CN112105641A

CN112105641A - Compositions and methods for modified B cell expression of redistributed biological agents

Info

Publication number: CN112105641A
Application number: CN201980030908.8A
Authority: CN
Inventors: 罗德里克·A·海德; 韦恩·R·金斯沃格尔; 盖瑞·L·麦克奈特
Original assignee: Kota Biotherapy Co Ltd
Current assignee: Invention Science Fund II LLC
Priority date: 2018-03-19
Filing date: 2019-03-15
Publication date: 2020-12-18
Also published as: EP3768707A1; WO2019182910A1; EP3768707A4

Abstract

Disclosed herein are compositions and methods for producing one or more immunoglobulins in an isolated cytotoxic B lymphocyte cell line. The isolated cell line comprises an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin capable of binding to a first antigen and at least one endogenously secreted immunoglobulin capable of binding to a second antigen, and further capable of expressing at least one exogenously incorporated recombinant B cell receptor for signaling expression of a cytotoxic effector molecule.

Description

Compositions and methods for modified B cell expression of redistributed biological agents

All subject matter of the priority application is incorporated herein by reference to the extent such subject matter is not inconsistent herewith.

Disclosure of Invention

Disclosed herein are compositions and methods for the production of immunoglobulins in recombinant B lymphocyte cell lines. Disclosed herein are compositions and methods for treating diseases in vertebrate subjects with immunotherapeutic products. The immunotherapeutic product may comprise a recombinant B lymphocyte cell line that produces one or more antibodies. Immunotherapeutic products may comprise recombinant B lymphocyte cell lines, a special type of antigen presenting cell.

Disclosed are compositions and methods for providing a therapeutically effective amount of one or more modified B lymphocytes to a patient suspected or known to have a disease, disorder or condition of the immune system, including but not limited to an infectious disease, autoimmune disease, cancer, neurophysiologic disease or disorder, or other pathological condition. In one embodiment, a set of modified B lymphocytes is provided, as described herein. In one embodiment, monoclonal administration of modified B lymphocytes is provided, as described herein. In one embodiment, polyclonal administration of modified B lymphocytes is provided, as described herein.

Disclosed herein are compositions and methods for producing one or more immunoglobulins in an isolated modified B cell, which may be part of a B lymphocyte cell line. Disclosed herein are compositions and methods for producing one or more immunoglobulins in an isolated modified B cell or B lymphocyte cell line that direct cell signaling through membrane immunoglobulins in the isolated modified B cell or B lymphocyte cell line. An immune cell therapy in a vertebrate subject can include administering to the vertebrate subject an isolated modified B cell or B lymphocyte cell line that synthesizes secreted and membrane immunoglobulins, each with a different target antigen. Immune cell therapy in a vertebrate subject can comprise administering to the vertebrate subject antigen-presenting cells comprising isolated modified B cells or B lymphocyte cell lines that direct antigen internalization and processing to produce a particular antigen-presenting cell. Isolated modified B cells or B lymphocyte cell lines can produce antigen presenting cells that are superior or superior in capturing, internalizing, and presenting antigens recognized by membrane immunoglobulins derived either endogenously or exogenously. Disclosed herein are compositions and methods for treating diseases in vertebrate subjects with immunotherapeutic products. The immunotherapeutic product may comprise an isolated modified B cell or B lymphocyte cell line having membrane immunoglobulins endogenously or exogenously derived that react with (e.g., are capable of binding to) a first antigen, wherein the isolated modified B cell or B lymphocyte cell line produces one or more secreted immunoglobulins that react with a second antigen or produces a redistributed (reassorted) biologic agent. The immunotherapeutic product may comprise an isolated modified B cell or B lymphocyte cell line, which may be a monoclonal or polyclonal B lymphocyte cell line that produces one or more secreted immunoglobulins. Immunotherapeutic products may comprise an isolated B lymphocyte cell line that produces one or more secreted antibodies, e.g., antibodies that recognize different epitopes on the same antigen. The immunotherapeutic product may comprise an isolated B lymphocyte cell line that produces redistributed biological agents (e.g., cytokines, cytotoxins, chemokines, receptors, ligands, immune modulators, immune effector molecules, transcription factors, etc.). The immunotherapeutic product may comprise an isolated B lymphocyte cell line as one or more antigen presenting cells.

Thus, the redistributed biological agent may comprise an agent (e.g., a cytokine or ligand) that is not normally expressed by, or under certain circumstances or conditions expressed by, naturally occurring B lymphocytes. Our modified B lymphocytes are capable of expressing the redistributed biological agent as a result of engineering of the B lymphocyte, which may include constitutive expression of the redistributed biological agent, or may include induced expression under specific circumstances or conditions (e.g., expression "triggered" on the surface of the modified B lymphocyte by receptor/ligand binding).

The isolated B lymphocyte cell line can comprise an immunotherapeutic product administered to a vertebrate subject to develop long-lived isolated B lymphocytes in the vertebrate subject for immune surveillance of chronic diseases. Immunotherapeutic products may include isolated B lymphocyte cell lines with redistributed biologics to modulate immunity to treat chronic or acute diseases (e.g., IL-10 for multiple sclerosis or IL-2 for influenza). Immunotherapeutic products can comprise isolated B lymphocyte cell lines with endogenously or exogenously derived membrane immunoglobulins that can be administered to a vertebrate subject to provide antigen presenting cells to the vertebrate subject.

In one embodiment, the isolated modified B cell comprises at least one redistributed biological agent that incorporates an active Ig gene position (e.g., H or L chain) in a memory B cell. In one embodiment, the redistributed biological agent is under the control of the Ig promoter/enhancer element and the endogenous antibodies (secreted and/or membrane) of the isolated modified B cell are disrupted. In one embodiment, the isolated modified B cell has an exogenous membrane Ig (B cell receptor) that binds to the antigen and induces expression of the redistributed biological agent. In one embodiment, both the exogenous membrane Ig (B cell receptor) and the redistributed biological agent may be expressed on the same active Ig locus (e.g., H chain).

An isolated cell line as described herein can include an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin reactive with a first antigen and at least one endogenously secreted immunoglobulin reactive with a second antigen. The isolated B lymphocyte cell line is capable of expressing at least one endogenous membrane immunoglobulin reactive with a second antigen. The at least one exogenously incorporated membrane immunoglobulin can comprise one or more exogenously incorporated membrane immunoglobulin polypeptides. The at least one exogenously incorporated membrane immunoglobulin can comprise at least one exogenously incorporated nucleic acid encoding the at least one membrane immunoglobulin, wherein the cell line is capable of expressing the at least one membrane immunoglobulin. The at least one exogenously incorporated membrane immunoglobulin comprises at least two exogenously incorporated nucleic acids encoding the at least one membrane immunoglobulin. The at least one exogenously incorporated membrane immunoglobulin can comprise nucleic acids encoding two heavy chain (H) immunoglobulins and two light chain (L) immunoglobulins. The at least one exogenously incorporated membrane immunoglobulin can comprise nucleic acids encoding one heavy (H) immunoglobulin and one light (L) immunoglobulin. The at least one exogenously incorporated membrane immunoglobulin can comprise nucleic acid encoding a single chain fv (SCFv) immunoglobulin (e.g., an SCFv fused to an immunoglobulin constant region domain). The exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be present in one or more chromosomal loci in an isolated B lymphocyte cell line. Exogenously incorporated nucleic acids in isolated B lymphocyte cell lines are capable of disrupting expression of endogenous immunoglobulins. For example, disruption of endogenous H or L chain expression can knock down the production of member IgG and/or secreted IgG, and more generally, synthesis of endogenous antibodies.

In one embodiment, insertion of exogenously incorporated membrane immunoglobulins, exogenously incorporated secreted immunoglobulins or exogenously incorporated cytotoxic effector molecules (e.g., using CRISPR techniques described herein) at the active site allows hijacking of endogenous machinery (e.g., in close proximity of promoter and enhancer elements upon rearrangement of regions).

At least two exogenously incorporated nucleic acids encoding at least one of the membrane immunoglobulins can be present in the Ig H chain and Ig L chain chromosomal loci of an isolated B lymphocyte cell line. The at least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be present in one or more non-Ig L chain or non-Ig H chain chromosomal loci in an isolated B lymphocyte cell line. At least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be present in an extrachromosomally replicating genetic element of an isolated B lymphocyte cell line. The at least one exogenously incorporated nucleic acid encoding the at least one membrane immunoglobulin can be derived from a B lymphocyte cell line. The at least one exogenously incorporated membrane immunoglobulin activated by the first antigen is capable of controlling the expression of at least one endogenously secreted immunoglobulin reactive with the second antigen. The isolated B lymphocyte cell line can comprise at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a B1B lymphocyte, a marginal zone B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell. The isolated B lymphocyte cell line may comprise a polyclonal population of B lymphocytes. An isolated B lymphocyte cell line can comprise a monoclonal population of B lymphocytes. The membrane immunoglobulin may comprise at least one of a membrane anchor, a cytoplasmic domain, a hinge region, and an extracellular ligand binding domain.

In one embodiment, the exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be integrated at one or more locations along the gene encoding the B cell receptor. In one embodiment, the exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be integrated into one or more positions of an Ig locus (e.g., a heavy or light chain immunoglobulin as described above).

An isolated recombinant cell line as described herein can comprise an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin reactive with a first antigen and at least one exogenously incorporated nucleic acid encoding a secreted immunoglobulin reactive with a second antigen. The isolated B lymphocyte cell line is capable of expressing at least one exogenously incorporated nucleic acid encoding a membrane immunoglobulin reactive with a second antigen. The isolated B lymphocyte cell line is capable of expressing at least one exogenously incorporated nucleic acid encoding a secreted immunoglobulin reactive with a third antigen. The second antigen and the third antigen may be different epitopes of a single antigen polypeptide. The at least one exogenously incorporated membrane immunoglobulin can comprise at least one exogenously incorporated membrane immunoglobulin polypeptide. The at least one exogenously incorporated membrane immunoglobulin can comprise at least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin polypeptide, wherein the cell line is capable of expressing the at least one membrane immunoglobulin polypeptide. The at least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be present in one or more chromosomal loci of an isolated B lymphocyte cell line. At least two exogenously incorporated nucleic acids encoding at least one membrane immunoglobulin can be present in the Ig H chain and Ig L chain chromosomal loci of an isolated B lymphocyte cell line. The at least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be present in one or more non-Ig L or non-Ig H chromosomal loci in an isolated B lymphocyte cell line. At least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin can be present in an extrachromosomally replicating genetic element of an isolated B lymphocyte cell line. The nucleic acid encoding at least one membrane immunoglobulin may be derived from a B lymphocyte cell line. The at least one exogenously incorporated membrane immunoglobulin activated by the first antigen is capable of controlling the expression of the at least one exogenously incorporated secreted immunoglobulin reactive with the second antigen. The isolated B lymphocyte cell line can comprise at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a B1B lymphocyte, a marginal zone B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell. The isolated B lymphocyte cell line may comprise a polyclonal population of B lymphocytes. An isolated B lymphocyte cell line can comprise a monoclonal population of B lymphocytes. The membrane immunoglobulin may comprise at least one of a membrane anchor, a cytoplasmic domain, a hinge region, and an extracellular ligand binding domain.

In one embodiment, the modified B lymphocyte comprises a structural or functional feature for exhibiting cytotoxicity. For example, in one embodiment, the modified B lymphocyte cell or cell line produces one or more antibodies and has one or more B cell receptors (such as membrane immunoglobulins as described herein) specific for a target antigen (including but not limited to antigens that are mutated forms of "normal" cell antigens, as well as antigens modified by post-translational modifications, and antigens expressed in aberrant manners or at aberrant levels), such as a tumor antigen. In one embodiment, the modified B lymphocyte cell or cell line is capable of generating a complete immune response with both humoral and cellular immune components. Thus, the cytotoxic expression may comprise one or more secreted antibodies. Cytotoxic expression may also include direct intercellular contact that induces death (e.g., death by lysis, necrosis, apoptosis, etc.). It is an object of various embodiments to provide highly specific, highly potent lethality of target cells by the modified B lymphocytes described herein. In one embodiment, the target cell comprises a cancer cell (e.g., a tumor or other cancer cell). In one embodiment, the target cell comprises a cell associated with an autoimmune disease or infection. In one embodiment, the target cells comprise donor or host cells that are reactive to donor cells from a cell, tissue, or organ transplant (e.g., graft versus host disease). In one embodiment, the target cells comprise cells associated with pathological inflammation or infection.

A method of producing immunoglobulins in an isolated B lymphocyte cell line as described herein may comprise: isolating a B lymphocyte cell line from a vertebrate subject, e.g., exposed to or immunized with at least one second antigen by infection, the B lymphocyte cell line expressing at least one endogenous secreted immunoglobulin reactive with the at least one second antigen; introducing at least one exogenous membrane immunoglobulin reactive to at least one first antigen into an isolated B lymphocyte cell line to produce a recombinant B lymphocyte cell line; and selecting an isolated B lymphocyte cell line that expresses membrane immunoglobulin reactive to at least one first antigen and expresses at least one endogenously secreted immunoglobulin reactive to at least one second antigen. The method of claim may include: administering at least one first antigen to stimulate a recombinant B lymphocyte cell line; and assessing the production of at least one endogenous secreted immunoglobulin reactive with the at least one second antigen in the recombinant B lymphocyte cell line. In the method, introducing into at least one isolated recombinant B lymphocyte cell line at least one exogenous membrane immunoglobulin reactive with at least one first antigen may comprise: introducing at least one exogenous membrane immunoglobulin polypeptide reactive to at least one first antigen. Introducing at least one exogenous membrane immunoglobulin reactive with at least one first antigen into at least one isolated recombinant B lymphocyte cell line may comprise: introducing at least one exogenous nucleic acid encoding at least one membrane immunoglobulin reactive to at least one first antigen. The method can comprise the following steps: exposing the recombinant B lymphocyte cell line to at least one first antigen to activate the recombinant B lymphocyte cell line to express endogenously secreted immunoglobulin reactive with at least one second antigen. The method can comprise the following steps: isolating endogenously secreted immunoglobulins reactive to at least one second antigen from the recombinant B lymphocyte cell line or culture of the recombinant B lymphocyte cell line. In this method, activation of at least one exogenously incorporated membrane immunoglobulin with a first antigen can control expression of at least one exogenously incorporated nucleic acid encoding at least one secreted immunoglobulin reactive with a second antigen. The isolated B lymphocyte cell line can comprise at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell. The isolated B lymphocyte cell line can comprise at least one memory B lymphocyte cell.

In one embodiment, an isolated B lymphocyte cell line that has been modified by a chimeric B cell receptor or a recombinant B cell receptor comprises the use of an scFv fragment in the construction of the modified B cell receptor. In one embodiment, transcription factors may also be constructed (e.g., on a separate vector) as part of a modified B cell line, as described herein.

Methods for treating a subject having a disease or disorder (e.g., an autoimmune disease, cancer, or infection, etc.) include: administering a therapeutically effective amount of an isolated modified B lymphocyte cell line disclosed herein. It is recognized that a therapeutically effective amount of cells to be administered to a subject can be determined using standard methods for immunotherapy and cell therapy procedures.

Methods of treating a disease in a vertebrate subject with an immunotherapeutic product described herein can comprise: isolating a B lymphocyte cell line from a vertebrate subject, e.g., exposed to or immunized with at least one second antigen by infection, the B lymphocyte cell line expressing at least one endogenous secreted immunoglobulin reactive with the at least one second antigen; introducing at least one exogenous membrane immunoglobulin reactive to at least one first antigen into an isolated B lymphocyte cell line to produce a recombinant B lymphocyte cell line; and selecting a recombinant B lymphocyte cell line for administration to one or more vertebrate subjects that expresses membrane immunoglobulins reactive with at least one first antigen and expresses at least one endogenously secreted immunoglobulin reactive with at least one second antigen. The method can comprise the following steps: administering at least one first antigen to stimulate a recombinant B lymphocyte cell line; and testing the recombinant B lymphocyte cell line for the presence of at least one endogenous secreted immunoglobulin reactive with the at least one second antigen. The method can comprise the following steps: administering to a vertebrate subject a pharmaceutical composition comprising an isolated B lymphocyte cell line; and administering at least one first antigen to the vertebrate subject to stimulate an isolated B lymphocyte cell line to produce at least one endogenous secreted immunoglobulin reactive with the at least one second antigen. The method can comprise the following steps: confirming the presence of at least one endogenously secreted immunoglobulin reactive to at least one second antigen in the bloodstream of the vertebrate subject. The method can comprise the following steps: administering at least one first antigen to stimulate a recombinant B lymphocyte cell line; testing for the presence of at least one endogenously secreted immunoglobulin reactive with at least one second antigen; and administering to the vertebrate subject a pharmaceutical composition comprising the stimulated recombinant B lymphocyte cell line. The recombinant B lymphocyte cell line can be autologous to one of the one or more vertebrate subjects. The recombinant B lymphocyte cell line can be allogeneic to one or more vertebrate subjects.

A method of producing at least one immunoglobulin in an isolated cell line as described herein may comprise: introducing at least one exogenous membrane immunoglobulin reactive with at least one first antigen into at least one isolated B lymphocyte cell line to produce at least one first isolated B lymphocyte cell line; selecting at least one first isolated B lymphocyte that expresses a membrane immunoglobulin reactive with at least one first antigen; introducing at least one exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one second antigen into at least one first isolated B lymphocyte cell line to produce at least one isolated recombinant B lymphocyte cell line; and selecting the at least one isolated recombinant B lymphocyte cell line that expresses one or more secreted immunoglobulins reactive with the at least one second antigen. The method can comprise the following steps: selecting at least one isolated recombinant B lymphocyte cell line expressing at least one exogenous membrane immunoglobulin reactive to at least one first antigen. The method can comprise the following steps: administering at least one first antigen to stimulate at least one isolated recombinant B lymphocyte cell line; and testing for the presence of one or more secreted immunoglobulins reactive with the at least one second antigen in the at least one isolated recombinant B lymphocyte cell line. The method can comprise the following steps: introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one first isolated B lymphocyte cell line. The method can comprise the following steps: introducing at least one exogenous nucleic acid sequence encoding one or more secreted immunoglobulins reactive with at least one third antigen into at least one isolated recombinant B lymphocyte cell line to produce at least one isolated second recombinant B lymphocyte cell line; and selecting at least one isolated second recombinant B lymphocyte cell line that expresses at least one secreted immunoglobulin reactive with the at least one second antigen and at least one secreted immunoglobulin reactive with the at least one third antigen. The method can comprise the following steps: administering at least one first antigen to stimulate at least one isolated second recombinant B lymphocyte cell line; and detecting the presence of at least one exogenously secreted immunoglobulin reactive with at least one third antigen in the recombinant B lymphocyte cell line. In the method, introducing at least one exogenous membrane immunoglobulin reactive to at least one first antigen into at least one isolated B lymphocyte cell line can comprise: introducing at least one exogenous membrane immunoglobulin reactive to at least one first antigen. Introducing at least one exogenous membrane immunoglobulin reactive to at least one first antigen into at least one isolated B lymphocyte cell line can comprise: introducing an exogenous nucleic acid encoding at least one membrane immunoglobulin reactive to at least one first antigen. Introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one first isolated B lymphocyte cell line can comprise: introducing at least one exogenous membrane immunoglobulin polypeptide reactive to at least one second antigen. Introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one first isolated B lymphocyte cell line can comprise: introducing at least one exogenous nucleic acid encoding at least one membrane immunoglobulin reactive to at least one second antigen. The method can comprise the following steps: exposing at least one isolated recombinant B lymphocyte cell line to at least one first antigen, and testing the at least one isolated recombinant B lymphocyte cell line for activation to express exogenously secreted immunoglobulin reactive with at least one second antigen. The method can comprise the following steps: exogenously secreted immunoglobulins reactive with at least one second antigen are isolated from at least one isolated recombinant B lymphocyte cell line or from a culture of at least one isolated recombinant B lymphocyte cell line. In this method, activation of at least one exogenously incorporated membrane immunoglobulin with a first antigen can control: expression of at least one exogenously incorporated nucleic acid encoding at least one secreted immunoglobulin reactive with a second antigen. The at least one isolated B lymphocyte cell line can comprise at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a marginal zone B lymphocyte, a B1B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell. The at least one isolated B lymphocyte cell line can comprise at least one memory B lymphocyte cell.

Methods of treating a disease in a vertebrate subject with an immunotherapeutic product described herein can comprise: introducing at least one exogenous membrane immunoglobulin reactive with at least one first antigen into at least one isolated B lymphocyte cell line to produce at least one first isolated B lymphocyte cell line; selecting at least one first isolated B lymphocyte that expresses a membrane immunoglobulin reactive with at least one first antigen; introducing at least one exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one second antigen into at least one first isolated B lymphocyte cell line to produce at least one isolated recombinant B lymphocyte cell line; selecting the at least one isolated recombinant B lymphocyte cell line expressing secreted one or more immunoglobulins reactive with the at least one second antigen for administration to one or more vertebrate subjects. The method can comprise the following steps: selecting at least one isolated recombinant B lymphocyte cell line expressing at least one exogenous membrane immunoglobulin reactive to at least one first antigen. The method can comprise the following steps: administering at least one first antigen to stimulate at least one isolated recombinant B lymphocyte cell line; and testing for the presence of one or more secreted immunoglobulins reactive with the at least one second antigen in the at least one isolated recombinant B lymphocyte cell line. The method can comprise the following steps: administering to a vertebrate subject a pharmaceutical composition comprising at least one isolated recombinant B lymphocyte cell line; and administering at least one first antigen to the vertebrate subject to stimulate at least one isolated recombinant B lymphocyte cell line to produce one or more exogenously secreted immunoglobulins reactive with the at least one second antigen. The method can comprise the following steps: confirming the presence of at least one exogenously secreted immunoglobulin reactive with the at least one second antigen in the bloodstream of the vertebrate subject. The method can comprise the following steps: administering at least one first antigen to stimulate at least one isolated recombinant B lymphocyte cell line to produce one or more exogenously secreted immunoglobulins reactive with at least one second antigen; and administering to the vertebrate subject a pharmaceutical composition comprising the stimulated at least one isolated recombinant B lymphocyte cell line. The method can comprise the following steps: introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one first isolated B lymphocyte cell line. The method can comprise the following steps: introducing at least one exogenous nucleic acid sequence encoding one or more secreted immunoglobulins reactive with at least one third antigen into at least one isolated recombinant B lymphocyte cell line to produce at least one isolated second recombinant B lymphocyte cell line; and selecting at least one isolated second recombinant B lymphocyte cell line that expresses at least one secreted immunoglobulin reactive with the at least one second antigen and a secreted immunoglobulin reactive with the at least one third antigen. The method can comprise the following steps: administering to the vertebrate subject a pharmaceutical composition comprising at least one isolated second recombinant B lymphocyte cell line; and administering the at least one first antigen to the vertebrate subject to stimulate at least one isolated second recombinant B lymphocyte cell line to produce one or more exogenously secreted immunoglobulins reactive with the at least one second antigen and one or more exogenously secreted immunoglobulins reactive with the at least one third antigen. The method can comprise confirming the presence of at least one exogenously secreted immunoglobulin reactive with at least one second antigen and one or more exogenously secreted immunoglobulins reactive with at least one third antigen in the blood stream of the vertebrate subject. The method can comprise the following steps: administering at least one first antigen to a vertebrate subject to stimulate at least one isolated second recombinant B lymphocyte cell line to produce one or more exogenously secreted immunoglobulins reactive with the at least one second antigen and one or more exogenously secreted immunoglobulins reactive with at least one third antigen; and administering to the vertebrate subject a pharmaceutical composition comprising the stimulated at least one isolated second recombinant B lymphocyte cell line. The recombinant B lymphocyte cell line can be autologous to one of the one or more vertebrate subjects. The recombinant B lymphocyte cell line can be allogeneic to one or more vertebrate subjects.

A method of producing at least one immunoglobulin in an isolated cell line as described herein may comprise: introducing at least one exogenous nucleic acid into at least one first isolated B lymphocyte cell line, said exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one first antigen to produce at least one isolated recombinant B lymphocyte cell line; selecting at least one isolated recombinant B lymphocyte cell line that expresses one or more secreted immunoglobulins reactive with the at least one first antigen; introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one isolated B lymphocyte cell line to produce at least one first isolated B lymphocyte cell line; and selecting a first isolated B lymphocyte cell line that expresses at least one membrane immunoglobulin reactive with at least one second antigen.

Methods of treating a disease in a vertebrate subject with an immunotherapeutic product described herein can comprise: introducing at least one exogenous nucleic acid into at least one first isolated B lymphocyte cell line, said exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one first antigen to produce at least one isolated recombinant B lymphocyte cell line; selecting at least one isolated recombinant B lymphocyte cell line that expresses secreted immunoglobulin(s) reactive with said at least one first antigen; introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one isolated B lymphocyte cell line to produce at least one first isolated B lymphocyte cell line; and selecting a first isolated B lymphocyte cell line expressing at least one membrane immunoglobulin reactive with at least one second antigen for administration to a vertebrate subject.

The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

Drawings

FIG. 1 is a schematic diagram of a diagrammatic view of hypothetical immunoglobulin genes for memory B lymphocytes.

FIGS. 2A, 2B, 2C are schematic illustrations of a schematic view of non-functional and functional immunoglobulin heavy chain genes on chromosome 14.

Fig. 3A, 3B, 3C are schematic diagrams of diagrammatic views of replacement at an immunoglobulin locus with a heavy chain gene to express membrane IgG and secreted IgG.

Fig. 4A, 4B, 4C, 4D are schematic diagrams of diagrammatic views of protocols for producing recombinant B lymphocytes having membrane immunoglobulins directed against a first antigen and secreted immunoglobulins directed against a second antigen.

FIG. 5 is a schematic diagram of a diagrammatic view of a method of producing immunoglobulins in an isolated B lymphocyte cell line.

FIG. 6 is a schematic diagram of a diagrammatic view of a method of producing immunoglobulins in an isolated B lymphocyte cell line.

FIG. 7 is a schematic diagram of a diagrammatic view of a method of producing immunoglobulins in an isolated B lymphocyte cell line.

FIG. 8A is a schematic representation of a diagrammatic view of a recombinant B cell receptor protein.

FIG. 8B is a schematic representation of a diagrammatic view of a recombinant B cell receptor expression vector.

Fig. 8C is a schematic diagram of a diagrammatic view of chromosome 14 with an inserted gene.

FIG. 8D is a schematic diagram of a diagrammatic view of an expression vector with a transcription factor.

FIG. 9A is a schematic diagram of a diagrammatic view of an example of integration of a desired expression construct at an endogenous site (light chain Ig).

FIG. 9B is a schematic diagram of a diagrammatic view of an example of integration of a desired expression construct at an endogenous site (heavy chain Ig).

Fig. 10 is a schematic diagram of a diagrammatic view through a modified B cell engineered to selectively bind a surface immunoglobulin to a first target antigen, followed by secretion of a predetermined antibody against a second target antigen, and optionally secretion of a redistributed biologic agent and/or cytotoxic effector molecule.

FIG. 11 is a schematic representation of a diagrammatic view of a lymph node with modified B cells that have reactivity to a selective antigen, as determined by the particular modification of the B cells.

FIG. 12A is a schematic diagram of a diagrammatic view of an example of integration of a desired expression construct at an endogenous site (heavy chain Ig).

Fig. 12B is a schematic view of an example of an expression vector for modifying B cells as described herein.

FIG. 13 is a schematic representation of a diagrammatic view of an example of a bicistronic expression construct required for integration at the endogenous site (heavy chain Ig).

Detailed Description

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like reference numerals generally identify like parts, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter set forth herein.

