001;19(28-29):4081-5; Bauer et al., Vaccine. 2006;24(5):572-7). Therefore, a successful vaccine stimulates the generation of antibody-secreting B cells and long-lived memory B cells that can express antibodies that all bind with high affinity to epitopes on the pathogen.

[0023] Unfortunately, there are numerous infectious pathogens for which no vaccines are available. Examples of infectious pathogens for which no effective vaccine strategies are available include RSV, HIV, and Zika virus.

[0024] Regarding RSV, the tragic failure of the formalin-inactivated RSV vaccine in the 1960s was probably not due to its inability to induce antibody-secreting B cells and memory B cells targeting RSV. The vaccine induced the production of antibodies that did not neutralize RSV, but instead may have enhanced RSV infection (Figure 2A) (Blanco et al., Hum Vaccin. 2010;6(6):482-492; Broadbent et al., Influenza Other Respir Viruses. 2015;9(4):169-178). This emphasizes the delicate balance that a vaccine must achieve: avoiding stimulating the production of "pathogenic" antibodies that target incorrect epitopes while inducing the production of "protective" antibodies that target certain epitopes (Figure 2A).

[0025] In a 2015 analysis of the World Health Organization's International Clinical Trials Registry Platform, 9 candidate RSV vaccines that had been clinically evaluated since 2008 were identified, but none of them advanced beyond phase 2 trials (Broadbent et al., Influenza Other Respir Viruses. 2015;9(4):169-178). Among these, only 3 trials were completed and only 1 reported results. The vaccine, called MEDI-559, appeared to reduce RSV infection, but the respiratory symptoms were too high to further test (Malkin et al., PLoS One. 2013;8(10):e77104). These data suggest that MEDI-559 induced the production of protective antibodies but probably also induced the production of pathogenic antibodies (Figure 2B).

[0026] Other "improved" vaccination strategies involve changing the formulation administered to patients. These strategies include alternatives to inactivating / attenuating the virus and changes to adjuvants aimed at increasing the inflammatory response (Broadbent et al., Influenza Other Respir Viruses. 2015;9(4):169-78; Garg et al., The Journal of general virology. 2014;95(Pt 5):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 led to increased protective antibodies in animal models, but these "improved" RSV vaccines may follow the same fate as MEDI-559 because they may induce pathogenic antibodies.

[0027] In an effort to focus the immune response on epitopes targeted by protective antibodies, a recent approach has been to graft a single RSV epitope onto a non-RSV scaffold (Figure 2C). This approach removes the possibility of pathogenic antibodies specific for other RSV epitopes because such antibodies do not exist on the scaffold. RSV epitope scaffold vaccination in rhesus monkeys produced neutralizing antibodies in some animals, but only after 3-5 injections (Correia et al., Nature. 2014;507(7491):201-6).

[0028] Approaches have also been developed to bypass vaccination and directly provide protective antibodies. The only clinically approved preventive measure for RSV is the 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(3 Pt 1):531-7). Unfortunately, because the cost of palivizumab for 5 months is $10,000, its use has been restricted to children at high risk of severe RSV infection (Meissner & Kimberlin, Pediatrics. 2013;132(5):915-8). Other RSV-specific antibodies designed to last up to 1 year are currently under clinical evaluation (Influenza Other Respir Viruses. 2015;9(4):169-78). However, annual antibody reinjections are not feasible for lifelong protection.

[0029] To eliminate the need for lifelong injections, a method has been developed to transfer genes encoding protective antibodies into muscle cells using an adenovirus vector (Figure 3B) (Schnepp & Johnson, Curr Opin HIV AIDS. 2014;9(3):250-256). Promisingly, mice were partially protected from RSV infection by adenovirus-mediated expression of palivizumab (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 required to achieve protective levels of antibodies (24). High doses are needed because antibody expression 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;236:464-473; Hibi & Dosch, Eur J Immunol. 1986;16(2):139-145). B cells achieve this high rate of secretion by completely reprogramming their protein production machinery to focus on antibody secretion. Without a major revolution in manufacturing capabilities, adenovirus-mediated transfer of antibody genes into muscle cells is not a practical option for RSV prevention.

[0030] Another approach is to use a lentivirus vector to integrate genes encoding protective antibodies into the genome of hematopoietic stem cells and then induce these cells to differentiate into antibody-secreting B cells (Figure 3C). One limitation of this approach is that antibody gene insertion is random, bringing with it the risk of off-target gene effects that can cause disease. A second limitation of this approach is that long-term two-month in vitro culture conditions are required to induce differentiation of hematopoietic stem cells into antibody-secreting cells (Luo et al., Blood. 2009;113(7):1422-1431). The final limitation is that this strategy does not generate a source of antibodies that can be increased immediately upon infection. Thus, if there are not many antibody-secreting cells or they are not long-lived, protection against infection will be inadequate.

[0031] In certain embodiments, the present disclosure provides for avoiding vaccination and / or genetically engineering B cells to eliminate the need for repeated therapeutic antibody injections by expressing a selected antibody (e.g., an antibody against an infectious pathogen such as palivizumab; Figure 4A). 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 proliferative and functional potential and can enter the germinal center response to improve their binding ability. B1 B cells express BCRs and migrate to different locations such as the peritoneal cavity. B1 B cells rapidly differentiate into antibody-secreting cells upon stimulation through the BCR and do not require T cell signals for optimal function. Marginal zone B cells are mostly located in the marginal zone of the spleen and rapidly differentiate into antibody-secreting cells upon stimulation through the BCR. Marginal zone B cells also do not require signals from T cells for optimal function. Manipulating one or more of these subsets of B cells can create a long-lived source of baseline antibodies for treating ongoing or immediate infections and induced antibodies in the event of future reinfection. Figure 4B shows a related strategy for simultaneous protection against multiple pathogens utilizing the teachings of the present disclosure, and Figure 5 depicts the hypothesized secretion of palivizumab by exemplary B cell subtypes in the presence and absence of infection.

[0032] The present disclosure provides for genetic engineering of B cells by inserting a gene construct containing a transgene into an endogenous antibody locus that has been 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 enables robust production of the selected antibody by utilizing the endogenous antibody expression regulatory mechanisms. A transgene may refer to a portion of DNA encoding a heterologous (i.e., foreign) protein. A gene construct may refer to an artificially constructed segment of nucleic acid that is intended to be introduced into a cell to enable expression of a heterologous protein.

[0033] In certain embodiments, the present disclosure provides B cells that have been modified to express a selected antibody. Antibodies are produced from two genes, a heavy chain gene and a light chain gene. Generally, an antibody contains two identical copies of the heavy chain and two identical copies of the light chain (see, e.g., FIG. 6B). The heavy chain is composed of two larger subunits, and each heavy chain contains a VDJ segment and a constant region (shown as “C” in FIG. 6B). The VDJ segment (or VDJ) refers to a unique pairing of V, D, and J gene segments that encodes a unique portion of the antibody heavy chain that binds to an epitope on a pathogen. Thus, V refers to one of the gene segments that randomly pairs with D and J segments that encode a unique portion of the antibody heavy chain that binds to an epitope on a pathogen. Similarly, D refers to one of the gene segments that randomly pairs with V and J segments that encode a unique portion of the antibody heavy chain that binds to an epitope on a pathogen. Finally, J refers to one of the gene segments that randomly pairs with V and D segments that encode a unique portion of the antibody heavy chain that binds to an epitope on a pathogen. There are several V segments, D segments, and J segments that can combine in various different combinations to form a specific VDJ segment of a specific heavy chain (see, e.g., FIG. 7).

[0034] Each B cell pairs 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 change to express different C region segments following activation, and different C regions confer different functions to the antibody. For example, one C gene segment encodes ε, and an antibody expressing ε is “IgE”. IgE-type antibodies often bind to cells of the body and mediate allergic reactions. Antibodies that express the α C region are IgA antibodies, antibodies that express the γ C region are IgG antibodies, and antibodies that express the μ C region are IgM antibodies. The human genome contains a single heavy chain locus, which is located on chromosome 14.

[0035] Referring again to FIG. 6B, the light chain of the antibody (IgL) 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 can include a single immunoglobulin constant domain. Humans express two different light chains: Igκ is encoded by the immunoglobulin kappa locus on chromosome 2, and Igλ is encoded by the immunoglobulin lambda locus on chromosome 22.

[0036] FIG. 6 depicts a schematic diagram of the endogenous B cell genome encoding the IgH chain and IgL chain. FIGS. 8A and 8B depict a first schematic diagram of where to insert an exogenous gene construct according to the present disclosure to achieve expression of a selected antibody. FIG. 8A depicts inserting a gene construct containing [a stop signal, the IgL chain of the selected antibody (here, PV), a skipping element (here, 2A), and the VDJ segment of the heavy chain] into the endogenous IgH genome between the endogenous VDJ segment and the endogenous C region coding segment. This approach 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. Expression of an antibody containing the endogenous C region can be useful because, for example, B cells modified by this expression can regulate C region expression based on natural B cell activation and maturation states. For example, alternative splicing in the constant region of the heavy chain locus allows modified B cells to switch between expression of membrane-bound antibody and secreted antibody. This approach also allows expression of exogenous VDJ without the need for excision of endogenous VDJ. This feature is beneficial because VDJ is a relatively large segment of DNA and its excision can have a negative impact on cell function.

[0037] FIG. 9 depicts a more detailed similar schematic diagram regarding the structure and function of the endogenous B cell genome and how the present disclosure utilizes this structure and function to achieve expression of a selected antibody. The promoter region is necessary to achieve transcription of gene segments. The heavy chain variable region (V H)The promoter is selectively active in the B cell lineage and contains a TATA box, an Inr element, and an octamer element within 100 base pairs (bp) of the transcription start site. V H Promoter activity is under proximal-dependent regulation by the Eμ enhancer element (gray oval) of the endogenous B cell genome and an enhancer element (gray circle) located at the 3' end of the proximal heavy chain locus of the heavy chain α constant gene. The Eμ enhancer element is an intron region (40 - 1500 bp in length) of DNA within a 700 bp intron between the J heavy chain segment and the C mu (μ) segment of the immunoglobulin heavy chain locus. The Eμ enhancer element can bind to activator proteins to increase or activate the transcription of the heavy chain gene. The sequence of the human Eμ enhancer element is provided as SEQ ID NO: 85 in FIG. 11. The sequence of the mouse Eμ enhancer element is provided as SEQ ID NO: 86 in FIG. 13A.

[0038] Inserting a gene construct containing a V H promoter between the endogenous variable heavy region and the endogenous Eμ enhancer can reduce or block the transcriptional activation from the endogenous V H promoter. This is because the Eμ enhancer initiates transcription at the most proximal upstream promoter. In this way, the expression of endogenous VDJ can be blocked without the need for the removal of such large DNA segments (which, as shown, involves problems for cell function and survival). In certain embodiments, VDJ recombination removes the genetic material between the V H promoter and the Eμ enhancer, such that the enhancer is placed at an appropriate distance from the exogenous promoter of the gene construct disclosed herein and activates transcription starting from the promoter in the inserted gene construct. 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 HThe promoter includes a natural light chain promoter for IgK or IgL, a natural human IgH promoter, the spleen focus-forming virus promoter SFFV, the J558h10 promoter or the IgVH1-69 promoter.

[0039] Figure 10 provides a schematic diagram of the addition of target regions for gene construct insertion. These target regions encompass two conserved regions present in all B cells: from the terminal J gene segment (IGHJ6 in humans, IGHJ4 in mice) to the heavy chain intron enhancer (Eμ) and from Eμ to the repetitive sequences associated with DNA switch recombination.

[0040] In certain embodiments, the region of the endogenous B cell genome targeted for insertion of the gene construct is upstream of the Eμ enhancer of SEQ ID NO: 85 or 86. Figures 11A - 14B provide specific sequences that can be targeted for gene construct insertion to achieve the expression of selected antibodies disclosed herein.

[0041] Figure 11A shows the human DNA sequence from IGHJ6 to the Eμ intron enhancer (SEQ ID NO: 1; >hg38_dna range = chr14:105862523~105863244 5’pad = 0 3’pad = 0 strand = - repeat masking = none). Figure 11B provides an exemplary range up to the targets (e.g., gRNA sites) within this sequence including SEQ ID NOs: 5 - 24 and the associated gRNA sequences (SEQ ID NOs: 88, 89 and 290 - 307). By way of example, in certain embodiments, the sgRNA of SEQ ID NO: 88 (see also Figure 25A) can be used to target the gRNA site of SEQ ID NO: 7. In certain embodiments, the sgRNA of SEQ ID NO: 89 (see also Figure 25A) can be used to target the gRNA site of SEQ ID NO: 10.

[0042] Figure 12A provides the human DNA sequence for region 2: from the Eμ intron enhancer to the switch region (SEQ ID NO: 2; >hg38_dna range = chr14:105860383~105861690 5’pad = 0 3’pad = 0 strand = -). Figure 12B provides an exemplary range up to the target (e.g., gRNA site) within this sequence that includes SEQ ID NOs: 25 - 44 and the associated gRNA sequences (SEQ ID NOs: 308 - 327).

[0043] Figure 13A provides the mouse DNA sequence for region 1: from IGHJ4 to the Eμ intron enhancer (SEQ ID NO: 3; >mm10_dna range = chr12:113427973~113428554 5’pad = 0 3’pad = 0 strand = - repeat masking = none). Figure 13B provides an exemplary range up to the target (e.g., gRNA site) within this sequence that includes SEQ ID NOs: 45 - 64 and the associated gRNA sequences (SEQ ID NO: 87, and 328 - 346). As an example, in certain embodiments, the sgRNA of SEQ ID NO: 87 (see also Figure 25A) can be used to target the gRNA site of SEQ ID NO: 46.

[0044] Figure 14A provides the mouse DNA sequence for region 2: from the Eμ intron enhancer to the switch region (SEQ ID NO: 4; >mm10_dna range = chr12:113425446~113426973 5’pad = 0 3’pad = 0 strand = - repeat masking = none). Figure 14B provides an exemplary range up to the target (e.g., gRNA site) within this sequence that includes SEQ ID NOs: 65 - 84 and the associated gRNA sequences (SEQ ID NOs: 347 - 366).

[0045] Accordingly, in certain embodiments, the present disclosure provides for targeted insertion of a gene construct comprising (i) a promoter and (ii) a transgene encoding a portion of a selected antibody in an intron region that is constant (before and after recombination) in all B cells, wherein (i) the promoter is positioned relative to an enhancer element that interacts with the promoter, and (ii) the transgene is in an orientation such that the endogenous heavy chain VDJ sequences of the B cell are not expressed. In certain embodiments, the encoded portion of the selected antibody comprises the VDJ segments of the entire light and heavy chains of the antibody. 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. Certain embodiments of the gene construct can also comprise or encode a signal peptide, a flexible linker, a skipping element, and / or a splice junction.

[0046] 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) (Figures 6A, 6B). This means that in certain embodiments, the positions of both genes must be modified simultaneously in order to correctly express the selected antibody. However, the present disclosure also provides strategies for producing a functional selected antibody without the need to modify both gene positions. One approach that enables this is through the use of sequences that allow for antibody expression through a single construct. In certain embodiments, this is achieved by including a skipping element within the gene construct. One example of a skipping element is a self-cleaving peptide such as self-cleaving "2A". The 2A peptide functions by skipping the synthesis of a peptide bond at a limited position on the ribosome and producing two proteins from one mRNA. The 2A sequence is short (e.g., 20 amino acids), facilitating its use in constructs of limited size, and the proteins are produced in 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).

[0047] In certain embodiments, the gene construct includes an internal ribosome entry site (IRES) sequence. The IRES can be located upstream of the heavy chain VDJ of the gene construct. The IRES is a non-coding structural RNA sequence that causes the ribosome to initiate translation at a second internal site on the mRNA molecule, resulting in the production of two proteins from one mRNA. However, IRES-driven translation is not as efficient as 2A-driven translation, and the expression of the second protein in the transcript is lower.

