B cell production and expansion

Abstract

The present disclosure provides methods of producing and expanding populations of B cells (e.g., human B cells for clinical applications). Such expanded populations can be used to produce B cell vaccines containing B cells that express 4-1BBL and are capable of promoting tumor cell death.

Description

[0001] B CELL PRODUCTION AND EXPANSION

[0002] RELATED APPLICATION

[0003] This application claims the benefit of the filing date under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 590,202, filed on October 13, 2023, the entire contents of which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant Numbers R37CA258426 and 2P50CA221747, awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] BACKGROUND

[0007] There are many types of antigen-presenting cells (APCs) that can be used for clinical applications. Even though classic APCs are dendritic cells and macrophages, the role and utility of activated B cells as APC have increasingly been recognized. Under steady state, naive B cells are poor APCs. Also, they are fewer in number for any practical clinical utility. However, upon acquiring and upregulation of coreceptors though activation, B cells become effective APCs.

[0008] Furthermore, B -cells are an important player in brain tumor immunity that can serve both regulatory and anti-tumor functions. B -cells begin at a naive state and upon BCR and cytokine stimulation, undergo differentiation into non-switched IgM+memory B-cells within the germinal center. Further activation leads to isotype switching and development into switched memory B-cells, which can then mature into plasmablasts and plasma cells-the most activated B-cell state. The function of anti-tumoral B-cells involved in immune checkpoint blockade efficacy include differentiation into plasmablast and the subsequent production of tumor-reactive antibodies as well as T-cell activation via antigen-presentation or complement.

[0009] SUMMARY

[0010] The present disclosure provides at least in part, methods of producing and expanding populations of B cells which can be used to produce B cell vaccines containing B cells that express 4-1BBE and are capable of promoting tumor cell death, and compositions thereof. In some embodiments, methods of producing an expanded population of 4-1BBL+ B cells are provided.

[0011] In some embodiments, the method of producing an expanded population of 4-lBBL+B cells comprises (i) culturing in a bioreactor a population of 4-lBBL+B cells in the presence of Interleukin-4 (IL-4), 5-15% v / v serum and CD40 ligand (CD40L) for a period of time to produce an expanded population of 4-lBBL+B cells.

[0012] In some embodiments, the population of 4-lBBL+B cells is not cultured in the presence of BAFF.

[0013] In some embodiments, the population of 4-lBBL+B cells cultured in step (i) is prepared by culturing in a bioreactor a population of CD19+B cells in the presence of B-cell activating factor (BAFF) and CD40L.

[0014] In some embodiments, the population of CD19+B cells is cultured in the presence of 50 - 200 nM, 50 - 150 nM, or 50 - 100 nM BAFF.

[0015] In some embodiments, the population of CD19+B cells is cultured in the presence of 100 nM BAFF.

[0016] In some embodiments, the population of CD19+B cells is cultured in the presence of 100 nM recombinant human BAFF.

[0017] In some embodiments, the population of CD19+B cells is not cultured in the presence of IL-4.

[0018] In some embodiments, the population of CD19+B cells and the population of 4- 1BBL+B cells are cultured in the presence of different amounts of CD40L.

[0019] In some embodiments, the population of CD19+B cells and the population of 4- 1BBL+B cells are cultured in the presence of same amounts of CD40L.

[0020] In some embodiments, the population of CD19+B cells and the population of 4- 1BBL+B cells are cultured in the presence of 5 - 20 U / mL CD40L or 5 - 15 U / mL CD40L.

[0021] In some embodiments, the population of CD19+B cells and the population of 4- 1BBL+B cells are cultured in the presence of 8 - 12 U / mL CD40L.

[0022] In some embodiments, the population of CD19+B cells are cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L.

[0023] In some embodiments, the population of 4-lBBL+B cells are cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L.

[0024] In some embodiments, the population of CD19+B cells are cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L and the population of 4-lBBL+B cells are cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L. In some embodiments, the population of 4-lBBL+B cells are cultured in the presence of 20 - 400 U / mL IL-4, 20 - 300 U / mL IL-4, 20 - 200 U / mL IL-4, 40 - 400 U / mL IL-4, 40 - 300 U / mL IL-4, 40 - 200 U / mL IL-4, 40 - 100 U / mL IL-4, 40 - 60 lU / mL IL-4, 20 - 100 U / mL IL-4, 20 - 60 U / mL IL-4, 50 U / mL IL-4, 30-60 ng / mL IL-4, or 40-60 ng / mL IL-4.

[0025] In some embodiments, the method further comprises isolating 4-lBBL+B cells from the population of CD19+B cells.

[0026] In some embodiments, the period of time is at least 4 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 21 days.

[0027] In some embodiments, the period of time is 12 - 14 days.

[0028] In some embodiments, the expanded population of 4-lBBL+B cells in (i) comprises at least 5xl06or at least 10xl06B cells.

[0029] In some embodiments, at least 90%, at least 95%, or at least 98% of the cells of the expanded population are 4-lBBL+B cells.

[0030] In some embodiments, the population of 4-lBBL+B cells comprise at least 5xl06or at least 10xl06B cells.

[0031] In some embodiments, the expanded population of 4-lBBL+B cells does not comprise a detectable amount of non-B cells, optionally wherein the expanded population of 4-lBBL+B cells does not comprise a detectable amount of T cells.

[0032] In some embodiments, the concentration of serum is maintained at 5-15% v / v for the period of time.

[0033] In some embodiments, the population of CD19+B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum.

[0034] In some embodiments, the serum is AB serum, optionally human AB serum.

[0035] In some embodiments, the serum is heat-inactivated serum.

[0036] In some embodiments, the CD40L is a multimeric CD40L.

[0037] In some embodiments, the multimeric CD40L is soluble 4-trimer CD40L, optionally wherein the trimers are linked by surfactant protein D.