Disclosed herein are compositions and methods for producing one or more immunoglobulins in an isolated B lymphocyte cell line. Disclosed herein are compositions and methods for producing one or more immunoglobulins in an isolated B lymphocyte cell line, thereby directing cell signaling by membrane immunoglobulins in the isolated B lymphocyte cell line. An immune cell therapy in a vertebrate subject can include administering to the vertebrate subject an isolated B lymphocyte cell line that synthesizes secreted and membrane immunoglobulins, each with a different target antigen. Immune cell therapy in a vertebrate subject can comprise administering to the vertebrate subject antigen-presenting cells comprising an isolated B lymphocyte cell line that directs antigen internalization and processing to produce a particular antigen-presenting cell. Isolated B lymphocyte cell lines can produce antigen presenting cells that are superior or superior in capturing, internalizing, and presenting antigens recognized by membrane immunoglobulins derived either endogenously or exogenously. Disclosed herein are compositions and methods for treating diseases in vertebrate subjects with immunotherapeutic products. The immunotherapeutic product may comprise an isolated B lymphocyte cell line having endogenous or exogenously derived membrane immunoglobulin reactive with a first antigen, wherein the isolated B lymphocyte cell line produces one or more secreted immunoglobulin reactive with a second antigen or produces a redistributed biologic agent. The immunotherapeutic product may comprise an isolated B lymphocyte cell line, which may be a monoclonal or polyclonal B lymphocyte cell line that produces one or more secreted antibodies and/or redistributed biological agents. Immunotherapeutic products may comprise an isolated B lymphocyte cell line that produces one or more secreted antibodies, e.g., antibodies that recognize different epitopes on the same antigen. The immunotherapeutic product may comprise an isolated B lymphocyte cell line as one or more antigen presenting cells.

The isolated B lymphocyte cell line can comprise an immunotherapeutic product administered to a vertebrate subject to develop long-lived isolated B lymphocytes in the vertebrate subject for immune surveillance of chronic diseases. Immunotherapeutic products can comprise isolated B lymphocyte cell lines with endogenously or exogenously derived membrane immunoglobulins that can be administered to a vertebrate subject to provide antigen presenting cells to the vertebrate subject.

An isolated cell line as described herein can comprise an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin reactive with a first antigen and at least one endogenously secreted immunoglobulin reactive with a second antigen. The at least one exogenously incorporated membrane immunoglobulin can comprise an exogenously incorporated membrane immunoglobulin polypeptide. The at least one exogenously incorporated membrane immunoglobulin can comprise an exogenously incorporated nucleic acid encoding a membrane immunoglobulin polypeptide, wherein the cell line is capable of expressing the membrane immunoglobulin polypeptide.

An isolated recombinant cell line as described herein can comprise an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin reactive with a first antigen and at least one exogenously incorporated nucleic acid encoding a secreted immunoglobulin reactive with a second antigen.

An isolated recombinant cell line as described herein can comprise an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated gene integrated on an active rearranged immunoglobulin gene under the control of an immunoglobulin variable region promoter and an immunoglobulin enhancer. For example, genes integrated on rearranged immunoglobulin H chain genes for redistribution of biological agents are expressed under the control of a variable heavy chain promoter and immunoglobulin μ enhancer.

An isolated recombinant B cell line as described herein may comprise the ability to express redistributed biological agents, such as proteins, glycoproteins, proteoglycans, nucleic acids (RNA, DNA, PNA, etc.) or other biological agents that are not normally expressed from the Ig chromosome genes of Ig H and Ig L chains in naturally occurring B cells. For example, a recombinant B cell described herein may comprise the ability to express at least one cytokine, cytokine receptor, small molecule, protein, monosaccharide, disaccharide, polysaccharide, or other biological agent. In one embodiment, the redistributed biological agent may comprise at least one enzyme, G protein-coupled receptor, or ligand. In one embodiment, the redistributed biologic agent can comprise Tumor Necrosis Factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), OX-40, CD95(FasL/Apo-1L), gamma interferon ((alpha-IFN), perforin, interleukin 21(IL-21), H, IL-15, IL-10, IL-22, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-17, IL-18, IL-23, pathogen-associated molecular pattern (PAMP), injury-associated molecular pattern (DAMP), CXCL-1, CXC, CC, GM-CSF, G-CSF, M-CSF, stem cell factor, TGF-beta, INF gamma, INF alpha, TNF alpha, or other cytokine, in one embodiment, the redistributed biological agent can comprise at least one of a tumor associated antigen, a cell surface antigen, or a viral antigen. For example, a tumor-associated antigen may comprise: MUC-1, MUC-16, Prostate Cancer Membrane Antigen (PCMA), epidermal growth factor receptor 2(HER2), B Cell Maturation Antigen (BCMA), CD38, CD30, MAGEA1, NY-ESO-1, and CD44v 6. The viral antigen may comprise: HIV-1 protein: gag, env, gp41, gp120, RNA-dependent DNA polymerase; influenza hemagglutinin and hepatitis c virus proteins: NS3, NS4 and NS 5. In one embodiment, the redistributed biological agent comprises a bacterial component configured to induce other B cells to become IgA-producing plasma cells. In one embodiment, the isolated modified B cells are used in the intestine of a subject to work synergistically or competitively with the microbiome to maintain the health of the subject or to alter a disease state. See, e.g., Shi et al, mil.med.res.2017; 4:14, online Apr 27,2017, which is incorporated herein by reference. In one embodiment, the disease state comprises a disease affecting the intestine. In one embodiment, the disease state comprises a disease affecting another system of the subject's body, including a systemic disease.

In one embodiment, expression of the agent may be initiated by binding of a modified immunoglobulin receptor (BCR) of the B cell. For example, transcription of the redistributed biological agent is initiated in a series of events following appropriate antigen binding of the BCR of the modified B cell, as described above. Binding of antigen to membrane IgG leads to tyrosine phosphorylation on Ig α and Ig β (BCR-containing signal transduction proteins), thereby initiating signaling pathways that activate and transport intracellular messengers and transcription factors (e.g., NF-AT, Ras/Erk, Bright, and BTK), which leads to memory B cell activation and Ig gene expression. (see, e.g., Current Topics in Microbiology and Immunology 393: 107-.

In one embodiment, the modified B lymphocyte is modified to express the at least one redistributed biological agent without further modification. In one embodiment, the modified B lymphocyte is modified to constitutively express at least one redistributed biological agent. As described herein, the redistributed biological agent comprises secreted molecules (e.g., cytokines, chemokines, cytotoxins, etc.) and may play a role in a greater immune response (e.g., stimulation of T cells, NK cells, macrophages, epithelial cells, other B cells, neutrophils, basophils, eosinophils, etc.). In one embodiment, the modified B lymphocyte is modified to inducibly express the at least one redistributed biological agent (e.g., expression of the at least one redistributed biological agent may be driven by receptor-ligand binding, by a transcription factor, by protein production of another biological response, etc.). In one embodiment, the modified B lymphocyte is also modified in other ways as described herein (e.g., to express an exogenous membrane immunoglobulin receptor and/or to express an exogenous secreted immunoglobulin receptor and/or to express a cytotoxic agent, etc.).

In one embodiment, the redistributed biological agent may comprise: an agent, which is not normally expressed at all by naturally occurring B lymphocytes, but which, due to its modification, is capable of expressing a redistributed biological agent by modified B lymphocytes. In one embodiment, the redistributed biological agent may comprise: an agent, which is not normally expressed by naturally occurring B lymphocytes under particular circumstances or conditions, but which, due to its modification, is capable of expressing a redistributed biological agent under these particular circumstances or conditions. For example, in some cases, naturally occurring regulatory B lymphocytes express and secrete IL-10 or TFG- β 1, while naturally occurring effector B lymphocytes produce cytokines, such as IL-2, IL-4, TNF α, IL-6, or INF γ. The determination of regulatory or effector B lymphocytes is based on the exposure of these particular cells to antigens and/or other cytokines and immune modulators. (see, e.g., Lund, curr. Opin. Immunol.2008 June; 20(3):332-338, incorporated herein by reference.)

With respect to our modified B lymphocytes, for example, B lymphocytes that will (in nature) express IL-2 can be modified to secrete IL-10. Thus, IL-10 will fulfill the role of the redistributed biologic agent. Conversely, if a naturally occurring B lymphocyte (in the case of naturally occurring cells) expresses IL-10 but, regardless of the particular immunological conditions, is modified to constitutively or inducibly secrete IL-2, then IL-2 will assume the role of the redistributed biologic agent. Thus, modification of the redistributed biological agent serves as a powerful tool to direct the immune response in the modified B lymphocytes themselves as well as other participants in the immune response (epithelial cells, neurons, other immune cells, etc.). This may be particularly useful, for example, for "misplaced" immune responses, such as tumor immunology (e.g., tumor suppression of a standard immune response to a tumor antigen), infectious disease (e.g., a virus that evades standard immune monitoring), or autoimmunity (e.g., exacerbation of inflammation or highly reactive immune response to "self" or "non-dangerous" antigens), and the like.

A method of producing immunoglobulins in an isolated B lymphocyte cell line as described herein may comprise: isolating a B lymphocyte cell line from a vertebrate subject, e.g., exposed to or immunized with at least one second antigen by infection, the B lymphocyte cell line expressing at least one endogenous secreted immunoglobulin reactive with the at least one second antigen; introducing at least one exogenous membrane immunoglobulin reactive to at least one first antigen into an isolated B lymphocyte cell line to produce a recombinant B lymphocyte cell line; and selecting an isolated B lymphocyte cell line that expresses membrane immunoglobulin reactive to at least one first antigen and expresses at least one endogenously secreted immunoglobulin reactive to at least one second antigen.

The modifications are described herein

Methods of treating a disease in a vertebrate subject with an immunotherapeutic product described herein can comprise: isolating B lymphocyte cells from a vertebrate subject, e.g., exposed to or immunized with at least one second antigen by infection, for producing a cell line expressing at least one endogenous secreted immunoglobulin reactive with the at least one second antigen; introducing at least one exogenous membrane immunoglobulin reactive to at least one first antigen into an isolated B lymphocyte to produce a recombinant B lymphocyte cell line; and selecting a recombinant B lymphocyte cell line for administration to one or more vertebrate subjects that expresses membrane immunoglobulins reactive with at least one first antigen and expresses at least one endogenously secreted immunoglobulin reactive with at least one second antigen.

A method of producing at least one immunoglobulin in an isolated cell line as described herein may comprise: introducing at least one exogenous membrane immunoglobulin reactive with at least one first antigen into at least one isolated B lymphocyte to produce at least one first isolated B lymphocyte cell line; selecting at least one first isolated B lymphocyte that expresses a membrane immunoglobulin reactive with at least one first antigen; introducing at least one exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one second antigen into at least one first isolated B lymphocyte cell line to produce at least one isolated recombinant B lymphocyte cell line; and selecting the at least one isolated recombinant B lymphocyte cell line that expresses one or more secreted immunoglobulins reactive with the at least one second antigen.

Methods of treating a disease in a vertebrate subject with an immunotherapeutic product described herein can comprise: introducing at least one exogenous membrane immunoglobulin reactive with at least one first antigen into at least one isolated B lymphocyte to produce at least one first isolated B lymphocyte cell line; selecting at least one first isolated B lymphocyte that expresses a membrane immunoglobulin reactive with at least one first antigen; introducing at least one exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one second antigen into at least one first isolated B lymphocyte cell line to produce at least one isolated recombinant B lymphocyte cell line; selecting the at least one isolated recombinant B lymphocyte cell line expressing secreted one or more immunoglobulins reactive with the at least one second antigen for administration to one or more vertebrate subjects.

A method of producing at least one immunoglobulin in an isolated cell line as described herein may comprise: introducing at least one exogenous nucleic acid into at least one first isolated B lymphocyte, said exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one first antigen to produce at least one isolated recombinant B lymphocyte cell line; selecting at least one isolated recombinant B lymphocyte cell line that expresses one or more secreted immunoglobulins reactive with the at least one first antigen; introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one isolated B lymphocyte cell line to produce at least one first isolated B lymphocyte cell line; and selecting a first isolated B lymphocyte cell line that expresses at least one membrane immunoglobulin reactive with at least one second antigen.

Methods of treating a disease in a vertebrate subject with an immunotherapeutic product described herein can comprise: introducing at least one exogenous nucleic acid into at least one first isolated B lymphocyte, said exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one first antigen to produce at least one isolated recombinant B lymphocyte cell line; selecting at least one isolated recombinant B lymphocyte cell line that expresses secreted immunoglobulin(s) reactive with said at least one first antigen; introducing at least one exogenous membrane immunoglobulin reactive with at least one second antigen into at least one isolated B lymphocyte cell line to produce at least one first isolated B lymphocyte cell line; and selecting a first isolated B lymphocyte cell line expressing at least one membrane immunoglobulin reactive with at least one second antigen for administration to a vertebrate subject.

The isolated recombinant cell line comprises an isolated B lymphocyte cell line capable of expressing at least one endogenous membrane immunoglobulin reactive with a first antigen and at least one exogenous incorporated nucleic acid encoding at least one secreted immunoglobulin reactive with a second antigen.

In one embodiment, the modified B lymphocyte comprises a structural or functional feature for exhibiting cytotoxicity. For example, in one embodiment, the modified B lymphocyte cell or cell line produces one or more antibodies and has one or more B cell receptors (such as membrane immunoglobulins as described herein) specific for a target antigen (including but not limited to antigens that are mutated forms of "normal" cell antigens, as well as antigens modified by post-translational modifications, and antigens expressed in aberrant manners or at aberrant levels), such as a tumor antigen. In one embodiment, the modified B lymphocyte cell or cell line is capable of generating a complete immune response with both humoral and cellular immune components.

In one embodiment, the modified B lymphocyte exhibiting cytotoxicity is capable of expressing (directly or indirectly) at least one of perforin, granzyme and other cytotoxic components. For example, in one embodiment, when bound to a tumor cell (i.e., the B cell receptor binds to an antigen of the tumor cell), the B cell receptor (membrane-bound immunoglobulin) signals to trigger a cytotoxic effector. In one embodiment, the modified lymphocytes are capable of secreting cytotoxic antibodies against the same tumor cell (e.g., by fixed complement or conjugation ADCC [ antibody-dependent cell-mediated cytotoxicity ]).

In one embodiment, the modified B lymphocyte cell is derived, e.g., from a B cell after vaccination with a tumor antigen, or from a donor peripheral blood lymphocyte cell, by modifying expression of at least one of an antibody or a B cell receptor (e.g., a chimeric B cell receptor or a recombinant B cell receptor).

In one embodiment, the modified B lymphocyte is modified to express cytotoxicity by expressing a recombinant B cell receptor or chimeric receptor having scFv and membrane immunoglobulin as extracellular transmembrane and cytoplasmic domains with cytoplasmic domains from IL21 receptor and TLR or another signaling molecule to cause expression of granzyme, perforin, etc. in the modified B lymphocyte.

For example, in the case of HIV infection, modified B lymphocytes are capable of eliciting both humoral and cytotoxic immune responses. For example, modified B lymphocytes may secrete neutralizing antibodies against HIV particles or virus-infected cells. Optionally, in addition to neutralizing antibodies, the modified B lymphocytes may also directly induce apoptosis or otherwise directly kill HIV-infected cells (e.g., T cells infected in lymph nodes, known as "depots" of infected T cells, that are not destroyed at the current level of HIV anti-viral therapy).

Likewise, modified B lymphocytes can target autoimmune cells (e.g., multiple sclerosis cells, arthritis cells, etc.) that can be recognized as autoreactive. In one embodiment, the modified B lymphocytes induce apoptosis or otherwise directly kill such self-reactive cells. In one embodiment, the self-reactive cells comprise at least one of B cells, T cells, macrophages, or other immune cells. In one embodiment, the self-reactive cells comprise inflammatory cells.

In one embodiment, the modified B lymphocytes are modified to express a recombinant or chimeric B cell receptor specific for a first antigen and an antibody that recognizes a second antigen, thereby providing increased specificity as well as increased cytotoxicity and antibody-mediated killing of target cells (e.g., tumor cells, autoimmune cells, infected cells, inflammatory cells, necrotic cells, regulatory cells (e.g., regulatory T cells, regulatory B cells, and myeloid-derived suppressor cells)).

In one embodiment, a modified B lymphocyte having a chimeric B cell receptor or a recombinant B cell receptor has been modified to specifically react with and exhibit cytotoxicity to one or more target cells. In one embodiment, the modified B lymphocyte cell or cell line is specifically engineered to react with one or more tumor cells or tumor cell lines. In one embodiment, the modified B lymphocyte cell or cell line is engineered by laboratory techniques and optionally by modeling using computer data and/or various components of the B lymphocyte cell.

In one embodiment, a modified B lymphocyte cell having a chimeric B cell receptor or a recombinant B cell receptor comprises a modified receptor that, when engaged, is capable of transducing a signal that induces expression of a cytotoxic effector molecule.

In one embodiment, the recombinant B cell receptor or chimeric B cell receptor comprises heterologous extracellular, transmembrane and cytoplasmic signaling domains that cause expression of cytotoxic effector molecules.

In one embodiment, the recombinant B cell receptor or chimeric B cell receptor comprises a cytoplasmic domain derived from at least one of a common gamma chain, an IL-21R, Toll-like receptor (TLR), or CD 40. In one embodiment, the recombinant B cell receptor or chimeric B cell receptor is capable of causing expression of a cytotoxic effector molecule (e.g., perforin, granzyme B, Fas ligand, TRAIL, or others). In one embodiment, the recombinant or chimeric B cell receptor is capable of causing expression of a TNF family receptor, such as TNFR1, Fas receptor, DR4, DR5 or other "death domain" receptor, in a target cell.

In one embodiment, the modified B cell secretes or expresses at least one of a TNF- α ligand, lymphotoxin α or β, OX40L, CD154, LIGHT, TL1-A, CD70, Siva, CD153, 4-1BB, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR, TL1A, or EDA-A2.

For example, the optimal stimulation of efficient expression of CD70 on IFN- α -induced monocyte-derived dendritic cells has been studied by exposure to various maturation-inducing factors (Toll-like receptor ligands, CD40 ligands, and precursor-inflammatory mediators, including prostaglandin E2), which are widely used in tumor immunotherapy. See Immunol,2010 May 130(1):137-149, which is incorporated herein by reference. In addition, CD70-CD27 interactions reduced IL-10 production. As above (Id).

For example, in the presence of B cell receptor involvement and IL-4, expression of CD153 on B cells leads to Ig class switching, while CD154 expression contributes to CD153 expression. See Cerutti, et al, J Immunol 2000 July 15; 165(2) 786 and 794, which are incorporated herein by reference. CD153 "switches" for expression of alternative or additional genes or expression cassettes may be constructed into the modified B cells described herein.

For example, OX40-OX40L interaction has been found to play a role in the development of several different inflammatory and autoimmune diseases, and it may be the target of intervention. See, e.g., Croft, et al, Immunol Rev 2009 May,229(1):173-191, which is incorporated herein by reference.

In one embodiment, the recombinant B cell receptor or chimeric B cell receptor comprises at least one extracellular domain specific for one or more target antigens. In one embodiment, the recombinant B cell receptor or chimeric B cell receptor comprises a modified receptor that recognizes a first antigen and secretes an antibody that recognizes a second antigen. In one embodiment, a recombinant B cell receptor or chimeric B cell receptor comprises a modified receptor that recognizes a first epitope of one antigen and secretes an antibody that recognizes a second epitope of the same antigen. Thus, modified B cells (with recombinant or chimeric B cell receptors) can be designed and engineered by laboratory techniques to respond to tumor cells with higher specificity and higher cytotoxic activity.

In one embodiment, a modified B cell that exhibits cytotoxicity further comprises in embodiments of a B cell described herein, the B cell exhibits a chimeric B cell receptor or a recombinant B cell receptor specific for one antigen and secretes an antibody specific for an antigen different from its B cell receptor. Such memory B cells may also comprise targeted cytotoxic features as described herein.

In one embodiment, the target cells include cells that have been infected with a virus, mycoplasma, bacteria, yeast, or other microorganism. In one embodiment, the target cell comprises a tumor cell, such as a primary tumor cell, a circulating tumor cell, or a metastatic tumor cell. In one embodiment, the target cell comprises an autoimmune cell.

In one embodiment, the method of making a modified B lymphocyte comprises engineering a cell by laboratory techniques. In one embodiment, a method of treating a disease using modified B lymphocytes comprises administering to a subject a therapeutically effective amount of modified B lymphocytes. Specific examples of methods of making modified B lymphocytes are described in more detail in the prior examples section herein.

In one embodiment, the modified B lymphocyte cell or cell line described herein is engineered and used for administration to a subject for treatment. As described herein, in one embodiment, the subject has active disease. In one embodiment, the subject has a chronic disease. In one embodiment, the subject has been exposed to a pathogenic agent and may or may not have disease symptoms. In one embodiment, the subject has an underlying disease.

A method of producing immunoglobulins in a recombinant B lymphocyte cell line comprising: isolating a B lymphocyte cell line expressing at least one endogenous membrane immunoglobulin reactive to at least one first antigen from a vertebrate subject exposed to the first antigen or immunized with the at least one first antigen, e.g., by infection; introducing at least one exogenous nucleic acid encoding at least one secreted immunoglobulin reactive with at least one second antigen into an isolated B lymphocyte cell line to produce a recombinant B lymphocyte cell line; and detecting the presence of at least one exogenously secreted immunoglobulin reactive with the at least one second antigen to select a recombinant B lymphocyte cell line.

A method of treating a disease in a vertebrate subject with an immunotherapeutic product comprises: isolating a B lymphocyte cell line expressing at least one endogenous membrane immunoglobulin reactive to at least one first antigen from a vertebrate subject exposed to the first antigen or immunized with the at least one first antigen, e.g., by infection; introducing at least one exogenous nucleic acid encoding at least one secreted immunoglobulin reactive with at least one second antigen into an isolated B lymphocyte cell line; and detecting the presence of at least one exogenously secreted immunoglobulin reactive with the at least one second antigen to select a recombinant B lymphocyte cell line for administration to the vertebrate.

The isolated B lymphocytes are useful for immunotherapy:

long-lived isolated B lymphocytes are useful for immune monitoring of chronic diseases.

Isolated B lymphocytes with membrane immunoglobulin recognition antigens can serve as special antigen presenting cells to present antigens to T lymphocytes.

Immunotherapy with polyclonal autologous isolated B lymphocytes is a valuable approach. For example, influenza-immunized B lymphocytes can be transfected in large numbers with retroviral vectors. Alternatively, a vaccine may be used to immunize and transfect a plurality of isolated B lymphocytes, such as polyclonal B lymphocytes, to recognize different epitopes of the same antigen.

As set forth herein, a number of protocols can be used to generate isolated B lymphocyte cell lines, as described in more detail in the detailed description and examples. Isolated B lymphocyte cell lines capable of expressing at least one endogenous membrane immunoglobulin reactive to a first antigen or capable of expressing at least one endogenously secreted immunoglobulin reactive to a first antigen can be developed by immunizing an individual with a model antigen, such as Dinitrophenol (DNP) or an influenza antigen, to elicit memory B cells having endogenous membrane immunoglobulins (e.g., B Cell Receptor (BCR)) and/or endogenous soluble immunoglobulins (e.g., antibodies) reactive to a DNP model antigen or an influenza antigen.

An isolated B lymphocyte cell line capable of expressing at least one exogenously secreted immunoglobulin reactive with broadly neutralizing influenza antigens can be developed by: human B cells are isolated from an individual who is immune to influenza virus infection and immortalized by infecting the isolated B cells with epstein-barr virus (EBV). Methods for cloning immunoglobulin heavy (H) chain and light (L) chain genes from EBV immortalized B lymphocyte cell lines can be used. See, for example, U.S. Pat. No.7,741,077 and Early et al, Proc. Natl. Acad. Sci. USA 76:857-861,1979, issued to Grawunder et al at 22.6.2010, which is incorporated herein by reference. To facilitate homologous recombination, immunoglobulin genes encoding H and L chains for secreted anti-influenza antibodies were cloned into plasmid targeting vectors to achieve targeted integration into the corresponding nonfunctional germline Ig loci on chromosome 14 and chromosome 2, respectively. Alternatively, memory B cells obtained from patients with chronic viral infections can be genetically engineered by replacing their functional expressed Ig genes with foreign Ig genes encoding membrane immunoglobulins (e.g., anti-DNP antibodies). By using a method of homologous recombination, IgH and IgL chain genes encoding anti-DNP antibodies can be inserted into functionally expressed Ig gene loci on

chromosome

14 and 2. See, for example, U.S. patent nos. 5,202,238, 6,570,061, and 6,841,383.

Memory B cells expressing anti-DNP membrane IgG can be engineered to express an Ig gene encoding a secretory IgG antibody specific for influenza. Anti-influenza IgG can be engineered₁H chain gene (i.e.. gamma.)₁-H chain gene) to remove coding sequences for transmembrane domain (TM), cytoplasmic amino acids (Cyt) and polyA addition sites, thereby generating a gene encoding only secreted H chain γ₁-an H chain gene.

In order to obtain human immunoglobulin (Ig) genes encoding specific antibodies against cancer, such as PSA, or against infectious disease, hybridoma cell lines producing anti-PSA antibodies were constructed. For example, transgenic mice with human Ig genes (e.g., available from Abgenix inc., Fremont, CA)

) PSA is immunized and their B cells are fused with a myeloma cell fusion partner (e.g., SP2/0 cells (available from the American type culture Collection, Manassas, Va.) to produce hybridoma cell clones expressing human antibodies (see, e.g., U.S. Pat. No.8,013,128, supra). Supernatants from the hybrid clones were screened using an immunoassay to detect human IgG antibodies that bound to PSA protein. Amplification of hybridoma clones producing antibodies recognizing PSA and use of Biacore ^TMThe a100 instrument (available from GE Healthcare of Piscataway, new jersey) tests the antibodies of each clone to measure antibody affinity and specificity for PSA (see, e.g., GE Healthcare, Application Note 84, "Early kinetic screening of antibodies …," which is incorporated herein by reference). Hybridomas expressing high affinity antibodies to PSA are selected for cloning of their human Ig genes, e.g., by homologous recombination.

Engineered immunoglobulin genes encoding membrane immunoglobulins are expressed in mammalian cell lines, and membrane IgG is purified from the cell lines. For example, the kappa (κ) L chain gene and the modified γ -1H chain gene are inserted into a lentiviral expression vector using standard recombinant DNA methods (see, e.g., U.S. patent publication No.2007/0116690 to Yang et al, published 24/5/2007, which is incorporated herein by reference). The viral vectors are used to transfect Chinese Hamster Ovary (CHO) cells (available from the American type culture Collection, Manassas, Va.) that are engineered to express membrane immunoglobulins.

To ensure that recombinant memory B cells are safe for use in patients, a suicide gene may be introduced into the B cells. To prevent uncontrolled proliferation (and other adverse events) of recombinant memory B cells, a suicide gene (herpes simplex virus thymidine kinase gene (HSV-TK)) was introduced using a retroviral expression vector. Methods of inserting and expressing the HSV-TK gene and activating cytotoxic prodrugs such as ganciclovir are known (see, e.g., U.S. Pat. No.6,576,464 to Gold and Lebkowski on 10.6.2003 and U.S. Pat. No.5,997,859 to Barber et al on 7.12.7.1999, which are incorporated herein by reference). To prevent the growth of recombinant B cells that are considered unsafe or cause adverse events, HSV-TK expressing B cells were provided with 20. mu.M ganciclovir (commercially available as CytoveneIV from Roche laboratories, Nutley, N.J.). Ganciclovir is converted to a toxic metabolite by HSV-TK expressing B cells causing its death. Cells that do not express HSV-TK are not damaged by ganciclovir.

In another embodiment, a Chemical Inducer of Dimerization (CID) may be used, wherein the pro-apoptotic molecule suitably comprises one or more binding sites for CID that, once reached its target, cause it to oligomerize and subsequently activate the apoptotic pathway. In this way, different apoptotic pathways may function as suicide systems (including death receptor Fas and Caspase9 enzymes). In addition to the very low risk of immunogenicity, these suicide genes have the advantages of non-cell cycle dependence, complete clinical compatibility and optimal biodistribution, since CID is a small molecule well-designed for suicide purposes. See, e.g., J cancer.2011; 2: 378-.