[0048] In certain embodiments, the gene construct encodes a flexible linker between the light chain portion of a selected antibody and the heavy chain portion of the selected antibody. The linker can be a series of amino acids that flexibly link one protein domain to another such that the linked sequences interact to form a functional unit.

[0049] In certain embodiments, the flexible linker can comprise one or more combinations of glycine and serine that impart 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), where n = 1 to 100 and all integers therebetween. In certain 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 certain embodiments, the Gly-Ser linker comprises 50 to 80 amino acids. In certain embodiments, the Gly-Ser linker comprises 54, 57, or 60 amino acids. In certain embodiments, the Gly-Ser linker is encoded by SEQ ID NO: 116. In certain embodiments, the Gly-Ser linker comprises SEQ ID NO: 122.

[0050] 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, and Glu and Lys are added to improve solubility. Bird, RE et al., Science, 1988; 242:423-426. In certain embodiments, n = 1 to 100 and all integers therebetween. In certain 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 certain embodiments, these linkers comprise 50 to 80 amino acids.

[0051] Certain embodiments include splice junctions that enable splicing between RNA encoded by a gene construct and RNA encoded by an endogenous heavy chain constant region. In certain embodiments, the gene construct includes a splice junction sequence at its 3' end. Splicing can refer to the removal of introns and the joining of exons into a single unit by an RNA / protein complex known as the spliceosome. A splice junction refers to an intron sequence that is directly adjacent to an exon. The splice junction at the 3' end of an exon can include a splice donor site. The splice donor site sequence typically begins with "GU". In certain embodiments, the splice junction may include an intron of 40 - 80 bp following the last exon of VDJ. In certain embodiments, the splice junction includes an intron of 40 - 80 bp adjacent to the 3' end of the human IGHJ1 gene segment or the mouse IGHJ3 gene. In certain embodiments, the splice junction includes CAG / gtaagt, and cleavage and splicing occur after the capital letter G (indicated by the "splice" annotation). In certain embodiments, the splice junction includes CAG / gtgagt. CA forms the end of a serine codon, and G begins the first codon from the constant region. In certain embodiments, a splice junction with flanking sequences includes SEQ ID NO: 124 or 151 in the gene construct for insertion into the human locus. In certain embodiments, a splice junction with flanking sequences includes SEQ ID NO: 139 in the gene construct for insertion into the mouse locus.

[0052] The gene 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 MDLLHKNMKHLWFFLLLVAAPRWVLS (SEQ ID NO: 192); and signal peptides derived from human IgL light chains such as MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 193); and MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 194). In certain embodiments, the signal peptide is encoded by SEQ ID NO: 112 and the gene construct includes SEQ ID NO: 118 for insertion into the human locus. In certain embodiments, the signal peptide is encoded by SEQ ID NO: 129 and the gene construct includes SEQ ID NO: 134 for insertion into the mouse locus. See also FIGS. 25B - 25I and Haryadi R et al., PLoS One v.10(2); 2015 PMC4338144.

[0053] As shown, certain embodiments of the present disclosure utilize the insertion of an exogenous gene construct at a targeted location within the endogenous B cell genome. In certain embodiments, such targeted insertion can be facilitated by including homology regions at one or both ends of the gene construct. The homology regions (i.e., homology stretches or homology arms) are homologous to the sequences at the desired insertion site. In certain embodiments, a homology arm refers to a segment of DNA that is 100% identical to a region of DNA contained within and modified in the gene construct. In certain embodiments, 100% identity may not be required to achieve targeted insertion (e.g., at least 90% identity may be sufficient).

[0054] The homology region aligns a gene construct adjacent to the targeted gene region, and a portion of the DNA from the gene construct is exchanged into the region cleaved by the gene editing technique. In certain embodiments, the gene construct may include an upstream genomic homology end with genomic homology of 20 to 1,500 bp and a downstream genomic homology end with genomic homology of 20 to 1,500 bp. The homology region may provide, for example, a "homology stitch" as shown in FIG. 15A, and this stitch can mediate the insertion of the gene construct into the targeted insertion site. In certain embodiments, the upstream genomic homology end and the downstream genomic homology end may include sequences homologous to the genomic sequence between the heavy chain VDJ region and the heavy chain Eμ enhancer element. In certain embodiments, the homology region may particularly include 20 to 50 base pairs; 300 to 500 base pairs; 350 to 550 base pairs; 900 to 1,000 base pairs, or 400 to 600 base pairs. In certain embodiments, the homology region may particularly include 30 to 40 base pairs (e.g., 36 base pairs); 445 to 455 base pairs (e.g., 450 base pairs); 495 to 510 base pairs (e.g., 503 base pairs); and / or 960 to 980 base pairs (e.g., 968 base pairs). In certain embodiments, the homology regions for use in mouse gene constructs include SEQ ID NOs: 90, 91, 96, 97, 127, 140, 142, 143, 170, and 171. In certain embodiments, the homology regions for use in human gene constructs include SEQ ID NOs: 92-95, 98-101, 110, 125, 153, 173, and 174.

[0055] In certain embodiments, the gene construct also encodes a tag sequence. The tag sequence can be useful, for example, such that cells expressing the gene construct can be identified and / or sorted during the genetic modification process and / or such that the cells can be controlled following administration to a subject. For example, in certain embodiments, the tag sequence can be useful to track and / or terminate genetically modified cells following administration to a subject. Exemplary tags include STREPTAG® (GmbH, LLC, Gottingen, DE), STREP® Tag II (WSHPQFEK (SEQ ID NO: 195)) or any variant thereof; see, e.g., U.S. Patent No. 7,981,632), His tag, Flag tag (DYKDDDDK (SEQ ID NO: 196)), Xpress tag (DLYDDDDK (SEQ ID NO: 197)), Avi tag (GLNDIEAQKIEWHE (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)), Softag 3 (TQDPSRVG (SEQ ID NO: 203)), and V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 204)).

[0056] In certain embodiments, the present disclosure provides a gene construct for the expression of a selected antibody 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 skipping element and / or (vi) a splice junction and / or (vii) a homology arm and / or (viii) a linker and / or (ix) a tag.

[0057] Certain 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 a selected antibody heavy chain; and (vi) a splice junction.

[0058] Certain 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 a selected antibody heavy chain; (vi) a splice junction; and (vii) a homology arm.

[0059] Certain 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 a selected antibody heavy chain; (vi) a splice junction; (vii) a homology arm; and (viii) a tag.

[0060] Figure 15B depicts additional examples of DNA repair templates. Examples of DNA repair templates that can be used include synthetic DNA templates and adeno-associated viruses. In certain embodiments, the synthetic DNA template can comprise or encode double-stranded DNA (dsDNA) that includes a promoter and a selected antibody moiety flanked by 20 to 1,500 base pairs of homology to a target site in the genome. In certain embodiments, the synthetic DNA template can comprise or encode single-stranded DNA (ssDNA) that includes a promoter and a selected antibody moiety flanked by 10 to 80 base pairs, or 400 to 1,000 base pairs of homology to a target site in the genome. In certain embodiments, the synthetic DNA template can comprise both dsDNA and ssDNA that are ultimately modified by phosphorylation to increase DNA ligation efficiency. In certain embodiments, both the dsDNA and ssDNA are ultimately modified with phosphorothioate bonds to increase stability and prevent endonuclease digestion.

[0061] In certain embodiments, the adeno-associated virus can include a segment encoding a synthetic antibody moiety flanked by 20 to 1,500 base pairs of homology to a target site in the genome. In certain embodiments, the promoter and the synthetic antibody moiety coding sequence can be juxtaposed by adapting the homology sequence to the target site in the genome.

[0062] In certain embodiments, gene constructs (e.g., homology stitches, synthetic DNA templates) comprising DNA repair machinery can be delivered using gene editing systems such as CRISPR, TALEN, megaTAL, zinc finger nucleases, and / or adeno-associated virus, which are described in more detail below. For example, the genome targeting elements, genome cutting elements, and gene constructs described herein can be administered to B cells.

[0063] As a specific example of the application of the present disclosure, B cells may be modified to express a paritumab antibody. The B cells may be modified with a gene construct comprising an 80 bp homology arm linked to a heavy chain promoter upstream of the complete light chain (IgLPV) and VDJ heavy chain gene segment (VDJPV) derived from paritumab separated by a 2A peptide. Here, the 2A peptide is included to induce a ribosome skipping event (Donnelly et al., The Journal of general virology. 2001; 82(Pt 5):1013-25), by which the heavy and light chains can be produced as separate subunits that normally associate to form the selected antibody. In certain embodiments, a stop codon can be included upstream of the inserted heavy chain promoter such that any potential transcription of the endogenous heavy chain variable region is terminated.

[0064] FIG. 16 depicts an example of a "modified" B cell genome, and FIG. 17 depicts the resulting B cell population expressing the selected antibody.

[0065] The following paragraphs provide further details regarding (i) exemplary selected antibodies and sequences; (ii) gene editing techniques and cell sorting; (iii) formulation of modified B cells; and (iv) methods of use.

[0066] (i) Exemplary selected antibodies and sequences. In certain embodiments, the selected antibody is an antibody capable of providing a protective effect against a pathogen or condition (e.g., an autoimmune disease). In certain embodiments, the selected antibody is an anti-RSV antibody, an anti-HIV antibody, an anti-dengue virus antibody, an anti-Bordatella pertussis antibody, an anti-hepatitis C antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, an anti-metapneumovirus (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.

[0067] In certain embodiments, the selected antibody is a chimeric antibody. In certain embodiments, a chimeric antibody refers to a synthetic antibody that comprises (i) at least one portion encoded by the endogenous genome of a B cell, and (ii) at least one portion encoded by an inserted gene construct. In certain embodiments, a chimeric antibody comprises an endogenous heavy chain constant domain, foreign immunoglobulin variable and constant light chains, and a foreign variable heavy chain.

[0068] The following antibodies and sequences are useful for conferring target binding to a selected antibody against a target pathogen or antigen (Kabat numbering is intended unless otherwise specified).

[0069] An exemplary anti-RSV antibody is palivizumab, which targets the RSV fusion protein and is used to prevent or reduce RSV infection.

[0070] In certain embodiments, the anti-RSV antibody is

[0071] [Chemical formula] A variable heavy chain array containing, and

[0072] [Chemical formula] A murine palivizumab containing a variable light chain array containing.

[0073] An additional exemplary anti-RSV antibody is human palivizumab, and

[0074] [Chemical formula] A variable light chain array containing, and

[0075] [Chemical formula] Contains a variable heavy chain array containing.

[0076] Within the variable heavy and light chains, segments called complementarity-determining regions (CDRs) direct 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).

[0077] An additional exemplary anti-RSV antibody is described in U.S. Patent No. 9,403,900. This anti-RSV antibody includes a variable heavy chain containing a CDRH1 sequence containing GASINSDNYYWT (SEQ ID NO: 207), a CDRH2 sequence containing HISYTGNTYYTPSLKS (SEQ ID NO: 208), and a CDRH3 sequence containing CGAYVLISNCGWFDS (SEQ ID NO: 209); and a variable light chain containing a CDRL1 sequence containing QASQDISTYLN (SEQ ID NO: 210), a CDRL2 sequence containing GASNLET (SEQ ID NO: 211), and a CDRL3 sequence containing QQYQYLPYT (SEQ ID NO: 212).

[0078] Exemplary anti-RSV antibodies also include AB1128 (available from MILLIPORE) and ab20745 (available from ABCAM).

[0079] An example of an anti-HIV antibody is 10E8, which is a broad neutralizing antibody that binds to gp41. The 10E8 anti-HIV antibody comprises a variable heavy chain comprising a CDRH1 sequence comprising GFDFDNAW (SEQ ID NO: 213), a CDRH2 sequence comprising ITGPGEGWSV (SEQ ID NO: 214), and a CDRH3 sequence comprising TGKYYDFWSGYPPGEEYFQD (SEQ ID NO: 215); and a variable light chain comprising a CDRL1 sequence comprising TGDSLRSHYAS (SEQ ID NO: 216), a CDRL2 sequence comprising GKNNRPS (SEQ ID NO: 217), and a CDRL3 sequence comprising SSRDKSGSRLSV (SEQ ID NO: 218).

[0080] Another example of an anti-HIV antibody is VRC01, which is a broad neutralizing antibody that binds to the CD4 binding site of gp120. The VRC01 antibody comprises a variable heavy chain comprising a CDRH1 sequence comprising GYEFIDCT (SEQ ID NO: 219), a CDRH2 sequence comprising KPRGGAVN (SEQ ID NO: 220), and a CDRH3 sequence comprising RGKNCDYNWDFEHW (SEQ ID NO: 221); and a variable light chain comprising a CDRL1 sequence comprising QYGS, a CDRL2 sequence comprising SGS, and a CDRL3 sequence comprising QQYEF (SEQ ID NO: 222).

[0081] Exemplary anti-HIV antibodies also include ab18633 and 39 / 5.4A (available from ABCAM); and H81E (available from THERMOFISHER).

[0082] An example of an anti-dengue virus antibody is antibody 55 described in US Patent Application Publication No. 20170233460, a variable heavy chain comprising a CDRH1 sequence containing EVQLHQSGAELVKPGASVKLSCTVSGFNIK (SEQ ID NO: 223), a CDRH2 sequence containing WVKQRPEQGLEWI (SEQ ID NO: 224), and a CDRH3 sequence containing ATIKADTSSNTAYLQLISLTSEDTAVYYCAF (SEQ ID NO: 225); and a variable light chain comprising a CDRL1 sequence containing DIQMTQSPASLSVSVGETVTITC (SEQ ID NO: 226), a CDRL2 sequence containing WYQQKQGKSPQLLVY (SEQ ID NO: 227), and a CDRL3 sequence containing GVPSRFSGSGSGTQYSLKINSLQSEDFGTYYC (SEQ ID NO: 228).

[0083] An additional example of an anti-dengue virus antibody is DB2-3 described in US Patent No. 8,637,035, a variable heavy chain comprising a CDRH1 sequence containing YTFTDYAIT (SEQ ID NO: 229), a CDRH2 sequence containing GLISTYYGDSFYNQKFKG (SEQ ID NO: 230), and a CDRH3 sequence containing TIRDGKAMDY (SEQ ID NO: 231); and a variable light chain comprising a CDRL1 sequence containing RSSQSLVHSNGNTYLH (SEQ ID NO: 232), a CDRL2 sequence containing KVSNRFS (SEQ ID NO: 233), and a CDRL3 sequence containing SQSTHVPYT (SEQ ID NO: 234). Examples of anti-dengue virus antibodies also include ab155042 and ab80914 (both available from ABCAM).

[0084] Examples of anti-pertussis antibodies are described in US Patent No. 9,512,204,

[0085] [Chemical formula] a variable heavy chain comprising, and

[0086] [Chemical formula] a variable light chain comprising.

[0087] Examples of anti-hepatitis C antibodies include a variable heavy chain comprising a CDRH1 sequence comprising SYGMHW (SEQ ID NO: 237), a CDRH2 sequence comprising VIWLDGSNTYYADSVKGR (SEQ ID NO: 238), and a CDRH3 sequence comprising ARDIFTVARGVIIYFDY (SEQ ID NO: 239); and a variable light chain comprising a CDRL1 sequence comprising RASQSVSSYLA (SEQ ID NO: 240), a CDRL2 sequence comprising DASNRAT (SEQ ID NO: 241), and a CDRL3 sequence comprising QQRSNWVT (SEQ ID NO: 242). Examples of anti-hepatitis C antibodies also include MAB8694 (available from MILLIPORE) and C7-50 (available from ABCAM).