[0038] In some embodiments, the population of CD19+B cells is stimulated with CD40L at least one time on days 0-4 of the period of time and / or wherein the population of 4-lBBL+B cells are stimulated with CD40L at least once on days 4-11 of the period of time.

[0039] In some embodiments, the method comprises seeding the population of CD19+B cells at a density of 400,000 to 800,000 cells / mL prior to the culturing step.

[0040] In some embodiments, the method comprises seeding the population of CD19+B cells at a density of about 750,000 cells / mL prior to the culturing step. In some embodiments, the method comprises seeding the population of 4-lBBL+B cells at a density of 100,000 to 800,000 cells / mL prior to the culturing step.

[0041] In some embodiments, the method comprises seeding the population of 4-lBBL+B cells at a density of about 250,000 to 500,000 cells / mL prior to the culturing step.

[0042] In some embodiments, the population of CD19+B cells is isolated and expanded from a population of B cells obtained from a human subject.

[0043] In some embodiments, the population of CD19+B cells and the population of 4- 1BBL+B cells are cultured in the presence of a cell media, optionally a good manufacturing practice (GMP) grade cell media. In some embodiments, the same cell media is used throughout the culturing step. In some embodiments, the cell media is HSC-Brew GMP Medium with 10% Human AB Serum

[0044] In some embodiments, no more than 20%, 10%, 5%, or 3% of the cell media is removed from culturing the population of CD19+B cells and / or culturing the population of 4- 1BBL+B cells.

[0045] In some embodiments, the population of CD19+B cells and / or the population of 4- 1BBL+B cells is supplemented with growth media during the period of time, optionally on days 0, 4, and / or 7 of the period of time.

[0046] In some embodiments, the population of CD19+B cells and / or the population of 4- 1BBL+B cells are further cultured in the presence of cyclosporine.

[0047] In some embodiments, CD19+B cells and / or the population of 4-lBBL+B cells are supplemented with IL-4 during the period of time, optionally every 2-4 days and / or on days 5, 7, 9, and / or 12 of the period of time.

[0048] In some embodiments, the population of 4-1BBL cells are supplemented with IL-4 on days 5, 7, 9, and / or 12 of the period of time.

[0049] In some embodiments, wherein the bioreactor comprises a gas permeable rapid expansion cell culture membrane.

[0050] In some embodiments, the population of CD19+B cells is a population of human B cells.

[0051] In some embodiments, the population of human B cells is isolated from whole blood, and optionally wherein the population of human B cells is isolated using a microbead, wherein the microbead comprises an antibody, optionally an anti-CD19 antibody.

[0052] In some embodiments, the population of B cells is not treated with IFN-y.

[0053] In some embodiments, a composition comprising an expanded population of 4- 1BBL+ B cells is provided. In some embodiments, the composition comprises an expanded population of 4- 1BBL+B cells produced according to a method disclosed herein.

[0054] In some embodiments, a composition comprising a population of at least 50 million cells is provided.

[0055] In some embodiments, at least 95% of the cells in the population are CD19+B cells, at least 90% of the cells in the population express HLA-DR (HLA-DR+), at least 75% of the cells in the population express CD80 (CD80+); and at least 75% of the cells in the population express CD86 (CD86+).

[0056] In some embodiments, 20% to 95% of B cells in the cell population express 4-1BBL.

[0057] In some embodiments, the population of cells comprises at least 75 million cells, at least 100 million cells, at least 150 million cells, at least 200 million cells, at least 500 million cells, at least 750 million cells or at least 1 billion cells.

[0058] In some embodiments, a composition comprising a population of at least 25 million cells is provided.

[0059] In some embodiment, at least 95% of the cells in the population are CD19+B cells, at least 90% of the cells in the population express HLA-DR, at least 75% of the cells in the population express CD80, at least 75% of the cells in the population express CD86, and at least 80% of B cells in the cell population express 4-1BBL.

[0060] In some embodiments, a composition comprising a population of at least 50 million cells is provided.

[0061] In some embodiments, at least 98% or 99% of B cells in the population express MHC- class I.

[0062] In some embodiments, at least 98% or 99% of B cells in the population express MHC- class II.

[0063] In some embodiments, the composition further comprises one or more tumor antigens.

[0064] In some embodiments, at least 98% or 99% of the cells in the population are CD19+B cells.

[0065] In some embodiments, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population express HLA-DR and optionally wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the population express CD80.

[0066] In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the population express CD86. In some embodiments, no more than 2%, no more than 1%, or no more than 05% of the cells in the population are T cells.

[0067] In some embodiments, the composition does not contain feeder cells.

[0068] In some embodiments, the population of cells was expanded from a cell population obtained from a patient, optionally wherein the patient has cancer and the cancer is glioma.

[0069] In some embodiments, methods of producing a B cell vaccine are provided.

[0070] In some embodiments, the method of producing a B cell vaccine comprises:

[0071] (i) culturing in a bioreactor a population of CD19+B cells in the presence of B-cell activating factor (BAFF), serum, and CD40 ligand (CD40L) to produce an expanded population of CD19+B cells;

[0072] (ii) isolating 4-lBBL+B cells from the expanded population of CD19+B cells in (i);

[0073] (iii) culturing in a bioreactor the isolated 4-lBBL+B cells from (ii) in the presence of Interleukin-4 (IL-4) and CD40L to produce an expanded population of 4-lBBL+B cells, and

[0074] (iv) incubating the expanded population of 4-lBBL+B cells from (iii) with a tumor lysate comprising at least one tumor antigen to produce an expanded population of 4-lBBL+B cells comprising at least one tumor antigen thereby producing a B cell vaccine.

[0075] In some embodiments, the population of CD19+B cells in (i) are cultured for 0 - 4 days.

[0076] In some embodiments, the isolated 4-lBBL+B cells in (iii) are cultured for 4 - 11 days.