The isolated cell line may comprise an isolated B lymphocyte cell line or an isolated recombinant B lymphocyte cell line that recognizes one or more antigens of an infectious bacterial or viral disease (e.g., an influenza antigen). Table 1 contains examples of protocols for constructing isolated B lymphocyte cell lines or isolated recombinant B lymphocyte cell lines comprising exogenous and/or endogenous membrane immunoglobulins and exogenous and/or endogenous secreted immunoglobulins. The secreted immunoglobulin from the isolated recombinant B lymphocyte cell line may comprise one or more secreted anti-influenza-extensively neutralizing antibodies (Flu BNAb). Broadly neutralizing antibodies against influenza viruses can be directed against two or more epitopes on the same influenza antigen (Flu BNAb1 and Flu BNAb 2). The secreted anti-influenza immunoglobulin from the isolated recombinant B lymphocyte cell line may comprise one or more secreted polyclonal antibodies (Flu Abn) against an influenza antigen.

B lymphocyte protocol 1 is a protocol for producing isolated recombinant B lymphocytes. Scheme 1 vertebrates were immunized with DNP-KLH (dinitrophenyl-Keyhole Limpet Hemocyanin) and memory B lymphocytes comprising membrane immunoglobulins recognizing DNP and secreted immunoglobulins recognizing DNP were selected. anti-DNPB lymphocytes can be transfected with a nucleic acid vector comprising membrane-encoding immunoglobulin genes and secreted anti-influenza broadly neutralizing antibodies (bnabs).

Isolated recombinant anti-influenza B lymphocytes can be transferred to a vertebrate subject to protect the vertebrate subject from influenza infection. When symptoms of influenza appear or a pandemic appears, long-lived anti-influenza B lymphocytes can be activated ad libitum by injecting DNP-KLH into a vertebrate subject.

B lymphocyte protocol 2 is a protocol for producing isolated recombinant B lymphocytes. Protocol 2 memory B lymphocytes are isolated from vertebrate subjects. Isolated memory B lymphocytes are transfected with a nucleic acid vector comprising only immunoglobulin genes encoding anti-DNP membrane immunoglobulins and not anti-DNP secreted immunoglobulins. B lymphocytes with anti-DNP membrane immunoglobulins can be selected and transfected with immunoglobulin genes encoding two anti-influenza bnabs directed against two different epitopes of influenza antigen. The immunoglobulin genes encoding each BNAb may encode the membrane and secreted form of the BNAb.

Isolated recombinant anti-influenza B lymphocytes can be transferred to a vertebrate subject to protect the vertebrate subject from influenza infection. When symptoms of influenza appear or pandemics occur, long-lived anti-influenza B lymphocytes can be activated at will by injecting DNP-KLH into a vertebrate subject, thereby producing two anti-BNAb antibodies. Long-lived anti-influenza B lymphocytes can also be activated at will to produce two anti-BNAb's by injecting influenza antigen into a vertebrate subject. Unlike scheme 1, when B lymphocytes are activated by DNP-KLH or influenza antigen, secreted immunoglobulins against DNP-KLH are not produced.

B lymphocyte protocol 3 is a protocol for generating polyclonal isolated recombinant B lymphocytes. Scheme 3 vertebrates are immunized with an influenza vaccine, such as a trivalent seasonal influenza vaccine. Selecting memory B lymphocytes expressing membrane immunoglobulins that recognize an influenza vaccine antigen in a vertebrate subject. Selected polyclonal anti-influenza memory B lymphocytes are transfected with immunoglobulin genes encoding anti-DNP membrane immunoglobulins.

Polyclonal anti-influenza B lymphocytes can be transferred to a vertebrate subject to protect the vertebrate from influenza infection. Polyclonal long-lived anti-B cells can be activated by injecting DNP-KLH into a vertebrate subject when symptoms of influenza appear or a pandemic occurs. In addition, individual B lymphocyte clones can be activated by their cognate influenza antigen.

B lymphocyte protocol 4 is a protocol for generating polyclonal isolated recombinant B lymphocytes. Scheme 4 vertebrates are immunized with an influenza vaccine, such as a trivalent seasonal vaccine. Selecting memory B lymphocytes expressing membrane immunoglobulins that recognize an influenza vaccine antigen in a vertebrate subject. Polyclonal anti-influenza B lymphocytes are transfected with immunoglobulin genes encoding membrane and secreted forms of anti-influenza bnabs.

The isolated recombinant polyclonal anti-influenza B lymphocytes can be transferred to a vertebrate subject to protect the vertebrate subject from influenza infection. Polyclonal long-lived anti-influenza B lymphocytes can be activated in large numbers by injecting a full range of influenza vaccine antigens into vertebrate subjects when influenza symptoms appear or a pandemic occurs. Each B lymphocyte produces a BNAb and clone-specific immunoglobulin that reacts with influenza.

In some aspects, an isolated B lymphocyte cell line comprising at least one exogenously incorporated membrane immunoglobulin activated by a first antigen is capable of controlling the expression of at least one endogenously secreted immunoglobulin reactive to a second antigen. Exogenously incorporated membrane immunoglobulins act as receptors for specific ligands (e.g., primary antigens). Binding of the first antigen to the exogenously incorporated membrane immunoglobulin controls signal transduction by the exogenously incorporated membrane immunoglobulin to control expression from at least one endogenously secreted immunoglobulin reactive with the second antigen. Binding of the primary antigen to the exogenously incorporated membrane immunoglobulin controls signal transduction through the membrane immunoglobulin to control activation of B lymphocytes or differentiation of B lymphocytes.

FIG. 1 is a schematic diagram of a diagrammatic view of hypothetical immunoglobulin genes for memory B lymphocytes. The heavy (H) chain gene is on chromosome 14. The kappa (. kappa.) L chain gene is on chromosome 2. The lambda (. lamda.) L chain gene is on chromosome 22. Functional and non-functional alleles are present on

chromosomes

14 and 2. Both λ L chain alleles are described as non-functional. As shown in example 3, immunoglobulin genes encoding the H and L chains of anti-PSA membrane antibodies were cloned into targeting plasmid vectors to enable targeted integration into the corresponding non-functional Ig loci on

chromosome

14 and 2, respectively.

FIGS. 2A, 2B and 2C are schematic illustrations of a schematic view of non-functional and functional immunoglobulin heavy chain genes on chromosome 14. Fig. 2A shows the genetic structure of maternal chromosome 14 germline configuration. Variable region (V)_H) D section (D), J section (J)_H) IgM constant region (C)_Hμ), secreted Tail (TP) and μmembrane anchor (TM and Cyt) exons are shown in FIG. 2B. As shown in FIG. 2C, the genetic structure of functionally recombined chromosome 14 of the male parent has recombined V, D and J segments (V)_HD₁J₂). The genetic structure of the secreted and membrane μ -H chain coding and the alternating polyadenylation sites is shown. Note that the Ig gene structure is simplified, showing only one constant region (C) _H) An exon. Promoter and enhancer sequences are also omitted.

As shown in example 3, anti-PCLA immunoglobulin H and L chain genes are integrated into the Ig loci of mature B cells that are functionally rearranged on

chromosome

14 and 2, respectively. Functionally rearranged H chain loci are shown in FIG. 2B.

Fig. 3A, 3B and 3C are schematic diagrams of diagrammatic views of the replacement of immunoglobulin genes with heavy chain genes engineered to express membrane IgG and secreted IgG. The genetic structure of the secreted and membrane gamma-H chain genes with alternating polyadenylation sites is shown in FIG. 3A. The genetic structure of maternal chromosome 14 with the engineered membrane gamma-H chain gene is shown in fig. 3B. The genetic structure of the male parent chromosome 14 with the engineered secreted γ -H chain gene is shown in figure 3C. Note that the Ig gene structure is simplified, showing only one constant region (C)_H) An exon. Promoter and enhancer sequences are also omitted.

As shown in example 3, the anti-PCLA IgGH chain gene (i.e., the γ -H chain gene) can be engineered to remove the coding sequence for the transmembrane domain (TM) and cytoplasmic amino acids (Cyt) to produce a γ -H chain gene that encodes only the secreted H chain (fig. 3C).

Fig. 4A, 4B, 4C, 4D are schematic diagrams of a schematic view of a protocol for producing recombinant B lymphocytes having membrane immunoglobulins against a first antigen and secreted immunoglobulins against a second antigen. Figure 4A shows isolated memory B lymphocytes having endogenous DNA encoding anti-DNP membrane immunoglobulins and exogenous DNA encoding anti-influenza broadly neutralizing antibody (BNAb) secreted immunoglobulins. Figure 4B shows isolated memory B lymphocytes having exogenous DNA encoding anti-DNP membrane immunoglobulin and exogenous DNA encoding anti-influenza broadly neutralizing antibody (BNAb) secreted immunoglobulin. Figure 4C shows isolated memory B lymphocytes having endogenous DNA encoding an immunoglobulin secreted against influenza Abs and exogenous DNA encoding an anti-DNP membrane immunoglobulin. Figure 4D shows isolated memory B lymphocytes with exogenous DNA encoding liposome-delivered anti-influenza BNAb secreted immunoglobulin and exogenous anti-DNP membrane immunoglobulin polypeptides.

Fig. 5 is a schematic diagram of a diagrammatic view of a method 500 for producing at least one immunoglobulin in an isolated B lymphocyte cell line 501, the method comprising isolating 502B lymphocytes expressing at least one endogenously secreted immunoglobulin reactive to at least one second antigen from a vertebrate subject immunized with the at least one second antigen; introducing 503 at least one exogenous membrane immunoglobulin reactive to at least one first antigen into the isolated B lymphocyte to produce a recombinant B lymphocyte cell line; an isolated B lymphocyte cell line is expanded and selected 504 that expresses membrane immunoglobulins reactive with at least one first antigen and expresses at least one endogenously secreted immunoglobulin reactive with at least one second antigen.

FIG. 6 is a schematic diagram of a diagrammatic view of a method 600 for producing at least one immunoglobulin protein in an isolated B lymphocyte cell line 601, the method comprising: introducing 602 at least one exogenous membrane immunoglobulin reactive with at least one first antigen into at least one isolated B lymphocyte to produce at least one first isolated B lymphocyte cell line; expanding and selecting 603 at least one first isolated B lymphocyte cell line expressing a membrane immunoglobulin reactive to at least one first antigen; introducing 604 at least one exogenous nucleic acid encoding one or more secreted immunoglobulins reactive with at least one second antigen into at least one first isolated B lymphocyte cell line to produce at least one isolated recombinant B lymphocyte cell line; and selecting 605 at least one isolated recombinant B lymphocyte cell line that expresses one or more secreted immunoglobulins reactive with at least one second antigen.

Fig. 7 is a schematic diagram of a diagrammatic view of a method 700 for producing at least one immunoglobulin in a recombinant B lymphocyte cell line 701, the method comprising: isolating 702B lymphocytes from a vertebrate subject immunized with at least one first antigen, the B lymphocytes expressing at least one endogenous membrane immunoglobulin reactive with the at least one first antigen; introducing 703 at least one exogenous nucleic acid into the isolated B lymphocyte, the exogenous nucleic acid encoding at least one of the secreted immunoglobulins reactive with the at least one second antigen, and expanding the cell to produce a recombinant B lymphocyte cell line; detecting 704 the presence of at least one exogenously secreted immunoglobulin reactive with the at least one second antigen to select a recombinant B lymphocyte cell line.

Figure 8A shows a recombinant B cell receptor protein 1000 with a single chain variable fragment 1005 linked to an IgG heavy chain domain comprising: a hinge fragment 1008 linked to a CH3 domain 1010; a transmembrane 1015; and a cytoplasmic 1020 domain linked to the IL-21 receptor cytoplasmic domain 1025.

FIG. 8B contains a diagram of a recombinant B cell receptor expression vector 1055, which vector 1055 contains a CMV promoter 1028 linked to a recombinant B cell receptor 1030 linked to a poly-A site 1035 and a neomycin resistance gene 1040.

Fig. 8C is a schematic representation of active gamma heavy chain locus 1105 having a perforin gene inserted on chromosome 14, comprising Ig variable gene promoter 1045, Ig variable gene promoter 1045 linked to perforin cDNA1048, and downstream of Ig α constant region 1049, having transmembrane domain 1051 and cytoplasmic domain 1052.

FIG. 8D contains a schematic representation of a Sendai virus expression vector 1150 with a transcription factor gene inserted therein, comprising a viral NP gene 1065 linked to a viral P/V gene 1062, with three transcription factors T-beta 1064, RunX 31066 and Eomes 1068, and a viral L gene 1075 at the 3' end.

Fig. 9A shows the integration of Single Chain (SC) antibody genes at the active, rearranged kappa light chain locus on human chromosome 2. Adeno-associated virus (AAV) vectors carry genes for anti-Prostate Cancer Lipid Antigen (PCLA) SC antibodies flanked by Homology Arms (HA) targeted to integrate into the kappa constant region gene (ck). The results of CRISPR-mediated integration of the anti-PCLASC antibody gene in the ck gene on chromosome 2 are shown.

Fig. 9B shows targeted integration of a chemokine receptor gene (CXCR3) at the active, rearranged immunoglobulin (Ig) μ heavy chain locus. The AAV vector carries a gene for CXCR3, CXCR3 is flanked by HA, which targets integration into the first exon (C) of the Ig μ constant region gene (C)_Hμ 1). The Ig μ heavy chain gene edited on chromosome 14 is shown.

Fig. 10 illustrates a method 1000, the method 1000 comprising: the modified B-cell 1070, engineered with a predetermined exogenous or endogenous membrane immunoglobulin 1060, is activated to selectively engage the first target antigen 1050, and subsequently secrete a predetermined antibody 1080 and/or secrete redistributed biological agents and/or cytotoxic effector molecules (1310) against the second target antigen 1220, resulting in death of the target cell (1210).

Referring to fig. 10, a modified B cell includes a binding or capable of binding 1100 an antigen (engineered or naturally occurring) to a predetermined surface immunoglobulin 1060. In the next step 1200, receptor engagement 1100 results in secretion of a predetermined (exogenous or endogenous binding) antibody 1080, the antibody 1080 being configured to engage a second target antigen 1220 on the target cell 1210. Finally, the next step 1300 includes optionally secreting one or more cytotoxic molecules (which may be redistributed biological agents) 1310 for additional target cell destruction.

Fig. 11 shows lymph node 1111 with modified B cells 1117, which modified B cells 1117 recognize specific target antigens 1118 that contribute to the activation of the modified B cells. As shown, medulla oblongata 1115 is enriched in macrophages and plasma cells, while cortex 1114 contains primarily inactivated B and T cells as well as dendritic cells and macrophages. In one embodiment, modified B cells are implanted into lymph node 1111, for example in cortical region 1114 to be activated, or even if they have been activated. As shown in fig. 11, afferent 1113 and efferent 1112 lymphatic vessels allow immune system cells to enter and leave lymph node 1111. In fig. 11, germinal center 1116 comprises the location where mature B cells proliferate, differentiate, and produce antibodies. In one embodiment, modified B cells may be implanted into one or more germinal centers 1116 of a subject lymph node 1111.

In one embodiment, B-cell stimulation device 1205 can be implanted, for example, in the cortical region or capsular space of lymph node 1111. The B cell stimulation device 1205 may include an array of holes 1208 that may include, for example, different antigens and/or different concentrations of antigens and/or different adjuvants and/or different concentrations of adjuvants. For example, in a particular well 1206, predetermined antigen 1207 (optionally with adjuvant) is remotely triggered for release in the lymph node, and eventually predetermined antigen 1118 reaches modified B cell 1117 for conjugation. Such engagement may comprise the initiation of (or become a "booster" for) the modified B cell, or may be the initial activation of the modified B cell, or if desired, the tolerance of the modified B cell (e.g., "autoantigens" associated with autoimmune diseases). As shown in fig. 11, B cell stimulation device 1205 may include 1119 remote triggers of different antigens and/or concentrations over time and may act on both naturally occurring B cells as well as modified B cells as described herein. For example, the 1119B cell stimulation apparatus 1205 can release three different concentrations and/or antigens into the bloodstream or lymph nodes over time.

FIG. 12A shows the insertion of the interleukin 10(IL-10) gene at the first exon of the active, rearranged Ig heavy chain gene on chromosome 14. Shows a rearranged Ig mu heavy chain gene having a variable region (V)_HDJ) exon, intron and mu constant region (C)_Hμ) exons. An AAV vector encoding an interleukin 10(IL-10) gene is shown with a splice acceptor Site (SA), a polyA addition site (pA), and flanking Homology Arms (HA) for targeted integration at the μ heavy chain gene. The edited Ig μ heavy chain gene is shown, with the IL-10 gene located downstream of the variable region and interrupting the μ constant region gene.

FIG. 12B shows a lentiviral expression vector encoding a single chain membrane antibody specific for the autoantigen Myelin Oligodendrocyte Glycoprotein (MOG). The vector contains a cytomegalovirus promoter element (CMV) that directs transcription of single chain antibodies. Single chain antibodies comprise a single chain fv (scfv) fragment fused to an Ig γ constant region gene.

FIG. 13 illustrates V_HIntegration of the promoter and the bicistronic construct downstream (3') of the μ enhancer (μ Enh) resulted in disruption of the active rearranged Ig γ heavy chain gene on chromosome 14. The location of guide rnas (grnas) targeting CRISPR-mediated integration in the intron of the γ -heavy chain gene and the γ CH1 exon is indicated. A Class Switch Reassembly Site (CSRS) is indicated. AAV vectors encoding the bicistronic constructs are shown. The bicistronic construct comprises genes for recombinant B cell receptor (recBCR) and interleukin 21(IL-21), wherein each gene is preceded by a self-cleaving peptide (P2 a). The edited gamma heavy chain gene is shown with its bicistronic construct expressed under the control of the VH promoter and μ enhancer. Expression of the gamma H chain gene was disrupted by the bicistronic construct.

In a method of treating a disease in a vertebrate subject with an immunotherapeutic product, the recombinant B lymphocyte cell line and one of the one or more vertebrate subjects can be autologous. Alternatively, in a method of treating a disease in a vertebrate subject with an immunotherapeutic product, the recombinant B lymphocyte cell line may be allogeneic with one of the one or more vertebrate subjects. In the case where the recombinant B lymphocyte cell line is allogeneic with one of the one or more vertebrate subjects. In each case, the recombinant B lymphocyte cell line can be modified, if necessary, to reduce or eliminate expression of mhc class i (mhc i) proteins or mismatched HLA antigens in the recombinant B lymphocyte cell line to avoid allograft rejection and to reduce or eliminate graft-versus-host disease in the recipient of the allogeneic recombinant B lymphocytes. See, for example, U.S. application serial No. 12/804,650 and U.S. application serial No. 12/804,647, which are incorporated herein by reference.

Vertebrate subjects are treated with unmatched allogeneic donor recombinant B lymphocytes engineered to prevent presentation of major histocompatibility class I (mhc I) proteins on their cell surfaces. Transfecting allogeneic donor recombinant B lymphocytes with a lentiviral expression vector that directs inhibition of beta 2-microglobulin (beta) ₂M) protein translation and prevention of MHC I assembly and expression of microrna (mirna) presented on the cell surface. The genetically engineered recombinant B lymphocytes are injected into a patient. The inhibition of MHCI production by transplanted recombinant B lymphocytes is controlled by regulatory components and the effector molecule, doxycycline. In cases where it is necessary to eliminate recombinant B lymphocytes, doxycycline is administered to inhibit the expression of miRNA such that beta₂M and MHC I are expressed on the cell surface and elicit an allogeneic immune response.

To avoid immunological rejection by the transplanted cells, the vertebrate subject is treated with recombinant B lymphocytes having reduced expression of major histocompatibility class I (MHC I) proteins on their cell surface. Engineered recombinant B lymphocytes also contain a suicide mechanism that can be activated by administration of the prodrug ganciclovir when uncontrolled proliferation or other adverse events associated with recombinant B lymphocytes occur.

Vertebrates are treated with recombinant B lymphocytes modified to reduce expression of mismatched HLA antigens, thereby avoiding allograft rejection. Recombinant B lymphocyte cells are infected with a lentiviral vector encoding microrna (mirna) that inhibits expression of a specific donor HLA allele not shared by the recipient. The production of mismatched HLA-A, HLA-B, HLA-C, HLA-DRBI and HLA-DQB1 alleles was blocked by mirnas and the modified donor recombinant B lymphocytes did not express the corresponding HLA proteins.

A vertebrate subject is treated by transplantation of recombinant B lymphocytes. Allogeneic recombinant B lymphocytes are modified to reduce expression of MHC class I (MHC I) proteins by expressing viral genes targeted to disrupt the MHC I proteins. Recombinant B lymphocytes are transduced with lentiviral expression vectors encoding Cytomegalovirus (CMV) proteins (unique sequence 11(US11)) to target disruption of MHC I proteins and avoid allograft rejection (see, e.g., Lin et al, Cellular and Molecular Immunology 4:91-98, (2007), which is incorporated herein by reference).

As described herein, in addition to secreting antibodies, in one embodiment, one or more modified B lymphocytes have been engineered to secrete non-antibody proteins (e.g., glycoproteins, proteoglycans, amino acids, etc.) when stimulated by binding to a surface immunoglobulin with a specifically designed "trigger" antigen. Surface immunoglobulins and specially designed "trigger" antigens. For example, the non-antibody protein may comprise a neurotransmitter, hormone, cytokine, fat, vitamin, mineral, or anti-inflammatory agent.

In one embodiment, one or more neurotransmitters, such as dopamine, 5-hydroxytryptamine, acetylcholine, GABA, norepinephrine, oxytocin, and the like, are secreted by the modified B lymphocytes.

In one embodiment, one or more neurotransmitters are secreted by one or more modified B lymphocytes upon uptake, injection, implantation, or otherwise transfer of the modified B lymphocytes to a subject. For example, it has been documented that naturally-ingested B lymphocytes, when taken orally (e.g., via breast milk), home to the lymphoid mass (Peyer's patch) and other areas of the gastrointestinal tract. See, e.g., cabin et al, PLoS one.2016; 11(6) e0156762, which is incorporated herein by reference.

In addition, microbiomes are known to play a role in cancer development, progression and treatment. See, e.g., Bhatt et al, CA Cancer J Clin 2017; 67: 326-344, which is incorporated herein by reference.

Thus, in one embodiment, an oral or other formulation of a composition comprising one or more modified B lymphocytes described herein can be provided to a subject to treat a disease or indication. In one embodiment, the composition may be delivered by another route, for example, by intramuscular injection, subcutaneous injection, sublingual administration, buccal administration, parenteral administration, anal administration, intralymphatic administration, or another route of administration sufficient to deliver the composition to a subject for treatment.

In one embodiment, the oral or other formulation of the composition comprising one or more modified B lymphocytes further comprises one or more viable, dead or preserved strains of microorganisms, such as e.coli (escherichia), Bacteroides (Bacteroides), Bifidobacterium (Bifidobacterium), Bacillus (Bacillus), Saccharomyces cerevisiae (Saccharomyces), Lactobacillus tannatis (Prevotella tannerie), Neisseria lactis (Neisseria lacticum), Streptococcus (Streptococcus), Staphylococcus (Staphylococcus), Serratia (Serratia), corynebacterium (corebacter), Lactobacillus (Lactobacillus), or others. For example, in one embodiment, the strain is, for example, lactobacillus acidophilus (l.acidophilus), bifidobacterium longum (b.longum), bifidobacterium bifidum (b.bifidum), bifidobacterium lactis (b.lactis), bifidobacterium infantis (b.infarnatis), bifidobacterium animalis (b.animalis), lactobacillus rhamnosus (l.rhamnosus), lactobacillus fermentum (l.fermentum), lactobacillus plantarum (l.plantarum), lactobacillus brevis (l.brevis), lactobacillus salivarius (l.salivariaus), lactobacillus paracasei (l.paracasei), lactobacillus gasseri (l.gasseri), lactobacillus reuteri (l.reuteri), bacillus coagulans (b.coagulomains), streptococcus salivarius (s.salivariarius), or the like.

In one embodiment, the modified B cell provides in vivo monitoring of a subject such that when the cell's surface immunoglobulin receptor is triggered by an antigen selected during engineering of the cell, the cell is directed to secrete the particular antigen.

To selectively stimulate and restimulate modified B cells, a specific antigen (e.g., DNP-KLH) is administered without adjuvant. The endogenous response of these modified B cells should be limited to stimulation and not over-stimulated, which may result in tolerance to antigens rather than antibody secretion. Furthermore, DNP conjugates of peptides from KLH will likely elicit limited endogenous responses.

In one embodiment, the modified B cells are stimulated or restimulated to use an alternative carrier (e.g., DNP-human serum albumin) that should not elicit an immune response, but will provide "booster" antigen stimulation to the modified B cells.

In one embodiment, a B cell stimulation device is implanted in or on a subject to provide stimulation or restimulation of modified B cells (or the patient's own innate B cells). For example, the B cell stimulation device may comprise a microarray or microchip device with the antigen (and optionally adjuvant, cytokine, chemokine, etc.) on or in the device, and may be injected into a subject, thereby activating the antigen carrying device such that the antigen and/or adjuvant can be released to "enhance" the activation of the modified B cells.

For example, a microchip containing one or more airtight compartments may be activated by a remote control (e.g., a wireless signal) to trigger the release of one or more compartments. In one embodiment, one or more compartments may be triggered to release antigens and/or adjuvants and/or other molecules (e.g., cytokines, chemokines, growth factors, etc.) based on a pre-set dosing schedule. In one embodiment, one or more compartments may be triggered as desired. In one embodiment, the subject may control the triggering of the B cell stimulation device. In one embodiment, another entity may control the triggering of the B cell stimulation device of the subject. In one embodiment, the B cell stimulation device is placed in a lymphoid tissue of a subject, including but not limited to, for example, lymph nodes, GALT, MALT, spleen, liver, and the like. See fig. 11.

In one embodiment, the B cell stimulation device can be easily implanted and/or removed in a medical facility. In an embodiment, the B cell stimulation device may comprise at least ten doses, at least twenty doses, at least fifty doses, at least one hundred doses, at least two hundred doses, or more doses to stimulate the B cells. In one embodiment, each dose is the same in a particular B cell stimulation device. In one embodiment, the plurality of different doses comprise different internal contents (e.g., different antigens and/or different adjuvants). In an embodiment, the compartment of the B-cell stimulation device is configured such that each dose can be released at a specific time (e.g., by a predetermined procedure, by a sensor detecting a specific physiological parameter that causes or warrants the release of the dose, or by active intervention of the subject or other entity (e.g., a healthcare worker or computing device)).

In one embodiment, the B cell stimulation device comprises a plurality of different antigens and/or a plurality of different adjuvants and/or cytokines or other molecules, such as ligands or transcription factors (configured each in its own compartment or as a mixture of two or more in a single compartment) and can be released wirelessly into the subject. In one embodiment, each compartment can be independently addressed and independently activated in any desired release sequence. In one embodiment, a B-cell stimulation device comprises: electronic circuitry, including wireless communications (e.g., radio frequency); circuitry in electronic communication with each compartment for independently releasing its contents; a timer or clock that intervals and/or releases the contents of the compartment at precise intervals; and a controller in electronic communication with the various electrical components to effect normal operation.

For example, implantable microchip-based drug delivery devices for wireless control of delivery of fragments of human parathyroid hormone have been successfully tested in human clinical trials to be bioequivalent to daily injections. See, e.g., Farra, Science relative Med 22 Feb 2012, vol.4, Issue 122, pp.122ra21, which is incorporated herein by reference.

In one embodiment, a B cell stimulation device similar to the microchip devices described in the above cited documents is configured to stimulate B cells in a subject, including modified B cells described throughout this document. For example, microchip array devices having discrete compartments with impermeable thin metal membranes are configured to retain the contents in lyophilized or other activatable form. For example, the metal film may be removed by electrothermal ablation, which releases the contents of the compartment in a controlled manner.