[0088] Examples of anti-influenza virus antibodies are described in U.S. Patent No. 9,469,685 and include a variable heavy chain comprising a CDRH1 sequence comprising GMTSNSLA (SEQ ID NO: 243), a CDRH2 sequence comprising IIPVFETP (SEQ ID NO: 244), and a CDRH3 sequence comprising ATSAGGIVNYYLSFNI (SEQ ID NO: 245); and a variable light chain comprising a CDRL1 sequence comprising QTITTW (SEQ ID NO: 246), a CDRL2 sequence comprising KTS, and a CDRL3 sequence comprising QQYSTYSGT (SEQ ID NO: 247). Examples of anti-influenza virus antibodies also include C102 (available from THERMOFISHER).

[0089] Exemplary anti-MPV antibodies include MPE8.

[0090] Exemplary anti-CMV antibodies include MCMV5322A, MCMV3068A, LJP538, and LJP539. RG7667 comprises a mixture of MCMV5322A and MCMV3068A, and CSJ148 comprises a mixture of LJP538 and LJP539. See, 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).

[0091] Examples of anti-EBV antibodies include a variable heavy chain comprising an AMM01 CDRH1 sequence containing YTFIHFGISW (SEQ ID NO: 248), an AMM01 CDRH2 sequence containing IDTNNGNTNYAQSLQG (SEQ ID NO: 249), and an AMM01 CDRH3 sequence containing RALEMGHRSGFPFDY (SEQ ID NO: 250); and a variable light chain comprising an AMM01 CDRL1 sequence containing GGHNIGAKNVH (SEQ ID NO: 251), an AMM01 CDRL2 sequence containing YDSDRPS (SEQ ID NO: 252), and an AMM01 CDRL3 sequence containing CQVWDSGRGHPLYV (SEQ ID NO: 253).

[0092] Examples of anti-HSV antibodies include HSV8-N and MB66.

[0093] Examples of anti-Clostridium difficile antibodies include actoxumab and bezlotoxumab. See, e.g., Wilcox et al., N Engl J Med 376(4):305-317 (2017).

[0094] Commercially available anti-TNF antibodies include infliximab (Remicade® Centocor, Inc., Malvern, PA, with Inflectra® Pfizer, Kent, UK, and Ixifi® Pfizer, New York, NY), adalimumab (Humira® Abbott Laboratories, Abbott Park, IL, with Amjevita® Amgen, Thousand Oaks, CA, and Cyltezo® Boehringer Ingelheim Int’l, Ingelheim, DE), golimumab (Simponi® Johnson & Johnson Corp., New Brunswick, NJ), etanercept (Enbrel® Immunex Corp, Thousand Oaks, CA, with Erelzi® Novartis AG, Basel, CH), and certolizumab pegol (Cimzia® UCB Pharma, Brussels, BE).

[0095] In certain embodiments, 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 certain embodiments, the heavy chain of infliximab begins with EVKLEESGGGLVQPGGSMK (SEQ ID NO: 254) and the light chain begins with DILLTQSPAILSVSPGER (SEQ ID NO: 255).

[0096] In certain embodiments, infliximab includes a variable heavy chain comprising a CDRH1 sequence including IFSNHW (SEQ ID NO: 256), a CDRH2 sequence including RSKSINSATH (SEQ ID NO: 257), and a CDRH3 sequence including NYYGSTY (SEQ ID NO: 258); and a variable light chain comprising a CDRL1 sequence including FVGSSIH (SEQ ID NO: 259), a CDRL2 sequence including KYASESM (SEQ ID NO: 260), and a CDRL3 sequence including QSHSW (SEQ ID NO: 261).

[0097] In certain embodiments, 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).

[0098] In certain embodiments, certolizumab 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).

[0099] Numerous additional antibody sequences available for use within the teachings of the present disclosure are available and are known to those of skill in the art. Sequence information for commercially available antibodies can be found in the Drug Bank database, CAS Registry, and / or the RSCB Protein DATA Bank. Additionally, nucleic acid sequences encoding selected portions of the antibodies described herein can be readily derived by one of skill in the art.

[0100] (ii) Gene editing techniques and cell sorting. Gene editing systems enable control over the target site of gene therapy. Within the teachings of the present disclosure, any gene editing system capable of accurate sequence targeting and modification can be used. These systems typically include a targeting element for accurate targeting and a cutting element for cleaving the target gene 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 can be linked, for example, by nanoparticles. Alternatively, the targeting element and the cutting element can be joined together to form a single dual-purpose molecule. If the insertion of a therapeutic nucleic acid sequence is intended, the system can also include a homologous recombination repair template associated with the gene construct (i.e., the homology arms described above). However, as will be detailed further below, different gene editing systems can incorporate different components and configurations while maintaining the ability to accurately target, cleave, and modify a selected genomic site.

[0101] Certain embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a type of site-specific nuclease engineered to bind and cleave DNA at specific locations. Using ZFNs, double-strand breaks (DSBs) can be introduced at specific sites within a DNA sequence, thereby enabling ZFNs to target unique sequences within the genome in a variety of different cells. Further, following the double-strand break, homologous recombination repair (HDR) or non-homologous end joining (NHEJ) occurs to repair the DSB, and thus genome editing becomes possible.

[0102] ZFNs are synthesized by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. The DNA-binding domain contains 3 to 6 zinc finger proteins that are transcription factors. The DNA cleavage domain contains, for example, the catalytic domain of the FokI endonuclease. The FokI domain functions as a dimer that requires two constructs with unique DNA-binding domains to a site on the target sequence. The FokI cleavage domain cleaves within a 5- to 6-base pair spacer sequence that separates two inverted half-sites.

[0103] Additional information regarding ZFNs can be found in Kim et al., Proceedings of the National Academy of Sciences of the United States of America 93, pp. 1156-1160 (1996); Wolfe et al., Annual review of biophysics and biomolecular structure 29, pp. 183-212 (2000); Bibikova et al., Science 300, 764 (2003); Bibikova et al., Genetics 161, pp. 1169-1175 (2002); Miller et al., The EMBO journal 4, pp. 1609-1614 (1985); and Miller et al., Nature biotechnology 25, pp. 778-785 (2007).

[0104] Certain embodiments can use transcription activator-like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins that contain a transcription activator-like effector (TALE) DNA-binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing DSBs in DNA, thereby inducing repair mechanisms in cells. Generally, two TALENs must bind and be adjacent to each side of the target DNA site for the DNA cleavage domains to dimerize and induce a DSB. When an exogenous double-stranded donor DNA fragment is present, the DSB is repaired in the cell by NHEJ or HDR.

[0105] As shown, TALENs are engineered, for example, to bind to a target sequence in the endogenous genome and cleave DNA at the position of the target sequence. The TALE of TALEN is a DNA-binding protein secreted by Xanthomonas bacteria. The DNA-binding domain of TALE contains highly conserved 33 or 34 amino acid repeats, with diverse residues at the 12th and 13th positions of each repeat. These two positions are called repeat variable diresidues (RVDs) and show a strong correlation with specific nucleotide recognition. Thus, targeting specificity can be improved by changing the amino acids of the RVDs and incorporating non-conventional RVD amino acids.

[0106] Examples of DNA cleavage domains that can be used in TALEN fusions are wild-type and mutant FokI endonucleases. For additional information regarding TALENs, see Boch et al., Science 326, pages 1509 - 1512 (2009); Moscou & Bogdanove, Science 326, 1501 (2009); Christian et al., Genetics 186, pages 757 - 761 (2010); and Miller et al., Nature biotechnology 29, pages 143 - 148 (2011).

[0107] Certain embodiments utilize MegaTAL as a gene editing agent. MegaTAL has a single-stranded rare-cut nuclease structure in which the TALE is fused to the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single polypeptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to TALENs, MegaTAL requires only delivery of a single polypeptide chain for functional activity.

[0108] In certain embodiments, the endogenous B cell genome can be targeted by a 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 repeats of base sequences. In the context of the prokaryotic immune system, each repeat is followed by a short segment of spacer DNA belonging to a foreign genetic element to which the prokaryote has been exposed. This CRISPR array of repeats separated by spacers can be transcribed into RNA. The RNA is processed to a mature form and can associate with nucleases such as cas (CRISPR-associated) nucleases. A CRISPR-Cas system comprising an RNA having a sequence capable of hybridizing to a foreign genetic element and a Cas nuclease can then recognize and cleave these foreign genetic elements in the genome.

[0109] The CRISPR-Cas system does not require the generation of customized proteins to target specific sequences. Rather, a single Cas enzyme can be programmed by short guide RNA molecules (crRNAs) to recognize specific DNA targets. The CRISPR-Cas systems of bacterial and archaeal adaptive immunity exhibit extreme diversity in protein composition and genomic locus structure. The CRISPR-Cas system loci have over 50 gene families and no strictly universal genes, showing rapid evolution and extreme diversity in locus structure. To date, a wide range of cas genes with 395 profiles for 93 Cas proteins have been identified using a multi-faceted approach. The classification includes signature gene profiles plus signatures of locus structure. A new classification of CRISPR-Cas systems has been proposed, which broadly divides these systems into two classes: class 1, which has multi-subunit effector complexes, and class 2, which has single-subunit effector modules exemplified by the Cas9 protein.

[0110] 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 and activates the DSB repair mechanism. DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination repair (HDR), or microhomology-mediated end joining (MMEJ). NHEJ can repair DSBs with no homology (<5 bp) between the two ends that bind during repair, HDR can repair DSBs with a large region (100 or more nucleotides) of homology between the ends bound during repair, and MMEJ can repair DSBs with a small (5-50 bp) region of homology between the ends bound during repair. Another type of Cas9, known as Cas9D10A, includes a mutant Cas9 with only nickase activity, which means that this mutant Cas9 cleaves only one DNA strand and does not activate NHEJ. Thus, DNA repair proceeds only through the HDR pathway. The third is nuclease-deficient Cas9 (dCas9), which has no cleavage activity but can bind to DNA. Thus, dCas9 can target specific sequences in the genome without cleavage. By fusing dCas9 with various effector domains, dCas9 can be used as a gene silencing tool or an activation tool.

[0111] In addition to class 1 and class 2 CRISPR-Cas systems, a putative class 2, type V CRISPR-Cas class exemplified by Cpf1 has recently been identified by Zetsche et al. (2015) Cell 163(3):759-771. The Cpf1 nuclease can provide additional flexibility in target site selection, particularly by a short 3-base pair recognition sequence (TTN), known as the protospacer adjacent motif or PAM. The cleavage site of Cpf1 is at least 18 bp away from the PAM sequence, and thus this enzyme can repeatedly cleave a specific locus after indel (insertion and deletion) formation to increase the efficiency of HDR. Furthermore, staggered DSBs with sticky ends allow for orientation-specific donor template insertion.

[0112] Additional information regarding the CRISPR-Cas system and its components is described in U.S. Patent No. 8,697,359, U.S. Patent No. 8,771,945, U.S. Patent No. 8,795,965, U.S. Patent No. 8,865,406, U.S. Patent No. 8,871,445, U.S. Patent No. 8,889,356, U.S. Patent No. 8,889,418, U.S. Patent No. 8,895,308, U.S. Patent No. 8,906,616, U.S. Patent No. 8,932,814, U.S. Patent No. 8,945,839, U.S. Patent No. 8,993,233, and U.S. Patent No. 8,999,641, and applications related thereto, as well as International Publication No. WO 2014 / 018423, International Publication No. WO 2014 / 093595, International Publication No. WO 2014 / 093622, International Publication No. WO 2014 / 093635, International Publication No. WO 2014 / 093655, International Publication No. WO 2014 / 093661, International Publication No. WO 2014 / 093694, International Publication No. WO 2014 / 093701, International Publication No. WO 2014 / 093709, International Publication No. WO 2014 / 093712, International Publication No. WO 2014 / 093718, International Publication No. WO 2014 / 145599, International Publication No. WO 2014 / 204723, International Publication No. WO 2014 / 204724, International Publication No. WO 2014 / 204725, International Publication No. WO 2014 / 204726, International Publication No. WO 2014 / 204727, International Publication No. WO 2014 / 204728, International Publication No. WO 2014 / 204729, International Publication No. WO 2015 / 065964, International Publication No. WO 2015 / 089351, International Publication No. WO 2015 / 089354, International Publication No. WO 2015 / 089364, International Publication No. WO 2015 / 089419, International Publication No. WO 2015 / 089427, International Publication No. WO 2015 / 089462, International Publication No. WO 2015 / 089465, International Publication No. WO 2015 / 089473, International Publication No. WO 2015 / 089486, International Publication No. WO 2016 / 205711, International Publication No. WO 2017 / 106657, International Publication No. WO 2017 / 127807, and applications related thereto.

[0113] Certain embodiments combine a tracrRNA and a crRNA into a single synthetic single guide RNA (e.g., sgRNA utilizing SEQ ID NOs: 87-89, or 290-366). In certain embodiments, the sgRNA can include a 20-nucleotide sequence similar to the crRNA and tracrRNA sequences. In certain gene editing systems, the target sequence may be adjacent to a PAM (e.g., 5'-20nt target-NGG-3'). In certain embodiments, the target sequence can include a PAM (SEQ ID NOs: 5-84). In certain embodiments, the guide RNA (gRNA) includes a target site adjacent to the PAM targeted by the genome editing complex. The gRNA can include at least 16, 17, 18, 19, 20, 21, or 22 nucleotides adjacent to the PAM.

[0114] In certain embodiments, the cutting element is directed to the target DNA location with the aid of the engineered gRNA (FIG. 25A (Sternberg et al., Mol Cell. 2015;58(4):568-74)). A gene construct with homology arms adjacent to the cut genomic region is efficiently inserted at this location by the homologous recombination DNA repair mechanism (e.g., see FIG. 15B (Elliott et al., Mol Cell Biol. 1998;18(1):93-101)). Using this approach, the gene encoding the selected antibody is inserted at the target gene location, except for the expression of the endogenous antibody. This targeted insertion eliminates or significantly reduces the likelihood of off-target effects resulting from random gene insertion.

[0115] In certain embodiments, the srRNAs targeting the mouse or human IgH of each endogenous antibody target the region 100 bp downstream of the J region (Figure 9). In experimental examples, this region was targeted and expressed a selected antibody paritumab version containing the C region from the endogenous genome (Figures 7 and 9). The crispr.mit.edu algorithm (Hsu et al., Nat Biotechnol. 2013;31(9):827-32) identified 22 targeting sequences for this region that are predicted to have little to no off-target binding. Individual targeting sequences are inserted into the full-length sgRNA and mixed with a nuclease such as Cas9 immediately prior to incubation as described (Schumann et al., Proc Natl Acad Sci U S A. 2015;112(33):10437-42) and can be electroporated into B cells (Kim et al., J Immunol. 1979;122(2):549-54). Since gene function is often lost by cellular repair of DNA cleaved by Cas9 (Symington & Gautier, Annu Rev Genet. 2011;45:247-71), an efficient sgRNA targeting the antibody coding region is expected to result in the appearance of several B cells lacking the antibody, which can be readily evaluated by flow cytometry. The activity of sgRNAs targeting intron sequences can be evaluated by sequencing or through enzymatic assays such as the T7 endonuclease assay.

[0116] In certain embodiments, the genome targeting and cutting elements can be administered through electroporation, nanoparticle-mediated delivery, and / or viral vector delivery. Electroporation can be useful, for example, for delivering the targeting and / or cutting elements. This is because cell membranes normally do not allow such foreign molecules to enter the cell. Electroporation delivers an electric shock to the cell, which temporarily enables such foreign molecules to pass through the cell membrane.

[0117] In certain embodiments, the gene construct for insertion can be administered through electroporation, nanoparticle-mediated delivery, and / or viral vector delivery. Adeno-associated virus vectors include, for example, vectors derived from 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, e.g., U.S. Patent No. 5,604,090; Kay et al., Nat. Genet. 24:257 (2000); Nakai et al., Blood 91:4600 (1998)).