[0077] In some embodiments, the population of CD19+B cells in (i) is cultured in the presence of 100 nM BAFF or 100 ng / mL BAFF and 8 U / mL CD40L or about 8 U / mL CD40L.

[0078] In some embodiments, the isolated 4-lBBL+B cells in (iii) are cultured in the presence of 50 U / mL or about 50 U / mL IL-4 and 12 U / mL or about 12 U / mL CD40L._

[0079] In some embodiments, the population of CD19+B cells in (i) is isolated from a population of B cells using a microbead, wherein the microbead comprises an antibody, optionally an anti-CD19 antibody.

[0080] In some embodiments, the isolated 4-lBBL+B cells in (ii) are isolated from the expanded population of CD19+B cells using a labeled anti-4-lBBL antibody.

[0081] In some embodiments, a B cell vaccine comprising an expanded population of B cells which are at least 25% viable 4-lBBL+B cells is provided. In some embodiments, the B cell vaccine comprises an expanded population of B cells which are 25% - 75% viable 4-lBBL+B cells. In some embodiments, the B cell vaccine comprises an expanded population of B cells which are 40% - 60% viable 4-lBBL+B cells.

[0082] In some embodiments, the B cell vaccine further comprising an antigen, optionally a tumor antigen.

[0083] In some embodiments, the tumor antigen is from a tumor lysate comprising the tumor antigen, optionally, wherein the tumor antigen is associated with glioblastoma.

[0084] In some embodiments, further comprising a CD40 agonist.

[0085] In some embodiments, methods of treating a disease or disorder in a human subject comprising administering a B cell vaccine are provided.

[0086] In some embodiments, the method of treating a disease or disorder in a human subject comprises administering a B cell vaccine disclosed herein.

[0087] In some embodiments, the disease or disorder is a cancer.

[0088] In some embodiments, the cancer is a brain cancer; optionally, wherein the cancer is glioblastoma.

[0089] In some embodiments, methods comprising administering a B cell vaccine to a human subject are disclosed.

[0090] In some embodiments, the method comprises administering a B cell vaccine disclosed herein to a human subject.

[0091] In some embodiments, the human subject has cancer.

[0092] In some embodiments, the human subject has brain cancer, optionally glioblastoma.

[0093] In some embodiments, the method further comprises administering a therapeutic agent, optionally wherein the therapeutic agent is an immune checkpoint inhibitor.

[0094] In some embodiments, methods of producing a B cell vaccine is provided.

[0095] In some embodiments, the method of producing a B cell vaccine comprises

[0096] (i) producing an expanded population of 4-lBBL+B cells according to the method of any one of claims 1-38; and

[0097] (ii) incubating the expanded population of 4-1 BBL+B cells in (i) with a tumor lysate comprising at least one tumor antigen.

[0098] In some embodiments, the tumor antigen in (ii) is associated with glioblastoma.

[0099] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0100] BRIEF DESCRIPTION OF THE DRAWINGS

[0101] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. For purposes of clarity, not every component may be labeled in every drawing. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

[0102] FIG. 1 provides a graph showing the total number of expanded B cells in the presence of varying concentrations of IL-4.

[0103] FIGs. 2A-2D provide depictions showing total number of expanded B cells and expression of relevant phenotypic markers following different B cell isolation protocols.

[0104] FIG. 3 provides a graph showing expansion of B cell populations obtained using different isolation and culture protocols.

[0105] FIG. 4 provides a graph showing the ability of B cell populations obtained from different isolation and culture protocols to express phenotypic markers.

[0106] FIGs. 5A-5B provide graphs showing the ability of B cell populations to expand in a bioreactor culture condition.

[0107] FIGs. 6A-6C provide depictions showing the ability of B cell populations expanded in a bioreactor culture condition to express phenotypic markers.

[0108] FIGs. 7A-7B provide representative flow cytometry data from a single B cell expansion experiment.

[0109] FIGs. 8-9 provide exemplary methods of B cell expansion of the disclosure.

[0110] FIGs. 10 provides an overview of the development of the tumor lysate protocol, and the optimization of tumor lysate production under GMP conditions.

[0111] FIGs. 11A-1 IB provide an analysis of the cellular components of BVax and the limitations identified in the original protocol. FIG. 11 A illustrates the cellular makeup of BVax,. FIG. 11B addresses the limitations encountered with the original protocol, including low cell count. Factors contributing to the low cell count include the downregulation of 4- 1BBL during 4°C storage and transportation of the leukopak sample, as well as immunodeficient patients presenting with low B cell counts.

[0112] FIGs. 12A-12F provide an overview of BVax components, CD40L titration, IL4 and cell density optimization, and B cell expansion. FIG. 12A shows the components of BVax, while FIG. 12B and FIG. 12C illustrate the small and large scale CD40L titrations, respectively. FIG. 12D provides data on IL4 optimization, and FIG. 12E focuses on cell density optimization. The conclusion of these optimizations suggests reducing CD40L from 24U to 8U-12U per mL and lowering IL4 from 360U to 50U per mL results in over 80% cost savings. Additionally, cell density can be readjusted to 0.25 x 106per mL throughout expansion, and the G-Rex product can be utilized with an increased frequency of fresh media replacements.

[0113] FIGs. 13A-13C provide details of BVax components and issues regarding 4-1BBL+ cell isolations. FIG. 13A shows the components of BVax, while FIG. 13B presents the original goal for 4-1BBL+ isolation. FIG. 13C details unsuccessful attempts at 4-1BBL+ isolations, and FIG. 13D provides cell viability staining with Trypan Blue Solution 0.4% on Countess, illustrating issues encountered during 4-1BBL+ isolations. This data suggests that biotinylated antibodies successfully select cells, but the viability of 4-1BBL+ cells after selection is less than 20%. Additionally, CD19+ B cells become enlarged and fragile after a 7-12 day expansion period, and high pressure in magnetic columns contributes to membrane rupture.