In one embodiment, a B cell stimulation device is subcutaneously inserted into a subject that has received or will receive modified B cells as described herein. In one embodiment, the B cell stimulation device is implanted in lymphoid tissue of a subject that has received or will receive modified B cells as described. The lymphoid tissue may include, for example, lymph nodes, tonsils, spleens, lymph nodes, mucosa-associated lymphoid tissue, bone marrow, or thymus.

In one embodiment, the B cell stimulation device may be about 50mm x 30mm x 10mm (l x w x h), as described by Farra herein above, with two microchips, each having 10 reservoirs. In one embodiment, the stimulation device may be about half this size, with a single microchip having 10 reservoirs per microchip. In an embodiment, the stimulation device may be much smaller, such as a single microchip having 1, 2, 3, 4, 5, 6, 7, 8, or 9 reservoirs.

In one embodiment, the B cell stimulation device comprises at least one compartment with an enzyme (e.g., collagenase) to aid in the penetration of a fibromembranous sac that may form around any implant in the subject. Typically, the fibrous tissue capsule is less than one millimeter thick and allows molecules to pass through. And Id. However, if additional penetration is desired, enzymatic release may be performed prior to release of the B cell stimulating agent. In this way, one or more compartments containing collagenase or other enzymes are released prior to the release of the B cell stimulus (e.g., antigen, adjuvant, cytokine, other immune stimulus, etc.).

In the report of patients undergoing CAR-T cell immunotherapy, it was indicated that the patient had complete remission of brain metastases, and after biopsy of recurrent subcutaneous lesions, CAR-T cells were activated or reactivated, and as a result, the subcutaneous tumors also regressed. See Science News, August 28,2017, online report from Massachusetts General Hospital, incorporated herein by reference. In one embodiment, the modified B cells described herein are stimulated or restimulated by physical biopsy sampling of suspicious tumor tissue or lymph nodes.

Prophetic examples

Example 1

Recombinant memory B lymphocytes expressing two different antibodies: 1) recognition of model antigen, B Cell Receptor (BCR) for dinitrophenol-keyhole limpet hemocyanin (DNP-KLH), and 2) neutralizing secreted antibodies of various influenza virus strains.

Isolated recombinant B lymphocyte cell lines that produce secreted, broadly neutralizing immunoglobulins against influenza viruses and membrane immunoglobulins against model antigens may be used for cell therapy in mammalian subjects. The recombinant B lymphocyte cell line can be injected into a mammalian subject as a cell therapy to provide immune protection against influenza virus infection. Recombinant B lymphocyte cell lines can be activated in vivo or ex vivo to produce broadly neutralizing influenza antibodies by injecting a mammalian subject (or in vitro cell culture) with a model antigen, dinitrophenol-keyhole limpet hemocyanin (DNP-KLM). The time to stimulate immune protection from influenza virus infection in a mammalian subject can be selected based on the time to influenza infection outbreak in the entire population.

An individual is immunized with the model antigen Dinitrophenol (DNP) to elicit memory B cells with B Cell Receptors (BCRs) specific for the DNP model antigen. Memory B cells develop in response to immunization with DNP conjugated with a carrier protein, Keyhole Limpet Hemocyanin (KLH). 1mg of primary immunisation of DNP-KLH (see e.g. Biosearch Technologies DNP-KLH Product Info Sheet, incorporated herein by reference) was injected into the right arm. See, e.g., Rentenaar et al, Kidney International 62: 319-. Approximately 12-14 days after immunization, memory B cells were identified using dinitrophenol-human serum albumin-biotin (DNP-HSA-biotin) and phycoerythrin-streptavidin (available from Biosearch Technologies, Novato, CA) and fluorescein-anti-CD 27 antibodies to isolate memory B cells expressing BCR specific for DNP. By activating the cell sorter with fluorescence (e.g., as available from Becton Dickinson, Franklin Lakes, N.J.)

) Cell sorting was performed to isolate DNP-specific memory B cells. See, for example, U.S. patent No.7,378,276 to Ettinger et al at 27.5.2008 and U.S. patent No.7,993,864 to Brown et al at 9.8.2011, which are incorporated herein by reference.

Memory B cells expressing DNP-binding BCR are genetically engineered to express secreted antibodies that are broadly neutralizing antibodies reactive with multiple influenza virus strains. anti-DNP BCR-expressing memory B cells containing membrane IgG antibodies have a highly rearranged and expressed membrane immunoglobulin heavy (H) chain gene located on chromosome 14 (one of the two parental chromosome 14 copies). However, the other parent chromosome 14 has an immunoglobulin (Ig) H chain gene that is not subject to efficient rearrangement and expression. See fig. 1, 2A and 2B. This phenomenon (termed "allelic exclusion") results in a single B cell that expresses only one Ig heavy chain (and one Ig light chain (L)), and thus only one antibody (see, e.g., Abbas et al, Cellular and Molecular Immunology, 7)^thEd, Elsevier Saunders, philiadelphia, PA,2012, incorporated herein by reference). To generate B cells producing two different antibodies, memory B cells expressing anti-DNP BCR were modified by replacing non-functional, non-expressed immunoglobulin genes with functionally expressed immunoglobulin genes (for H and L chains). For example, the alternative immunoglobulin genes may encode secreted antibodies that are broadly neutralizing anti-influenza antibodies.

In one embodiment, the IgH chain or IgL chain chromosomal locus is an unexpressed Ig allele, wherein, for example, an endogenously rearranged VH promoter proximal to the μ enhancer is used for expression (the unexpressed allele is likely to have no VH promoter near the constant region due to VJ ligation).

Immunoglobulin genes encoding broadly neutralizing antibodies reactive against multiple influenza strains can be isolated from chromosomal DNA of antibody-producing human B cell clones. For example, human B cells isolated from individuals having immunity to influenza virus infection are immortalized by infecting isolated B cells with epstein-barr virus (EBV). Supernatants from individual EBV-transformed B cell clones were tested in an immunoassay for antibodies recognizing influenza virus. Methods of immortalizing B cells and detecting anti-viral antibodies are described (see, e.g., Zhang et al, proc.natl.acad.sci.usa 107:732-737,2010and Corti et al, j.clin.invasion 120:1663-1673,2010, incorporated herein by reference).

Methods for cloning heavy (H) chain and light (L) chain genes of Ig may be used. See, for example, U.S. Pat. No.7,741,077 to Grawunder et al, on 22.6.2010, and Early et al, Proc. Natl. Acad. Sci. USA 76: 857-. For example, expression of human anti-influenza antibody IgG ₁The (κ) EBV transformed B cell line was grown in culture and used as a source for isolation of messenger rna (mrna) and genomic DNA using standard methods using phenol/chloroform. See, e.g., Sambrook et al, In Molecular Cloning: A Laboratory Manual,2^ndEd, Cold Spring Harbor Press, Cold Spring Harbor, n.y., 1989. Encoding IgG₁The H-chain and κ L-chain mrnas were molecularly cloned after amplification using Polymerase Chain Reaction (PCR) and Reverse Transcriptase (RT). Methods and Ig gene primers for amplifying IgH chain mRNA and IgL chain mRNA are described in U.S. Pat. No.7,741,077, supra. Cloning of H and L chain mRNA (amplified to complementary DNA) into a plasmid vector (e.g., available from Invitrogen Corp., Carlsbad, Calif.)

Plasmid). The DNA sequences of the variable (V) region of the IgH chain (comprising Vh, D and J segments) and the V region of the kappa L chain (comprising Vk and Jk segments) were determined. The V region DNA sequence can be determined by automated DNA sequencing (DNA sequencing services are available from Charles River Laboratories International, inc., Wilmington, MA).

To isolate the corresponding genomic Ig gene, genomic DNA isolated from an anti-influenza B cell line (see above) was used as a template for PCR amplification of the human H chain gene and the kappa L chain geneThus, the method is simple and easy to operate. PCR primers (oligonucleotides) that amplify the V-region genes, including their respective promoters and upstream flanking regions (i.e., the 5' end of the V-gene), were determined by searching the human genome database for V-region DNA sequences established from cloned Ig mrnas. For example, the human genome nucleotide database available from the National Center for Biotechnology Information (National Center for Biotechnology Information) can be searched using the computer program BLAST to obtain sequences that match the H chain and L chain V regions. The Human RefSeq Genome database and BLAST software are available online (see, e.g., world wide web BLAST. ncbi. nlm. nih. gov/BLAST. cgi). Primers, enhancer sequences, H chain membrane anchors, polyA addition sites and downstream flanking regions (i.e., 3' of the Ig gene) are described for amplifying Ig constant regions (see, e.g., U.S. Pat. No.7,741,077, supra), and PCR amplified genomic fragments can be cloned into plasmid vectors (such as those available from Invitrogen Corp., Carlsbad, Calif.)

) In (1).

Memory B cells expressing anti-DNP membrane IgG are engineered to express an Ig gene encoding a secreted IgG antibody specific for influenza. Anti-influenza IgG can be engineered₁H chain gene (i.e.. gamma.)₁-H chain gene) to remove the coding sequence of the transmembrane domain (TM), cytoplasmic amino acids (Cyt) and polyA addition sites, thereby generating gamma encoding only the secreted H chain₁-an H chain gene. See fig. 3C and Abbas et al, supra. Ig genes were engineered using standard methods In Molecular biology (see, e.g., Sambrook et al, In: Molecular Cloning: A Laboratory Manual, 2)^ndEd., Cold Spring Harbor Press, Cold Spring Harbor, NY,1989, which is incorporated herein by reference) to remove membrane exons and retain promoter and enhancer sequences associated with a functional anti-influenza Ig gene (see, e.g., Abbas et al, supra). By using a method of homologous recombination, IgH chain and IgL chain genes encoding an antiviral antibody can be inserted into the unexpressed Ig gene locus (see, for example, U.S. Pat. No.5,202,238 to Perry et al on 4.13.1993; U.S. Pat. No.6,570 to Rajewsky and Zou on 5.27.2003Us patent No.6,841,383 to Reff et al, 061 and 11/1/2005, which is incorporated herein by reference). Methods for identifying and targeting the DNA sequence of a single Ig locus in memory B cells are known (see, e.g., Suk et al, Genome Research published online August 3,2011.DOI/10.1101/gr.125047.111, which is incorporated herein by reference). DNA sequences determined from the unexpressed immunoglobulin loci (i.e., non-functional immunoglobulin genes) are used to target recombination with anti-influenza immunoglobulin genes.

To facilitate homologous recombination, Ig genes encoding the H and L chains of anti-influenza antibodies for secretion were cloned into plasmid targeting vectors to achieve targeted integration into the corresponding non-functional germline Ig loci on

chromosome

14 and 2, respectively. See fig. 1 and 2A. For example, J_HAnti-influenza gamma cloning of the DNA sequence of the fragment 5' (see FIG. 2A) into the targeting plasmid₁Upstream of the H chain gene (5'), and downstream of the u-H chain membrane anchoring exon (3') (sequence cloned into γ)₁Downstream of the H chain gene (3') to promote recombination of the germline H chain locus on chromosome 14. Methods of constructing targeting vectors comprising a target sequence, a replacement gene, and a selectable marker are described (see, e.g., U.S. Pat. No.5,202,238, supra, U.S. Pat. No.6,570,061, supra, and U.S. Pat. No.6,841,383, supra).

Targeting vectors encoding secreted anti-influenza antibodies were used to replace non-functional germline μ -H chain genes and non-functional κ L chain genes in memory B cells expressing membrane anti-dnpig. The targeting vector plasmid was linearized by restriction enzyme digestion and transferred into memory B cells by electroporation, and subsequently selected for use in targeting vector plasmids. Methods and reagents for Electroporation of mammalian primary cells are described (see, e.g., "Electroporation Guide" available from biorad inc, Hercules, CA, which is incorporated herein by reference). After electroporation, memory B cells are cultured in tissue culture medium containing a selection drug (e.g., G418 and methotrexate) to select for selectable marker genes present on the H and L chain targeting vectors, i.e., the neomycin resistance gene and the dihydrofolate reductase, respectively. Selectable marker genes and their use are described (see, e.g., U.S. Pat. No.6,841,383, supra). Electroporated memory B cells resistant to both G418 and methotrexate were tested for expression of secreted IgG that binds influenza. After transfection and selection of memory B cells, those cells that produce secreted IgG antibodies to influenza are identified using standard immunoassays to evaluate B cell supernatants (see, e.g., Zhang et al, proc.natl.acad.sci.usa 107: 732-737.

To ensure that recombinant memory B cells are safe for use in patients, a suicide gene is introduced into the B cells. To prevent uncontrolled proliferation (and other adverse events) of recombinant memory B cells, a suicide gene (herpes simplex virus thymidine kinase gene (HSV-TK)) is introduced using a retroviral expression vector. Methods of inserting and expressing the HSV-TK gene and activating cytotoxic prodrugs such as ganciclovir are known (see, e.g., U.S. Pat. No.6,576,464 to Gold and Lebkowski on 10.6.2003 and U.S. Pat. No.5,997,859 to Barber et al on 7.12.1999, the contents of which are incorporated herein by reference). To prevent the growth of recombinant B cells that are considered unsafe or cause adverse events, HSV-TK expressing B cells were provided with 20 μ M ganciclovir (cytovine IV available from Roche Laboratories, Nutley, NJ). Ganciclovir is converted to a toxic metabolite by HSV-TK expressing B cells causing its death. Cells that do not express HSV-TK are not damaged by ganciclovir.

Recombinant memory B cells can be activated and expanded in vitro to assess their proliferation, activation and production of secreted anti-influenza antibodies. Engineered anti-DNP memory B cells isolated as described above were cultured in vitro with DNP-HSA to activate the cells. For example, at about 10 ⁵-10⁶Individual cells/mL of memory B cells are cultured in tissue culture flasks in standard media (e.g., RPMI 1640 serum-free media available from Sigma-Aldrich chem.co., st.louis, mo.) containing about 1 μ g/mL of DNP-HSA. Methods of activating memory B cells are described (see, e.g., U.S. patent No.7,378,276, supra). To assess activation, cells were tested in a proliferation assay after 3-5 days in culture. For aliquots of cultures³H-chestGlycosides were supplemented and cultured for an additional 16 hours. Measured by using a liquid scintillation counter (see, e.g., U.S. Pat. No.7,378,276, supra)³Uptake of H-thymidine. Equivalent cultures of memory B cells without DNP-HSA were used as negative controls for the proliferation assay. To evaluate the antibodies produced by activated memory B cells, supernatants from 3-5 day cultures were tested by enzyme-linked immunosorbent assay (ELISA) to detect and quantify anti-influenza antibodies. Methods for detecting and quantifying anti-influenza antibodies using ELISA are described (see, e.g., Khurana et al, PLoS Med. published online April 21,2009; doi:10.1371/journal. pmed.1000049 and Corti et al, Science 333: 850-. Purified anti-influenza antibodies derived from recombinant cell lines (see, e.g., Wrammert et al, Nature 453:667-671,2008, incorporated herein by reference) can be used to generate standard curves relating absorbance and antibody concentration in ELISA assays. Supernatants from non-activated (i.e., cultured in the absence of DNP-HA) recombinant memory B cells can be used as negative control samples for anti-influenza antibody ELISA.

Recombinant B lymphocyte cell lines can be activated in vivo or ex vivo to produce secreted broadly neutralizing influenza antibodies by injecting a mammalian subject (or in vitro cell culture) with a model antigen, DNP-KLH, to activate the production of secreted antibodies in recombinant B lymphocyte cell lines. The time to stimulate immunological protection of a mammalian subject from an influenza virus infection may be selected based on the time to an influenza infection outbreak in the entire population.

Example 2

Memory B lymphocytes are engineered to express a B Cell Receptor (BCR) that recognizes Dinitrophenol (DNP) and a secretory antibody that recognizes hepatitis c virus.

Isolated recombinant B lymphocyte cell lines that produce secreted immunoglobulins directed against Hepatitis C Virus (HCV) and produce membrane immunoglobulins directed against a model antigen can be used for cell therapy in mammalian subjects. The recombinant B lymphocyte cell line can be injected into a mammalian subject as an adoptive cell therapy to provide immune protection against hepatitis c virus infection. Recombinant B lymphocyte cell lines can be activated in vivo or ex vivo to produce secreted anti-HCV antibodies by injecting a mammalian subject (or in vitro cell culture) with the model antigen dinitrophenol-keyhole limpet hemocyanin (DNP-KLH). The timing of stimulating immunological protection of a mammalian subject from HCV infection can be selected based on the time the mammalian subject is exposed to HCV or based on the occurrence of symptoms in the subject.

Memory B cells expressing membrane IgG (also known as surface IgG or B Cell Receptor (BCR)) were isolated from peripheral blood of patients infected with chronic Hepatitis C Virus (HCV). Polyclonal memory B cells are isolated from the peripheral blood of a patient by: 1) peripheral blood mononuclear cells were isolated using a Ficoll Hypaque density gradient (available from Sigma Aldrich, st. louis, MO); 2) total B cells were negatively selected using magnetic beads (available from Stem Cell Technology, Vancouver, BC), and 3) cells were labeled with fluorescent monoclonal antibodies (memory B Cell marker) that recognize membrane IgG and CD27 and fluorescence activated Cell sorting was performed. See, for example, U.S. patent No.7,378,276 to Ettinger et al at 27.5.2008 and U.S. patent No.7,993,864 to Brown et al at 9.8.2011, which are incorporated herein by reference.

Immunoglobulin (Ig) genes encoding membrane IgG antibodies specific for DNP can be obtained from healthy volunteers immunized with DNP-KLH (see, e.g., Biosearch Technologies: DNP-KLH Product Info Sheet, incorporated herein by reference). By activating the cell sorter with fluorescence (e.g., as available from Becton Dickinson, Franklin Lakes, N.J.)

) Cell sorting was performed to isolate memory B cells with membrane IgG that recognized DNP. See, for example, the above-identified U.S. patent No.7,378,276. And the same U.S. patent No.7,993,864, supra. The Ig gene encoding anti-DNP antibodies was isolated from individual B cells (see, e.g., Tiller et al, J.Immunol. methods 329:112-124,2008, incorporated herein by reference). For anti-DNP B cells alone, use

Reverse transcriptase (available from Invitrogen corp., Carlsbad, CA) and taq dna polymerase (available from Qiagen, Valencia, CA) were used to amplify Ig heavy (H) chain gene transcripts and corresponding Ig light (L) chain gene transcripts by reverse transcriptase polymerase chain reaction (RT-PCR). Reaction conditions and oligonucleotide primers for amplifying IgH and IgL chains are known (see, e.g., Tiller et al, supra). DNA fragments encoding IgH and L chain variable region genes are isolated and cloned into IgH and L chain constant region-containing genes (e.g., Cgamma₁And Ck) in a mammalian expression vector. The DNA sequence (C.gamma.for the cloned DNP-resistant Ig gene) was determined using a DNA sequencer (e.g., using 3130Genetic Analyzer available from Applied Biosystems, Carlsbad, Calif.)₁Chains and Ck chains). IgG₁H chain gene (i.e.. gamma.)₁-H chain gene) is engineered to remove the "tail" and polyadenylation site encoding the secreted form of the H chain, thus encoding only membrane gamma by engineering ₁H chain gene (see, e.g., FIG. 3B and Abbas et al, Cellular and Molecular Immunology, 7)^thEd, Elsevier Saunders, philiadelphia, PA,2012, incorporated herein by reference). For example, cloned γ₁the-H chain gene can be amplified by PCR using primers that amplify γ₁-H chain constant region gene but omitting gene encoding gamma₁-the tail segment of the secreted form of the H chain and the polyadenylation site (see FIG. 3B). The primer may also be at gamma₁The 3' end of the H chain gene is added with an RNA splice donor site and a unique restriction enzyme site (e.g.a site for Not I; an enzyme available from New England Biolabs, Ipswich, Mass.). Coding for RNA splice acceptor sites, membrane anchor exons and the remaining gamma using PCR primer pairs containing restriction sites₁PCR amplification of the individual DNA fragments of the H chain gene to enable recombination of the gamma gene encoding the membrane form₁-gamma of H chain₁-an H gene. See fig. 3B. Methods of amplifying and assembling Ig genes are described (see, e.g., U.S. Pat. No.7,741,077, supra).

Genetic engineering of memory B cells obtained from patients with chronic HCV infection by replacing their functionally expressed Ig gene with an Ig gene encoding a Membrane IgG (kappa) recognizing DNP (see above)Text). IgH and L chain genes encoding anti-DNP antibodies can be inserted into functionally expressed Ig gene loci on chromosome 14 and chromosome 2 by methods using homologous recombination (see, e.g., U.S. Pat. No.5,202,238 issued to Perry et al on 4.13.1993; U.S. Pat. No.6,570,061 issued on 27.5.2003 to Rajewsky and Zou and U.S. Pat. No.6,841,383 issued on 11.1.2005 to Reff et al, which are incorporated herein by reference). For targeted integration into functional γ ₁In the H chain locus, will be from J_HTargeting sequence of an intron between Cluster and mu constant region genes (C)_Hμ, see FIG. 2A) was placed at the 5' end of the anti-DNP γ -H chain gene and would be located at γ₁The sequence downstream of the membrane-anchored exon was placed at the 3' end of the γ -H chain gene (see FIG. 3A). Similar targeting sequences (i.e., from the Jk-Ck intron and the 3' end of the Ck gene) were used to target the anti-DNP κ L chain gene into a functional Ck gene. Targeting vectors for anti-DNP H and L chains contain selectable marker genes such as hygromycin resistance and histidinol dehydrogenase, respectively. The engineered mature B cells expressing the secreted IgG anti-DNP antibodies were selected using a medium containing hygromycin and histidinol. Essential transcriptional promoter sequences and enhancer sequences necessary for Ig gene expression remain in the Ig gene complex (see Abbas et al, supra). After transfection and selection of memory B cells, those cells that produce membrane IgG antibodies specific for DNP were isolated using DNP-KLH attached to magnetic beads (protocols and isolation equipment available from Miltenyi Biotec, Auburn, CA).

To generate B cells producing two different antibodies, engineered memory B cells expressing anti-DNP membrane IgG were engineered to replace their non-functional germline Ig genes with functional Ig genes (for H and L chains). For example, the replaced Ig gene may encode a secreted antibody, an anti-HCV antibody. The Ig gene encoding anti-viral HCV antibodies can be isolated from chromosomal DNA of human B cell clones producing anti-viral antibodies. For example, human B cells from an individual immunized against HCV are immortalized by infection with epstein-barr virus (EBV), and supernatants from B cell clones from the individual are tested in an immunoassay for antibodies that recognize HCV. Methods of immortalizing B cells and detecting anti-viral antibodies are described (see, e.g., Zhang et al, supra and Corti et al, j.clin.immunization 120:1663-1673,2010, incorporated herein by reference).

Methods for cloning heavy (H) and light (L) chain genes of Ig may be used (see, e.g., U.S. Pat. No.7,741,077 to Grawunder et al, 6.2010, 22, and Early et al, Proc. Natl. Acad. Sci. USA 76: 857-. For example, expressing human anti-HCV antibody IgG₁(κ) EBV-transformed B cell lines were grown In culture and used as a source for isolation of messenger RNA (mRNA) and genomic DNA using standard methods using phenol/chloroform (see, e.g., Sambrook et al, In: Molecular Cloning: A Laboratory Manual, 2)^ndEd., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). Encoding IgG₁The H-chain and κ L-chain mrnas were molecularly cloned after amplification using Polymerase Chain Reaction (PCR) and Reverse Transcriptase (RT). Methods and Ig gene primers for amplifying H and L chain mRNA are described in U.S. Pat. No.7,741,077, supra. Cloning of H and L chain mRNA (amplified to complementary DNA) into a plasmid vector (e.g., available from Invitrogen Corp., Carlsbad, Calif.)

Plasmid), and the DNA sequences of the IgH chain variable (V) region (containing Vh, D and J segments) and the κ L chain V region (containing Vk and Jk segments). The V region DNA sequence can be determined by automated DNA sequencing (DNA sequencing services are available from Charles River Laboratories International, inc., Wilmington, MA).

To isolate the corresponding genomic Ig genes, genomic DNA isolated from anti-HCV B cell lines (see above) was used as a template for PCR amplification of the human H chain gene and the κ L chain gene. PCR primers (oligonucleotides) that amplify the V-region genes, including their respective promoters and upstream flanking regions (i.e., the 5' end of the V-gene), were determined by searching the human genome database for V-region DNA sequences established from cloned Ig mrnas. For example, the computer program BLAST can be used to search the nucleotide database of the human genome available from the National Center for Biotechnology Information to obtain matchesSequences of H chain and L chain V regions. The Human RefSeq Genome database and BLAST software are available online (see, e.g., http:// BLAST. ncbi. nlm. nih. gov/BLAST. cgi). Primers, enhancer sequences, H chain membrane anchors, polyA addition sites and downstream flanking regions (i.e., 3' of the Ig gene) are described for amplifying Ig constant regions (see, e.g., U.S. Pat. No.7,741,077, supra), and PCR amplified genomic fragments can be cloned into plasmid vectors (such as those available from Invitrogen Corp., Carlsbad, Calif.)

) In (1). Memory B cells expressing anti-DNP membrane IgG antibodies were engineered to express the Ig gene encoding secreted IgG antibodies specific for HCV. anti-HCV IgG H chain genes (i.e., γ -H chain genes) can be engineered to remove the coding sequence for the transmembrane domain (TM), cytoplasmic amino acids (Cyt), and polyA addition sites, thereby producing γ -H chain genes that encode only the secreted H chain. See figure 3 and Abbas et al, supra. Ig genes were engineered using standard methods In Molecular biology (see, e.g., Sambrook et al, In: Molecular Cloning: A Laboratory Manual, 2) ^ndEd., Cold Spring Harbor Press, Cold Spring Harbor, NY,1989, which is incorporated herein by reference) to remove membrane exons and retain promoter and enhancer sequences associated with a functional anti-HCV Ig gene (see, e.g., Abbas et al, supra). By using a method of homologous recombination, IgH chain and L chain genes encoding an anti-viral antibody can be inserted into the unexpressed Ig gene locus (see, e.g., U.S. Pat. No.5,202,238, supra; U.S. Pat. No.6,570,061, supra; and U.S. Pat. No.6,841,383, supra).

To facilitate homologous recombination, Ig genes encoding the H and L chains of anti-HCV antibodies for secretion are cloned into plasmid targeting vectors to achieve targeted integration into the corresponding germline Ig loci on chromosome 14 and chromosome 2, respectively. See fig. 1. For example, J from upstream of germline u-H chain gene_HThe 5 'sequence of the fragment (see FIG. 2: "Maternal Chromosome 14 Germine Configuration") was cloned upstream (5') of the anti-HCV gamma-H chain gene in the targeting plasmid and the mu-H chain membrane was anchored to the exonThe downstream (3') sequence was cloned downstream (3') of the gamma-H chain gene to facilitate recombination of the germline H chain locus on chromosome 14. Methods for constructing targeting vectors comprising a target sequence, a replacement gene, and a selectable marker have been described (see, e.g., U.S. Pat. No.5,202,238, supra, U.S. Pat. No.6,570,061, supra, and U.S. Pat. No.6,841,383, supra).

Targeting vectors encoding secreted anti-HCV antibodies are used to replace non-functional germline μ -H chain genes and non-functional κ L chain genes in memory B cells expressing membrane anti-DNPs. The targeting vector plasmid was linearized by restriction enzyme digestion and transferred into memory B cells by electroporation, and subsequently selected for use in targeting vector plasmids. Methods and reagents for Electroporation of mammalian primary cells are described (see, e.g., "Electroporation Guide" available from biorad inc, Hercules, CA, which is incorporated herein by reference). Following electroporation, memory B cells are cultured in tissue culture medium containing drugs (e.g., G418 and methotrexate) to select for selectable marker genes (i.e., neomycin resistance gene and dihydrofolate reductase, respectively) present on the H and L chain targeting vectors. Selectable marker genes and their use are described (see, e.g., U.S. Pat. No.6,841,383, supra). Electroporated memory B cells resistant to both G418 and methotrexate were tested for expression of secreted IgG that binds HCV. After transfection and selection of memory B cells, those cells that produce secreted IgG antibodies specific for HCV are identified using standard immunoassays to evaluate B cell supernatants (see, e.g., Zhang et al, proc.natl.acad.sci.usa 107: 732-.