[0118] In certain embodiments, the genome targeting and cutting element can be administered through electroporation, and the gene construct for insertion can be administered through AAV-mediated delivery. In certain embodiments, the genome targeting and cutting element can be administered through nanoparticle-mediated delivery, and the gene construct for insertion can be administered through AAV-mediated delivery.

[0119] In certain embodiments, the gene construct containing the transgene can be mixed with a targeting element (e.g., sgRNA) and a cutting element (e.g., Cas9 or cpf1) immediately before or shortly before electroporation. The selected antibody expression can be confirmed later (e.g., 3 days later) by measuring cell binding to a fluorescently tagged target protein 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 enable the detection of antibody-expressing B cells selected at a frequency as low as 0.00002% of the total B cell population (Taylor et al., Science. 2015;347(6223):784-7).

[0120] In certain embodiments, cells can be identified and / or sorted based on marker expression, either before or after delivery of the gene construct. For example, prior to delivery of the gene construct, it may be useful to isolate a particular type of B cell (e.g., memory B cell, antibody-secreting B cell, naive B cell, B1 B cell, marginal zone B cell) from a sample. As another example, it may be useful to isolate B cells from other cells present in a blood sample. CD19 is an example of a protein expressed by B cells but hardly expressed by other cells in the body. By labeling CD19 with a fluorescent molecule, B cells can be specifically identified. B220 is a marker useful for identifying mouse B cells.

[0121] CD27 is an example of a protein expressed by memory B cells but not expressed by naive human B cells. By labeling CD27 with a fluorescent molecule, memory B cells can be identified.

[0122] CD21 is an example of a protein not (or poorly) expressed by some memory human B cells that have the ability to rapidly secrete antibodies following infection. Low CD21 expression can be used to define B cells primed for plasma cell differentiation. By labeling CD21 with a fluorescent molecule, these B cells can be specifically identified, for example, by negative selection.

[0123] 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+ B220LOW. 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-.

[0124] Certain embodiments can utilize a CD19 + CD27 + CD21 lo marker profile.

[0125] CD45 is a marker used to identify and / or isolate cell types used in the experiments described herein. Different versions of the protein called CD45 are expressed depending on the mouse strain, and are named CD45.1 and CD45.2. In the experiments disclosed herein, B cells from mice expressing CD45.2 are harvested and transferred into mice expressing CD45.1. By labeling CD45.1 and CD45.2 with different fluorescent molecules, cells from the donor animal can be identified, because these cells express CD45.2 but not CD45.1.

[0126] Certain embodiments include sorting genetically modified B cells based on the expression of an exogenous light chain. For example, B cells that naturally express a kappa light chain can be modified to express a selected antibody that includes a lambda light chain. B cells that naturally express a lambda light chain can be modified to express a selected antibody that includes a kappa light chain. Sorting based on the expression of an exogenous light chain enables the isolation of only those B cells that express the selected antibody. In certain embodiments, only B cells that completely lack surface expression of their endogenous light chain are isolated for formulation and administration to a subject.

[0127] In certain embodiments, cells can be identified and / or isolated using flow cytometry. Flow cytometry is a sensitive and powerful analytical approach that uses laser light 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 a selected antibody (e.g., palivizumab) within these cells.

[0128] In certain embodiments, methods of modifying B cells can include obtaining hematopoietic stem cells (HSCs) and / or delivering a gene construct to the HSCs. HSCs can refer to a type of stem cell that naturally produces B cells as well as all other cells of the immune system. HSCs are obtainable, for example, from umbilical cord blood.

[0129] Certain experimental results described herein developed a gene modification method using A20 cells prior to transferring to freshly isolated B cells. A20 is an immortalized cell line made from mouse B cells.

[0130] In certain embodiments, B cells may be obtained from a human subject, and the obtained B cells or a subset thereof may be modified ex vivo.

[0131] Formulation of modified B cells. After modification, the cells can be harvested from the culture medium, washed, and concentrated in a carrier at a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hank's solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0132] 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 carrier for injection comprises buffered saline containing 5% sodium hyaluronate (HAS) or glucose. Additional isotonic agents include polyhydric sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0133] The carrier can contain buffers such as citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer, and / or triethylamine salt.

[0134] The stabilizer refers to a broad category of excipients whose functional range can extend from additives that help prevent cell adhesion from the filler to the container wall. Typical stabilizers include polyhydric 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 cyclitols like lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, and inositol; PEG; amino acid polymers; sulfur-containing reducing agents such as urea, glutathione, thiocctic acid, sodium thioglycolate, thioglycerol, alpha-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.

[0135] If necessary or beneficial, the formulation can contain local anesthetics such as lidocaine to relieve pain at the injection site.

[0136] Exemplary preservatives include phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0137] The preparation can contain, 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.

[0138] Method of use. The methods disclosed herein include treating a subject (e.g., human, veterinary animal (dog, cat, reptile, bird), livestock (e.g., horse, cow, goat, pig, chicken) and research animals (e.g., monkey, rat, mouse, fish)) with the preparations disclosed herein. Treating the subject includes delivering a therapeutically effective amount. A therapeutically effective amount includes an effective amount, an amount that provides prophylactic treatment and / or therapeutic treatment.

[0139] An "effective amount" is the amount of the composition required to effect a desired physiological change in a subject. An effective amount is often administered for research purposes. The effective amounts disclosed herein can cause a statistically significant effect in an animal model relevant to the assessment of the onset, progression and / or resolution of a condition or in an in vitro assay.

[0140] "Preventive treatment" includes treatment administered to a subject that does not exhibit signs or symptoms of a condition or exhibits only initial signs or symptoms of a condition, such that the treatment is administered for the purpose of reducing or decreasing the risk that the condition will develop. Thus, preventive treatment functions as prophylaxis against a condition. In certain embodiments, preventive treatment reduces, delays, or prevents the worsening of a condition. Certain embodiments include administration of a formulation described herein as a preventive defense when there is currently no effective vaccine. Certain embodiments include administration of a formulation described herein as a preventive defense as an alternative to conventional vaccination strategies. Certain embodiments include administration of a formulation described herein as a preventive defense as an adjunct to conventional vaccination strategies.

[0141] "Therapeutic treatment" includes treatment administered to a subject that exhibits signs or symptoms of a condition and is administered to the subject for the purpose of reducing or eliminating those signs or symptoms of the condition. Therapeutic treatment can reduce, control, or eliminate the presence or activity of a condition and / or reduce, control, or eliminate the side effects of a condition.

[0142] In certain embodiments, the condition is an infectious disease.

[0143] Functioning as an effective amount, preventive treatment or therapeutic treatment are not mutually exclusive, and in certain embodiments, a regulated dosage can achieve more than one type of treatment.

[0144] In certain embodiments, a therapeutically effective amount provides an anti-pathogen effect. The anti-pathogen effect can include an anti-infective effect. The anti-infective effect can include a decrease in the occurrence of an infectious disease, a decrease in the severity of an infectious disease, a decrease in the duration of an infectious disease, a decrease in the number of infected cells, a decrease in the amount of infected tissue, an increase in mean lifespan, an induced susceptibility of infected cells to immune clearance, a decrease in infection-related pain, and / or a reduction or elimination of symptoms associated with the treated infectious disease.

[0145] In certain embodiments, a therapeutically effective amount provides an anti-inflammatory effect. The anti-inflammatory effect can include a reduction in inflammation-related pain, fever, erythema, swelling, and / or loss of function.

[0146] In certain embodiments, a therapeutically effective amount provides an anti-Crohn's disease effect or an anti-ulcerative colitis effect. The anti-Crohn's disease effect or anti-ulcerative colitis effect can include a reduction in diarrhea, rectal bleeding, unexplained weight loss, fever, abdominal pain and muscle spasms, fatigue and lethargy, and / or a restoration of appetite.

[0147] In certain embodiments, a therapeutically effective amount provides an anti-arthritis effect. The anti-arthritis effect can include a reduction in joint pain, stiffness, swelling, erythema, and / or a restoration of range of motion. Types of arthritis include rheumatoid arthritis (RA), ankylosing spondylitis, and psoriatic arthritis.

[0148] In certain embodiments, a therapeutically effective amount provides an anti-plaque psoriasis effect. The anti-plaque psoriasis effect can include a reduction in erythema, scaly patches, itching, burning pain, stinging pain, nail bed abnormalities, and / or swelling or stiffened joints.

[0149] In certain embodiments, B cells may be obtained from a subject, a subset of the B cells may be modified ex vivo, and then the modified B cells may be formulated and administered to the subject. In certain embodiments, a first subset of the subject's B cells may be modified with a first gene construct to produce a selected antibody against a first pathogen, and a second subset of the subject's B cells may be modified with a second gene construct to produce a selected antibody against a second pathogen, thereby providing protective antibodies against two pathogens. As shown, it is possible to generate B cells against any number of pathogens and administer them to a subject. In certain embodiments, the selected antibody may be an anti-RSV antibody, an anti-HIV antibody, an anti-dengue virus antibody, an anti-Bordatella pertussis antibody, an anti-hepatitis C antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, an anti-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, and / or an anti-TNF antibody. In certain embodiments, the selected antibody may be one or more of an anti-RSV antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, and / or an anti-MPV antibody. 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.

[0150] In certain embodiments, the B cells can be obtained from a bone marrow donor or hematopoietic stem cell donor who is immunologically compatible with the recipient. In certain embodiments, a first subset of the donor's B cells may be modified with a first gene construct to produce a selected antibody against a first pathogen, and a second subset of the donor's B cells may be modified with a second gene construct to produce a selected antibody against a second pathogen, thereby providing protective antibodies against two pathogens. As shown, it is possible to generate B cells against any number of pathogens and administer them to a subject. In certain embodiments, the selected antibody can be an anti-RSV antibody, an anti-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-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, and / or an anti-TNF antibody. In certain embodiments, the selected antibody can be one or more of an anti-RSV antibody, an anti-influenza virus antibody, an anti-parainfluenza virus antibody, and / or an anti-MPV antibody. In certain embodiments, the selected antibody can 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. The genetically modified B cells can be administered to the recipient to provide protection against infection (e.g., an anti-infective effect) until the transplanted cells re-colonize the recipient's own immune system.

[0151] In certain embodiments, the recipient has received a donor-derived bone marrow or hematopoietic stem cell graft as a treatment for a hematologic malignancy. Examples of hematologic malignancies include acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, Burkitt lymphoma / leukemia, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, follicular lymphoma (grades I, II, III, or IV), Hodgkin lymphoma, intravascular large B-cell lymphoma, lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, marginal zone lymphoma (extranodal or nodal), mediastinal (thymic) large B-cell lymphoma, multiple myeloma, non-Hodgkin lymphoma, POEMS syndrome / osteosclerotic myeloma, primary effusion lymphoma, splenic marginal zone lymphoma, small lymphocytic lymphoma, smoldering multiple myeloma (SMM), and Waldenström macroglobulinemia.

[0152] In certain embodiments, the recipient has received genetically modified hematopoietic stem cells that provide a gene the recipient lacks. These recipients may have primary or secondary immunodeficiencies that can be treated by the delivery of a therapeutic gene through the hematopoietic stem cells. Over 80 primary immunodeficiency diseases are recognized by the World Health Organization. These diseases are, in some cases, characterized by an inherent defect in the immune system such that the body cannot produce any or sufficient antibodies against infection. In other cases, the cellular defenses against infection do not function properly. Typically, primary immunodeficiencies are genetic disorders. X-linked severe combined immunodeficiency (SCID-X1) is another example of a primary immunodeficiency. X-linked SCID results in both cellular and humoral immune depletion caused by a mutation in the common gamma chain gene (γC), which eliminates T and natural killer (NK) lymphocytes.

[0153] Secondary or acquired immunodeficiency occurs not as a result of inherited genetic abnormalities, but rather in individuals in whom the immune system has been impaired by factors other than the immune system itself. Examples include trauma, viruses, chemotherapy, toxins, and contamination. Acquired immunodeficiency syndrome (AIDS) is an example of a secondary immunodeficiency disorder caused by the virus, human immunodeficiency virus (HIV), which depletes T lymphocytes, rendering the body unable to fight infection.

[0154] In certain embodiments, B cells may be obtained from a subject, a subset of the B cells may be modified ex vivo, and then the modified B cells may be formulated and administered to the subject. In certain embodiments, a first subset of the subject's B cells may be modified with a first gene construct to produce selected antibodies against inflammatory molecules such as inflammatory cytokines. In certain embodiments, the selected antibodies may be anti-TNF antibodies and / or anti-IL-1 antibodies. In certain embodiments, the selected antibodies may be infliximab, adalimumab, and / or golimumab, and / or approved biosimilars thereof.

[0155] In terms of administration, a therapeutically effective amount (also referred to herein as a dose) can initially be evaluated based on the results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine a useful dose in the subject of interest. The actual dose administered to a particular subject can be determined by a physician, veterinarian, or researcher taking into account parameters such as age, previous vaccinations (if any), target, body weight, severity of the condition, type of condition, stage of the condition, previous or current therapeutic interventions, idiopathic diseases of the subject, and physical and physiological factors including the route of administration.

[0156] As shown, in certain embodiments, the modified B cells express a tag that enables, for example, tracking and / or removal after administration to the subject.

[0157] Exemplary doses are more than 10 2 modified B cells, more than 10 3 modified B cells, more than 104 more modified B cells, 10 5 more modified B cells, 10 6 more modified B cells, 10 7 more modified B cells, 10 8 more modified B cells, 10 9 more modified B cells, 10 10 more modified B cells, or 10 11 can include more modified B cells.

[0158] In certain embodiments, the effect of the selected antibody is measurable using viral titer. Viral titer refers to the amount of virus detectable. A high viral titer means a high level of infection. The optimal defensive response is observed at a titer that drops to zero.

[0159] As will be understood by those skilled in the art, while certain embodiments have been described, additional embodiments may also be utilized within the scope of the present disclosure. The following description provides an illustration and feasibility of representative additional embodiments.