[0114] FIG. 14 provides a schematic representation of the principle of B cell isolation.

[0115] FIG. 15 provides a schematic on the daily monitoring of microbead attachment to B cells.

[0116] FIG. 16 provides insights into limiting B cell growth and fragility during the expansion process.

[0117] FIG. 17 provides a schematic on the 4-day priming culture using IL-4 or BAFF.

[0118] FIG. 18 provides a comparative analysis of 4-lBBL+B cell isolation on Day 4 using BAFF versus IL-4.

[0119] FIG. 19 provides data showing effect of IL-4 on 4-1BBL+ cell recovery.

[0120] FIG. 20 provides data showing the increased recovery of 4-lBBL+cells using BAFF.

[0121] FIG. 21 provides results of the 4-lBBL+B cell expansion.

[0122] FIG. 22 provides a schematic illustrating the newly developed protocol. FIG. 23 provides a summary of the challenges encountered during GMP optimization of BVax.

[0123] DETAILED DESCRIPTION

[0124] Previous studies have demonstrated that B cells can be isolated and efficiently expanded. The inventors of the present disclosure have realized that the currently available methods and commercially available kits for B cell expansion are limited by their low scale (or impracticality of using on a large scale), low yield and throughput, use for basic research, and low reproducibility (e.g., high variability from one B cell expansion to another). Furthermore, many of these B cell expansion protocols rely on transfected feeder cell lines that may limit their clinical use. Accordingly, the inventors of the present disclosure have identified novel methods of B cell expansion that are capable of providing higher yields of B cells at large-scale suitable for clinical applications with a high degree of reproducibility. The novel approaches described herein, in some embodiments, further provide the ability of B cells to be grown in culture more easily and reliably (compared to other APCs), the ability of B cells to expand to large numbers needed for clinical applications, the ability to be compatible with Good Manufacturing Practices (GMP), and do not require the use of transfected cells (which has raised regulatory concerns, e.g., with the Federal Drug Administration). The disclosure provides, in some embodiments, that the methods disclosed can be used to produce B cell vaccines (e.g., cancer vaccines) that are derived from the expanded B cell populations generated using the expansion and culturing protocols of the disclosure. Such B cell vaccines may be useful for treating cancer in human patients in need thereof. The disclosure described herein further provides populations of cells produced using the methods provided herein.

[0125] The methods described herein, in some embodiments, utilize multimerized CD40L in presence of B cell growth factors to stimulate B cells for expansion, utilize GMP reagents, ensure that desirable B cell surface markers are expressed on the B cells, and can be used to expand B cells which are later incubated with an antigen (e.g., proteins and protein fragments from cancer cells, autoantigens, infectious agents, etc.) to which an immune response is desired.

[0126] The methods described herein involve, in some embodiments, the specific variation of the number, concentration, or purity of starting cells (e.g., purified B cells versus total peripheral blood mononuclear cells (PMBCs), culture media and supplements, stimulants, restimulation requirements, cytokine combinations, culture vessels, and duration of the expansion culture. In some embodiments, PMBCs are isolated from whole blood (leukopheresis product can also be used for extra large-scale expansions). In some embodiments, B cells are isolated using a pulldown reagent (e.g., anti-CD19 microbeads, e.g., GMP-grade anti-CD19 microbeads). In some embodiments, B cells are purified (e.g., from PMBCs) using a cell sorting platform (e.g., CliniMACS Plus closed system).

[0127] Methods of the disclosure

[0128] Some aspects of the disclosure provide a method of producing a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells and / or a method of producing an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells).

[0129] In some embodiments, a method of producing an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells, etc.) comprises (i) culturing in a bioreactor a population of 4-lBBL+B cells in the presence of Interleukin-4 (IL-4), 5-15% v / v serum and CD40L for a period of time to produce an expanded population of 4-lBBL+B cells. In some embodiments, a method of producing an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) comprises culturing a population of B cells (e.g., population of CD19+ B cells, population of 4-lBBL+B cells) in the presence of cell media, one or more cytokines, CD40 ligand (CD40L), vitamins and / or growth factors. In some embodiments, culturing a population of B cells in the presence of cell media or any molecule (e.g., one or more cytokines, CD40 ligand (CD40L), vitamins and / or growth factors) involves addition of said cell media or any molecule to the population of B cells at a given time. In some embodiments, culturing a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+ B cells, expanded population of 4-lBBL+B cells) in the presence of cell media or any molecule (e.g., one or more cytokines, CD40 ligand (CD40L), vitamins and / or growth factors) involves maintaining a specific amount or concentration of said cell media or any molecule to the population of B cells (e.g., population of CD19+B cells, population of 4- 1BBL+B cells) for a period of time.

[0130] A method of producing an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) comprises, in some embodiments, the following steps: (a) addition of a cell media, CD40 ligand (CD40L), and Interleukin-4 (IL-4) to a B cell population (e.g., a cell population on a culture plate, a cell population in a suspension culture, or a cell population in a bioreactor) on Day 0; (b) addition of IL-4 (and optionally growth media) to the B cell population on Day 5; (c) addition of CD40L, IL-4 (and optionally growth media) to the B cell population on Day 7 ; (d) addition of CD40L, IL-4 (and optionally growth media) to the B cell population on Day 9; (e) addition of IL-4 (and optionally growth media) to the B cell population on Day 12; and (f) harvesting of the B cell population on Day 14, thereby producing the expanded B cell population (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells).

[0131] The one or more cytokines, in some embodiments, comprise Interleukin- 1 (IL-1), Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin- 12 (IL-12), and / or Interleukin- 15 (IL-15). In some embodiments, the one or more cytokines comprises Interleukin-4 (IL-4). In some embodiments, the one or more cytokines comprises Interleukin-4 (IL-4) and at least one of IL-2, IL-6, IL-7, IL- 12, and / or IL- 15. In some embodiment, the one or more cytokines are added to a population of B cells at the onset of their culturing and / or at intervals during culturing (e.g., on Days 0, 5, 7, 9, 12, 14, 16, and / or 18 of the culture period). The one or more cytokines are, in some embodiments, useful for stimulating, promoting, and / or activating B cell growth or survival.