Engineered memory B cells expressing two different antibodies can be activated in vitro and assayed for proliferation and production of secreted anti-HCV antibodies. Engineered anti-DNP memory B cells were cultured in vitro with dinitrophenol-human serum albumin (DNP-HSA available from Biosearch Technologies, Novato, CA) to activate the cells. For example, at about 10⁵-10⁶Individual cells/mL of memory B cells are obtained in tissue culture flasks at 37 ℃ in standard media (e.g., available from Sigma-Aldrich chem.co., st.louis, mo.)RPMI 1640 serum-free medium) containing about 1 μ g/ml of DNP-HSA. In addition, memory B cell cultures may contain 1. mu.g/ml of anti-CD 40 antibody and 100ng/ml of interleukin 21 (both available from R)&D Systems, Minneapolis, MN acquired) to activate cells and promote antibody production. Methods of activating memory B cells are described (see, e.g., U.S. patent No.7,378,276, supra). To assess activation, cells were tested in a proliferation assay after 3-5 days in culture. For aliquots of cultures³H-thymidine was supplemented and cultured for another 16 hours. Measured by using a liquid scintillation counter (see, e.g., U.S. Pat. No.7,378,276, supra)³Uptake of H-thymidine. An equivalent culture of memory B cells incubated without DNP-HSA served as a negative control for the proliferation assay. To evaluate the anti-HCV antibodies produced by activated memory B cells, supernatants from approximately 3-5 day cultures were tested by enzyme-linked immunosorbent assay (ELISA) to detect and quantify anti-HCV antibodies. Methods for the detection and quantification of anti-viral antibodies using ELISA are described (see, e.g., Corti et al, Science 333: 850-. Viral particles or viral proteins are adsorbed onto the microtiter plate to capture the anti-viral antibodies, and the anti-viral antibodies are detected using a second antibody (e.g., anti-IgG). Anti-viral antibodies can be detected using an ELISA at a concentration ranging from about 1ng/ml to 10,000 ng/ml. Purified anti-HCV antibodies produced by recombinant cell lines (see, e.g., Wrammert et al, Nature 453:667-671,2008, incorporated herein by reference) can be used to generate standard curves for determining antibody concentrations in ELISA assays. Supernatants from non-activated (i.e., not cultured with DNP-HSA) engineered memory B cells can be used as negative control samples for anti-HCV antibody ELISA.

To ensure that the engineered memory B cells are safe for use in patients, a suicide gene is introduced into the B cells. To prevent uncontrolled proliferation (and/or other adverse events), a suicide gene, the herpes simplex virus thymidine kinase gene (HSV-TK), is introduced into engineered memory B cells using a retroviral expression vector. Methods of inserting and expressing the HSV-TK gene and activating cytotoxic prodrugs such as ganciclovir are known (see, e.g., U.S. Pat. No.6,576,464 to Gold and Lebkowski on 10.6.2003 and U.S. Pat. No.5,997,859 to Barber et al on 7.12.1999, the contents of which are incorporated herein by reference). If the engineered memory B cells are deemed unsafe or cause adverse events, HSV-TK expressing B cells are treated with 20 μ M ganciclovir (Cytoven IV, available from Roche Laboratories, Nutley, NJ). Ganciclovir is converted to a toxic metabolite by HSV-TK expressing B cells causing its death. Cells that do not express HSV-TK are not damaged by ganciclovir.

To ensure that engineered memory B cells are safe for use in patients, suicide genes are introduced into B cells. To prevent uncontrolled proliferation (and/or other adverse events), a suicide gene, herpes simplex virus thymidine kinase gene (HSV-TK), is introduced into engineered memory B cells using a retroviral expression vector. Methods of inserting and expressing the HSV-TK gene and activating cytotoxic prodrugs such as ganciclovir are known (see, e.g., U.S. Pat. No.6,576,464 to Gold and Lebkowski at 10/6/2003 and U.S. Pat. No.5,997,859 to Barber et al (7/12/1999), the contents of which are incorporated herein by reference. if it is believed that the engineered B cells are unsafe or cause adverse events, then the HSV-TK expressing B cells are treated with 20 μ M ganciclovir (CytoveneIV available from Roche laboratories of Nutley, N.J.. the conversion of ganciclovir by HSV-TK expressing B cells to toxic metabolites causes their death.

Engineered memory B cells expressing anti-DNP BCR and anti-viral (anti-HCV) secretory antibodies can be expanded and used for adoptive cell therapy in chronic HCV infected patients. B cells may be activated in vitro (as described above) or in vivo by administering DNP-HSA to a patient. Immunization can be performed with subcutaneous administration of about 100mg of DNP-KLH to activate engineered memory B cells (see, e.g., renternaar et al, supra). Multiple activations can be stimulated in response to HCV infection.

Example 3

Mature B lymphocytes engineered to express a membrane antibody specific for prostate specific antigen and a second secreted antibody specific for prostate cancer lipid antigen.

Isolated recombinant B lymphocyte cell lines that produce secreted immunoglobulins directed to Prostate Cancer Lipid Antigen (PCLA) and produce membrane immunoglobulins directed to Prostate Specific Antigen (PSA) are useful for cell therapy to treat prostate cancer in a mammalian subject. The recombinant B lymphocyte cell line can be injected into a mammalian subject as an adoptive cell therapy to provide immunoreactivity for PSA on prostate cancer cells, and to process and present PSA to T lymphocytes. The recombinant B lymphocyte cell line can be activated by endogenous PSA produced in the subject, thereby producing a secreted anti-PCLA antibody. Recombinant B lymphocyte cell lines can also be activated in vivo or ex vivo by injection of exogenous Prostate Specific Antigen (PSA) into a mammalian subject (or in vitro cell culture) to produce secreted anti-PCLA antibodies. The timing of stimulating an immune response to prostate cells in a mammalian subject can be selected based on the detection of prostate cancer cells in the mammalian subject.

Polyclonal mature B cells expressing B Cell Receptors (BCR) comprising membrane IgM and IgD are isolated from prostate cancer patients. Mature B cells can be obtained from peripheral blood leukocytes of a patient. For example, about 10 can be collected using leukapheresis⁹Individual white Blood cells (see, e.g., Bensinger et al, Blood 81:3158-⁷Individual cells) are B cells. Mature B cells were isolated from patient leukocytes using antibodies specific for the B cell markers CD19, IgD, CD38, and CD21 (available from Becton Dickinson/Pharmingen, San Diego, CA). Methods for purifying mature B cells using magnetic beads (available from Miltenyi Biotech, Auburn, CA) and a Fluorescence Activated Cell Sorter (FACS) are described (see, e.g., U.S. patent No.7,378,276, supra). Mature B cells expressing membrane IgM and IgD were cultured in vitro and genetically engineered to express two different antibodies.

Mature B cells were genetically engineered to express membrane IgG antibodies specific for Prostate Specific Antigen (PSA). PSA is with prostateCancer-associated protein antigens that can be produced and purified using recombinant DNA methods for use as antigens (see, e.g., U.S. patent No.8,013,128 issued to Gudas et al on 6/9/2011, which is incorporated herein by reference). To obtain human immunoglobulin (Ig) genes encoding antibodies specific for PSA, hybridoma cell lines producing anti-PSA antibodies were constructed. For example, transgenic mice with human Ig genes (e.g., available from Abgenix inc., Fremont, CA)

) PSA were immunized and their B cells fused with myeloma cell fusion partners (e.g., SP2/0 cells (available from the american type culture collection, manassas, va)) to produce hybridoma cell clones expressing human antibodies (see, e.g., U.S. patent No.8,013,128, supra). Supernatants from the hybrid clones were screened using an immunoassay to detect human IgG antibodies that bound to PSA protein. Amplification of hybridoma clones producing antibodies recognizing PSA and use of Biacore^TMThe a100 instrument (available from GE Healthcare, Piscataway, NJ) tests the antibodies of each clone to measure antibody affinity and specificity for PSA (see, e.g., GE Healthcare, Application Note 84, "Early kinetic screening of hybrids …," which is incorporated herein by reference). Hybridomas expressing high affinity antibodies against PSA were selected to clone their human Ig genes. Methods for cloning heavy (H) chain and light (L) chain genes of Ig may be used. See, for example, U.S. Pat. No.7,741,077 and Early et al, Proc. Natl. Acad. Sci. USA 76:857-861,1979, issued to Grawunder et al at 22.6.2010, which is incorporated herein by reference. For example, IgG antibody expressing human anti-PSA ₁(kappa) hybridoma cell lines are grown In culture and used as a source for isolating messenger RNA (mRNA) and genomic DNA using standard methods using phenol/chloroform (see, e.g., Sambrook et al, In: Molecular Cloning: A Laboratory Manual, 2)^ndEd., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). Encoding IgG₁H chain and kappa L chain mRNAs were amplified using Polymerase Chain Reaction (PCR) and Reverse Transcriptase (RT) and then expressed in gramsAnd (4) a long ring. Methods and Ig gene primers for amplifying H and L chain mRNA are described in U.S. Pat. No.7,741,077, supra. Cloning of H and L chain mRNA (amplified to complementary DNA) into a plasmid vector (e.g., available from Invitrogen Corp., Carlsbad, Calif.)

Plasmid), and the DNA sequences of the IgH chain variable (V) region (containing Vh, D and J segments) and the κ L chain V region (containing Vk and Jk segments). The V region DNA sequence can be determined by automated DNA sequencing (DNA sequencing services are available from Charles River Laboratories International, inc., Wilmington, MA). To isolate the corresponding genomic Ig genes, genomic DNA isolated from anti-PSA hybridoma cell lines (see above) was used as a template for PCR amplification of the human H chain gene and the κ L chain gene. PCR primers (oligonucleotides) that amplify the V-region genes, including their respective promoters and upstream flanking regions (i.e., the 5' end of the V-gene), were determined by searching the human genome database for V-region DNA sequences established from cloned Ig mrnas. For example, the human genome nucleotide database available from the National Center for Biotechnology Information (National Center for Biotechnology Information) can be searched using the computer program BLAST to obtain sequences that match the H chain and L chain V regions. The Human RefSeq Genome database and BLAST software are available online (see, e.g., world wide web BLAST. ncbi. nlm. nih. gov/BLAST. cgi). Primers, enhancer sequences, H chain membrane anchors, polyA addition sites and downstream flanking regions (i.e., 3' of the Ig gene) are described for amplifying Ig constant regions (see, e.g., U.S. Pat. No.7,741,077, supra), and PCR amplified genomic fragments can be cloned into plasmid vectors (such as those available from Invitrogen Corp., Carlsbad, Calif.)

) In (1). Can be used for IgG₁H chain gene (i.e.. gamma.)₁-H chain gene) to remove the "tail" and polyadenylation site encoding the secreted form of the H chain, so that only membrane gamma is present₁the-H chain is encoded by an engineered gene (see, e.g., FIG. 3, and Abbas et al, Cellular and Molecular Immunology, 7)^th Ed.Elsevier Saunders, philiadelphia, PA,2011, which is incorporated herein by reference). For example, cloned γ₁the-H chain gene can be amplified by PCR using primers which amplify γ₁-H chain constant region gene, but omitting γ encoding secreted form₁-the tail segment of the H chain and the polyadenylation site. The primer may also be at gamma₁The 3' end of the H chain gene is added with an RNA splice donor site and a unique restriction enzyme site (e.g., a site for NotI; an enzyme available from New England Biolabs, Ipswich, Mass.). Encoding membrane-anchored exons and gamma using PCR primer pairs containing restriction sites₁PCR amplification of a separate DNA fragment of the remainder of the H chain gene, thus enabling recombination of the gene coding for gamma₁-gamma in the form of H membrane₁-the H gene chain. See fig. 3. Methods of amplifying and assembling Ig genes are described (see, e.g., U.S. Pat. No.7,741,077, supra).

The H and L chain encoding Ig genes directed against anti-PSA membrane antibodies were cloned into plasmid targeting vectors to achieve targeted integration into the corresponding non-functional germline Ig loci on chromosome 14 and chromosome 2, respectively. For example, J _Hanti-PSA Gamma Gene sequence 5' (see FIG. 2A) cloning into targeting plasmid₁Upstream of the H chain gene (5'), and the sequence downstream of the μ -H chain membrane anchoring exon (3') (TM and Cyt) was cloned into γ₁Downstream of the H chain gene (3') to promote recombination of the germline H chain locus on chromosome 14. Methods for constructing targeting vectors comprising a target sequence, a replacement gene, and a selectable marker have been described (see, e.g., U.S. Pat. No.5,202,238 to Perry et al at 4/13/1993, U.S. Pat. No.6,570,061 to Rajewsky and Zou at 5/27/2003, and U.S. Pat. No.6,841,383 to Reff et al at 11/1/2005, which are incorporated herein by reference). Targeting vectors constructed to replace non-functional germline μ -H chain genes and non-functional κ L chain genes in mature B cells are transferred in vitro into mature B cells. Targeting vector plasmids were linearized by restriction enzyme digestion (injection) and transferred into mature B cells by electroporation, and subsequently selected for use in targeting vector plasmids. Methods for electroporation of mammalian primary cells are describedAnd reagents (see, e.g., "electrophoresis Guide" available from biorad inc., Hercules, CA, which is incorporated herein by reference). Following electroporation, mature B cells are cultured in tissue culture medium containing drugs (e.g., G418 and methotrexate) to select for selectable marker genes (i.e., neomycin resistance gene and dihydrofolate reductase, respectively) present on the H and L chain targeting vectors. Selectable marker genes and their use are described (see, e.g., U.S. Pat. No.6,841,383, supra). Electroporated mature B cells resistant to both G418 and methotrexate were tested for expression of membrane IgG bound to PSA. For example, engineered mature B cells expressing membrane IgG specific for PSA are isolated using magnetic beads with PSA attached and propagated in vitro prior to transfection with Ig genes for secondary antibodies specific for different prostate tumor-associated antigens.

Mature B cells expressing anti-PSA membrane IgG antibodies were engineered to express an Ig gene encoding a secreted IgG antibody specific for Prostate Cancer Lipid Antigen (PCLA). Methods for extracting PCLA and obtaining monoclonal antibodies specific for PCLA are known (see, e.g., Zhang et al, proc. natl. acad. sci. usa 107: 732-. Human IgG antibodies specific for PCLA and the corresponding Ig gene can be obtained as described above (see, e.g., U.S. patent No.7,741,077 and Early et al, supra). The anti-pclaeggh chain gene (i.e., the γ -H chain gene) can be engineered to remove the coding sequence for the transmembrane domain and cytoplasmic amino acids, thereby producing a γ -H chain gene that encodes only the secreted H chain. See figure 3 and Abbas et al, supra. The anti-PCLA Ig gene was integrated into functionally rearranged Ig gene loci of mature B cells, which contained the mu-H chain gene on chromosome 14 and the kappa L chain gene on chromosome 2 (see, e.g., FIG. 2; only the H chain gene is shown). The PCLA-resistant γ -H chain gene and L chain gene are targeted for integration into the corresponding functional H and L chain gene loci (i.e., into

chromosome

14 and 2, respectively) using methods of homologous recombination as described above (see U.S. patent No.6,570,061 supra, and U.S. patent No.6,841,383 supra). For targeted integration into functional μ -H In the chain locus, will be from J_HTargeting sequence of an intron between Cluster and mu constant region genes (C)_Hμ, see fig. 2A) was placed at the 5 'end of the PCLA-resistant γ -H chain gene and the sequence located downstream of the μmembrane anchoring exon was placed at the 3' end of the γ -H chain gene (see fig. 2). Similar targeting sequences (i.e. from the Jk-Ck intron and the 3' end of the Ck gene) were used to target the anti-PCLA kappa light chain gene into a functional Ck gene. Targeting vectors for PCLA-resistant H and L chains contain different selectable marker genes, hygromycin resistance and histidinol dehydrogenase, respectively. The engineered mature B cells expressing the secreted IgG anti-PCLA antibody were selected using media containing hygromycin and histidinol. Essential transcriptional promoter sequences and enhancer sequences necessary for Ig gene expression remain in the Ig gene complex (see Abbas et al, supra). After transfection and selection of mature B cells, those cells that produce secreted IgG antibodies specific for PCLA are identified using standard immunoassays to evaluate B cell supernatants (see, e.g., Zhang et al, supra). Engineered mature B cells were cultured in vitro and stimulated with PSA to activate the cells and stimulate secretion of anti-pclaagg antibodies.

To ensure that engineered mature B cells are safe for use in patients, suicide genes are introduced into B cells. To prevent uncontrolled proliferation (and/or other adverse events), a suicide gene, herpes simplex virus thymidine kinase gene (HSV-TK), is introduced into engineered memory B cells using a retroviral expression vector. Methods of inserting and expressing the HSV-TK gene and activating cytotoxic prodrugs such as ganciclovir are known (see, e.g., U.S. Pat. No.6,576,464 to Gold and Lebkowski at 10/6/2003 and U.S. Pat. No.5,997,859 to Barber et al (7/12/1999), the contents of which are incorporated herein by reference. if it is believed that the engineered B cells are unsafe or cause adverse events, then the HSV-TK expressing B cells are treated with 20 μ M ganciclovir (CytoveneIV available from Roche laboratories of Nutley, N.J.. the conversion of ganciclovir by HSV-TK expressing B cells to toxic metabolites causes their death.

Administering the isolated recombinant B lymphocytesUsed in prostate cancer patients to provide anti-PCLA antibodies and to process and present PSA to T cells. Autologous B cells engineered to express anti-PSA membrane IgG and anti-PCLA secreted IgG were cultured in vitro for about 3-5 days at about 1 μ g/ml PSA, and then washed in serum-free medium prior to injection. About 5-10X 10 for intravenous injection ⁸B cells and monitoring the concentration of anti-PCLA antibodies in the peripheral blood of the patient and the number of engineered B cells by immunoassay and flow cytometry, respectively.

Example 4

Memory B lymphocytes of influenza vaccine vaccinated patients are provided with membrane antibodies specific for DNP and activated by administration of DNP-HSA.

Isolated recombinant B lymphocyte cell lines that produce secreted, broadly neutralizing immunoglobulins against influenza viruses and membrane immunoglobulins against model antigens may be used for cell therapy in mammalian subjects. The recombinant B lymphocyte cell line can be injected into a mammalian subject as a cell therapy to provide immune protection against influenza virus infection. Recombinant B lymphocyte cell lines can be activated in vivo or ex vivo to produce broadly neutralizing influenza antibodies by injecting a mammalian subject (or in vitro cell culture) with a model antigen, dinitrophenol-keyhole limpet hemocyanin (DNP-KLH). The time to stimulate immune protection from influenza virus infection in a mammalian subject can be selected based on the time to influenza infection outbreak in the entire population.

Individuals are immunized with an influenza vaccine to obtain memory B cells having a B Cell Receptor (BCR) against influenza virus. Memory B cells generated in response to immunization with subunit vaccines against influenza virus can elicit broadly neutralizing antibodies (see, e.g., Ekiert et al, Science 324: 246-. Primary immunization with 1mg of influenza virus vaccine (e.g., a conserved epitope from the viral Hemagglutinin (HA) protein) was injected subcutaneously into the right arm. About 12-14 days after immunization, BCR memory B cells expressing specificity for influenza were isolated using influenza HA protein-biotin and Phycoerythrin (PE) -streptavidin and fluorescein-anti-CD 27 antibody to identify memory B cells (biotin, streptavidin, and antibodies available from Becton Dickinson/Pharmingen, San Diego, CA). By activating the cell sorter with fluorescence (e.g., as available from Becton Dickinson, Franklin Lakes, N.J.)

) Cell sorting was performed to isolate influenza-specific memory B cells. See, for example, U.S. patent No.7,378,276 to Ettinger et al at 27.5.2008 and U.S. patent No.7,993,864 to Brown et al at 9.8.2011, which are incorporated herein by reference. Memory B cells expressing membrane IgG specific for influenza HA epitopes were cultured and expanded in vitro and then transfected with membrane immunoglobulin specific for Dinitrophenol (DNP). For example, at about 10⁵-10⁶Individual cells/mL of memory B cells are cultured in tissue culture flasks at 37 ℃ in standard media (e.g., RPMI 1640 serum-free media available from Sigma-Aldrich chem.co., st.louis, mo.) containing about 1 μ g/mL of influenza HA peptide (see, e.g., Ekiert et al, supra). In addition, memory B cell cultures may contain 1. mu.g/ml anti-CD 40 antibody and 100ng/ml interleukin 21 (both from R)&D Systems, Minneapolis, obtained from MN) to activate cells (see, e.g., U.S. patent No.7,378,276, supra). Membrane immunoglobulins specific for DNP are produced using recombinant DNA methods and inserted into the membrane of memory B cells producing anti-influenza antibodies. Immunoglobulin (Ig) genes encoding membrane IgG antibodies specific for DNP can be obtained from healthy volunteers immunized with DNP-KLH (see, e.g., Biosearch Technologies: DNP-KLH Product Info Sheet, incorporated herein by reference). By activating the cell sorter with fluorescence (e.g., as available from Becton Dickinson, Franklin Lakes, N.J.)

) Cell sorting was performed to isolate memory B cells with membrane IgG that recognized DNP. See, for example, the above-identified U.S. patent No.7,378,276. And the same U.S. patent No.7,993,864, supra.

Slave sheetIndividual B cells isolated immunoglobulin genes encoding anti-DNP antibodies (see, e.g., Tiller et al, j. immunological methods 329:112-124,2008, incorporated herein by reference). For each individual anti-DNP B cell, use was made

Reverse transcriptase (available from Invitrogen corp., Carlsbad, CA) and taq dna polymerase (available from Qiagen, Valencia, CA) were used to amplify Ig heavy (H) chain gene transcripts and corresponding Ig light (L) chain gene transcripts by reverse transcriptase polymerase chain reaction (RT-PCR). Reaction conditions and oligonucleotide primers for amplifying IgH and IgL chains are known (see, e.g., Tiller et al, supra). DNA fragments encoding the IgH and IgL chain variable region genes are isolated and cloned into IgH and IgL chain constant region-containing genes (e.g., C.gamma.₁And Ck) in a mammalian expression vector. The DNA sequence (C.gamma.for the cloned DNP-resistant Ig gene) was determined using a DNA sequencer (e.g., using 3130Genetic Analyzer available from Applied Biosystems, Carlsbad, Calif.)₁Chains and Ck chains). IgG ₁H chain gene (i.e.. gamma.)₁-H chain gene) is engineered to remove the "tail" and polyadenylation site encoding the secreted form of the H chain, thus encoding only membrane gamma by engineering₁H chain gene (see, e.g., FIG. 3B and Abbas et al, Cellular and Molecular Immunology, 7)^thEd, Elsevier Saunders, philiadelphia, PA,2012, incorporated herein by reference). For example, cloned γ₁the-H chain gene can be amplified by PCR using primers that amplify γ₁-H chain constant region gene but omitting gene encoding gamma₁-a tail segment of the secreted form of the H chain and a polyadenylation site. Encoding gamma using PCR primer pairs containing restriction sites₁Membrane anchor exons and remaining gamma₁PCR amplification of the individual DNA fragments of the H chain gene to enable recombination of the gamma gene encoding the membrane form₁-gamma of H chain₁-an H gene. See fig. 3B. Methods of amplifying and assembling Ig genes are described (see, e.g., U.S. Pat. No.7,741,077, supra).

Genetically engineered immunoglobulin genes encoding anti-DNP membrane antibodies are expressed in mammalian cell lines and membrane IgG is purified from the cell lines. For example, the kappa (κ) L chain gene and the modified γ -1H chain gene are inserted into a lentiviral expression vector using standard recombinant DNA methods (see, e.g., U.S. patent publication No.2007/0116690 to Yang et al, published 5/24 of 2007, which is incorporated herein by reference). The viral vectors are used to transfect Chinese Hamster Ovary (CHO) cells (available from the American type culture Collection, Manassas, Va.). Methods of expressing membrane immunoglobulins can be used. See, e.g., Price et al, j.immunol.methods 343:28-41,2009, which is incorporated herein by reference. To identify and isolate CHO clones expressing anti-DNP membrane IgG, phycoerythrin-conjugated anti-human IgG antibody was used to label CHO cells and sorted using FACS (see, e.g., Price et al, supra). CHO cell lines producing anti-DNP membrane IgG were isolated and expanded and membrane IgG was purified from CHO cell lysates using an immunoaffinity column. Affinity columns constructed from protein a-agarose (available from Sigma-Aldrich co., st.louis, MO) were used to purify membrane IgG from lysates of engineered CHO cells. For example, cells can be lysed in a buffer containing 0.15M NaCl, 0.01M TrisHCl, pH8.2, 1mM EDTA, 2mM phenylmethylsulfonyl fluoride, 0.5% NonidetP-40, and 1mg/mL HSA (see, e.g., Schneider et al, J.biol.chem.257:10766-10769,1982, which is incorporated herein by reference). Purified anti-DNP membrane IgG was used to construct liposomes fused to memory B cells specific for influenza antigens (see above).

Liposomes containing anti-DNP membrane IgG were constructed from phospholipids and purified anti-DNP membrane IgG antibodies. Liposomes incorporating anti-DNP membrane IgG antibodies can be fused with memory B cells specific for influenza virus to obtain memory B cells in which anti-DNP membrane immunoglobulins are incorporated into the B cell membrane. Liposomes can be prepared from cholesterol and L- α -phosphatidylcholine. See, e.g., U.S. patent publication No.2005/0208120, which is incorporated herein by reference. Cholesterol and L- α -phosphatidylcholine were mixed in chloroform at a ratio of 2: 7 and the chloroform was evaporated off using a stream of argon. The liposomes were resuspended in 140mM NaCl, 10mM Tris HCI, 0.5% deoxycholate at pH8 and sonicated for three minutes. Purified anti-DNP membrane antibodies (see above) were inserted into liposomes by mixing membrane IgG with liposomes at a 1:10 molar ratio and dialyzing against phosphate buffered saline at 4 ℃ for 72 hours. Liposomes were characterized to assess liposome size and the amount of anti-DNP membrane IgG protein incorporated into the liposomes. The size of the liposomes was determined using dynamic light scattering and flow cytometry (see, e.g., U.S. patent application No.2005/0208120 to Albani, which is incorporated herein by reference). For example, the liposomes containing the anti-DNP antibody may have an average diameter of about 50 nanometers. To measure anti-DNP IgG protein on liposomes, the liposomes were analyzed on a flow cytometer after staining with FITC-labeled anti-IgG antibody. Liposomes were classified according to FITC fluorescence, forward scatter and side scatter to isolate and count liposomes with IgG. anti-DNP IgG protein on liposomes was measured using enzyme-linked immunosorbent assay (ELISA). Methods of analyzing liposomes by flow cytometry and measuring IgG and other proteins by ELISA are known (see, e.g., U.S. patent application No.2005/0208120, supra).

Liposomes containing anti-DNP membrane IgG were fused to memory B cells specific for influenza virus (see above) to obtain memory B cells with anti-DNP B cell receptors. Purified liposomes having anti-DNP BCR On their surface are electrofused with memory B cells (see, e.g., Zimmermann et al, IEEE Transactions On Plasma Science 28:72-82,2000, incorporated herein by reference). For example, liposomes and memory B cells are suspended in a 1: 1 ratio in hypotonic buffer containing 0.1mM calcium acetate, 0.5mM magnesium acetate, and 1mg/ml bovine serum albumin. The osmolality is adjusted to about 75mOsm and about 200. mu.L of a solution containing about 2X 10⁴To 2X 10⁵A cell suspension of individual cells is placed in an electrofusion chamber (electrofusion generators and chambers are available from BTX Instrument Division, Harvard Apparatus, inc. Cells were aligned by applying an alternating magnetic field of 5V amplitude and 2MHz frequency for approximately 30 seconds. Fusion is then initiated by applying a rectangular fusion pulse of 20V to 40V in amplitude and 15 sec in duration. The alternating electric field was again applied for 30 seconds to fuse the cells andthe liposomes are held in place. Cells were transferred to culture flasks and grown for 2 to 5 weeks.