[0160] Exemplary embodiments 1. A method of genetically engineering B cells to express a selected antibody, comprising targeted insertion of a gene construct comprising (i) a promoter and (ii) a transgene encoding a portion of the selected antibody in an intron region that is constant in all B cells, wherein (i) the promoter is positioned relative to an enhancer element that interacts with the promoter to drive expression of the transgene, and (ii) the transgene is in an orientation such that a portion of the endogenous antibody-encoding genome of the B cell is not expressed. 2. A method of genetically engineering B cells to express a selected antibody, comprising inserting into SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 a gene construct comprising (i) a heavy chain promoter, (ii) a signal peptide, (iii) the full-length light chain of the selected antibody; (iv) a flexible linker or a skipping element; (v) the variable region of the heavy chain of the selected antibody; and (vi) a splice junction, thereby genetically engineering the B cells to express the selected antibody. 3. The method of embodiment 1 or 2, wherein the endogenous variable heavy chain coding genome of the B cell is not excised during genetic modification. 4. The method of any one of embodiments 1 to 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-metapneumovirus (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. 5. The method of any one of embodiments 1 to 4, wherein the gene construct comprises SEQ ID NOs: 102-175, 278, 279, or 280-289. 6. The method of any one of embodiments 2 to 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. 7. The method of any one of embodiments 2 to 6, wherein the flexible linker is selected from SEQ ID NOs: 180-184. 8. The method of any one of embodiments 2 to 7, wherein the flexible linker is a Gly-Ser linker comprising 50-80 amino acids. 9. The method of any one of embodiments 2 to 8, wherein the flexible linker is a Gly-Ser linker comprising 57 amino acids. 10. The method of any one of embodiments 2 to 6, 8, or 9, wherein the flexible linker is SEQ ID NO: 122. 11. The method of any one of embodiments 2 to 10, wherein the skipping element is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody. 12. The method according to any one of embodiments 2 to 11, wherein the skipping element is a self-cleaving peptide. 13. The method according to embodiment 12, wherein the self-cleaving peptide is selected from SEQ ID NOs: 176 to 179. 14. The method according to any one of embodiments 2 to 13, wherein the skipping element is an internal ribosome entry site (IRES). 15. The method according to any one of embodiments 2 to 14, wherein the heavy chain promoter is selected from SEQ ID NOs: 111 and 128. 16. The method according to any one of embodiments 2 to 15, wherein the heavy chain promoter is IgVH1-69 or J558H10. 17. The method according to any one of embodiments 2 to 16, wherein the signal peptide is selected from SEQ ID NOs: 118, 134, and 185 to 194. 18. The method according to any one of embodiments 2 to 17, wherein the signal peptide is derived from a human IgH heavy chain or a human IgL light chain. 19. The method according to any one of embodiments 1 to 18, wherein the gene construct comprises homology arms. 20. The method according to embodiment 19, wherein the homology arms comprise SEQ ID NOs: 90 to 101, 110, 125, 127, 140, 142, 143, 153, 170, 171, 173, 174, 278, or 279. 21. The method according to any one of embodiments 1 to 20, wherein the gene construct encodes a tag. 22. The method according to embodiment 21, wherein the tag comprises STREPTAG (registered trademark), STREP (registered trademark) tag II, His tag, Flag tag, Xpress tag, Avi tag, calmodulin tag, polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Sof tag 1, Sof tag 3, or V5 tag. 23. The method according to embodiment 21 or 22, wherein the tag comprises SEQ ID NO: 122 or SEQ ID NOs: 195 to 204. 24. The method according to any one of embodiments 1 to 23, further comprising delivering a guide RNA (gRNA) sequence selected from one or more of SEQ ID NOs: 87 to 89 and 290 to 366 and a nuclease to a B cell. 25. The method of embodiment 24, wherein delivery is through electroporation, nanoparticle, or viral-mediated delivery. 26. The method of any one of embodiments 1-25, wherein the gene construct is part of an adeno-associated virus vector. 27. The method of any one of embodiments 24-26, wherein the gRNA and nuclease are delivered through electroporation and the gene construct is delivered as part of an adeno-associated virus vector. 28. The method of any one of embodiments 24-27, wherein the nuclease is Cas9 or Cpf1. 29. The method of 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. 30. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-RSV antibody comprising palivizumab, AB1128, or ab20745. 31. The method of any one of embodiments 1-30, wherein the selected antibody is palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 136; palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 205; an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 120; or an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 206. 32. The method of 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. 33. The method of any one of embodiments 1-29, wherein the selected antibody is an anti-HIV antibody comprising 10E8, VRC01, ab18633, or 39 / 5.4A. 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. 35. The method according to any one of embodiments 1 to 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. 36. The method according to any one of embodiments 1 to 29, wherein the selected antibody is an anti-dengue virus antibody comprising antibody 55, DB2-3, ab155042 or ab80914. 37. The method according to any one of embodiments 1 to 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. 38. The method according to any one of embodiments 1 to 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. 39. The method according to any one of embodiments 1 to 29, wherein the selected antibody is an anti-hepatitis C antibody comprising MAB8694 or C7-50. 40. The method according to any one of embodiments 1 to 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. 41. The method according to any one of embodiments 1 to 29, wherein the selected antibody is an anti-influenza virus antibody comprising C102. Any of the methods 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. Any of the methods of Embodiments 1-29 or 41, 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. Any of the methods of Embodiments 1-29, wherein the selected antibody is an anti-MPV antibody comprising MPE8. Any of the methods of Embodiments 1-29, wherein the selected antibody is an anti-CMV antibody comprising MCMV5322A, MCMV3068A, LJP538 or LJP539. Any of the methods of Embodiments 1-29, 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. Any of the methods 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. Any of the methods of Embodiments 1-29, wherein the selected antibody is an anti-HSV antibody comprising HSV8-N and MB66. Any of the methods of Embodiments 1-29, wherein the selected antibody is an anti-Clostridium difficile antibody comprising actoxumab or bezlotoxumab. Any of the methods of Embodiments 1-29, wherein the selected antibody is an anti-TNF antibody comprising infliximab, adalimumab, etanercept, certolizumab or an approved biosimilar thereof. 51. The method according to any one of embodiments 1-29 or 50, wherein the selected antibody is an anti-TNF antibody comprising a heavy chain comprising SEQ ID NO: 254 and a light chain comprising SEQ ID NO: 255; CDRH1 comprising SEQ ID NO: 256, CDRH2 comprising SEQ ID NO: 257, and CDRH3 comprising SEQ ID NO: 258; CDRL1 comprising SEQ ID NO: 259, CDRL2 comprising SEQ ID NO: 260, and CDRL3 comprising SEQ ID NO: 261; CDRH1 comprising SEQ ID NO: 262, CDRH2 comprising SEQ ID NO: 263, and CDRH3 comprising SEQ ID NO: 264; CDRL1 comprising SEQ ID NO: 265, CDRL2 comprising SEQ ID NO: 266, and CDRL3 comprising SEQ ID NO: 267; or CDRH1 comprising SEQ ID NO: 268, CDRH2 comprising SEQ ID NO: 269, and CDRH3 comprising SEQ ID NO: 270; CDRL1 comprising SEQ ID NO: 271, CDRL2 comprising SEQ ID NO: 272, and CDRL3 comprising SEQ ID NO: 273. 52. The method according to 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. 53. The method according to 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. 54. A B cell modified according to the method of any one of embodiments 1-53. 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. 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 cell of embodiment 54 or 55, thereby providing an anti-infective effect. 57. The method of embodiment 56, which removes the need for vaccination by providing. 58. The method of embodiment 56 or 57, wherein the administration replaces a vaccination protocol. 59. The method according to any one of embodiments 56-58, wherein the subject is immunosuppressed. 60. The method according to any one of embodiments 56-59, wherein the subject is immunosuppressed as part of a treatment regimen comprising bone marrow transplantation, hematopoietic stem cell transplantation or administration of genetically modified hematopoietic stem cells. 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. 62. A gene construct for modifying B cells to express a selected antibody, the gene 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 skipping element; (v) a variable region of the heavy chain of the selected antibody; and (vi) a splice junction. 63. The gene construct of embodiment 62, comprising SEQ ID NOs: 102-175 or 280-289. 64. The gene construct of 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. 65. The gene construct of any one of embodiments 62-64, wherein the flexible linker is selected from SEQ ID NOs: 180-184. 66. The gene construct of any one of embodiments 62-65, wherein the flexible linker is a Gly-Ser linker comprising 50-80 amino acids. 67. The gene construct of any one of embodiments 62-66, wherein the flexible linker is a Gly-Ser linker comprising 57 amino acids. 68. The gene construct of any one of embodiments 62-64, 66 or 67, wherein the flexible linker is SEQ ID NO: 122. 69. The gene construct of any one of embodiments 62-68, wherein the skipping element is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody. 70. The gene construct of any one of embodiments 62-69, wherein the skipping element is a self-cleaving peptide. 71. The gene construct of embodiment 70, wherein the self-cleaving peptide is selected from SEQ ID NOs: 176-179. 72. The gene construct of any one of embodiments 62-69, wherein the skipping element is an internal ribosome entry site (IRES). 73. A gene construct according to any one of embodiments 62 to 72, wherein the heavy chain promoter is selected from SEQ ID NOs: 111 and 128. 74. A gene construct according to any one of embodiments 62 to 73, wherein the heavy chain promoter is IgVH1-69 or J558H10. 75. A gene construct according to any one of embodiments 62 to 74, wherein the signal peptide is selected from SEQ ID NOs: 118, 134, and 185 to 194. 76. A gene construct according to any one of embodiments 62 to 75, wherein the signal peptide is derived from a human IgH heavy chain or a human IgL light chain. 77. A gene construct according to any one of embodiments 62 to 76, comprising a homology arm. 78. A gene construct according to embodiment 77, wherein the homology arm comprises SEQ ID NOs: 90 to 101, 110, 125, 127, 140, 142, 143, 153, 170, 171, 173, 174, 278, or 279. 79. A gene construct according to any one of embodiments 62 to 78, encoding a tag. 80. A gene construct according to embodiment 79 or 80, wherein the tag comprises STREPTAG (registered trademark), STREP (registered trademark) tag II, His tag, Flag tag, Xpress tag, Avi tag, calmodulin tag, polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Sof tag 1, Sof tag 3, or V5 tag. 81. A gene construct according to embodiment 79 or 80, wherein the tag comprises SEQ ID NO: 122 or SEQ ID NOs: 195 to 204. 82. A gene construct according to any one of embodiments 62 to 81, wherein the selected antibody is an anti-RSV antibody comprising palivizumab, AB1128, or ab20745. 83. A gene construct according to any one of embodiments 62 to 82, wherein the selected antibody is palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 136; palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 205; an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 120; or an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 206. 93. The gene construct of any one of embodiments 62 to 82, wherein the selected antibody is an anti-RSV antibody comprising CDRH1 containing SEQ ID NO: 207, CDRH2 containing SEQ ID NO: 208, CDRH3 containing SEQ ID NO: 209; CDRL1 containing SEQ ID NO: 210, CDRL2 containing SEQ ID NO: 211, and CDRL3 containing SEQ ID NO: 212. 94. The gene construct of any one of embodiments 62 to 81, wherein the selected antibody is an anti-HIV antibody comprising 10E8, VRC01, ab18633 or 39 / 5.4A. 95. The gene construct of any one of embodiments 62 to 81 or 85, wherein the selected antibody is an anti-HIV antibody comprising a heavy chain containing SEQ ID NO: 150 and a light chain containing SEQ ID NO: 149. 96. The gene construct of any one of embodiments 62 to 81 or 85, wherein the selected antibody is an anti-HIV antibody comprising CDRH1 containing SEQ ID NO: 213, CDRH2 containing SEQ ID NO: 214, CDRH3 containing SEQ ID NO: 215, CDRL1 containing SEQ ID NO: 216, CDRL2 containing SEQ ID NO: 217, and CDRL3 containing SEQ ID NO: 218 or CDRH1 containing SEQ ID NO: 219, CDRH2 containing SEQ ID NO: 220, CDRH3 containing SEQ ID NO: 221, CDRL1 containing QYGS, CDRL2 containing SGS, and CDRL3 containing SEQ ID NO: 222. 97. The gene construct of any one of embodiments 62 to 81, wherein the selected antibody is an anti-dengue virus antibody comprising antibody 55, DB2-3, ab155042 or ab80914. 98. The gene construct of any one of embodiments 62 to 81 or 88, wherein the selected antibody is an anti-dengue virus antibody comprising CDRH1 containing SEQ ID NO: 223, CDRH2 containing SEQ ID NO: 224, CDRH3 containing SEQ ID NO: 225; CDRL1 containing SEQ ID NO: 226, CDRL2 containing SEQ ID NO: 227, and CDRKL3 containing SEQ ID NO: 228 or CDRH1 containing SEQ ID NO: 229, CDRH2 containing SEQ ID NO: 230, CDRH3 containing SEQ ID NO: 231, CDRL1 containing SEQ ID NO: 232, CDRL2 containing SEQ ID NO: 233, and CDRL3 containing SEQ ID NO: 234. 90. A gene construct according to any one of embodiments 62 - 81, wherein the selected antibody is an anti - pertussis antibody comprising a heavy chain containing SEQ ID NO: 235 and a light chain containing SEQ ID NO: 236. 91. A gene construct according to any one of embodiments 62 - 81, wherein the selected antibody is an anti - hepatitis C antibody comprising MAB8694 or C7 - 50. 92. A gene construct according to any one of embodiments 62 - 81 or 91, wherein the selected antibody is an anti - hepatitis C antibody comprising CDRH1 containing SEQ ID NO: 237, CDRH2 containing SEQ ID NO: 238, CDRH3 containing SEQ ID NO: 239, CDRL1 containing SEQ ID NO: 240, CDRL2 containing SEQ ID NO: 241, and CDRL3 containing SEQ ID NO: 242. 93. A gene construct according to any one of embodiments 62 - 81, wherein the selected antibody is an anti - influenza virus antibody comprising C102. 94. A gene construct according to any one of embodiments 62 - 81 or 93, wherein the selected antibody is an anti - influenza virus antibody comprising a heavy chain containing SEQ ID NO: 159 and a light chain containing SEQ ID NO: 158. 95. A gene construct according to any one of embodiments 62 - 81 or 93, wherein the selected antibody is an anti - influenza virus antibody comprising CDRH1 containing SEQ ID NO: 243, CDRH2 containing SEQ ID NO: 244, CDRH3 containing SEQ ID NO: 245, CDRL1 containing SEQ ID NO: 246, CDRL2 containing KTS, and CDRL3 containing SEQ ID NO: 247. 96. A gene construct according to any one of embodiments 62 - 81, wherein the selected antibody is an anti - MPV antibody comprising MPE8. 97. A gene construct according to any one of embodiments 62 - 81, wherein the selected antibody is an anti - CMV antibody comprising MCMV5322A, MCMV3068A, LJP538 or LJP539. 98. A gene construct according to any one of embodiments 62 - 81, wherein the selected antibody is an anti - EBV antibody comprising a heavy chain containing SEQ ID NO: 168 and a light chain containing SEQ ID NO: 166. 99. A gene construct according to any of embodiments 62 - 81, wherein the selected antibody is an anti - EBV antibody comprising CDRH1 containing SEQ ID NO: 248, CDRH2 containing SEQ ID NO: 249, CDRH3 containing SEQ ID NO: 250, CDRL1 containing SEQ ID NO: 251, CDRL2 containing SEQ ID NO: 252, and CDRL3 containing SEQ ID NO: 253. 100. A gene construct according to any of embodiments 62 - 81, wherein the selected antibody is an anti - HSV antibody comprising HSV8 - N and MB66. 101. A gene construct according to any of embodiments 62 - 81, wherein the selected antibody is an anti - Clostridium difficile antibody comprising actoxumab or bezlotoxumab. 102. A gene construct according to any of embodiments 62 - 81, wherein the selected antibody is an anti - TNF antibody comprising infliximab, adalimumab, etanercept, certolizumab, or an approved biosimilar thereof. 103. A gene construct according to any of embodiments 62 - 81 or 102, wherein the selected antibody is an anti - TNF antibody comprising a heavy chain containing SEQ ID NO: 254 and a light chain containing SEQ ID NO: 255; CDRH1 containing SEQ ID NO: 256, CDRH2 containing SEQ ID NO: 257, and CDRH3 containing SEQ ID NO: 258; CDRL1 containing SEQ ID NO: 259, CDRL2 containing SEQ ID NO: 260, and CDRL3 containing SEQ ID NO: 261; CDRH1 containing SEQ ID NO: 262, CDRH2 containing SEQ ID NO: 263, and CDRH3 containing SEQ ID NO: 264; CDRL1 containing SEQ ID NO: 265, CDRL2 containing SEQ ID NO: 266, and CDRL3 containing SEQ ID NO: 267; or CDRH1 containing SEQ ID NO: 268, CDRH2 containing SEQ ID NO: 269, and CDRH3 containing SEQ ID NO: 270; CDRL1 containing SEQ ID NO: 271, CDRL2 containing SEQ ID NO: 272, and CDRL3 containing SEQ ID NO: 273. 104. A kit for genetically modifying B cells, comprising a gene construct according to any 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. 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. 106. The kit of embodiment 104 or 105, further comprising a nuclease. 107. The kit of embodiment 106, wherein the nuclease is Cas9 or Cpf1. 108. The kit of any one of embodiments 104 - 107, further comprising nanoparticles or adeno-associated virus vectors. 109. The kit of any one of embodiments 104 - 108, wherein the gRNA and the nuclease are associated with the nanoparticles. 110. The kit of any one of embodiments 104 - 109, wherein the gene construct is part of an adeno-associated virus vector.