[0132] In some embodiments, the one or more cytokines are added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10-100 ng / mL. In some embodiments, the one or more cytokines are added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10-50 ng / mL, 20-60 ng / mL, 30-70 ng / mL, 40-80 ng / mL, or 50-100 ng / mL. In some embodiments, the concentration of the one or more cytokines is maintained within the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or in an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10-50 ng / mL, 20-60 ng / mL, 30-70 ng / mL, 40-80 ng / mL, or 50-100 ng / mL for a period of time (e.g., a period of cell culturing and expansion). In some embodiments, the one or more cytokines are added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL. In some embodiments, IL-4 is added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10-50 ng / mL, 20-60 ng / mL, 30-70 ng / mL, 40-80 ng / mL, or 50-100 ng / mL. In some embodiments, the concentration of IL-4 is maintained within the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or within the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10-50 ng / mL, 20-60 ng / mL, 30-70 ng / mL, 40-80 ng / mL, or 50-100 ng / mL for a period of time (e.g., culture period). In some embodiments, IL-4 is added to the population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL.

[0133] In some embodiments, IL-4 is added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 5 lU / mL or about 5 lU / mL, 10 lU / mL or about 10 lU / mL, 15 lU / mL or about 15 lU / mL, 20 lU / mL or about 20 lU / mL, 25 lU / mL or about 25 lU / mL, 30 lU / mL or about 30 lU / mL, 35 lU / mL or about 35 lU / mL, 40 lU / mL or about 40 lU / mL, 45 lU / mL or about 45 lU / mL, 50 lU / mL or about 50 lU / mL, 60 lU / mL or about 60 lU / mL, 70 lU / mL or about 70 lU / mL, 80 lU / mL or about 80 lU / mL, 90 lU / mL or about 90 lU / mL, 100 lU / mL or about 100 lU / mL.

[0134] In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 20 - 400 U / mL, 20 - 300 U / mL, 20 - 200 U / mL, 20 - 100 U / mL, 20 - 60 U / mL, 20 - 50 U / mL, 30 - 400 U / mL, 30 - 300 U / mL, 30 - 200 U / mL, 30 - 100 U / mL, 30 - 60 U / mL, 30 - 50 U / mL, 30 - 70 lU / mL, 40 - 400 U / mL , 40 - 300 U / mL, 40 - 200 U / mL, 40 - 100 U / mL, 40 - 60 lU / mL, 20 - 50 U / mL, 100 - 1000 lU / mL, 100-750 lU / mL, 100 - 500 lU / mL, 100 - 250 lU / mL, 200 - 1000 lU / mL, 200 - 750 lU / mL, 200 - 600 lU / mL, 200 - 500 lU / mL, or 250 - 400 lU / mL.

[0135] In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 20 - 400 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 20 - 300 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 20 - 200 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 20 - 100 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 20 - 60 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 20 - 50 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 30 - 400 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 30 - 300 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 30 - 200 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 30 - 100 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 30 - 60 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 30 - 50 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 30 - 70 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 40 - 400 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 40 - 300 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 40 - 200 U / mL In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 40 - 100 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 40 - 60 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 40 - 50 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 100 - 1000 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 100 - 750 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 100 - 500 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 100 - 250 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 200 - 1000 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 200 - 750 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 200 - 600 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 200 - 500 lU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 250 - 400 lU / mL.In some embodiments, the concentration of IL-4 is maintained within the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4- 1BBL+B cells) at a concentration of 5 lU / mL, 10 lU / mL, 15 lU / mL, 20 lU / mL, 25 lU / mL, 30 lU / mL, 35 lU / mL, 40 lU / mL, 45 lU / mL, 50 lU / mL, 60 lU / mL, 70 lU / mL, 80 lU / mL, 90 lU / mL, or 100 lU / mL. In some embodiments, the concentration of IL-4 is maintained within the population of B cells (e.g., population of CD19+B cells) and / or within the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4- 1BBL+B cells) at a concentration of 100-1000 lU / mL, 100-750 lU / mL, 200-600 lU / mL, 200- 500 lU / mL, or 250-400 lU / mL for a period of time (e.g., culture period).

[0136] In some embodiments, IL-4 is added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 100 lU / mL, 200 lU / mL, 300 lU / mL, 400 lU / mL, 500 lU / mL, 600 lU / mL, 700 lU / mL, 800 lU / mL, 900 lU / mL, or 1000 lU / mL. In some embodiments, the one or more cytokines (e.g., IL-4) are added to the population of B cells on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the culture period (e.g., period of time). In some embodiments, the one or more cytokines (e.g., IL-4) are added to the population of B cells (e.g., population of CD19+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) every 2-4 days. In some embodiments, the one or more cytokines (e.g., IL-4) are added to the population of B cells on days 5, 7, 9, and / or 12 of the culture period.

[0137] In some embodiments, CD40L (e.g., multimeric CD40L) is added to the population of B cells at a concentration of 0.1-1 mg / mL. In some embodiments, CD40L is added to the population of B cells at a concentration of 0.1-0.5 mg / mL, 0.2-0.6 mg / mL, 0.3-0.7 mg / mL, 0.4-0.8 mg / mL, 0.5-0.9 mg / mL, or 0.6-1 mg / mL. In some embodiments, the concentration of CD40L is maintained in the population of B cells at a concentration of 0.1-0.5 mg / mL, 0.2- 0.6 mg / mL, 0.3-0.7 mg / mL, 0.4-0.8 mg / mL, 0.5-0.9 mg / mL, or 0.6-1 mg / mL for a period of time (e.g., culture period). In some embodiments, CD40L is added to the population of B cells at a concentration of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL.