Fused memory B cells are characterized by assessing their production of anti-DNP BCR and its anti-influenza antibodies. Fused memory B cells were tested for membrane anti-DNP antibodies using fluorescent DNP-HSA and FACS analysis. Methods for assessing membrane anti-DNP IgG antibodies using flow cytometry are described above (see embodiment 2). Fused memory B cells can be activated in vitro to produce anti-influenza antibodies when stimulated with DNP-HSA, and production of secreted anti-influenza antibody can be measured using an ELISA based on influenza virus hemagglutinin protein or influenza virus particles. Methods for measuring anti-influenza antibody and memory B cell activation are known (see, e.g., U.S. patent No.7,378,276, supra and example 1).

Recombinant fused memory B cells are provided to human patients at risk for influenza virus infection as a therapeutic and prophylactic cell therapy that can be activated in vivo. When an anti-influenza antibody response is desired, recombinant memory B cells are activated in vivo by administering DNP-HAS to the patient. For example, when the patient is healthy before the "flu season," about 10 may be injected⁸-10⁹Individual fused B cells serve as prophylactic agents. When needed, fused memory B cells can be activated by intradermal injection of 100 μ g DNP-HSA into the patient. For example, fused memory B cells can be activated after exposure of a patient to influenza virus or upon initial signs of infection. The production of anti-influenza antibodies can be monitored by sampling the patient's peripheral blood and performing an ELISA with influenza virus as antigen. In addition, the presence of broadly neutralizing antibodies against multiple influenza strains can be determined by ELISA based on conserved epitopes from influenza virus (see, e.g., Ekiert et al, supra).

Example 5

Construction of autologous memory B lymphocytes engineered to produce two different anti-staphylococcus aureus antibodies.

The isolated recombinant B lymphocyte cell line, which produces two different secreted immunoglobulins against methicillin-resistant staphylococcus aureus (MRSA) and produces a membrane immunoglobulin against a third staphylococcus aureus antigen, can be used for cell therapy in a mammalian subject. The recombinant B lymphocyte cell line can be injected into a mammalian subject as a cell therapy to provide immune protection against MRSA infection. Recombinant B lymphocyte cell lines can be activated in vivo or ex vivo to produce antibodies against MRSA by injecting a mammalian subject (or cell culture in vitro) with a staphylococcus aureus antigen. The time to stimulate immunological protection from MRSA infection in a mammalian subject may be selected based on the exposure of the subject to MRSA or the appearance of symptoms of MRSA infection.

A patient infected with methicillin-resistant staphylococcus aureus (MRSA), which has recurrent episodes of infection, will be treated with his own long-lived memory B-cells that have been genetically engineered to express two different anti-staphylococcus aureus monoclonal antibodies (mabs). Memory B cells expressing membrane IgG (also known as surface IgG or B Cell Receptor (BCR)) were isolated from peripheral blood of patients with recurrent MRSA infection. Polyclonal memory B cells of unknown antigen specificity are isolated from the peripheral blood of a patient by: 1) peripheral blood mononuclear cells were isolated using a Ficoll Hypaque density gradient (available from Sigma Aldrich, st. louis, MO); 2) negative selection of all B cells was performed using magnetic beads (available from Stem Cell Technology, Vancouver, BC), and 3) cells were labeled with fluorescent monoclonal antibodies recognizing IgG and CD27 (memory B Cell marker) and fluorescence activated Cell sorting was performed. See, for example, U.S. patent No.7,378,276 to Ettinger et al at 27.5.2008 and U.S. patent No.7,993,864 to Brown et al at 9.8.2011, which are incorporated herein by reference. Purified memory B cells were modified using genetic engineering methods to introduce immunoglobulin (Ig) genes encoding two different anti-staphylococcus aureus antibodies.

The Ig gene encoding the first anti-Staphylococcus aureus gene was isolated from an antibody-producing hybridoma cell line. Methods of constructing hybridoma cell lines that produce an IgG antibody specific for poly-N-acetylglucosamine (PNAG), which has a protective effect against Staphylococcus aureus, are describedBodies (see, e.g., Kelly-Quintos et al, Infection and Immunity 74:2742-2750,2006, which is incorporated herein by reference). For example, transgenic mice with human Ig genes (e.g., available from Abgenix inc., Fremont, CA)

) PNAG was used to immunize and their B cells were fused with myeloma cell fusion partners (e.g., SP2/0 cells (available from the american type culture collection, manassas, va)) to produce hybridoma cell clones expressing human antibodies (see, e.g., U.S. patent No.8,013,128, supra). Hybridomas expressing high affinity antibodies to PNAG were selected for cloning their Ig genes. Methods for cloning heavy (H) and light (L) chain genes for Ig are known (see, e.g., U.S. Pat. No.7,741,077 and Early et al, Proc. Natl. Acad. Sci. USA 76: 857-. For example, expression of anti-PNAG antibody IgG ₁(kappa) hybridoma cell lines are grown In culture and used as a source for isolating messenger RNA (mRNA) and genomic DNA using standard methods using phenol/chloroform (see, e.g., Sambrook et al, In: Molecular Cloning: A Laboratory Manual, 2)^ndEd., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). Encoding IgG₁The H-chain and κ L-chain mrnas were molecularly cloned after amplification using Polymerase Chain Reaction (PCR) and Reverse Transcriptase (RT). Methods and Ig gene primers for amplifying H and L chain mRNA are described in U.S. Pat. No.7,741,077, supra. Cloning of H and L chain mRNA (amplified to complementary DNA) into a plasmid vector (e.g., available from Invitrogen Corp., Carlsbad, Calif.)

In order to isolate the corresponding genomic Ig genes,genomic DNA isolated from the anti-PNAG hybridoma cell line isolated as above (see above) was used as a template for PCR amplification of the H chain gene and the κ L chain gene. PCR primers (oligonucleotides) that amplify the V-region genes, including their respective promoters and upstream flanking regions (i.e., the 5' end of the V-gene), were determined by searching the human genome database for V-region DNA sequences established from cloned Ig mrnas. For example, the human genome nucleotide database available from the National Center for Biotechnology Information (National Center for Biotechnology Information) can be searched using the computer program BLAST to obtain sequences that match the H chain and L chain V regions. The Human RefSeq Genome database and BLAST software are available online (see, e.g., world wide web BLAST. ncbi. nlm. nih/gov/BLAST. cgi). Primers, enhancer sequences, H chain membrane anchors, polyA addition sites and downstream flanking regions (i.e., 3' of the Ig gene) are described for amplifying Ig constant regions (see, e.g., U.S. Pat. No.7,741,077, supra), and PCR amplified genomic fragments can be cloned into plasmid vectors (such as those available from Invitrogen Corp., Carlsbad, Calif.)

) In (1). Can be used for IgG₁H chain gene (i.e.. gamma.)₁-H chain gene) to remove the "tail" and polyadenylation site encoding the secreted form of the H chain, so that only membrane gamma is present₁the-H chain is encoded by an engineered gene (see, e.g., FIG. 3B, and Abbas et al, Cellular and Molecular Immunology, 7)^thEd, Elsevier Saunders, philiadelphia, PA,2011, which is incorporated herein by reference). For example, cloned γ₁the-H chain gene can be amplified by PCR using primers which amplify γ₁-H chain constant region gene, but omitting γ encoding secreted form₁-the tail segment of the H chain and the polyadenylation site. Encoding membrane-anchored exons and gamma using PCR primer pairs containing restriction sites₁PCR amplification of a separate DNA fragment of the remainder of the H chain gene, thus enabling recombination of the gene coding for gamma₁-gamma in the form of H membrane₁-the H gene chain. See fig. 3B. Methods for amplifying and assembling IgH and L chain genes are described (see, e.g., Meimei)National patent No.7,741,077, supra).

Ig genes encoding the heavy (H) and light (L) chains of the anti-PNAG antibody were cloned into targeting plasmid vectors to achieve targeted integration and replacement of the corresponding functionally rearranged IgH and IgL chain genes on

chromosome

14 and 2, respectively (see, e.g., fig. 1). Methods for targeting genes to Ig loci using homologous recombination are known (see, e.g., U.S. Pat. No.5,202,238 to Perry et al at 4/13/1993, U.S. Pat. No.6,570,061 to Rajewsky and Zou at 5/27/2003, and U.S. Pat. No.6,841,383 to Reff et al at 11/1/2005, which are incorporated herein by reference). For targeted integration into functional γ ₁In the H chain locus, will be from J_HTargeting sequence of an intron between Cluster and mu constant region genes (C)_Hμ, see fig. 2A) was placed at the 5 'end of the anti-PNAG γ -H chain gene and the sequence located downstream of the γ 1 membrane anchoring exon was placed at the 3' end of the γ -H chain gene (see fig. 3A). Similar targeting sequences (i.e., from J)_k-C_kIntron and C_k3' of the gene) for targeting the anti-PNAG κ light chain gene to functional C_kIn a gene. Targeting vectors for anti-PNAG H and L chains contain selectable marker genes such as hygromycin resistance and Zeocin, respectively^TMResistance to bleomycin. Comprising hygromycin B and Zeocin^TMBleomycin medium was used to select engineered memory B cells expressing membrane IgG anti-PNAG antibodies (protocols, selection agents and selectable markers available from Invitrogen, Carlsbad, CA). Essential transcriptional promoter sequences and enhancer sequences necessary for Ig gene expression remain in the Ig replacement gene (see Abbas et al, supra). After transfection, homologous recombination and selection, recombinant memory B cells expressing membrane IgG specific for staphylococcus aureus PNAG were isolated using magnetic beads with PNAG attached (magnetic beads and protocols available from Miltenyi Biotech inc. Memory B cells expressing anti-PNAG membrane antibodies were cultured in vitro prior to transfection with an Ig gene encoding a second antibody specific for a different s.

Producing memory B cells using recombinant DNA methods to express membrane antibodies specific for PNAG,and further genetically engineered to express a second anti-staphylococcus aureus MAb. Monoclonal antibodies (mabs) specific for the immunodominant staphylococcal antigen a (isaa) are expressed in memory B cells. To obtain anti-IsaA mabs with prophylactic and therapeutic activity against s.aureus infections, hybridoma cell lines were constructed using purified recombinant IsaA protein to immunize mice and hybridoma clones were selected (see, e.g., Lorenz et al, antimicrob. agents chemith.55: 165-173,2011, which is incorporated herein by reference). To clone the anti-IsaA antibody variable region gene, messenger RNA was extracted from the selected hybridoma cell line and used as a template for complementary DNA synthesis by Reverse Transcriptase (RT) and amplification using the Polymerase Chain Reaction (PCR) (i.e., RT-PCR). Methods of amplifying and cloning the heavy and light chain variable regions in antibody expression vectors are described (see, e.g., Kelly-Quintos et al, supra). For example, plasmid expression vectors encoding the complete gamma (γ) -1 and lambda (λ) L chains can be used to construct the H chain using restriction enzymes and standard molecular biology methods (see, e.g., Sambrook et al, supra). To facilitate transfection and expression of anti-IsaA antibodies in memory B cells, γ can be used ₁the-H chain gene and the. lamda. -L chain gene are transferred into a lentiviral vector (see, for example, U.S. Pat. No.7,939,059 to Yang et al, 5/10/2011). Infection of memory B cells with recombinant lentiviruses results in integration of the vector sequence into random sites of genomic DNA of memory B cells (i.e., untargeted) and production of secreted IgG1(λ) anti-IsaA antibodies. Protocols and lentiviral expression vectors are available from Invitrogen corp, Carlsbad, CA; see e.g., User Manual, "ViraPower^TMHiPerfom^TMLentiviral Expression System ", which is incorporated herein by reference. For example, a vial of memory B cells is infected with a titrated stock of recombinant lentivirus to produce a multiple infection of about 1.0 transduction units per cell. Cells and lentiviruses were incubated at 5% CO₂Incubation at 37 ℃ overnight; the lentiviral-containing medium was then replaced with fresh medium and incubated overnight. On day three, cells were placed in selective media (e.g., blasticidin-containing media available from Invitrogen corp., Carlsbad, CA) to select for stable transduction containingA cell of a lentiviral vector. Clones of memory B cells resistant to blasticidin were also placed on hygromycin B and Zeocin^TMTo select clones expressing two anti-MRSA antibodies. To identify and purify memory B cells expressing both anti-staphylococcus aureus antibodies, memory B cells with surface IgG specific for PNAG were purified using magnetic beads (available from Miltenyi Biotec inc., Auburn, CA) and cultured in vitro with PNAG. Methods of obtaining PNAG and culture conditions for human memory B cells are described (see, e.g., Kelly-Quintos et al, supra; and U.S. patent No.7,378,276, supra). Supernatants from the cultures were tested for anti-IsaA IgG antibodies by ELISA (see, e.g., Lorenz et al, supra) and memory B cells producing anti-IsaA antibodies were selected and expanded for adoptive immunotherapy.

Providing recombinant memory B cells to a human patient at risk of MRSA infection as a therapeutic and prophylactic cell therapy that can be activated in vivo. When an anti-MRSA antibody response is required, recombinant memory B cells are activated in vivo by administering a staphylococcus aureus antigen to the patient. For example, when a patient is healthy and recently infected with MRSA, about 10 may be injected⁸-10⁹Individual recombinant B cells serve as prophylactic agents. When needed, recombinant memory B cells can be activated by intradermal injection of 100 μ g of staphylococcus aureus into a patient. For example, memory B cells may be activated after a patient is exposed to MRSA or at the first sign of infection. The production of anti-influenza antibodies can be monitored by sampling the patient's peripheral blood and performing an ELISA with MRSA antigen as the target antigen.

Example 6

Adoptive immunotherapy of drug-resistant bacterial infections with autologous memory B lymphocytes engineered to produce two different anti-staphylococcus aureus antibodies.

The isolated recombinant B lymphocyte cell line, which produces two different secreted immunoglobulins against methicillin-resistant staphylococcus aureus (MRSA) and produces a membrane immunoglobulin against a third staphylococcus aureus antigen, can be used for cell therapy in a mammalian subject. The recombinant B lymphocyte cell line can be injected into a mammalian subject as a cell therapy to provide immune protection against MRSA infection. Recombinant B lymphocyte cell lines can be activated in vivo or ex vivo to produce antibodies against MRSA by injecting a mammalian subject (or cell culture in vitro) with a staphylococcus aureus antigen. The time to stimulate immunological protection from MRSA infection in a mammalian subject may be selected based on the time to an outbreak of MRSA infection in the overall population.

To protect and treat patients with recurrent MRSA infections, patients are provided with autologous recombinant B cells. The patient's memory B cells were genetically engineered to express two antibodies that recognize two staphylococcus aureus antigens: poly-N-acetylglucosamine (PNAG) and immunodominant staphylococcus aureus antigen (IsaA). Recombinant memory B cells are activated and expanded in vitro in culture medium (e.g., RPMI 1640, Sigma-Aldrich, st.louis, MO) containing: the cognate antigen PNAG, at a concentration of about 100ng/mL, and activates cytokines such as interleukin-2 (Roche, Indianapolis, IN), interleukin 4, interleukin 21, and anti-CD 40 antibody (R)&D Systems, Minneapolis, MN). After about 5 days of culture, memory B cells were collected, washed and concentrated prior to infusion into the patient. Will be about 5X 10⁸The recombinant B cells are injected into a patient, then expanded and persisted, followed by sampling of the patient's peripheral blood. Methods of infusing and tracking genetically engineered lymphocytes are described (see, e.g., Kalos et al, sci. trans. med.3,95ra73,2011; DOI: 10.1126/scitranslim.3002842, incorporated herein by reference). For example, quantitative PCR analysis of genomic DNA obtained from patient whole blood can be used to determine the copy number of anti-PNAG Ig gene and anti-IsaA Ig gene per microgram of genomic DNA. About 100-200ng of genomic DNA was analyzed using ABI Taqman technology (available from Life Technologies Corp., Carlsbad, Calif.). PCR primers specific for the transfected Ig gene were verified by analysis of control genomic DNA spiked with known copy number of anti-Staphylococcus aureus Ig gene. Peripheral blood can also be assessed using flow cytometry and fluorescently labeled PNAG-binding anti-IgG antibodies The number of genetically engineered B cells that persist. For example, Phycoerythrin (PE) conjugated PNAG and Fluorescein Isothiocyanate (FITC) conjugated anti-IgG were used to stain and count recombinant B cells. Protocols, reagents and instruments for flow cytometry are available from Becton Dickinson, Franklin Lakes, NJ. In addition, ELISA can be used to analyze the level of anti-IsaA IgG (λ) antibodies in the peripheral blood of patients. ELISA can be constructed with recombinant purified IsaA protein and anti-IgG or anti- λ L chain antibodies. Methods of constructing and performing ELISAs are known (see, e.g., Kelly-Quintos et al, supra).

Recombinant memory B cells can be activated in vivo as well as in vitro to produce anti-staphylococcus aureus antibodies. Memory B cells can be activated in vivo by PNAG released from staphylococcus aureus infecting patients or by injection of purified PNAG. Methods of purifying PNAG from staphylococcus aureus are known (see, e.g., Lorenz et al, supra). Memory B cells are activated in vivo by allowing PNAG to bind to its B Cell Receptor (BCR) and by interacting with T cells and cytokines (see, e.g., Abbas et al, supra). To enhance the activation of recombinant memory B cells, PNAG may be administered with an immunological adjuvant (e.g., aluminum hydroxide). Repeated activation of memory B cells may be performed in response to recurrent MRSA infection.

Administering recombinant memory B cells to a human patient at risk of MRSA infection as a therapeutic and prophylactic cell therapy that can be activated in vivo. When an anti-MRSA antibody response is required, recombinant memory B cells are activated in vivo by administering PNAG antigen to the patient. For example, when a patient is healthy and recently infected with MRSA, about 10 may be injected⁸-10⁹Individual recombinant B cells serve as prophylactic agents. When needed, recombinant memory B cells can be activated by intradermal injection of 100 μ g PNAG into the patient. For example, memory B cells may be activated after a patient is exposed to MRSA or at the first sign of infection. The production of anti-influenza antibodies can be monitored by sampling the patient's peripheral blood and performing an ELISA with MRSA antigen as the target antigen. In addition, the presence of antibodies for MRSA can be determined by ELISA based on conserved epitopes from MRSA (see, for example,ekiert et al, supra).

Example 7

Construction of cytotoxic B cells with recombinant B cell receptors.

B lymphocytes produce antibodies in response to binding to antigens from infectious disease microorganisms or cancer cells, but they may also produce cytotoxic molecules when co-stimulated with antigens and selected cytokines. For example, stimulation of B cells with cytokines, interleukin-21 (IL-21), and antigens may result in the production of cytotoxic molecules (e.g., granzyme B), which may cause cell death. Cytotoxic B cells useful for adoptive cell therapy are constructed by engineering recombinant B Cell Receptors (BCRs) that recognize antigens associated with disease and signal the expression of cytotoxic effector functions.

Recombinant B cell receptors are constructed using single chain antibodies, membrane immunoglobulin (Ig) domains, and cytoplasmic domains of the IL-21 receptor. In this case, a single chain antibody (single chain variable fragment (SCFv)) is specific for a tumor associated antigen (prostate cancer lipid antigen (PCLA)). The SCFv is linked to a membrane Ig heavy chain domain, which comprises: hinge (H), constant zone 3 (C)_H3) The Transmembrane (TM) and cytoplasmic (Cyto) domains that are involved in signaling B cell activation and are ultimately linked to the IL-21 receptor cytoplasmic domain, signaling the B cell to exert a cytotoxic effect. See fig. 8A. Thus, gene transfer and expression of recombinant B cell receptors in these engineered B cells results in modified B cells responsive to the antigen PCLA. Upon exposure to PCLA on cancer cells, the modified B cells produce cytotoxic effector molecules, such as granzyme B, and kill PCLA-expressing prostate cancer cells.

Immunoglobulin (Ig) genes encoding antibodies that bind PCLA are isolated and engineered to construct recombinant BCR genes for transfer and expression in the patient's own B cells. The glycolipid antigen PCLA associated with prostate cancer was obtained from a prostate cancer cell line and used as an antigen. The glycolipid antigen was used to select single chain antibody variable fragments (SCFv) that bind PCLA. Scfvs comprising Ig variable region genes linked by a linker peptide have been described and can be adapted to this embodiment, and methods of selecting antibodies from phage display single chain variable fragment (SCFv) libraries can be used. The SCFv protein (and corresponding SCFv gene) that binds tightly to PCLA on prostate cancer cells was selected to construct recombinant BCR.

The anti-PCLA SCFv gene was ligated to a fragment encoding the domain of the membrane IgG1 heavy (H) chain to create a recombinant B cell receptor. Hinge fragment 1008, carboxy-terminal heavy chain constant region domain (C) of membrane IgG1 heavy chain_H3)1010, transmembrane domain (TM)1015, and cytoplasmic domain 1020 are encoded at the 3' end of the SCFv1005 gene. See fig. 8A. Detailed methods for constructing membrane IgGH chains may be applied to this embodiment. The IgG1 transmembrane and cytoplasmic domain is a 52 amino acid fragment that interacts with the associated transmembrane B cell signaling proteins Ig alpha and Ig beta that make up the B cell receptor (see, e.g., Abbas et al, Cellular and Molecular Immunology,7th Edition, pp.159-161,2012, Elsevier, Philadelphia, Pa.)]。

The final fragment of the recombinant B cell receptor comprises the cytoplasmic domain of interleukin 21(IL-21) receptor 1025. The IL-21 receptor can signal to trigger the cytotoxic effector function of B cells. For example, co-stimulation of human B cells with anti-Ig antibodies (i.e., stimulation of membrane IgG) and IL-21 cause expression of cytotoxic effectors such as granzyme B and perforin. The cytoplasmic domain of the IL-21 receptor protein has been identified, and the structure and signaling of the IL-21 receptor has been described. A model of the recombinant B cell receptor protein is shown in figure 8A.

DNA fragments encoding the SCFv, IgG constant domain, and IL-21 receptor cytoplasmic domain were amplified from DNA clones using polymerase chain reaction or synthesized (e.g., Custom DNA synthesis is available from Life Technologies corp., Grand Island, NY 14072). Fragments encoding anti-PCLA SCFv can be amplified from the phage clones selected above. Immunoglobulin constant region, transmembrane domain and cytoplasmic domain sequences can be synthesized by automated DNA synthesis based on publicly available sequences, and IL-21 receptor sequences and subdomains are available. Genes encoding recombinant B cell receptors can be assembled from DNA fragments using splicing overlap extension methods.

The recombinant B cell receptor gene is inserted into a mammalian cell expression vector for transfer into a human B cell. Expression vectors having a Cytomegalovirus (CMV) promoter element, a selectable marker gene and a polyA addition signal are described. See fig. 8B. The plasmid vector directs the expression of the recombinant B cell receptor under the control of the CMV promoter and carries a selectable marker gene to enable selection of B cells expressing the vector with a drug (e.g., Neo expression confers resistance to G418; both the resistance gene Neo and the drug G418 are available from InVivoGen, San Diego, Calif.). Plasmid vectors encoding recombinant B Cell receptors are transfected into primary Human B cells (isolated from peripheral blood) (see, e.g., Human B Cell Protocol) using equipment, kits, and protocols available from Lonza inc

Kit, herein incorporated by reference). Transfected G418 resistant B cells expressing recombinant B cell receptors were identified by flow cytometry using antibodies specific for SCFvs present in recombinant B cell receptors. Alternatively, PCLA antigens may be used to identify B cells expressing recombinant B cell receptors, for example by flow cytometry to identify and sort cells.

Following stimulation, B cells expressing recombinant B cell receptors are tested in vitro for cytotoxic effector function. Cytotoxic B cells expressing anti-PCLA recombinant BCR are stimulated with antibodies specific for the SCFv or IgH chain constant region component of the recombinant BCR (e.g., anti-human IgG), and the cell culture supernatant is analyzed for the presence of granzyme B by using an immunoassay. The number of Granzyme B producing cells in culture can be determined using the Granzyme B ELIspot assay kit (available from Cell Sciences, Canton, MA). An Immunospot analyzer and Immunospot 3 software (CTL Cellular Technology ltd., Cleveland, OH) can be used to detect and count granzyme B that produces modified B cells.

To determine the killing effect of recombinant cytotoxic B cells on target cells, flow cytometry based assays were used to determine the percentage of apoptosis following exposure of target cells to recombinant cytotoxic B cells. For example, about 250,000 recombinant cytotoxic B cells are added to 10,000 prostate cancer cells (e.g., PCLA-expressing PC3 cell line). After approximately 3 days of co-culture, apoptosis of the target cells (i.e., PC3) was determined by staining with annexin V and propidium iodide. The percentage of apoptotic cells was determined by flow cytometry. Matched negative control cultures with target cell lines that do not express PCLA (e.g., HeLa) were compared to PC3 cultures. Furthermore, cultures with different ratios of effector cells (recombinant cytotoxic B cells) to target cells (PC3 cells) were analyzed. For example, the analysis has a ratio of 5: 1. 10: 1. 25: 1 and 50: 1, effector of: target apoptosis and viability of a culture of target cells. Target cell viability versus effector: a map of target cell ratios may indicate cytotoxic effector function of recombinant cytotoxic B cells.

Example 8

Construction of cytotoxic B cells with recombinant B cell receptors and coordinated perforin expression.

Modified B cells that are cytotoxic to prostate cancer tumor antigens are engineered with a recombinant B cell receptor and a perforin-inducing gene that promotes cytotoxicity of the target cell. Stimulation of B cells with cytokines, interleukin 21(IL-21) and antigens may lead to the production of cytotoxic molecules (e.g., granzyme B), resulting in cell death. However, B cells may lack expression of the important cytotoxic effector molecule perforin. Thus, to provide for coordinated production of perforin, the expression cassette for perforin was placed in the Ig heavy chain variable region (V)_H) Under the control of promoter/enhancer sequences. Engineered cytotoxic B cells with coordinated expression of granzyme B and perforin are cytotoxic to target cells (i.e., prostate cancer cells).

Recombinant B cell receptors are constructed using single chain antibodies, membrane immunoglobulin (Ig) domains, and cytoplasmic domains of the IL-21 receptor. The single chain antibody (single chain variable fragment (SCFv)) is specific for a tumor associated antigen (prostate cancer lipid antigen (PCLA)). The SCFv is linked to a membrane Ig heavy chain domain, which comprises: hinge (H), constant zone 3 (C) _H3) Transmembrane (TM) and cytoplasmic domains involved in signal transductionActivation of B cells, and finally attachment to the IL-21 receptor cytoplasmic domain, signaling to cause B cells to exert cytotoxic effects. See fig. 8A. Gene transfer and expression of the recombinant B cell receptor expression vector (see figure 8B) results in B cells that express the recombinant receptor, respond to PCLA and produce cytotoxic effector molecules (e.g., granzyme B). A detailed description of the recombinant B cell receptor is given in prophetic example 1 above.

Furthermore, expression vectors and transfection methods are given to obtain B cells expressing recombinant B cell receptors (see prophetic example 1). Recombinant B cells respond to antigen (i.e., PCLA or tumor cells bearing PCLA on their surface) by signaling through recombinant BCR to activate transcription at the IgH and L sites of the antigen. For example, signaling of recombinant BCR via interaction with Ig α and Ig β can lead to B cell activation and differentiation, while signaling through the IL-21 cytoplasmic domain can lead to granzyme B production. In addition, to further promote cytotoxicity of recombinant B cells, human perforin gene was used in IgV_HThe functional IgH chain locus for activity is introduced under the control of promoter and Ig enhancer elements.