Examples

[0161] [Example 1] Provide 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 in those with chronic lung disease, congenital heart disease, or premature infants. Humoral immunity can mediate effective protection against RSV, as demonstrated by the therapeutic effect of the recombinant antibody Synagis® (Medlmmune, Inc.) / palivizumab. However, neither natural infection nor previous vaccine trials have been able to induce a complete protective immune response against RSV.

[0162] In diseases like RSV where vaccination is difficult, bypassing vaccination by manipulating primary B cells to induce the expression of desired therapeutic antibodies is extremely attractive. The immunoglobulin (Ig) locus is extremely large, diverse, and susceptible to extensive genomic recombination and editing. Furthermore, the transcription of immunoglobulin genes to produce both membrane and secreted forms utilizes the regulation of mRNA splicing and polyadenylation by regulatory DNA elements. This complexity makes viral transduction, a conventional approach for lymphocyte cell engineering, technically infeasible for producing therapeutic B cells.

[0163] Certain embodiments include a platform for rapid and selective reprogramming of primary B cell antibody specificity by single-hit immune gene editing. This platform utilizes the high activity of the microhomology-mediated end-joining DNA repair pathway in primary B cells to insert a fully synthetic hybrid double-stranded / single-stranded DNA template after creation of a DNA break by Cas9 / sgRNA ribonucleoprotein. Key to this approach is the preservation of endogenous regulatory elements, which enables natural control of surface-bound and secreted antibody expression. Furthermore, the strategy is not limited to RSV. With just a single blood draw and subsequent cell infusion a few days later, it is possible to express antibodies that protect against virtually any pathogen.

[0164] Materials and methods. Design of sgRNAs. sgRNAs targeting intron sequences in the mouse and human IgH loci were designed using CrispRGold (crisprgold.mdc-berlin.de) and produced in synthetic form incorporating 2’-O-methyl analogs and 3’ phosphorothioate nucleotide internucleotide linkages at the first three 5’ and 3’ terminal RNA residues (Synthego). The genomic targeting sequences are as follows:

[0165]

Chemical formula

[0166] Design and assembly of template sequences Human: The antibody construct includes the IgVH1-69 heavy chain promoter region (SEQ ID NO: 111), the full-length codon-optimized light chain of palivizumab (SEQ ID NO: 113 (nucleotide) and SEQ ID NO: 120 (amino acid)), a 57 amino acid glycine-serine linker containing three tandem copies of the Strep tag II motif (SEQ ID NO: 116 (nucleotide) and SEQ ID NO: 122 (amino acid)), the codon-optimized variable region of the palivizumab heavy chain (SEQ ID NO: 117 (nucleotide) and SEQ ID NO: 123 (amino acid)), and a splice junction with a 60 bp flanking sequence from the human IgHJ1 gene segment (SEQ ID NO: 124).

[0167] Mouse: The mCherry template contains the J5558H10 heavy chain promoter, the fully codon-optimized mCherry open reading frame, and the sv40 polyadenylation site. The antibody construct contains the J5558H10 heavy chain promoter (SEQ ID NO: 128, V.A Love et al., Molecular Immunology 2000), the 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 the Strep tag II sequence (SEQ ID NO: 116 (nucleotide) and SEQ ID NO: 122 (amino acid)), the codon-optimized variable region of the heavy antibody chain (SEQ ID NO: 133 (nucleotide) and SEQ ID NO: 138 (amino acid)), and a splice junction with a 60bp adjacent sequence derived from the mouse IgH J3 gene segment (SEQ ID NO: 139).

[0168] Annealing of stitching oligonucleotides (i.e., splicing oligonucleotides). Stitching oligonucleotides with 36bp complementarity to the PAM terminus and PAM-proximal non-target DNA strand and 50 - 100bp complementarity to the inserted template were synthetically produced and pre-annealed to the DNA template (e.g., SEQ ID NOs: 96 - 101) before use. As shown in Figure 15, the splicing oligonucleotides can provide "homology stitches".

[0169] Adeno-associated virus (AAV) viral vector template delivery. The AAV viral vector contained the MND promoter, the fully codon-optimized mCherry open reading frame, and two mouse sgRNA recognition sites or the sv40 polyadenylation site flanked by 400bp of homology. AAV virions were produced in pseudotyped 293T cells with the AAV6 viral capsid, purified by sucrose gradient centrifugation, and stored at -80°C. For viral delivery of the template DNA, the concentrated AAV virus was added to the final volume of 10% of the total culture volume 12 hours before electroporation.

[0170] Mouse B cells and electroporation. The basal B cell medium contained RPMI medium supplemented with 10% fetal bovine serum (Hyclone), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 55 μM β-mercaptoethanol (Sigma), and, except for the indicated antibiotic-free step, 100 U / ml penicillin plus 100 μg / mL streptomycin (Gibco).

[0171] B cells were isolated from the spleen and lymph nodes by negative selection using magnetic beads (Miltenyi) and cultured at 2×10 6 / ml for 24 h in B cell medium supplemented with 100 ng / ml of recombinant carrier HA-tagged mouse CD40L (R&D systems), 100 ng / ml of anti-HA antibody (clone 543851, R&D systems), and 4 ng / ml of mouse IL-4 (R&D systems). Next, the B cells were electroporated using a Neon transfection system and a 10 μl tip as follows. Cas9 protein (Invitrogen) and synthetic sgRNA (Synthego) were mixed at a ratio of 3 μg of Cas9 / 900 ng of sgRNA and incubated at room temperature for at least 10 min. The B cells were washed with PBS and suspended in Neon buffer T at a final density of 2.5×10 7 cells / ml together with Cas9 / sgRNA and pre-assembled DNA template. The cells were electroporated (1675 V, 10 milliseconds, 3 pulses) and immediately dispensed into pre-warmed antibiotic-free medium.

[0172] For cell expansion, the B cells were co-cultured with irradiated (80 Gy) 3T3-CD40L feeder cells in the presence of 20 ng / ml of mouse IL-21.

[0173] Evaluation of sgRNA activity by tracking of indels by decomposition (TIDE). Genomic DNA was isolated from mock and Cas9-treated cells on days 3 to 5 after electroporation. The 500 - 600 bp region adjacent to the cleavage site was amplified using the following oligos:

[0174] [Chemical formula] was amplified by PCR using

[0175] Results. The results are shown in FIGS. 18A - 18C, FIGS. 22A & 22B, FIGS. 23B - 23E, and FIGS. 24A - 24C. FIGS. 18A, 18B, and 18C demonstrate successful cleavage at the targeted IgH locus in mouse B cell line A20, primary mouse B cells, and human B cell line RAMOS, respectively, after electroporation of cells with Cas9 / sgRNA ribonucleoprotein. FIGS. 22A and 22B depict the insertion of the mCherry fluorescent protein reporter into the IgH locus of primary mouse B cells. FIGS. 23B and 23C demonstrate surface expression of anti - RSV antibody after insertion of a partial antibody cassette into A20 mouse B cell line (FIG. 23B) and RAMOS human B cell line (FIG. 23C). FIGS. 23D and 23E demonstrate secretion of anti - RSV antibody after insertion of a partial antibody cassette into A20 mouse B cell line (FIG. 23D) and RAMOS human B cell line (FIG. 23E). FIG. 24A demonstrates surface expression of anti - RSV antibody after insertion of a partial antibody cassette into primary mouse B cells, initially (left panel) and after in vitro enrichment and expansion (right panel). FIG. 24B demonstrates secretion of anti - RSV antibody after insertion of a partial antibody cassette into primary mouse B cell line. FIG. 24C shows the in vitro proliferation potential of the engineered B cells.

[0176] [Example 2] The goal of this example was to generate genetically modified B cells with limited specificity that maintain the natural control of secreted Ig and surface Ig expression through genomic manipulation of the IgH locus. The IgH locus in B cells is a difficult region to target for genomic manipulation due to the highly variable sequences present in B cells. During B cell development, V, D, and J elements are recombined on DNA exceeding 1 megabase to generate the VDJ variable region essential for antibody diversity. Later, during the process of B cell ontogeny, DNA is lost over a similar sequence range due to class switching between different constant regions (Reviewed in Watson et al., (2017). Trends Immunol 38(7):459-470).

[0177] Due to this sequence diversity, it is not feasible to directly target the antibody coding region. However, in all B cells, there is a small DNA region between the last J gene segment and the switch region involved in class switching. This universal target contains the important intronic Eμ enhancer, which is one of several strong enhancer elements that, despite its weak promoter, cooperate to drive high-level expression of the IgH gene. The activity of these enhancers is regulated in part by the proximity of the promoter to the Eμ enhancer, and introduction of a transgene between the recombined 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 example targeted the region upstream of the Eμ enhancer to insert a new antibody cassette (Figure 26A). By targeting this region, the inserted emAb gene can be driven by the native (but inserted) IgH promoter, maximizing the natural control of immunoglobulin expression.

[0178] To enable one - hit insertion and minimize off - target interactions, the emAb construct was expressed as a single - chain fusion. This fusion consists of a full - length light - chain sequence linked to the variable region of the heavy chain by a 57 - amino - acid glycine - serine linker (Figure 26A) as described for single - chain F(ab) fragments (Koerber et al., (2015). J Mol Biol 427(2):576 - 586). This linker contains three tandem repeats of the Strep - tag II motif (Schmidt & Skerra (2007). Nat Protoc 2(6):1528 - 1535) to facilitate the detection and enrichment of genetically modified cells. Physically linking the light and heavy chains minimizes the potential for mispairing between the inserted emAb and the endogenous light chain. Optimized splice junctions splice the emAb to the downstream endogenous IgH constant region. This results in the emAb being expressed as one of the heavy - chain isotype classes.

[0179] The strategy was tested in the RAMOS human B - cell line. This Burkitt lymphoma - derived B - cell line naturally expresses surface and secreted IgM paired with lambda light chains. In these experiments, the expression of the engineered αRSV - emAb derived from palivizumab was detected using streptactin, a modified streptavidin with high affinity for the monomeric RSV - F protein and the Strep - tag II motif in the linker. αRSV - emAb - genetically modified RAMOS cells expressed the engineered RSV - specific antibody, which could be detected on the cell surface (Figure 26B) and as secreted form in the supernatant (Figure 26C). To confirm that the emAb BCR associates with secondary protein complexes important for BCR signaling, RAMOS cells were exposed to stimulation with multimerized RSV - F antigen. αRSV - emAb - engineered but not control cells showed rapid and sustained calcium signaling in response to the protein antigen (Figure 26D). These data helped to confirm the feasibility of the emAb - engineering approach.

[0180] Next, human primary B cells were genetically modified using a multi-step process of expansion and differentiation (Figure 27A). By electroporation of pre-complexed guide RNA and Cas9, genomic DNA was highly efficiently cleaved, and incomplete repair occurred in this region in 70% of the targeted alleles analyzed across multiple independent donors (Figure 27B). The sgRNA target sites were strongly conserved in humans, and none were reported at frequencies exceeding 1% of the reported single nucleotide polymorphisms (Figure 27C). Addition of the AAV-delivered αRSV-emAb cassette efficiently reprogrammed human B cells to bind the RSV-F protein (Figure 27D). Notably, emAb B cells were successfully produced from all human donors tested, and the average manipulation rate was 24% (Figure 27E). In vitro culture and differentiation during emAb production increased antibody-secreting potential. Cells primed on day 2 expressed high levels of CD19, as well as small amounts of the plasma cell markers CD138, CD27, and CD138, and were in contrast to cells on day 18 with lower levels of CD19, as well as increased CD38, CD27, and CD138 (Figure 27F). Corresponding to these changes in cell surface markers, differentiated emAb-manipulated B cells secrete substantial amounts of the target antibody (Figure 27G). Collectively, these data demonstrate the ability to rapidly and efficiently manipulate primary B cells to produce specific protective antibodies.

[0181] To demonstrate the flexible aspect of the platform, emAb cassettes from three additional broad neutralizing antiviral antibodies, including the anti-HIV target VRC01, EBV target AMM01, and influenza HA-stem target MEDI8852, were tested. Primary B cells were efficiently reprogrammed using all four constructs, which included antibodies with both kappa (paritumab, VRC01, MEDi8852) and lambda (AMM01) light chains (Figure 28). These data demonstrate the flexible and broadly applicable nature of the emAb platform.

[0182] Blocking the production of endogenous Ig heavy chains is important to maximize the production of emAbs and minimize the potential for the production of unknown endogenous antibodies from genetically modified cells. The RAMOS B cell line endogenously expresses IgH paired with lambda light chains. Manipulating these cells with αRSV-emAb linked to the kappa light chain enables the use of surface lambda light chain expression as an effective means of IgH expression. Furthermore, RAMOS cells have undergone a c-myc translocation that disrupts one IgH allele such that any emAb insertion would be in the productive allele (Figure 29A). Input RAMOS cells express high levels of lambda light chain on the surface, and cells expressing αRSV-emAb have almost completely lost lambda expression (Figure 29B). These data indicate that emAb insertion on the productive allele can effectively block the expression of endogenous IgH. In almost all primary B cells, one IgH allele has productive VDJ rearrangement and the other allele has not undergone VDJ recombination or has been rearranged non-productively. However, both of these alleles have potential sites for emAb insertion (Figure 29C). To test the effect of emAb insertion, purified lambda light chain-expressing primary B cells were genetically modified with αRSV-emAb. Input cells continued to express endogenous antibodies paired with lambda light chain on the surface. In contrast, half of the αRSV-emAb-manipulated B cells lost lambda light chain expression (Figure 29D). The differential expression patterns seen in RAMOS and primary B cells suggest that emAb insertion can block endogenous IgH expression when inserted into the productive allele. Differential expression of surface light chains is a means for the purification of cells that exclusively express the emAb construct. Instead, the possibility of insertion into either allele provides the potential for the production of dual antibody-expressing emAb cells by the selection of the initial pool of antiviral memory B cells for manipulation or by the insertion of different cassettes onto each allele.

[0183] Since we demonstrated the ability to engineer B cells, we next sought to confirm the cellular protective capacity in a murine model of viral infection. Murine emAb B cells were generated using expansion similar to that used for priming, electroporation + emAb cassette delivery, and expansion in human primary B cells (Figure 30A). Electroporation combined with pre-complexed guide RNA and Cas9 cleavage was highly efficient, with imperfect repair occurring in this region in 80% of the DNA analyzed (Figure 30B). Delivery of the murine αRSV-emAb cassette via AAV reproducibly modified murine B cells, with 8 - 24% of murine B cells binding RSV-F (Figures 30C, 30D). Insertion in 1 - 7% of the cells was also achieved using double-stranded DNA (dsDNA) containing short homology regions instead of AAV (Figures 30C, 30D (see also Example 1)), offering the possibility of emAb engineering of B cells using purely synthetic components. High titers of the secreted engineered antibodies were also detectable in the culture supernatants produced by both methods (Figure 30E).

[0184] To test the potential for antiviral protection, 1.5×10 7 Genetically modified murine B cells were injected into wild-type Balbc / byJ mice, followed by blood collection and RSV challenge (Figure 31A). RSV-specific antibodies and genetically modified B cells were present in the blood 6 days after transfer of the genetically modified murine B cells (Figures 31B, 31C). Importantly, mice that received genetically modified murine B cells were almost completely protected against RSV infection (Figure 31D). This protection approached that provided by injection of palivizumab 2 days prior to infection (Figure 31D). Transfer of mixed human emAb cells targeting RSV and influenza into NOD-scidIL2R gamma null (NSG) mice results in serum titers of antibodies targeting both viruses (Figures 32A, 32B). These results demonstrate that the genetically modified B cells disclosed herein protect against viral infection.