[0138] In some embodiments, the concentration of CD40L is maintained in the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or in the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 0.1-0.5 mg / mL, 0.2-0.6 mg / mL, 0.3-0.7 mg / mL, 0.4-0.8 mg / mL, 0.5-0.9 mg / mL, or 0.6-1 mg / mL for a period of time (e.g., culture period). In some embodiments, CD40L is added to the population of B cells (e.g., population of CD19+B cells) and / or to the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL.

[0139] In some embodiments, CD40L is useful for stimulating, promoting, and / or activating B cell growth or survival. In some embodiments, CD40L is added to the population of B cells (e.g., population of CD19+B cells) and / or to the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20 of the culture period. In some embodiments, CD40L is added to the population of B cells on days 0, 7, 9, and / or 14 of the culture period (e.g., period of time).

[0140] In some embodiments, CD40L (e.g., multimeric CD40L) is added to the population of B cells (e.g., population of CD19+B cells) and / or to the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of at least 5 lU / mL. In some embodiments, CD40L is added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 6 lU / mL, 7 lU / mL, 8 lU / mL, 9 lU / mL, 10 lU / mL, 11 lU / mL, 12 lU / mL, 13 lU / mL, 14 lU / mL, 15 lU / mL, 16 lU / mL, 17 lU / mL, 18 lU / mL, 19 lU / mL, 20 lU / mL, 25 lU / mL, 30 lU / mL, 35 lU / mL, 40 lU / mL, 45 lU / mL, or 50 lU / mL, or any ranges or combinations thereof. In some embodiments, CD40L is added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 5 - 100 lU / mL, 5-90 lU / mL, 5-80 lU / mL, 5-70 lU / mL, 5-60 lU / mL, 5-50 lU / mL, 5-40 lU / mL, 5-30 lU / mL, 5 - 20 lU / mL, 5-15 lU / mL, 5-10 lU / mL, 8 - 100 lU / mL, 8-90 lU / mL, 8-80 lU / mL, 8-70 lU / mL, 8-60 lU / mL, 8-50 lU / mL, 8-40 lU / mL, 8-30 lU / mL, 8-20 lU / mL, 8-15 lU / mL, 8-10 lU / mL, 8-12 lU / mL, 8 - 100 lU / mL, 10 - 100 lU / mL, 10 - 90 lU / mL, 10 - 80 lU / mL, 10 - 70 lU / mL, 10 - 60 lU / mL, 10 - 50 lU / mL, 10 - 40 lU / mL, 10 - 30 lU / mL, 10 - 20 lU / mL, or 10 - 15 lU / mL.

[0141] In some embodiments, the concentration of CD40L is maintained in the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or in the expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of at least 5 lU / mL. In some embodiments, CD40L is added to the population of B cells at a concentration of 6 lU / mL, 7 lU / mL, 8 lU / mL, 9 lU / mL, 10 lU / mL, 11 lU / mL, 12 lU / mL, 13 lU / mL, 14 lU / mL, 15 lU / mL, 16 lU / mL, 17 lU / mL, 18 lU / mL, 19 lU / mL, 20 lU / mL, 25 lU / mL, 30 lU / mL, 35 lU / mL, 40 lU / mL, 45 lU / mL, or 50 lU / mL, or any ranges or combinations thereof. In some embodiments, CD40L is added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 5 - 100 lU / mL, 5-90 lU / mL, 5-80 lU / mL, 5-70 lU / mL, 5-60 lU / mL, 5-50 lU / mL, 5-40 lU / mL, 5-30 lU / mL, 5-20 lU / mL, 5-15 lU / mL, 5-10 lU / mL, 8 - 100 lU / mL, 8-90 lU / mL, 8-80 lU / mL, 8-70 lU / mL, 8-60 lU / mL, 8-50 lU / mL, 8-40 lU / mL, 8-30 lU / mL, 8-20 lU / mL, 8-15 lU / mL, 8-10 lU / mL, 8-12 lU / mL, 8 - 100 lU / mL, 10 - 100 lU / mL, 10 - 90 lU / mL, 10 - 80 lU / mL, 10 - 70 lU / mL, 10 - 60 lU / mL, 10 - 50 lU / mL, 10 - 40 lU / mL, 10 - 30 lU / mL, 10 - 20 lU / mL, or 10 - 15 lU / mL.

[0142] In some embodiments, the population of CD19+B cells and the population of 4- 1BBL+B cells are cultured in the presence of different amounts of CD40L disclosed herein. In some embodiments, the population of CD19+B cells and the population of 4-lBBL+B cells are cultured in the presence of same amounts of CD40L disclosed herein. In some embodiments, the population of CD19+B cells are cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L and the population of 4-lBBL+B cells are cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L. In some embodiments, the population of CD19+B cells are cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L for 4 days and the population of 4-lBBL+B cells are cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L for 7 days. In some embodiments, the population of CD19+B cells are cultured for 4 days using a low dose of CD40L (e.g., 8 U / mL) to prevent CD19+ B Cell activation / expansion and the isolated 4-lBBL+B cells are cultured at a higher dose of CD40L (12 U / mL) to induce higher proliferation and activation. In some embodiments, CD40L is useful for stimulating, promoting, and / or activating B cell growth or survival. In some embodiments, CD40L is added to the population of B cells on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20 of the culture period. In some embodiments, CD40L is added to the population of B cells on days 0, 7, 9, and / or 14 of the culture period (e.g., period of time).