Cytotoxic B cells were engineered to switch from active functional Ig heavy chain loci and in IgV_HThe human perforin gene is expressed under the control of the promoter sequence and the Ig enhancer. The gene encoding human perforin is publicly available. Polymerase Chain Reaction (PCR) and oligonucleotide primers were used to amplify approximately 1668 nucleotide complementary dna (cdna) encoding human perforin to add terminal sequences homologous to the 5 'and 3' flanking sequences of the active, rearranged Ig γ H chain gene in recombinant B cell lines. See fig. 3A. For example, with an inclusion compound having an activity V_H5' primers of about 30 nucleotides homologous to the upstream sequences flanking the gene (see, e.g., V in FIG. 3A)_H1D₁J₂) And a 3' primer comprising about 30 nucleotides homologous to a sequence downstream of the active constant region gene to amplify the perforin cDNA (see, e.g., C in FIG. 3A)_Hγ). The amplified perforin gene having an end homologous to the active IgH chain gene on chromosome 14 was integrated by homologous recombination in place of the γ H chain gene (see fig. 8C). Description of the inventionEngineering and site-specific integration of genes at the active Ig heavy chain locus in isolated recombinant cell lines is described (see, e.g., U.S. Pat. No.9,175,072, supra) ]. Perforin expression in transfected B cells can be determined using the Elispot assay, which lists cells that produce perforin vitro following stimulation of the cells. Materials and protocols for Human Perforin Elispot assays are available from Cell Sciences, inc., Canton, MA (see Data sheet: Human Perforin Elispot Kit, available online at www.cellsciences.com, incorporated herein by reference).

To determine the killing effect of recombinant cytotoxic B cells expressing perforin on target cells, a flow cytometer based assay was used to determine the percentage of apoptosis of target cells following exposure to recombinant cytotoxic B cells. For example, about 250,000 recombinant cytotoxic B cells are added to 10,000 prostate cancer cells (e.g., PCLA-expressing PC3 cell line). After approximately 3 days of co-culture, apoptosis of the target cells (i.e., PC3) was determined by staining with annexin V and propidium iodide. The percentage of apoptotic cells was determined by flow cytometry. Matched negative control cultures with target cell lines that do not express PCLA (e.g., HeLa) were compared to PC3 cultures. In addition, cytotoxic B cells not transfected with the perforin gene were compared in the cytotoxicity assay. Cultures with different ratios of effector cells (recombinant cytotoxic B cells) to target cells (PC3 cells) were analyzed. For example, the analysis has a ratio of 5: 1. 10: 1. 25: 1 and 50: 1, effector of: target apoptosis and viability of the culture of the target. Target cell viability versus effector: a map of target cell ratios may indicate cytotoxic effector function of recombinant cytotoxic B cells.

Example 9

The engineered B lymphocytes express recombinant B cell receptors specific for prostate cancer lipid antigens and secreted antibodies specific for prostate specific stem cell antigens.

An isolated recombinant B lymphocyte cell line for treating prostate cancer is constructed that expresses a recombinant B Cell Receptor (BCR) specific for Prostate Cancer Lipid Antigen (PCLA) and secretes antibodies that recognize Prostate Stem Cell Antigen (PSCA). The recombinant B lymphocyte cell line is infused into a subject to provide B cells that are not only cytotoxic to tumor cells but also produce therapeutic antibodies that recognize prostate cancer cells. Recombinant B lymphocytes bind PCLA on prostate cancer cells by engineering recombinant BCR and are activated to produce cytotoxic effectors (e.g., granzyme B) and secrete anti-PSCA antibodies.

Recombinant B cells provide cellular and humoral immunity that targets prostate cancer cells. Recombinant B lymphocyte cell lines can also be stimulated in vivo or ex vivo by injecting exogenous PCLA into a mammalian subject (or in vitro cell culture) to elicit cytotoxic effectors and produce secreted anti-PSCA antibodies. The time determined to stimulate an immune response to prostate cells in a mammalian subject can be selected based on the detection of prostate cancer cells in the mammalian subject.

Polyclonal memory B cells expressing the B Cell Receptor (BCR) for membrane IgG are isolated from prostate cancer patients. Polyclonal memory B cells are isolated from the peripheral blood of a patient by: 1) peripheral blood mononuclear cells were isolated using a Ficoll Hypaque density gradient (available from Sigma aldrich.st.louis.mo.); 2) all B cells were negatively selected using magnetic beads (available from Stem Cell Technology Vancouver, BC), and 3) cells were labeled with fluorescent monoclonal antibodies recognizing membrane IgG and CD27 (memory B Cell marker) and fluorescence activated Cell sorting was performed. Memory B cells expressing membrane IgG are cultured in vitro and genetically engineered to express recombinant B cell receptors and secreted anti-PCSA antibodies.

Memory B cells were genetically engineered to express recombinant BCR specific for PCLA and secreted IgG antibodies specific for Prostate Stem Cell Antigen (PSCA). An isolated recombinant cell line comprising a recombinant BCR is constructed. The recombinant BCR binds PCLA on prostate cancer cells and signals intracellularly to B cells to induce and release cytotoxic effector molecules, e.g., granzyme B, as described in prophetic example 6. Recombinant cell lines can be selected using drug selection (e.g., with G418) and flow cytometry to identify clones expressing recombinant BCR (see, e.g., prophetic example 6 above).

Immunoglobulin genes encoding anti-PSCA antibodies (i.e., Ig heavy and light chain genes) are integrated at the active, rearranged Ig heavy chain locus on chromosome 14 (see fig. 3A), and the active, rearranged kappa light chain locus on chromosome 2, respectively. Methods and materials for obtaining anti-PSCA antibodies are available. For example, to target integration of an anti-PSCA antibody gene into a functional γ l-H chain locus, one would come from J_HTargeting sequence for an intron between an exon and a gamma constant region gene (C)_HGamma; see FIG. 3A) is placed at the 5' end of the anti-PSCA γ 1-H chain gene, while a targeting sequence from downstream (3') of the γ l-H chain cytoplasmic exon is placed at the 3' end of the γ l-H chain gene (see FIG. 3A). Similar targeting sequences (i.e., from the Jk-Ck intron and the 3' end of the Ck gene) were used to target the anti-PSCA κ L chain gene into a functional Ck gene. Targeting vectors against PSCA H and L chains contain selectable marker genes such as hygromycin resistance and histidinol dehydrogenase, respectively. The hygromycin and tissue sterol containing medium was used to select engineered B cells expressing targeting vectors encoding secreted IgG anti-PSCA antibodies.

After transfection and selection of recombinant memory B cells, B cell supernatants were evaluated using standard immunoassays to identify those cells that produced secreted IgG antibodies specific for PSCA. Engineered memory B cells were cultured in vitro and stimulated with PCLA to activate cellular cytotoxicity and stimulate secretion of anti-PSCA IgG antibodies. Laboratory methods for purification of PCLA (lipid antigen) are applicable to this embodiment. Furthermore, isolated recombinant cell lines expressing recombinant B cell receptor and anti-PSCA antibodies were tested in cytotoxicity assays of PCLA and PSCA expressing target cells. For example, PC3, a PCLA-bearing prostate tumor cell line, can be transduced with a PSCA-encoding vector and subjected to cytotoxicity assays in vitro with an engineered cytotoxic B cell line. Methods and materials for transducing PC3 cells are described, and details of cytotoxicity assays are described above. See prophetic example 6.

Example 10

Cytotoxic B cells of prostate cancer were constructed by transfecting B cells with transcription factors.

Cytotoxic B cells are generated by memory B cells engineered to express a B receptor (BCR) specific for Prostate Cancer Lipid Antigen (PCLA). B cells are transfected with a viral vector encoding a transcription factor that controls the expression of cytotoxic effector molecules including granzyme B and perforin. Engineered cytotoxic B cells that recognize and kill prostate cancer cells are useful for adoptive cell therapy in prostate cancer patients.

Memory B cells expressing PCLA-recognizing B cell receptors were constructed using immunoglobulin (Ig) genes encoding anti-PCLA antibodies. The engineered Ig genes are inserted into the actively transcribed heavy (H) and light (L) chain loci of

human chromosomes

14 and 2. Polyclonal memory B cells expressing the B Cell Receptor (BCR) for membrane IgG are isolated from prostate cancer patients. Polyclonal memory B cells are isolated from the peripheral blood of a patient by: 1) peripheral blood mononuclear cells were isolated using a Ficoll Hypaque density gradient (available from Sigma aldrich.st.louis.mo.); 2) all B cells were negatively selected using magnetic beads (available from Stem Cell Technology Vancouver, BC), and 3) cells were labeled with fluorescent monoclonal antibodies recognizing membrane IgG and CD27 (memory B Cell marker) and fluorescence activated Cell sorting was performed. Memory B cells expressing membrane IgG are cultured in vitro and genetically engineered to express B cell receptors specific for PCLA.

Immunoglobulin genes encoding anti-PCLA antibodies (i.e., Ig heavy and light chain genes) are integrated at the active rearranged Ig heavy chain locus on chromosome 14 (see fig. 3A) and the active rearranged kappa light chain locus on chromosome 2, respectively. anti-PCLA antibodies were obtained and the corresponding Ig genes were isolated, engineered and site-specifically integrated at the active Ig heavy and light chain loci in isolated recombinant cell lines. For example, to target integration of anti-PCLA membrane IgH chain genes into functional rearranged γ -H chain loci: targeting sequences located upstream (5') of the variable region exon (VH1D1J 2; see FIG. 3A) and downstream (3') of the gamma-H chain constant region exon (CH gamma; see FIG. 3A) were placed at the 5 'and 3' ends, respectively, of the engineered membrane gamma-H chain gene (see FIG. 3B). Similar targeting sequences (i.e. 5 'from VkJk exon and 3' from Ck gene) were used to target PCLA κ L chain resistant genes to functional Ck genes on chromosome 2.

Engineered B cells expressing membrane IgG specific for PCLA were transduced with viral vectors encoding three transcription factors essential for cytotoxic B cell function. Three transcription factors regulate the expression of cytotoxic effector molecules such as granzyme B. Transcription factors: t-beta, Runx3 and Eomes are critical for granzyme B and perforin expression in the context of cytotoxic T cell differentiation. Sendai virus vectors for transduction of human B cells were used to construct tricistronic vectors encoding T-beta, Runx3 and Eomes. See fig. 8D. Transfection and expression of vectors in engineered B cells can be monitored by immunostaining with antibody-enzyme conjugates specific for T-beta, Runx3, and Eomes (e.g., anti-Eomes, anti-T-beta, and anti-Runx 3 antibodies are available from Abcam, Cambridge, MA), and expression of granzyme B and perforin can be detected by reverse transcription polymerase chain reaction (RT-PCR) of RNA in transfected cells.

Engineered cytotoxic B cells of membrane IgG specific for PCLA were tested for cytotoxic effector function relative to prostate cancer cell lines. For example, PCLA bearing prostate tumor cell line PC3 can be used as a target cell in a flow cytometry analysis that detects apoptotic cells after exposure to engineered cytotoxic B cells (see prophetic example 2 above).

Engineered cytotoxic B cells expressing PCLA-recognizing membrane IgG can also be tested in vivo in xenogenic mouse models of human prostate cancer. For example, PC3 (human prostate tumor cells) was implanted subcutaneously into immunodeficient mice (e.g., NSG mice available from Jackson Labs, Bar Harbor, ME) and the mice were treated with engineered cytotoxic B cells. Mice were evaluated for tumor size, body weight and survival. The control tumor cell line can comprise a tumor that does not express PCLA. In addition, the survival or expansion of cytotoxic B cells in mice following infusion or injection in control mice or mice bearing the PC3 tumor was evaluated and recorded.

Example 11

Modified memory B lymphocytes for the treatment of prostate cancer

Memory B lymphocytes were isolated from prostate cancer patients and engineered to express anti-PCLA (prostate cancer lipid antigen) membrane Ab (antibody) and chemokine receptor CXCR 3. The gene encoding single-chain anti-PCLA Ab was integrated at the active rearranged kappa light (L) chain locus on chromosome 2 and disrupted endogenous Ig kappa L chain expression. The CXCR3 gene was integrated at the endogenously active rearranged immunoglobulin (Ig) heavy (H) chain locus and disrupts endogenous IgH chain expression. Engineered B cells that home to prostate cancer tumors through CXCR3 mediated chemotaxis (see, e.g., Sackstein et al, Laboratory Investigation 97:669-697,2017, incorporated herein by reference) are useful for adoptive cell therapy of prostate cancer. Tumor-localized engineered B cells provide therapeutic anti-PCLA antibodies to tumor cells at higher local concentrations.

Memory B cells are engineered to express single chain membrane antibodies to PCLA (prostate cancer lipid antigen) under the control of endogenous Ig kappa promoter and/or enhancer sequences by insertion into the endogenously rearranged active kappa light chain gene locus using CRISPR technology (Clustered Regularly spaced Short Palindromic Repeats). Immunoglobulin (Ig) genes encoding antibodies that bind to PCLA are isolated and engineered to construct recombinant Ig genes for transfer and expression in the B cells of the patient himself. The glycolipid antigen PCLA associated with prostate cancer was obtained from a prostate cancer cell line and used as an antigen. The glycolipid antigen was used to select single chain antibody variable fragments (SCFv) that bind PCLA. Scfvs comprising Ig variable region genes linked by a linker peptide have been described and may be suitable for use in this embodiment.

Methods for selecting antibodies from phage display single chain variable fragment (SCFv) libraries can be utilized. The SCFv protein (and corresponding SCFv gene) that binds tightly to PCLA on prostate cancer cells was selected to construct recombinant single chain abs. The anti-PCLA SCFv is linked to a gamma 1-H chain constant region gene encoding the transmembrane and cytoplasmic domains of gamma 1-H chain. The anti-PCLA single chain Ab gene was incorporated into an AAV vector (adeno-associated viral vector) and flanked by homology arms for integration at the κ L chain locus on chromosome 2 (see, e.g., Eyquem et al, Nature 543, 113-. Homology arms of the endogenous Ig kappa chain gene targeted for activity flank the anti-PCLAAb gene to integrate directly into the kappa L chain locus and disrupt endogenous kappa L chain expression. For more details, please refer to fig. 9A.

Synthetic RNA encoding a guide RNA targeting the integration site and a messenger RNA encoding Cas9 endonuclease was introduced into memory B cells by electroporation. After about 2 hours, cells were transduced with the AAV-PCLA Ab vector. (synthetic guide RNA and Cas9 mRNA are available from Trilink Biotechnologies, San Diego, Calif., whereas AAV vectors are available from Cell Biolabs, Inc., San Diego, Calif.).

Memory B cells are engineered to also express the CXCR3 gene at the active Ig H chain locus on chromosome 14. anti-PCLA Ab and CXCR3 genes can be integrated simultaneously using CRISPR techniques to accelerate and optimize the engineering of memory B cells (see, e.g., Le Cong et al, Science 339: 819. sup. 823,2013, which is incorporated herein by reference). Genes encoding CXCR3 are available from GenScript, Piscataway, NJ. Specific integration of the CXCR3 gene at the active H chain gene locus was accomplished using CRISPR technology (see, e.g., Eyquem et al, Nature 543: 113-. Adeno-associated virus (AAV) vectors were designed to encode the human CXCR3 gene, flanked by homologous arms that target the IgH chain CH1 exon on chromosome 14, to disrupt the active IgH chain gene and insert a functional CXCR3 gene. For more details, please refer to fig. 9B.

Synthetic RNA encoding a guide RNA targeting the integration site and messenger RNA encoding Cas9 endonuclease were introduced by electroporation into memory B cells isolated from prostate cancer patients. After about 2 hours, cells were transduced with the AAV-CXCR3 vector. (synthetic guide RNA and Cas9 mRNA are available from Trilink Biotechnologies, San Diego, Calif., whereas AAV vectors are available from Cell Biolabs, Inc., San Diego, Calif.). Genetically engineered B cells are grown in vitro using media containing cytokines (e.g., IL-21) and co-stimulators (e.g., oligodeoxynucleotides, CpG, and anti-CD 40 Ab) (see, e.g., Kwakkenbos et al, Nature Medicine 16:123-128,2010, incorporated herein by reference).

B cells expressing CXCR3 from a disrupted IgH chain locus were identified and isolated using flow cytometry. B cells stained with fluorescent anti-CXCR 3 Ab but not with fluorescent anti-IgM antibody were selected by cell sorting and cultured in vitro. Fluorescent anti-CXCR 3 and anti-human IgM antibodies are available from ABCAM, Cambridge, MA. B cells positive for CXCR3 Ab staining were tested in vitro for CXCR3 mRNA expression using RT-PCR; CXCR3 negative and/or IgM positive B cells were tested as negative controls. Each B cell sample is tested for positive and negative stimulation with B cell activators (e.g., IL-21, anti-CD 40, and CpG).

To assess the biological function of the CXCR3 gene in engineered B cells, an in vitro chemotaxis assay was performed using CXCR10 (a ligand for CXCR 3). Engineered B cells are introduced onto a permeable membrane in a well plate. The compartment under the membrane was filled with medium containing CXCL10 and after several hours of incubation, the migration of engineered B cells into the lower compartment was scored by staining the B cells in situ. Control plates omit CXCL10, or use untransfected B cells or IgM positive B cells. Methods and materials for performing chemotaxis assays are described (see, e.g., Conley-LaComb et al, Ibid, and Xia et al, Oncotarget 7: 60461-.

Recombinant memory B cells are activated and expanded in vitro in culture medium (e.g., RPMI 1640, Sigma-Aldrich, st.louis, MO) containing: the cognate antigen, PCLA, at a concentration of about 100ng/mL, and activating cytokines such as interleukin-2 (Roche, Indianapolis, IN), interleukin 4, interleukin 21, and anti-CD 40 antibodies (R & D Systems, Minneapolis, MN).

After about 5 days of culture, memory B cells were collected, washed and concentrated prior to infusion into the patient. Will be about 5X 10⁸Recombinant B cells are injected, then expanded and persisted, followed by sampling of the patient's peripheral blood. Infusion and chase are described Methods of tracking genetically engineered lymphocytes (see, e.g., Kalos et al, Sci. Transl. Med.3,95ra73,2011; DOI: 10.1126/scitranslim.3002842, incorporated herein by reference).

Example 12

Construction of engineered memory B lymphocytes for modulating autoimmunity

Memory B lymphocytes obtained from Multiple Sclerosis (MS) patients are engineered to express a membrane antibody (Ab) that recognizes Myelin Oligodendrocyte Glycoprotein (MOG) and produce the anti-inflammatory cytokine interleukin 10 (IL-10). Engineered B cells bind to MOG and respond by producing IL-10. The gene for anti-MOG membrane Ab is expressed under the control of a constitutive promoter, while the gene encoding IL-10 is integrated at the endogenous Ig heavy (H) chain locus of memory B cells and expressed under the control of an endogenously rearranged immunoglobulin variable heavy chain (VH) promoter and/or enhancer element.

Using CRISPR technology, a gene encoding IL-10 is inserted into the endogenous active IgH chain locus on chromosome 14. For example, complementary dna (cdna) encoding human IL-10 (available from Harvard Medical School, Boston, MA) is incorporated into an adeno-associated virus (AAV) vector (e.g., AAV vectors are available from Cell Biolabs, inc., San Diego, CA). The human IL-10cDNA is flanked by a Splice Acceptor (SA) sequence, a polyA addition site (pA) and a Homology Arm (HA) to target recombination at the IgH chain locus on chromosome 14. For more details, please refer to fig. 12A.

Targeting of guide RNA to the CH μ 1 exon of the μ H chain gene results in a double stranded DNA break, disrupting μ H chain expression and facilitating insertion of the IL-10 gene. For more details, please refer to fig. 12A.

Methods of constructing guide RNAs for targeted insertion are described (see, e.g., Eyquem et al, ibid, and Zheng et al, BioTechniques 57:115-124,2014, which is incorporated herein by reference), and synthetic guide RNAs and Cas9 mrnas are available from Trilink Biotechnologies, San Diego, CA. Memory B cells expressing membrane IgM are obtained and purified from peripheral blood of MS patients. Memory B cells were obtained by cell sorting using anti-IgM and anti-CD 27 antibodies. See, for example, Tangye et al, j.immunology 179,13-19,2007, which is incorporated herein by reference. Methods and materials for electroporation and viral transduction of lymphocytes are described (see, e.g., Eyquem et al, supra). Memory B cells lacking membrane IgM on the surface were tested for their ability to produce IL-10. Membrane IgM negative, IL-10 positive B cells were expanded and activated prior to lentivirus transduction.

Lentiviral vectors were constructed to direct the expression of anti-MOG membrane Ab on memory B cells. For example, a single chain membrane Ab gene can be constructed using a gene encoding a human single chain variable fragment (SCFv) specific for MOG (obtained from an SCFv phage library). The SCFv gene is linked to a human Ig γ 1 constant region gene comprising: hinge region, C _H1-C_H3. A transmembrane domain (TM) and a cytoplasmic tail (Cyto) (see, e.g., Xu et al, Cell Research 24,651-654,2014, which is incorporated herein by reference). See fig. 12B.

The anti-MOG membrane Ig gene is preceded by a constitutive promoter (e.g., human cytomegalovirus promoter (CMV) and cloned in a lentiviral vector suitable for B lymphocyte transduction (see, e.g., Bovia et al, Blood 101,1727-1733,2003, which is incorporated herein by reference.) methods and reagents for activating and transducing human B lymphocytes are described (Bovia et al, supra), B lymphocytes expressing membrane IgG are detected and purified using anti-human IgG antibodies and flow cytometry (available from ABCAM inc, Cambridge, MA).

Engineered B cells expressing membrane IgG specific for MOG were tested in vitro to assess their functionality. For example, engineered B cells are grown with media containing MOG glycoprotein to activate their membrane IgG receptors, and media samples are collected and analyzed for IL-10. Control cultures containing human serum albumin or no other proteins were also tested. Positive control cultures containing anti-human IgG were also tested for IL-10 concentration. Standard enzyme-linked immunosorbent assays for IL-10 and recombinant human MOG protein are available from R & D Systems, Inc., Minneapolis, MN. The initial in vitro culture used to verify the function of the engineered B cells established a dose response curve to MOG protein and kinetics of IL-10 production. Such dosages may be suitable for animal studies and clinical trials in human subjects.

Example 13

Construction of cytotoxic B cells for the treatment of HIV

Genes encoding recombinant B Cell Receptor (BCR) and interleukin 21(IL-21) are inserted into the immunoglobulin (Ig) heavy (H) chain locus in memory B cells to disrupt endogenous active gamma (γ) H chain genes and engage Ig variable region (VH) promoter and H chain enhancer elements. Genome targeting using CRISPR/Cas9 technology comprising specific guide RNAs restricts DNADS fragmentation and gene insertion into rearranged γ H chain genes expressed per IgG positive memory B cell.

A bicistronic gene comprising a recombinant BCR gene and an IL-21 gene was inserted into a rearranged active H-chain gene on chromosome 14 in IgG-positive memory B cells. Polyclonal memory B cells expressing membrane IgG are isolated from the peripheral blood of patients using magnetic bead isolation and flow cytometry to isolate IgG-positive, CD 27-positive B cells (see, e.g., Ettinger et al, supra and Brown et al, supra). Targeted disruption and integration on multiple different active Ig γ H chain genes is achieved using a CRISPR/Cas9 system with two guide RNAs and a template gene for homologous recombination. Methods and materials for deleting DNA fragments and replacing with a donor gene of interest are described (see, e.g., Zheng et al, biotechniques57,115-124,2014, incorporated herein by reference). For example, guide rnas (grnas) targeting two conserved sites in each rearranged γ H chain gene were designed to target: (1) a site in the VDJ-C γ intron that is 3 'downstream of the Ig μ enhancer but 5' upstream of the crossover-like recombination site (CSRS); (2) a site near the first exon (CH γ 1) of the γ constant region gene. See fig. 13 for more details.

The design of guide RNAs and selection of target sites to give optimal specificity and efficient deletion of genomic fragments is described (see e.g. Zheng et al, supra).

AAV vectors comprising a bicistronic DNA construct encoding recombinant BCR and IL-21 were constructed using a homology arm that flanks the deletion of the γ 1-H chain gene. See fig. 13 for more details.

The P2a peptide is encoded by the 5' end of the BCR and IL-21 genes. Bicistronic constructs with P2a self-cleaving peptide are described (see, e.g., Kim et al, PLoS ONE 6(4): e18556,2011; doi:10.1371/journal. bone.0018556, incorporated herein by reference). Recombinant BCR can be constructed from broadly neutralizing abs to HIV (see, e.g., Balazs et al, Nature 481,81-84,2012, incorporated herein by reference) and expressed as single-chain variable fragments (SCFV).

Engineered memory B cells are incubated with HIV envelope proteins and media samples are collected at 1, 2, 4, and 8 hours and the amount of IL-21 is determined using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA). The IL-21 ELISA kit is available from RayBiotech, Inc., Norcross, GA. Negative control cultures without HIV env protein or with a control protein (e.g. human serum albumin) were compared to HIVenv-stimulated cultures. Positive control cultures were performed using anti-IgG antibodies to stimulate engineered B cells.

Engineered memory B cells are tested in vitro for cytotoxic effector function using engineered mammalian cells expressing HIV env protein. Mammalian cell lines, such as HEK293, are transfected with an expression vector encoding HIV-1gp41 (gp41 cDNA is available from Bioclone inc., San Diego, CA) and cell lines expressing gp41 on the cell surface (gp41/HEK) are selected using anti-gp 41 monoclonal antibody Mab. Following co-culture of the engineered memory B cells with gp41/HEK target cells, cell viability is monitored, e.g., trypan blue staining, apoptosis is assessed and lactate dehydrogenase release is measured. In addition, ELISA can be used to measure granzyme B release into the culture medium. Cytotoxicity as measured against gp41/HEK cells was obtained at various ratios of cytotoxic B cell effector to target cells (e.g., 1: 1, 2: 1, 4: 1, 10: 1, 20: 1, 40: 1) relative to control HEK293 cells.

Each of the ranges includes all combinations and subcombinations of ranges, as well as specific numerals contained therein.

All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes and were not intended to be inconsistent with this specification.

Those skilled in the art will recognize that the state of the art has developed to the extent that: there is little distinction left between hardware and software implementations of aspects of systems; the use of hardware or software is typically (but not always, in that in some contexts the choice between hardware and software can become significant) a design choice representing a cost vs. benefit tradeoff. One of ordinary skill in the art will recognize that there are numerous vehicles (e.g., hardware, software, and/or firmware) that can embody the methods and/or systems and/or other techniques disclosed herein, and that the preferred vehicle will vary depending on the context in which the methods and/or systems and/or other techniques are deployed. For example, if the practitioner determines that speed and accuracy are paramount, the surgeon may select a carrier that is primarily hardware and/or firmware; alternatively, if flexibility is paramount, the surgeon may choose a mainly software implementation; alternatively, or in addition, an implementer may opt for some combination of hardware, software, and/or firmware. Thus, there are several possible vehicles by which the methods and/or apparatus and/or other techniques disclosed herein can be effected, none of which is inherently superior to the other in that any vehicle to be used is a choice dependent upon the context in which it is deployed and the particular concerns (e.g., speed, flexibility, or predictability) of the implementer, any of which may vary. Those of ordinary skill in the art will appreciate that the optical aspects of an implementation will typically employ optical aspects of hardware, software, and or firmware.

In a general sense, various aspects disclosed herein that may be implemented, individually and/or collectively, by a wide range of hardware, software, firmware, or any combination thereof, may be considered to comprise various types of "circuitry". Thus, "circuitry" as used herein includes, but is not limited to: a circuit having at least one discrete circuit, a circuit having at least one integrated circuit, a circuit having at least one application specific integrated circuit, a circuit forming a general purpose computing device configured by a computer program (e.g., a general purpose computer configured by a computer program that at least partially implements the methods and/or apparatus disclosed herein, or a micro-digital processing unit configured by a computer program that at least partially implements the methods and/or apparatus disclosed herein), a circuit forming a storage device (e.g., random access memory), and/or a circuit forming a communication device (e.g., a modem, a communication switch, or an optoelectronic device). The subject matter disclosed herein may be implemented in analog or digital fashion, or some combination thereof.