[0185] Method. Design of a single-chain antibody template sequence. Human: The antibody construct contains the IgVH1-69 heavy-chain promoter region (SEQ ID NO: 111), a full-length antibody light chain (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 Strep tag II 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 splice junction (e.g., SEQ ID NOs: 124 and 151) with a 60-base pair adjacent sequence derived from adapting the IgHJ variable region.

[0186] Mouse: The antibody construct contains the J5558H10 heavy-chain promoter (SEQ ID NO: 128, V.A 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 Strep tag II motif (SEQ ID NO: 116 (nucleotides) and SEQ ID NO: 122 (amino acids)), the codon-optimized variable region of the heavy antibody chain (SEQ ID NO: 133 (nucleotides) and SEQ ID NO: 138 (amino acids)), and a splice junction (e.g., SEQ ID NO: 139) with a 60-base pair adjacent sequence derived from the mouse IgHJ3 gene segment.

[0187] The complete sequences of the exemplary antibody constructs are available in FIGS. 25B - 25I.

[0188] Production of recombinant AAV vectors. AAV vectors were generated by triple transfection of AAV vectors, serotype 6 capsids, and adenovirus helper plasmid (pHelper) into HEK293T cells using PEI. At 24 hours after transfection, the medium was replaced with serum-free DMEM, and at 72 hours, the cells were harvested, lysed by freeze-thawing, treated with benzonase, purified on an iodixanol gradient, and subsequently concentrated into PBS using an Amicon Ultra-15 column (EMD Millipore) (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 ranged from 5×10 10 ~1×10 12 per microliter (Aurnhammer et al., (2012). Hum Gene Ther Methods 23(1):18 - 28).

[0189] Production of mouse dsDNA emAb template.

[0190] The αRSV-emAb template was amplified, and the short homology regions were modified with the following DNA oligos: Forward primer: (5' phosphate in bold, containing mouse genomic homology region)

[0191]

Chem.

[0192]

Chem.

[0193] Cell lines. 3T3-msCD40L was obtained from Dr. Mark Connors of the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Cat#12535. 3T3 was cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco), 100 U / ml penicillin plus 100 μg / ml streptomycin (Gibco), and G418 (350 μg / mL).

[0194] RAMOS cells were obtained from ATCC (CRL-1596 (trademark)). RAMOS cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (Gibco) and 100 U / ml penicillin plus 100 μg / ml streptomycin (Gibco).

[0195] Mouse B cell culture and electroporation. The basal B cell medium contained RPMI medium supplemented with 10% fetal bovine serum (Gemini Biosciences), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 55 μM beta-mercaptoethanol (Sigma), and 100 U / ml penicillin plus 100 μg / ml streptomycin (Gibco) except for the antibiotic-free step as indicated.

[0196] B cells were isolated from the spleen and lymph nodes by negative selection using magnetic beads (Miltenyi) and cultured at 2×10 6 / ml for 24 hours in B cell medium supplemented with 100 ng / ml of recombinant carrier HA-tagged mouse CD40L (R&D systems), 100 ng / ml of anti-HA antibody (clone 543851, R&D systems), and 4 ng / ml of mouse IL-4 (R&D systems). Next, the 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 of Cas9 to 300 ng of sgRNA and incubated at room temperature for at least 10 minutes. The B cells were washed with PBS and resuspended in 12 μg of Cas9 RNP / 108 Together with the cells, they were suspended in Neon Buffer T at a final density of 2.5×10 7 cells / ml. Under dsDNA conditions, 7.5 μg of dsDNA template / 10 6 cells were also included in the electroporation. The cells were electroporated (1675 V, 10 milliseconds, 3 pulses) and immediately dispensed into pre-warmed antibiotic-free medium. Under AAV conditions, concentrated AAV in PBS was added up to 15% of the final culture volume. After electroporation, B cells were further expanded for 48 hours using B cell medium supplemented with 100 ng / ml of recombinant carrier HA-tagged mouse CD40L (R&D systems), 100 ng / ml of anti-HA antibody (clone 543851, R&D systems), 4 ng / ml of mouse IL-4 (R&D systems) and 20 ng / ml of mouse IL-21 (Biolegend). In the secondary expansion, B cells were co-cultured with irradiated (80 Gy) NIH 3T3-CD40L feeder cells in the presence of 20 ng / ml of mouse IL-21 (Biolegend).

[0197] Human B cell culture and electroporation. The basal medium for human B cell culture (hBCM) was IMDM medium supplemented with 10% fetal bovine serum (Gemini Biosciences), 100 U / ml of penicillin and 100 μg / ml of streptomycin (Gibco), except for the antibiotic-free steps noted.

[0198] Human PBMCs were obtained through the Fred Hutchinson Cancer Research Center. The cells were thawed and isolated using negative selection with the Miltenyi B cell isolation kit II (Human) according to the manufacturer's protocol. The isolated cells were 0.5 - 1.0×10 6Resuspended in cells / mL.

[0199] After 48 hours of stimulation, the cells were electroporated using the Neon transfection system. Cas9 protein (Invitrogen) and H7 sgRNA (Synthego) were pre-complexed at a 2:1 ratio in buffer T for 20 minutes at room temperature. The cells were washed with PBS (Gibco) and resuspended in buffer T containing the pre-complexed Cas9 RNP at a final concentration of 2.5×10 7 cells / ml. The cell-RNP mixture was loaded into 10 μL of Neon transfection tips and electroporated at settings of 1750 V, 20 ms, and 1 pulse according to the manufacturer's protocol. Immediately after electroporation, the cells were seeded into 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 thoroughly mixed. After 2 - 4 hours, the cells were transferred to a larger culture dish for further expansion.

[0200] Two days after electroporation, the cells were stained with a fluorescent dye-labeled antigen or streptactin to select genetically modified cells. For secondary expansion, B cells were co-cultured with irradiated (80 Gy) NIH 3T3-CD40L feeder cells in hBCM containing 5 μg / mL of human recombinant insulin (Sigma), 50 μg / mL of transferrin (Sigma), 50 ng / mL of recombinant IL-2 (Biolegend), 20 ng / mL of IL-21 (Biolegend), and 10 ng / mL of IL-15 (Shenandoah Biotech).

[0201] To promote differentiation into plasma cells, the cells were transferred from the expansion conditions to fresh feeder-free culture conditions containing hBCM supplemented with 5 μg / mL of human recombinant insulin (Sigma), 50 μg / mL of transferrin (Sigma), 500 U / mL of universal type 1 IFN protein (R&D Systems), 50 ng / mL of IL-6 (Shenandoah Biotech), and 10 ng / mL of IL-15 (Shenandoah Biotech).

[0202] Evaluation of sGRNA activity by TIDE. Genomic DNA was isolated from mock and Cas9 / sgRNA-treated cells 3 - 5 days after electroporation. The 500 - 600 base pair region adjacent to the sgRNA target site was amplified by PCR using the following oligos: Mouse:

[0203]

Chemical formula

[0204] The purified PCR products were Sanger sequenced, and the frequency of indels in Cas9 / sgRNA electroporated cells compared to mock electroporated cells was determined using the ICE algorithm (Hsiau et al., (2018). "Inference of CRISPR Edits from Sanger Trace Data." bioRxiv).

[0205] Protein antigens. Pre-fusion RSV-F protein, EBV gh / gl complex, and modified HIV env antigen (426c TM4 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 - 811 e799). Stabilized influenza HA-stem was produced from VRC clone 3925 derived from strain H1 1999NC as described (Yassine et al., (2015). Nat Med 21(9):1065 - 1070). Monomeric pre-fusion RSV-F protein was labeled with Alexa-488 (Thermo Fisher). All other proteins were conjugated to biotin using a biotin to protein molar ratio of 0.8 - 2, followed by tetramerization using streptavidin-PE or -APC (prozyme).

[0206] Flow cytometry. Flow cytometry analysis was performed on a FACSymphony instrument (BD bioscience), cells were sorted on an Aria II (BD bioscience), and data were analyzed using Flowjo software (Tree Star).

[0207] EmAb treatment study in mice. Animal studies were approved by and conducted in accordance with the Fred Hutchinson Cancer Research Center Animal Care and Use Committee.

[0208] For RSV load, EmAb or control B cells were administered as a single intraperitoneal (IP) dose of 1.5×10 7 cells. For passive transfer of palivizumab, mice received a single dose of 15 mg / kg i.p. 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 intranasally with 10 6 pfu of sucrose-purified RSV in 40 μL of PBS. Lungs were harvested 5 days post-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 of 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 DMEM culture medium. 100 μL of each dilution was added to confluent Vero cells in 24-well plates at 37 °C for 2 hours. Next, an overlay of 0.8% methylcellulose was added and plates were incubated for 5 days prior to imaging with a Typhoon imager with filter settings for GFP. Titers in pfu / lung were calculated by counting the number of plaques in the highest positive dilution and correcting for the dilution factor.

[0209] For human cell engraftment, human emAb B cells were 5×106 Single IP dose of cells / emAb specificity (1×10 7 total) was administered to NOD-scid IL2R gamma ヌル (NSG) mice (produced by the FHCRC breeding facility). Seven days after transplantation, blood was collected, and human emAb titers against RSV-F and HA-stem in serum were determined by ELISA.

[0210] Statistical analysis. Statistical analysis was performed using GraphPad Prism 7. Pairwise comparisons were performed using an independent t-test with Welch's correction.

[0211] The nucleic acid sequences described herein are shown using standard character abbreviations representing nucleotide bases as defined in 37 C.F.R.§1.822. In some examples, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in embodiments where it is considered appropriate. As an example, sequences complementary to the target sites including SEQ ID NOs: 5-84 provide gRNA targeting sequences that target these sites.

[0212] Any nucleic acid encoding a selected antibody construct described herein can be utilized. Variants of the nucleic acid sequences disclosed herein include various sequence polymorphisms, mutations, and changes that do not substantially affect the function of the encoded protein. The term nucleic acid or "gene" may include not only the coding sequence but also regulatory regions such as promoters, enhancers, and termination regions. The term can further include all introns and other DNA sequences spliced from mRNA transcripts, as well as variants resulting from alternative splicing sites. The coding nucleic acid can be DNA or RNA that directs the expression of one or more selected antibody constructs. These nucleic acid sequences can be either the DNA strand sequence transcribed into RNA or the RNA sequence translated into protein. The nucleic acid sequences include both full-length nucleic acid sequences and non-full-length sequences derived from full-length proteins. The sequences can also include natural sequences or degenerate codons of sequences that may be introduced to provide codon selection in a particular cell type. The nucleic acid sequences encoding the selected antibody constructs can be readily prepared from the associated amino acid sequences of the selected antibody constructs.

[0213] A "variant" of a protein sequence includes a protein sequence having one or more amino acid additions, deletions, stop positions, or substitutions as compared to the protein sequences disclosed elsewhere herein.

[0214] The amino acid substitutions can be conservative or non-conservative substitutions. Variants of the protein sequences disclosed herein can include protein sequences having one or more conservative amino acid substitutions. "Conservative substitution" or "conservative amino acid substitution" includes substitutions found in one of the following groups of conservative substitutions: 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.

[0215] Furthermore, amino acids can be classified into conservative substitution groups based on similar functions, chemical structures or compositions (e.g., acidic, basic, aliphatic, aromatic or sulfur-containing). For example, the aliphatic classification may include G, A, V, L and I for substitution purposes. Other groups containing amino acids considered to be conservative substitutions for each other are: 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.

[0216] Non-conservative substitutions include substitutions that significantly affect the structure of the peptide backbone (e.g., alpha helix or beta sheet structure) in the region of change; the charge or hydrophobicity of the molecule at the target site; or the volume of the side chain. In general, non-conservative substitutions that are expected to produce the greatest change in the properties of a protein are those in which (i) a hydrophilic residue (e.g., S or T) can be replaced by (or with) a hydrophobic residue (e.g., L, I, F, V or A); (ii) C or P can be replaced by (or with) any other residue; (iii) a residue with a positively charged side chain (e.g., K, R or H) can be replaced by (or with) a residue with a negatively charged side chain (e.g., Q or D); or (iv) a residue with a bulky side chain (e.g., F) can be replaced by (or with) a residue without a bulky side chain (e.g., G). Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0217] 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 the reference sequences disclosed herein.

[0218] With respect to the sequences identified herein, "percent (%) sequence identity" is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to the nucleic acid or amino acid residues in a reference sequence, after the sequences have been aligned to achieve maximum percent sequence identity and gaps, if necessary, have been introduced. Alignment for the purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways within the skill in the art using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm not necessary to achieve maximum alignment over the full length of the sequences being compared. For example, the % sequence identity values generated using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology, 266:460-480 (1996)) use several search parameters, most of which are set to default values. The parameters that are not set to default values (i.e., adjustable parameters) are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11 and scoring matrix BLOSUM62.

[0219] Variants typically exhibit the same qualitative biological activity and elicit a biological response that is substantially similar to the reference nucleic acid or peptide sequence, but the variants can be selected to modify the required characteristics of the reference nucleic acid or peptide. Screening for variants can be performed using the experimental protocols described herein.

[0220] As will be understood by those skilled in the art, each of the embodiments disclosed herein can consist essentially of or consist of including its specific recited elements, steps, components or ingredients. Accordingly, the terms "include" or "including" should be construed as listing "comprise, consist of or consist essentially of". The transitional term "comprise" or "comprises" means includes, but is not limited to, and permits the inclusion of elements, steps, components or ingredients not specified, even in large amounts. The transitional phrase "consisting of" excludes any element, step, component or ingredient not specified. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, components or ingredients and those that do not materially affect the embodiment. A material effect is considered to cause a statistically significant decrease in B cell expression of the selected antibody.

[0221] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and other properties 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 attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Each numerical parameter should at least be construed in light of the reported number of significant digits and by applying ordinary rounding techniques. In circumstances where further clarity is required, the term "about" when used in conjunction with a stated numerical value or range has the meaning that would reasonably be attributed to that term by a person of ordinary skill in the art, i.e., within a range of somewhat more or less than the stated value, within a range of plus or minus 20% of the stated value, plus or minus 19% of the stated value, plus or minus 18% of the stated value, plus or minus 17% of the stated value, plus or minus 16% of the stated value, plus or minus 15% of the stated value, plus or minus 14% of the stated value, plus or minus 13% of the stated value, plus or minus 12% of the stated value, plus or minus 11% of the stated value, plus or minus 10% of the stated value, plus or minus 9% of the stated value, plus or minus 8% of the stated value, plus or minus 7% of the stated value, plus or minus 6% of the stated value, plus or minus 5% of the stated value, plus or minus 4% of the stated value, plus or minus 3% of the stated value, plus or minus 2% of the stated value, or plus or minus 1% of the stated value.

[0222] 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 its respective testing measurements.

[0223] The terms "a", "an", "the", and similar referents used in the context of describing the present invention (in particular, in the context of the following claims) are to be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. The recitation of a range of values herein is merely intended to serve as a shorthand way of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not limit the scope of the invention as otherwise claimed. The language of the specification should not be construed as indicating any non-claimed element essential to the practice of the invention.

[0224] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in or deleted from the group for convenience and / or for reasons of patentability. Even if any such inclusion or deletion occurs, the specification is considered to contain the modified group and thus to satisfy the written description of all Markush groups used in the appended claims.

[0225] Certain embodiments of the invention are described herein, including the best mode known to the inventors as of the time of filing this application for carrying out the invention. It will be apparent to those skilled in the art that variations of these described embodiments will become evident upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, any combination of the above-described elements in all possible variations thereof, unless otherwise indicated herein or otherwise clearly contradicted by context, is included within the invention.