[0143] In some embodiments, the CD40L is a multimeric CD40L. In some embodiments, the multimeric CD40L comprises CD40L trimers. In some embodiments, the multimeric CD40L comprises at least two CD40L trimers. The at least two trimers can be linked, e.g., by a surfactant. In some embodiments, the at least two timers are linked by surfactant protein D. In some embodiments, the multimeric CD40L comprises at least three CD40L timers. The at least three trimers can be linked, e.g., by a surfactant. In some embodiments, the at least three timers are linked by surfactant protein D. In some embodiments, the multimeric CD40L comprises at least four CD40L timers. The at least four trimers can be linked, e.g., by a surfactant. In some embodiments, the at least four timers are linked by surfactant protein D. In some embodiments, the multimeric CD40L comprises two to five CD40L trimers. The two to five trimers can be linked, e.g., by a surfactant. In some embodiments, the two to five timers are linked by surfactant protein D. In some embodiments, the multimeric CD40L comprises four CD40L trimers. The four trimers can be linked, e.g., by a surfactant. In some embodiments, the four timers are linked by surfactant protein D. In some embodiments, the multimeric CD40L is soluble 4-trimer CD40L.

[0144] In some embodiments, serum (e.g., human serum, e.g., human AB serum) is added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells). In some embodiments, serum (e.g., human serum, e.g., human AB serum) is added to the population of B cells at a concentration of 5-20% v / v (volume-to-volume ratio), 5-15% v / v, 5-12% v / v, 5-10% v / v, 7-15% v / v, 7-12% v / v, or 7- 10% v / v. In some embodiments, serum (e.g., human serum, e.g., human AB serum) is added to the population of B cells at a concentration of 5% v / v, 6% v / v, 7% v / v, 8% v / v, 9% v / v, 10% v / v, 11% v / v, 12% v / v, 13% v / v, 14% v / v, 15% v / v, 16% v / v, 17% v / v, 18% v / v, 19% v / v, or 20% v / v.

[0145] In some embodiments, B-cell activating factor (BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-1BBL+ B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells). In some embodiments, BAFF is recombinant human BAFF.

[0146] In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 100 nM or around 100 nM BAFF (e.g., recombinant human BAFF). In some embodiments, BAFF (e.g., recombinant human BAFF) is added at a concentration of 10 nM, around 10 nM or at least 10 nM; 20 nM, around 20 nM or at least 20 nM; 30 nM, around 30 nM or at least 30; 40 nM, around 40 nM or at least 40 nM; 50 nM, around 50 nM or at least 50 nM; 60 nM, around 60 nM or at least 60 nM; 70 nM, around 70 nM or at least 70 nM; 80 nM, around 80 nM or at least 80 nM; 90 nM, around 90 nM or at least 90 nM; 100 nM, around 100 nM or at least 100 nM; 120 nM, around 120 nM or at least 120 nM; 140 nM, around 140 nM or at least 140 nM; 160 nM, around 160 nM or at least 160 nM; 180 nM, around 180 nM or at least 180 nM; 200 nM, around 200 nM or at least 200 nM; 300 nM, around 300 nM or at least 300 nM; or 400 nM, around 400 nM or at least 400 nM; or any ranges or combinations thereof.

[0147] In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10 - 400 nM, 50 - 400 nM, 10 - 300 nM, 50 - 300 nM, 50 - 200 nM, 50 - 150 nM, or 50 - 100 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4- 1BBL+B cells) at a concentration of 10 - 400 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 50 - 400 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4- 1BBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 10 - 300 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 50 - 300 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 50 - 200 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4- 1BBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 50 - 150 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a concentration of 50 - 100 nM.

[0148] In some embodiments, vitamins are added to the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or to an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4- 1BBL+B cells). Vitamins can function as enzyme cofactors (e.g., Vitamin K, select B vitamins), as biological antioxidants (e.g., Vitamin C, Vitamin E), or as hormones (e.g., Vitamin A, Vitamin D). In some embodiments, vitamins are fat-soluble or water-soluble. A vitamin to be added to the population of B cells may include Vitamin A, Vitamin D, Vitamin E, Vitamin K, Vitamin C, Riboflavin (Vitamin B2), Folic acid (Vitamin B9), Cyanocobalamin (Vitamin B12), Thiamine (Vitamin Bl), Pyridoxine (Vitamin B6), Biotin (Vitamin B7), Pantothenate (Vitamin B5), or Nicotinamide (Vitamin B3).

[0149] In some embodiments, cyclosporine is added to the population of cells. In some embodiments, the methods comprise culturing the population of cells in the presence of cyclosporin. In some embodiments, cyclosporin functions to limit T cell growth. In some embodiments, the concentration of cyclosporin is maintained in the population of cells at 200-1000 ng / mL, 200-800 ng / mL, 300-700 ng / mL, 300-600 ng / mL, 300-500 ng / mL, or 400- 500 ng / mL.

[0150] The population of B cells can be cultured for any reasonable period of time (e.g., any reasonable culture period). In some embodiments, the population of B cells is cultured for 10- 25 days. In some embodiments, the population of B cells is cultured for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. The population of B cells can be cultured at any reasonable temperature as known to a person skilled in the art. In some embodiments, the population of B cells is cultured at a temperature from 30-40 °C. In some embodiments, the population of B cells is cultured at a temperature of 37 °C.

[0151] The population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) can be seeded at any reasonable density in accordance with the culture technology being used (e.g., culture on plates, suspension culture, culture in a bioreactor). In some embodiments, the method comprises seeding the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a density of 100,000 to 1,000,000 cells / mL (e.g., on day 0). In some embodiments, the method comprises seeding the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a density of 100,000 to 500,000 cells / mL, 100,000 to 400,000 cells / mL, 100,000 to 300,000 cells / mL, 100,000 to 200,000 cells / mL, 400,000 to 1,000,000 cells / mL, 500,000 to 900,000 cells / mL, 600,000 to 800,000 cells / mL, or 400,000 to 600,000 cells / mL (e.g., on day 0). In some embodiments, the method comprises seeding the population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) at a density of about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, or about 1,000,000 cells / mL.