Those skilled in the art will recognize that at least a portion of the devices and/or methods described herein may be integrated into a data processing system. Those skilled in the art will recognize that data processing systems typically include one or more of the following: a system unit housing, a video display device, a memory such as a volatile or non-volatile memory, a processor such as a microprocessor or digital signal processor, a computing entity such as an operating system, a driver, a graphical user interface, an application program, one or more interaction devices (e.g., a touch pad, a touch screen, an antenna, etc.), and/or a control system including a feedback loop and a control motor (e.g., a feedback for sensing position and/or velocity, a control motor for moving and/or adjusting a component and/or quantity). The data processing system may be implemented using suitable commercially available components such as those commonly found in data computing/communication and/or network computing/communication systems.

The foregoing detailed description has set forth various embodiments of the devices and/or methods via the use of block diagrams, flowcharts, and/or examples. Insofar as such block diagrams, flowcharts, and/or embodiments contain one or more functions and/or operations, it will be understood by those within the art that each function and/or operation within such block diagrams, flowcharts, or embodiments can be implemented, individually and/or collectively, by a wide range of hardware, software, firmware, or virtually any combination thereof. In an embodiment, portions of the subject matter described herein may be implemented by Application Specific Integrated Circuits (ASICs), Field Programmable Gate Arrays (FPGAs), Digital Signal Processors (DSPs), or other integrated forms. Some aspects of the embodiments disclosed herein may be implemented efficiently, in whole or in part, in integrated circuits, as one or more computer programs running on one or more computers (e.g., as one or more programs running on one or more computer systems), as one or more programs running on one or more processors (e.g., as one or more programs running on one or more microprocessors), as firmware, or as virtually any combination thereof, and designing the circuitry and/or writing the code for the software and or firmware would be well within the skill of one of skill in the art in light of this disclosure. Moreover, those skilled in the art will appreciate that the mechanisms of the subject matter described herein are capable of being distributed as a program product in a variety of forms, and that an illustrative embodiment of the subject matter described herein applies regardless of the particular type of signal bearing media used to actually carry out the distribution. Examples of signal bearing media include, but are not limited to, the following: recordable type media such as floppy disks, hard disk drives, Compact Disks (CDs), Digital Video Disks (DVDs), digital tapes, computer memories, etc.; and a transmission type medium such as a digital and/or an analog communication medium (e.g., a fiber optic cable, a waveguide, a wired communications link, a wireless communication link (e.g., transmitter, receiver, transmit logic, receive logic, etc.).

For the sake of conceptual clarity, the components (e.g., steps), devices, and objects described herein and the descriptions accompanying them are used as embodiments, and various configuration modifications using the disclosure provided herein are within the ability of those skilled in the art. Accordingly, as used herein, the particular embodiments set forth and the accompanying description are intended to be representative of their more general categories. In general, the use of any particular embodiment herein is also intended to be representative of its class, and the absence of such specific components (e.g., steps), devices, and objects herein is not to be taken as an indication of the desired limitations.

For the use of substantially any plural and/or singular terms herein, the reader can convert the plural to the singular and vice versa depending on the context and/or application. For purposes of clarity, various singular/plural permutations are not expressly set forth herein.

The subject matter described herein sometimes sets forth different components contained within or connected with different other components. It is to be understood that such depicted architectures are merely examples, and that in fact many other architectures can be implemented which achieve the same functionality. In a conceptual sense, any arrangement of components to achieve the same functionality is effectively "associated" such that the desired functionality is achieved. Hence, any two components herein combined to achieve a particular functionality can be seen as "associated with" each other such that the desired functionality is achieved, irrespective of architectures or intermedial components. Likewise, any two components so associated can also be viewed as being "operably connected," or "operably coupled," to each other to achieve the desired functionality, and any two components capable of being so associated can also be viewed as being "operably couplable," to each other to achieve the desired functionality. Specific examples of operably couplable include but are not limited to physically mateable or physically interacting components, or wirelessly interactable or wirelessly interacting components, or logically interacting or logically interactable components.

While particular aspects of the subject matter described herein have been shown and described, changes and modifications may be made without departing from the subject matter described herein and its broader aspects, and therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of the subject matter described herein. Furthermore, it is to be understood that the invention is defined by the appended claims. It will be understood that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It should be further understood that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an"; the same holds true for the use of definite articles used to introduce claim recitations. Furthermore, even if a specific number of an introduced claim recitation is explicitly recited, such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations). Further, in those instances where a convention analogous to "A, B, at least one of C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand (e.g., "a system having at least one of A, B and C" would include, but not be limited to, an A only system, a B only system, a C only system, both A and B systems, both A and C systems, both B and C systems, or both A, B and C systems, etc.). In those instances where a convention analogous to "A, B or at least one of C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand (e.g., "a system having at least one of A, B or C" would include but not be limited to an A only system, a B only system, a C only system, both A and B systems, both A and C systems, both B and C systems, or both A, B and C systems, etc.). Nearly any optional words and/or phrases that suggest two or more alternative items, whether in the specification, claims, or drawings, should be understood to contemplate the possibilities of including one, either, or both. For example, the phrase "a or B" should be understood to include the possibility of "a" or "B" or "a and B".

Aspects of the subject matter described herein are set forth in the following numbered clauses:

1. an isolated modified B cell capable of expressing:

at least one exogenously incorporated membrane immunoglobulin or recombinant B cell receptor capable of binding a first antigen;

at least one exogenously incorporated redistributed biological agent; and

at least one endogenously secreted immunoglobulin reactive with a second antigen.

2. The isolated modified B cell of clause 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises one or more exogenously incorporated membrane immunoglobulin polypeptides.

3. The isolated modified B cell of clause 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises at least one exogenously incorporated nucleic acid encoding the at least one exogenous membrane immunoglobulin.

4. The isolated modified B cell of clause 3, wherein the exogenously incorporated nucleic acid encoding the at least one membrane immunoglobulin is integrated in one or more chromosomal loci in the isolated B lymphocyte cell line.

5. The isolated modified B cell of clause 4, wherein the chromosomal locus comprises at least one of an Ig H chain or an Ig L chain chromosomal locus.

6. The isolated modified B cell of clause 5, wherein the Ig H chain or IgL chain chromosomal locus is on the unexpressed Ig allele.

7. The isolated modified B cell of clause 6, wherein at least one of the Ig H chain or IgL chain chromosomal loci is under expression control of an endogenous promoter or enhancer element.

8. The isolated modified B cell of clause 6, wherein the Ig L chain locus comprises at least one of a kappa or lambda light (L) chain locus.

9. The isolated modified B cell of clause 8, wherein integration at the kappa or lambda light (L) chain locus disrupts endogenous B cell kappa or lambda light (L) chain expression.

10. The isolated modified B cell of clause 4, wherein the chromosomal locus comprises one or more non-Ig L or non-Ig H chromosomal loci in the isolated B lymphocyte cell line.

11. The isolated modified B cell of clause 4, wherein the at least one exogenously incorporated nucleic acid encoding the at least one exogenous membrane immunoglobulin is incorporated into an extrachromosomally replicating genetic element in the isolated modified B cell.

12. The isolated modified B cell of clause 4, wherein integration of the at least one exogenous nucleic acid encoding the exogenous membrane immunoglobulin comprises disrupting expression of an immunoglobulin endogenous to the B cell.

13. The isolated modified B cell of clause 4, wherein the at least one exogenously incorporated nucleic acid encoding the at least one exogenous membrane immunoglobulin is derived from a B cell.

14. The isolated modified B cell of clause 4, wherein the at least one exogenously incorporated membrane immunoglobulin activated by a first antigen is capable of controlling the production of at least one endogenously secreted immunoglobulin reactive to the second antigen.

15. The isolated modified B cell of clause 4, wherein the at least one exogenously incorporated membrane immunoglobulin nucleic acid comprises an exogenous bicistronic expression construct.

16. The isolated modified B cell of clause 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises at least two exogenously incorporated nucleic acids encoding the at least one exogenous membrane immunoglobulin.

17. The isolated modified B cell of clause 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises nucleic acid encoding one heavy chain (H) immunoglobulin and one light chain (L) immunoglobulin.

18. The isolated modified B cell of clause 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises nucleic acid encoding a single chain Fv immunoglobulin.

19. The isolated modified B cell of clause 1, wherein the isolated modified B cell comprises at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a B1B lymphocyte, a marginal zone B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell.

20. The isolated modified B cell of clause 1, wherein the isolated modified B cell is part of a polyclonal population of B lymphocytes.

21. The isolated modified B cell of clause 1, wherein the isolated modified B cell is part of a monoclonal population of B lymphocytes.

22. The isolated modified B cell of clause 1, wherein the isolated modified B cell is part of a modified B lymphocyte cell line.

23. The isolated modified B cell of clause 1, wherein the membrane immunoglobulin comprises at least one of a membrane anchor, a cytoplasmic domain, a hinge region, or an extracellular ligand binding domain.

24. The isolated modified B cell of clause 1, wherein the isolated modified B cell bears at least one chimeric B cell receptor capable of transducing a signal to induce expression of a cytotoxic effector molecule.

25. The isolated modified B cell of clause 24, wherein the chimeric B cell receptor is derived from at least one of membrane IgG, CD19, BCMA, CD38, common gamma chain receptor, IL-21 receptor, Toll-like receptor, or CD 40.

26. The isolated modified B cell of clause 18, wherein the cytotoxic effector molecule comprises at least one of perforin, granzyme B, Fas ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

27. The isolated modified B cell of clause 24, wherein the cytotoxic effector molecule is expressed in response to a particular target cell or target antigen.

28. An isolated modified B cell capable of expressing:

at least one exogenously incorporated redistributed biological agent; and

at least one exogenously incorporated nucleic acid encoding a secreted immunoglobulin capable of binding a second antigen.

29. The isolated modified B cell of clause 28, wherein the at least one exogenously incorporated membrane immunoglobulin comprises at least one exogenously incorporated membrane immunoglobulin polypeptide.

30. The isolated modified B cell of clause 28, wherein the at least one exogenously incorporated membrane immunoglobulin is encoded by at least one exogenously incorporated nucleic acid.

31. The isolated modified B cell of clause 28, wherein the exogenously incorporated nucleic acid encoding the at least one membrane immunoglobulin is integrated in one or more chromosomal loci in the isolated modified B cell.

32. The isolated modified B cell of clause 31, wherein the chromosomal locus comprises at least one of an Ig H chain or an IgL chain chromosomal locus.

33. The isolated modified B cell of clause 32, wherein the Ig H chain or IgL chain chromosomal locus is an unexpressed Ig allele.

34. The isolated modified B cell of clause 32, wherein the Ig H chain or Ig L chain chromosomal locus is an expressed Ig allele.

35. The isolated modified B cell of clause 34, wherein at least one of the Ig H chain or IgL chain chromosomal loci is under expression control of an endogenous promoter or enhancer element.

36. The isolated modified B cell of clause 34, wherein the Ig L chain locus comprises at least one of a kappa or lambda light (L) chain locus.

37. The isolated modified B cell of clause 36, wherein integration at the kappa or lambda light (L) chain locus disrupts endogenous B cell kappa or lambda light (L) chain expression.

38. The isolated modified B cell of clause 32, wherein the chromosomal locus comprises one or more non-IgL or non-IgH chromosomal loci in the isolated modified B cell.

39. The isolated modified B cell of clause 32, wherein the at least one exogenously incorporated nucleic acid encoding the at least one exogenous membrane immunoglobulin is incorporated into an extrachromosomally replicating genetic element in the isolated modified B cell.

40. The isolated modified B cell of clause 32, wherein integration of the at least one exogenous nucleic acid encoding the exogenous membrane immunoglobulin comprises disrupting expression of an immunoglobulin endogenous to the B cell.

41. The isolated modified B cell of clause 32, wherein the at least one exogenously incorporated nucleic acid encoding the at least one exogenous membrane immunoglobulin is derived from a B cell line.

42. The isolated modified B cell of clause 32, wherein the at least one exogenously incorporated membrane immunoglobulin activated by a first antigen is capable of controlling the production of at least one exogenously secreted immunoglobulin reactive with the second antigen.

43. The isolated modified B cell of clause 32, wherein the at least one exogenously incorporated membrane immunoglobulin nucleic acid comprises an exogenous bicistronic expression construct.

44. The isolated modified B cell of clause 28, wherein the at least one exogenously incorporated membrane immunoglobulin comprises at least two exogenously incorporated nucleic acids encoding the at least one exogenous membrane immunoglobulin.

45. The isolated modified B cell of clause 44, wherein the at least one exogenously incorporated membrane immunoglobulin comprises nucleic acids encoding two heavy chain (H) immunoglobulins and two light chain (L) immunoglobulins.

46. The isolated modified B cell of clause 28, wherein the at least one exogenously incorporated membrane immunoglobulin comprises nucleic acid encoding one heavy chain (H) immunoglobulin and one light chain (L) immunoglobulin.

47. The isolated modified B cell of clause 28, wherein the at least one exogenously incorporated membrane immunoglobulin comprises nucleic acid encoding a single chain Fv immunoglobulin.

48. The isolated modified B cell of clause 28, wherein the isolated modified B cell comprises at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a B1B lymphocyte, a marginal zone B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell.

49. The isolated modified B cell of clause 48, wherein the isolated modified B cell comprises a polyclonal population of B lymphocytes.

50. The isolated modified B cell of clause 48, wherein the isolated modified B cell comprises a monoclonal population of B lymphocytes.

51. The isolated modified B cell of clause 48, wherein the isolated modified B cell is part of a modified B lymphocyte cell line.

52. The isolated modified B cell of clause 28, wherein the membrane immunoglobulin comprises at least one of a membrane anchor, a cytoplasmic domain, a hinge region, or an extracellular ligand binding domain.

53. The isolated modified B cell of clause 28, wherein the isolated modified B cell bears at least one chimeric B cell receptor capable of transducing a signal to induce expression of a cytotoxic effector molecule.

54. The isolated modified B cell of clause 53, wherein the chimeric B cell receptor is derived from at least one of membrane IgG, CD19, BCMA, CD38, common gamma chain receptor, IL-21 receptor, Toll-like receptor, or CD 40.

55. The isolated modified B cell of clause 54, wherein the cytotoxic effector molecule comprises at least one of perforin, granzyme B, Fas ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

56. The isolated modified B cell of clause 54, wherein the cytotoxic effector molecule is expressed in response to a particular target cell or target antigen.

57. The isolated modified B cell of clause 28, wherein the at least one exogenously incorporated secreted immunoglobulin comprises at least one exogenously incorporated secreted immunoglobulin polypeptide.

58. The isolated modified B cell of clause 57, wherein the at least one exogenously incorporated secreted immunoglobulin is encoded by at least one exogenously incorporated nucleic acid.

59. The isolated modified B cell of clause 58, wherein the exogenously incorporated nucleic acid encoding the at least one secreted immunoglobulin is integrated in one or more chromosomal loci in the isolated modified B cell.

60. The isolated modified B cell of clause 59, wherein the chromosomal locus comprises at least one of the Ig H chain or Ig L chain chromosomal loci.

61. The isolated modified B cell according to clause 60, wherein the Ig H chain or IgL chain chromosomal locus is an unexpressed Ig allele.

62. The isolated modified B cell of clause 59, wherein the Ig H chain or Ig L chain chromosomal locus is the expressed Ig allele.

63. The isolated modified B cell of clause 62, wherein at least one of the Ig H chain or Ig L chain chromosomal loci is under expression control of an endogenous promoter or enhancer element.

64. The isolated modified B cell of clause 63, wherein the endogenous promoter or enhancer element comprises a promoter and enhancer element that is proximal due to rearrangement of a VH-CH or VL-CL region.

65. The isolated modified B cell of clause 63, wherein the Ig L chain locus comprises at least one of a kappa or lambda light (L) chain locus.

66. The isolated modified B cell of clause 65, wherein integration at the kappa or lambda light (L) chain locus disrupts endogenous B cell kappa or lambda light (L) chain expression.

67. The isolated modified B cell of clause 59, wherein the chromosomal locus comprises one or more non-IgL or non-IgH chromosomal loci in the isolated modified B cell.

68. The isolated modified B cell of clause 67, wherein the at least one exogenously incorporated nucleic acid encoding the at least one exogenously secreted immunoglobulin is incorporated into an extrachromosomally replicating genetic element in the isolated modified B cell.

69. The isolated modified B cell of clause 67, wherein integration of at least one exogenous nucleic acid encoding the exogenously secreted immunoglobulin comprises disrupting expression of an immunoglobulin H-chain and/or L-chain endogenous to the B cell.

70. The isolated modified B cell of clause 59, wherein the at least one exogenously incorporated nucleic acid encoding the at least one exogenously secreted immunoglobulin is derived from a B lymphocyte.

71. The isolated modified B cell of clause 59, wherein the at least one exogenously incorporated secreted immunoglobulin nucleic acid comprises an exogenous bicistronic expression construct.

72. The isolated modified B cell of clause 59, wherein the at least one exogenously incorporated secreted immunoglobulin nucleic acid comprises at least two exogenously incorporated nucleic acids encoding the at least one exogenously secreted immunoglobulin.

73. The isolated modified B cell of clause 72, wherein the at least one exogenously incorporated secreted immunoglobulin comprises nucleic acids encoding two heavy chain (H) immunoglobulins and two light chain (L) immunoglobulins.

74. The isolated modified B cell of clause 72, wherein the at least one exogenously incorporated secreted immunoglobulin comprises nucleic acid encoding one heavy chain (H) immunoglobulin and one light chain (L) immunoglobulin.

72. The isolated modified B cell of clause 72, wherein the at least one exogenously incorporated secreted immunoglobulin comprises a nucleic acid encoding a single chain Fv immunoglobulin.

76. An isolated modified B cell capable of expressing at least one exogenously incorporated membrane immunoglobulin or recombinant B cell receptor reactive with a first antigen;

at least one endogenously secreted immunoglobulin reactive with a second antigen;

at least one exogenously incorporated redistribution biologic;

and at least one exogenously incorporated membrane receptor configured to directly or indirectly induce expression of at least one cytotoxic effector molecule.

77. The isolated modified B cell of clause 76, wherein the membrane receptor configured to directly or indirectly induce at least one cytotoxic effector molecule comprises at least one chimeric receptor exhibiting at least a portion of at least one cytokine receptor.

78. An isolated modified B cell capable of expressing at least one exogenously incorporated membrane immunoglobulin or recombinant B cell receptor reactive with a first antigen;

at least one exogenously secreted immunoglobulin reactive with a second antigen;

at least one exogenously incorporated redistribution biologic;

79. The isolated modified B cell of clause 78, wherein the membrane receptor configured to directly or indirectly induce at least one cytotoxic effector molecule comprises at least one chimeric receptor exhibiting at least a portion of at least one cytokine receptor.

80. An isolated modified B cell capable of expressing:

at least one exogenously incorporated redistributed biological agent, wherein the redistributed biological agent comprises at least one cytokine, chemokine, cytotoxin, receptor, or ligand.

81. The isolated modified B cell of clause 80, wherein the isolated modified B cell is capable of expressing at least one exogenously incorporated membrane receptor configured to directly or indirectly induce expression of at least one cytotoxic effector molecule.

82. The isolated modified B cell of clause 80, wherein the isolated modified B cell is capable of expressing at least one exogenously incorporated membrane immunoglobulin or recombinant B cell receptor reactive to a first antigen.

83. The isolated modified B cell of clause 80, wherein the isolated modified B cell is capable of expressing at least one exogenously incorporated secreted immunoglobulin reactive with a second antigen.

84. A method, comprising:

administering to a subject having a disease or disorder a therapeutically effective amount of an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin or recombinant B cell receptor capable of binding a first antigen and at least one endogenously secreted immunoglobulin capable of binding a second antigen; and

at least one exogenous nucleic acid encoding at least one redistributed biological agent.

85. The method of clause 84, wherein the isolated B lymphocyte cell line is further capable of expressing at least one cytotoxic effector molecule.

86. A method, comprising:

administering to a subject having a disease or disorder a therapeutically effective amount of an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin or recombinant B cell receptor capable of binding a first antigen and at least one exogenously secreted immunoglobulin capable of binding a second antigen; and

87. The method of clause 86, wherein the isolated B lymphocyte cell line is further capable of expressing at least one cytotoxic effector molecule.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. An isolated modified B cell capable of expressing:

at least one exogenously incorporated redistributed biological agent; and

2. The isolated modified B cell of claim 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises one or more exogenously incorporated membrane immunoglobulin polypeptides.

3. The isolated modified B cell of claim 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises at least one exogenously incorporated nucleic acid encoding the at least one exogenous membrane immunoglobulin.

4. The isolated modified B cell of claim 3, wherein the exogenously incorporated nucleic acid encoding the at least one membrane immunoglobulin is integrated in one or more chromosomal loci in the isolated B lymphocyte cell line.

5. The isolated modified B cell of claim 4, wherein the chromosomal locus comprises at least one of an Ig H chain or an Ig L chain chromosomal locus.

6. The isolated modified B cell of claim 5, wherein the Ig H chain or IgL chain chromosomal locus is located on an unexpressed Ig allele.

7. The isolated modified B cell of claim 6, wherein at least one of the Ig H chain or IgL chain chromosomal loci is under expression control of an endogenous promoter or enhancer element.

8. The isolated modified B cell of claim 6, wherein the Ig L chain locus comprises at least one of a kappa or lambda light (L) chain locus.

9. The isolated modified B cell of claim 8, wherein integration at the kappa or lambda light (L) chain locus disrupts endogenous B cell kappa or lambda light (L) chain expression.

10. The isolated modified B cell of claim 4, wherein the chromosomal locus comprises one or more non-Ig L or non-Ig H chromosomal loci in the isolated B lymphocyte cell line.

11. The isolated modified B cell of claim 4, wherein the at least one exogenously incorporated membrane immunoglobulin nucleic acid comprises an exogenous bicistronic expression construct.

12. The isolated modified B cell of claim 1, wherein the at least one exogenously incorporated membrane immunoglobulin comprises nucleic acid encoding a single chain Fv immunoglobulin.

13. The isolated modified B cell of claim 1, wherein the isolated modified B cell comprises at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a B1B lymphocyte, a marginal zone B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell.

14. The isolated modified B cell of claim 1, wherein the isolated modified B cell is part of a polyclonal population of B lymphocytes.

15. The isolated modified B cell of claim 1, wherein the isolated modified B cell is part of a monoclonal population of B lymphocytes.

16. The isolated modified B cell of claim 1, wherein the isolated modified B cell is part of a modified B lymphocyte cell line.

17. The isolated modified B cell of claim 1, wherein the membrane immunoglobulin comprises at least one of a membrane anchor, a cytoplasmic domain, a hinge region, or an extracellular ligand binding domain.

18. The isolated modified B cell of claim 1, wherein the isolated modified B cell bears at least one chimeric B cell receptor capable of transducing a signal to induce expression of a cytotoxic effector molecule.

19. The isolated modified B cell of claim 18, wherein the chimeric B cell receptor is derived from at least one of membrane IgG, CD19, BCMA, CD38, common gamma chain receptor, IL-21 receptor, Toll-like receptor, or CD 40.

20. The isolated modified B-cell of claim 18, wherein the cytotoxic effector molecule comprises at least one of perforin, granzyme B, Fas ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

21. The isolated modified B cell of claim 18, wherein the cytotoxic effector molecule is expressed in response to a particular target cell or target antigen.

22. An isolated modified B cell capable of expressing:

at least one exogenously incorporated redistributed biological agent; and

23. The isolated modified B cell of claim 22, wherein the at least one exogenously incorporated membrane immunoglobulin comprises at least one exogenously incorporated membrane immunoglobulin polypeptide.

24. The isolated modified B cell of claim 22, wherein the at least one exogenously incorporated membrane immunoglobulin is encoded by at least one exogenously incorporated nucleic acid.

25. The isolated modified B cell of claim 22, wherein the exogenously incorporated nucleic acid encoding the at least one membrane immunoglobulin is integrated in one or more chromosomal loci in the isolated modified B cell.

26. The isolated modified B cell of claim 25, wherein the chromosomal locus comprises at least one of an Ig H chain or an IgL chain chromosomal locus.

27. The isolated modified B cell of claim 26, wherein the Ig H chain or IgL chain chromosomal locus is an unexpressed Ig allele.

28. The isolated modified B cell of claim 26, wherein the Ig H chain or Ig L chain chromosomal locus is an expressed Ig allele.

29. The isolated modified B cell of claim 28, wherein at least one of the Ig H chain or IgL chain chromosomal loci is under expression control of an endogenous promoter or enhancer element.

30. The isolated modified B cell of claim 28, wherein the Ig L chain locus comprises at least one of a kappa or lambda light (L) chain locus.

31. The isolated modified B cell of claim 30, wherein integration at the kappa or lambda light (L) chain locus disrupts endogenous B cell kappa or lambda light (L) chain expression.

32. The isolated modified B cell of claim 26, wherein the at least one exogenously incorporated membrane immunoglobulin nucleic acid comprises an exogenous bicistronic expression construct.

33. The isolated modified B cell of claim 22, wherein the at least one exogenously incorporated membrane immunoglobulin comprises nucleic acid encoding a single chain Fv immunoglobulin.

34. The isolated modified B cell of claim 22, wherein the isolated modified B cell comprises at least one of a naive B lymphocyte, an immature B lymphocyte, a transitional B lymphocyte, a mature B lymphocyte, a B1B lymphocyte, a marginal zone B lymphocyte, a follicular B lymphocyte, a memory B lymphocyte, a plasmablast cell, or a plasma cell.

35. The isolated modified B cell of claim 22, wherein the membrane immunoglobulin comprises at least one of a membrane anchor, a cytoplasmic domain, a hinge region, or an extracellular ligand binding domain.

36. The isolated modified B cell of claim 22, wherein the isolated modified B cell bears at least one chimeric B cell receptor capable of transducing a signal to induce expression of a cytotoxic effector molecule.

37. The isolated modified B cell of claim 36, wherein the chimeric B cell receptor is derived from at least one of membrane IgG, CD19, BCMA, CD38, common gamma chain receptor, IL-21 receptor, Toll-like receptor, or CD 40.

38. The isolated modified B cell of claim 37, wherein the cytotoxic effector molecule comprises at least one of perforin, granzyme B, Fas ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

39. The isolated modified B cell of claim 37, wherein the cytotoxic effector molecule is expressed in response to a particular target cell or target antigen.

40. The isolated modified B cell of claim 22, wherein the at least one exogenously incorporated secreted immunoglobulin comprises at least one exogenously incorporated secreted immunoglobulin polypeptide.

41. The isolated modified B cell of claim 40, wherein the at least one exogenously incorporated secreted immunoglobulin is encoded by at least one exogenously incorporated nucleic acid.

42. The isolated modified B cell of claim 41, wherein the exogenously incorporated nucleic acid encoding the at least one secreted immunoglobulin is integrated in one or more chromosomal loci in the isolated modified B cell.

43. The isolated modified B cell of claim 42, wherein the chromosomal locus comprises at least one of the Ig H chain or Ig L chain chromosomal loci.

44. The isolated modified B cell of claim 43, wherein the Ig H chain or IgL chain chromosomal locus is an unexpressed Ig allele.

45. The isolated modified B cell of claim 42, wherein the Ig H chain or Ig L chain chromosomal locus is the expressed Ig allele.

46. The isolated modified B cell of claim 45, wherein at least one of the Ig H chain or Ig L chain chromosomal loci is under expression control of an endogenous promoter or enhancer element.

47. The isolated modified B cell of claim 42, wherein the at least one exogenously incorporated secreted immunoglobulin nucleic acid comprises an exogenous bicistronic expression construct.

48. The isolated modified B cell of claim 42, wherein the at least one exogenously incorporated secreted immunoglobulin comprises nucleic acid encoding a single chain Fv immunoglobulin.

49. An isolated modified B cell capable of expressing at least one exogenously incorporated membrane immunoglobulin or recombinant B cell receptor reactive with a first antigen;

at least one exogenously incorporated redistributed biological agent;

50. The isolated modified B cell of claim 49, wherein the membrane receptor configured to directly or indirectly induce at least one cytotoxic effector molecule comprises at least one chimeric receptor exhibiting at least a portion of at least one cytokine receptor.