[0226] Furthermore, throughout this specification, numerous patents, publications, journal articles, and other documents have been referenced (references herein). Each of the references is hereby incorporated by reference in its entirety for the teachings to which it is referred.

[0227] In conclusion, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be used are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to the invention precisely shown and described.

[0228] The features shown herein are presented by way of example and for purposes of illustrative discussion of the preferred embodiments of the invention only, and are provided to provide what is believed to be the most useful and readily understood description of the principles of the invention and the conceptual aspects of various embodiments. In this regard, no attempt has been made to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description incorporated with the figures and / or examples is to make apparent to those skilled in the art how some forms of the invention may be practiced in fact.

[0229] The definitions and explanations used in this disclosure are intended to be controlling in any future interpretation, provided they are not modified without clear ambiguity in the following examples, or rendered meaningless or essentially meaningless by any application of meaning. When an interpretation of a term is considered to render that interpretation meaningless or essentially meaningless, the definition should be drawn from a dictionary known to those of ordinary skill in the art, such as Webster's Third New International Dictionary or the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. 1. A method of genetically modifying B cells to express palivizumab, comprising inserting a genetic construct comprising a palivizumab heavy chain promoter and a nucleic acid sequence encoding the entire light chain, a Gly-Ser linker, and the variable region of the heavy chain of palivizumab in SEQ ID NO:1, thereby genetically engineering the B cells to express palivizumab.

2. 1. A method of genetically engineering B cells to express a selected antibody, comprising inserting 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 skipping element; (v) a variable region of the heavy chain of the selected antibody; and (vi) a splice junction in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, thereby genetically engineering B cells to express the selected antibody.

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

4. 3. The method of claim 2, 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-metapneumovirus (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.

5. The method of claim 2, wherein the genetic construct comprises SEQ ID NOs: 102-175, 278, 279 or 280-289.

6. The method of claim 2, wherein a 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.

7. The method of claim 2, wherein the flexible linker is selected from SEQ ID NOs: 180-184.

8. The method of claim 2, wherein the flexible linker is a Gly-Ser linker comprising 50 to 80 amino acids.

9. The method of claim 2, wherein the flexible linker is a Gly-Ser linker comprising 57 amino acids.

10. 3. The method of claim 2, wherein the flexible linker is SEQ ID NO:

122.

11. The method of claim 2, wherein the skipping element is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody.

12. The method of claim 2 , wherein the skipping element is a self-cleaving peptide.

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

14. The method of claim 2, wherein the skipping element is an internal ribosome entry site (IRES).

15. 3. The method of claim 2, wherein the heavy chain promoter is selected from SEQ ID NOs: 111 and 128.

16. The method of claim 2, wherein the heavy chain promoter is IgVH1-69 or J558H10.

17. The method of claim 2, wherein the signal peptide is selected from SEQ ID NOs: 118, 134 and 185-194.

18. The method of claim 2 , wherein the signal peptide is derived from a human IgH heavy chain or a human IgL light chain.

19. The method of claim 2 , wherein the genetic construct comprises homology arms.

20. 20. The method of claim 19, wherein the homology arms comprise SEQ ID NOs: 90-101, 110, 125, 127, 140, 142, 143, 153, 170, 171, 173, 174, 278 or 279.

21. The method of claim 2 , wherein the genetic construct encodes a tag.

22. 22. The method of claim 21, wherein the tag comprises STREPTAG®, STREP® tag II, His tag, Flag tag, Xpress tag, Avi tag, calmodulin tag, polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Sof tag 1, Sof tag 3 or V5 tag.

23. The method of claim 21, wherein the tag comprises SEQ ID NO: 122 or 195-204.

24. 3. The method of claim 2, further comprising delivering a guide RNA (gRNA) sequence selected from one or more of SEQ ID NOs: 87-89 and 290-366 and a nuclease to the B cell.

25. 25. The method of claim 24, wherein the delivering is through electroporation, nanoparticles or viral mediated delivery.

26. The method of claim 2, wherein the genetic construct is part of an adeno-associated viral vector.

27. 25. The method of claim 24, wherein the gRNA and nuclease are delivered via electroporation and the genetic construct is delivered as part of an adeno-associated viral vector.

28. 25. The method of claim 24, wherein the nuclease is Cas9 or Cpf1.

29. 25. The method of claim 24, 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.

30. 3. The method of claim 2, wherein the selected antibody is an anti-RSV antibody, including palivizumab, AB1128, or ab20745.

31. 3. The method of claim 2, wherein the selected antibody is palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 136; palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 205; an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 120; or an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO:

206.

32. The method of claim 2, 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.

33. 3. The method of claim 2, wherein the selected antibody is an anti-HIV antibody, including 10E8, VRC01, ab18633, or 39 / 5.4A.

34. The method of claim 2, 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.

35. The method of claim 2, wherein the selected antibody is an anti-HIV antibody comprising a CDRH1 comprising SEQ ID NO: 213, a CDRH2 comprising SEQ ID NO: 214, a CDRH3 comprising SEQ ID NO: 215, a CDRL1 comprising SEQ ID NO: 216, a CDRL2 comprising SEQ ID NO: 217, and a CDRL3 comprising SEQ ID NO: 218, or a CDRH1 comprising SEQ ID NO: 219, a CDRH2 comprising SEQ ID NO: 220, a CDRH3 comprising SEQ ID NO: 221, a CDRL1 comprising QYGS, a CDRL2 comprising SGS, and a CDRL3 comprising SEQ ID NO:

222.

36. The method of claim 2, wherein the selected antibody is an anti-dengue virus antibody, including antibody 55, DB2-3, ab155042, or ab80914.

37. The method of claim 2, 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 CDRL3 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.

38. The method of claim 2, 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.

39. The method of claim 2, wherein the selected antibody is an anti-Hepatitis C antibody, including MAB8694 or C7-50.

40. The method of claim 2, wherein the selected antibody is an anti-Hepatitis C antibody comprising a CDRH1 comprising SEQ ID NO: 237, a CDRH2 comprising SEQ ID NO: 238, a CDRH3 comprising SEQ ID NO: 239, a CDRL1 comprising SEQ ID NO: 240, a CDRL2 comprising SEQ ID NO: 241, and a CDRL3 comprising SEQ ID NO:

242.

41. The method of claim 2, wherein the selected antibody is an anti-influenza virus antibody, including C102.

42. The method of claim 2, 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.

43. The method of claim 2, 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.

44. The method of claim 2, wherein the selected antibody is an anti-MPV antibody that comprises MPE8.

45. 3. The method of claim 2, wherein the selected antibody is an anti-CMV antibody, including MCMV5322A, MCMV3068A, LJP538 or LJP539.

46. The method of claim 2, 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.

47. The method of claim 2, wherein the selected antibody is an anti-EBV antibody comprising a CDRH1 comprising SEQ ID NO: 248, a CDRH2 comprising SEQ ID NO: 249, a CDRH3 comprising SEQ ID NO: 250, a CDRL1 comprising SEQ ID NO: 251, a CDRL2 comprising SEQ ID NO: 252, and a CDRL3 comprising SEQ ID NO:

253.

48. The method of claim 2, wherein the selected antibodies are anti-HSV antibodies, including HSV8-N and MB66.

49. 3. The method of claim 2, wherein the selected antibody is an anti-Clostridium difficile antibody, including actoxumab or bezlotoxumab.

50. 3. The method of claim 2, wherein the selected antibody is an anti-TNF antibody, including infliximab, adalimumab, etanercept, certolizumab, or an approved biosimilar thereof.

51. 3. The method of claim 2, wherein the selected antibody is an anti-TNF antibody comprising 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; or a CDRH1 comprising SEQ ID NO:268, a CDRH2 comprising SEQ ID NO:269, and a CDRH3 comprising SEQ ID NO:270; a CDRL1 comprising SEQ ID NO:271, a CDRL2 comprising SEQ ID NO:272, and a CDRL3 comprising SEQ ID NO:

273.

52. The method of claim 2, wherein the genetic modification utilizes a sequence comprising any one of SEQ ID NOs: 87, 88, 89, 90-175, 278-366.

53. 3. The method of claim 2, 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.

54. A B cell modified according to the method of any one of claims 2 to 53.

55. 55. The B cell of claim 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.

56. 55. A method of providing an anti-infective effect in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a B cell of claim 54, thereby providing an anti-infective effect.

57. 57. The method of claim 56, wherein providing obviates the need for vaccination.

58. 57. The method of claim 56, wherein administering replaces a vaccination protocol.

59. 57. The method of claim 56, wherein the subject is immunosuppressed.

60. 57. The method of claim 56, wherein the subject is immunosuppressed as part of a treatment regimen comprising bone marrow transplantation, hematopoietic stem cell transplantation, or administration of genetically modified hematopoietic stem cells.

61. 55. A method of providing an anti-inflammatory effect in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a B cell described in claim 54, thereby providing an anti-inflammatory effect.

62. A genetic construct for modifying a B cell to express a selected antibody, 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 skipping element; (v) a variable region of the heavy chain of the selected antibody; and (vi) a splice junction.

63. 63. The genetic construct of claim 62, comprising SEQ ID NO: 102-175 or 280-289.

64. 63. The genetic construct of claim 62, wherein a 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.

65. 63. The genetic construct of claim 62, wherein the flexible linker is selected from SEQ ID NOs: 180 to 184.

66. 63. The genetic construct of claim 62, wherein the flexible linker is a Gly-Ser linker comprising 50 to 80 amino acids.

67. 63. The genetic construct of claim 62, wherein the flexible linker is a Gly-Ser linker comprising 57 amino acids.

68. 63. The genetic construct of claim 62, wherein the flexible linker is SEQ ID NO:

122.

69. 63. The genetic construct of claim 62, wherein the skipping element is between the full-length light chain of the selected antibody and the variable region of the heavy chain of the selected antibody.

70. 63. The genetic construct of claim 62, wherein the skipping element is a self-cleaving peptide.

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

72. 63. The genetic construct of claim 62, wherein the skipping element is an internal ribosome entry site (IRES).

73. 63. The genetic construct of claim 62, wherein the heavy chain promoter is selected from SEQ ID NOs: 111 and 128.

74. The genetic construct of claim 62, wherein the heavy chain promoter is IgVH1-69 or J558H10.

75. 63. The genetic construct of claim 62, wherein the signal peptide is selected from SEQ ID NOs: 118, 134 and 185-194.

76. 63. The genetic construct of claim 62, wherein the signal peptide is derived from a human IgH heavy chain or a human IgL light chain.

77. 63. The genetic construct of claim 62, comprising homology arms.

78. 78. The genetic construct of claim 77, wherein the homology arms comprise SEQ ID NOs: 90-101, 110, 125, 127, 140, 142, 143, 153, 170, 171, 173, 174, 278 or 279.

79. 63. The genetic construct of claim 62, which encodes a tag.

80. 80. The genetic construct of claim 79, wherein the tag comprises STREPTAG (registered trademark), STREP (registered trademark) tag II, His tag, Flag tag, Xpress tag, Avi tag, calmodulin tag, polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Sof tag 1, Sof tag 3 or V5 tag.

81. The genetic construct of claim 79, wherein the tag comprises SEQ ID NO: 122 or 195-204.

82. 63. The genetic construct of claim 62, wherein the selected antibody is an anti-RSV antibody, including palivizumab, AB1128 or ab20745.

83. The genetic construct of claim 62, wherein the selected antibody is palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 136; palivizumab comprising a heavy chain comprising SEQ ID NO: 138 and a light chain comprising SEQ ID NO: 205; an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO: 120; or an anti-RSV antibody comprising a heavy chain comprising SEQ ID NO: 123 and a light chain comprising SEQ ID NO:

206.

84. The genetic construct of claim 62, 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.

85. 63. The genetic construct of claim 62, wherein the selected antibody is an anti-HIV antibody comprising 10E8, VRC01, ab18633 or 39 / 5.4A.

86. 63. The genetic construct of claim 62, 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.

87. The genetic construct of claim 62, wherein the selected antibody is an anti-HIV antibody comprising a CDRH1 comprising SEQ ID NO: 213, a CDRH2 comprising SEQ ID NO: 214, a CDRH3 comprising SEQ ID NO: 215, a CDRL1 comprising SEQ ID NO: 216, a CDRL2 comprising SEQ ID NO: 217, and a CDRL3 comprising SEQ ID NO: 218 or a CDRH1 comprising SEQ ID NO: 219, a CDRH2 comprising SEQ ID NO: 220, a CDRH3 comprising SEQ ID NO: 221, a CDRL1 comprising QYGS, a CDRL2 comprising SGS, and a CDRL3 comprising SEQ ID NO:

222.

88. The genetic construct of claim 62, wherein the selected antibody is an anti-dengue virus antibody, including antibody 55, DB2-3, ab155042 or ab80914.

89. The genetic construct of claim 62, 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 CDRL3 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.

90. The genetic construct of claim 62, 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.

91. The genetic construct of claim 62, wherein the selected antibody is an anti-hepatitis C antibody, including MAB8694 or C7-50.

92. The genetic construct of claim 62, wherein the selected antibody is an anti-Hepatitis C antibody comprising a CDRH1 comprising SEQ ID NO: 237, a CDRH2 comprising SEQ ID NO: 238, a CDRH3 comprising SEQ ID NO: 239, a CDRL1 comprising SEQ ID NO: 240, a CDRL2 comprising SEQ ID NO: 241, and a CDRL3 comprising SEQ ID NO:

242.

93. The genetic construct of claim 62, wherein the selected antibody is an anti-influenza virus antibody, including C102.

94. The genetic construct of claim 62, 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.

95. The genetic construct of claim 62, 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.

96. 63. The genetic construct of claim 62, wherein the selected antibody is an anti-MPV antibody comprising MPE8.

97. 63. The genetic construct of claim 62, wherein the selected antibody is an anti-CMV antibody comprising MCMV5322A, MCMV3068A, LJP538 or LJP539.

98. The genetic construct of claim 62, 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.

99. The genetic construct of claim 62, wherein the selected antibody is an anti-EBV antibody comprising a CDRH1 comprising SEQ ID NO: 248, a CDRH2 comprising SEQ ID NO: 249, a CDRH3 comprising SEQ ID NO: 250, a CDRL1 comprising SEQ ID NO: 251, a CDRL2 comprising SEQ ID NO: 252, and a CDRL3 comprising SEQ ID NO:

253.

100. The genetic construct of claim 62, wherein the selected antibodies are anti-HSV antibodies including HSV8-N and MB66.

101. 63. The genetic construct of claim 62, wherein the selected antibody is an anti-Clostridium difficile antibody, including actoxumab or bezlotoxumab.

102. 63. The genetic construct of claim 62, wherein the selected antibody is an anti-TNF antibody, including infliximab, adalimumab, etanercept, certolizumab or an approved biosimilar thereof.

103. 63. The genetic construct of claim 62, wherein the selected antibody is an anti-TNF antibody comprising 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; or a CDRH1 comprising SEQ ID NO: 268, a CDRH2 comprising SEQ ID NO: 269, and a CDRH3 comprising SEQ ID NO: 270; a CDRL1 comprising SEQ ID NO: 271, a CDRL2 comprising SEQ ID NO: 272, and a CDRL3 comprising SEQ ID NO:

273.

104. A kit for genetically modifying B cells comprising a genetic construct according to any one of claims 62 to 103 and a gRNA targeting SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

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

106. 105. The kit of claim 104, further comprising a nuclease.

107. The kit of claim 106, wherein the nuclease is Cas9 or Cpf1.

108. 105. The kit of claim 104, further comprising a nanoparticle or an adeno-associated viral vector.

109. The kit of claim 106, wherein the gRNA and the nuclease are associated with a nanoparticle.

110. The kit of claim 104, wherein the genetic construct is part of an adeno-associated viral vector.

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