[0152] The population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) are further cultured in the presence of a cell media (also referred to as a “growth media” or “expansion media” or “culture media”). Any reasonable cell media that is known to a person of skill in the art can be used for the methods described herein. In some embodiments, the method of expanding a population of B cells can be performed without changing or removing the media during the culture period. In some embodiments, the method of expanding a population of B cells can be performed without changing or removing the media during the culture period and instead supplementing with fresh or “new” media during the culture period. In some embodiments, cell media is changed or supplemented if conditions require (e.g., if the glucose levels in the media fall below a threshold level). In some embodiments, the cell media is RPMI media.

[0153] In some embodiments, the method is performed using a bioreactor. In some embodiments, the bioreactor comprises a gas permeable rapid expansion cell culture membrane. The inventors have found that, in some embodiments, the use of a bioreactor enables large scale production of expanded B cell populations. In some embodiments, the bioreactor is the GRex® 10M bioreactor (Wilson Wolf). In some embodiments, the bioreactor is a rocking motion bioreactor. In some embodiments, the bioreactor is a stirred flask or stirred- tank bioreactor. In some embodiments, the bioreactor is a hollow-fiber bioreactor. In some embodiments, the bioreactor is as described in “Recent Advances in the Development of Bioreactors for Manufacturing of Adoptive Cell Immunotherapies” Bioengineering 2022, 9(12), 808; the content of which is incorporated herein by reference.

[0154] B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or a method of producing an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) for use in the methods described herein are generally human B cells. In some embodiments, a population of human B cells is isolated from whole blood. In such embodiments, the whole blood comprises total peripheral blood mononuclear cells (PMBCs). In some embodiments, PMBCs are obtained from the whole blood or an apheresis product (e.g., obtained using Ficoll density gradient separation). In some embodiments, the B cells are obtained or isolated using a microbead comprising an anti-CD19 antibody. In some embodiments, the B cells are obtained or isolated using cell separation, e.g., magnetic cell separation. For magnetic cell separation, monoclonal antibodies that bind to a target (e.g., CD19) on the B cells can be conjugated to magnetic particles and then separated using a magnetic matrix. In some embodiments, monoclonal antibodies that bind to a target (e.g., CD19) on the B cells can be conjugated to super paramagnetic iron dextran particles (e.g., MACS microbeads) and then separated using a matrix of super paramagnetic spheres. Exemplary cell separation systems include e.g., the CliniMACS® system. In some embodiments, 4-lBBL+B cells are isolated from the population of CD19+B cells using an antibody. In some embodiments, the antibody is labeled. In some embodiments, the label is biotin. In some embodiments, 4-lBBL+B cells are isolated using a labeling process that includes an anti-4- 1BBL biotinylated antibody and an anti-biotin microbead. In some embodiments, GMP grade CliniMACS® Anti-Biotin Microbeads are used to further label a cell or cell population disclosed herein with a magnetic bead to be detected during isolation. In some embodiments, the 4-lBBL+B cells are obtained or isolated using cell separation, e.g., magnetic cell separation. For magnetic cell separation, monoclonal antibodies that bind to a target (e.g., 4-1BBL) on the B cells can be conjugated to magnetic particles and then separated using a magnetic matrix. In some embodiments, monoclonal antibodies that bind to a target (e.g., 4-1BBL) on the B cells can be conjugated to super paramagnetic iron dextran particles (e.g., MACS microbeads) and then separated using a matrix of super paramagnetic spheres. Exemplary cell separation systems include e.g., the CliniMACS® system.

[0155] In some embodiments, a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) comprises B cells, T cells, NK cells monocytes, dendritic cells, red blood cells, white blood cells and / or platelets. In some embodiments, a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) comprises B cells, epithelial cells, neural cells, hormone- secreting cells, immune cells, secretory cells, blood cells, interstitial cells, and / or germ cells. In some embodiments, at least 20%, at least 30% at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of all cells in the population of B cells are B cells. In some embodiments, a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) comprises at least 100,000 B cells, at least 200,000 B cells, at least 300,000 B cells, at least 400,000, at least 500,000, at least 600,000, at least 700,000, at least 800,000, at least 900,000, or at least 1,000,000 B cells. In some embodiments, a population of B cells comprises 100,000 to 500,000 cells, 100,000 to 400,000 cells, 100,000 to 300,000 cells, 100,000 to 200,000 cells, 400,000 to 1,000,000 cells, 500,000 to 900,000 cells, 600,000 to 800,000 cells, or 400,000 to 600,000 cells. In some embodiments, a population of B cells does not include detectable amounts of non-B cells. In some embodiments, the methods described herein result in 2-20-fold expansions of B cells, which provides a sufficient number of cells for use in a clinical setting. In some embodiments, the methods described herein result in at least a 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold expansion of B cells. In some embodiments, the level of expansion of is determined by counting viable B cells. In some embodiments, viable B cells can be counted by cell staining with, for example Trypan blue (and light microscopy) or 7-amino- actinomycin D, vital dye emitting at 670 nm (or Via Probe a commercial ready-to-use solution of 7AAD) and flow cytometry, employing a technique known to those skilled in the art.

[0156] In some embodiments, a population of B cells (e.g., population of CD19+B cells, population of 4-lBBL+B cells) and / or a method of producing an expanded population of B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) is subjected to a cryopreservation procedure. In some embodiments, a population of cells is frozen following expansion. In some embodiments, a population of cells is maintained in a frozen state for a period of time (e.g., at least 1, 2, 4, 8, 12, or more weeks). In some embodiments, a population of cells has been previously frozen and thawed (e.g., 1, 2, 3, 4, 5, or more freeze / thaw cycles). In some embodiments, a population of cells are maintained in liquid culture media. In some embodiments, a population of cells have been passaged 1, 2, 3, 4, 5, or more times, using any known method.

[0157] Aspects of the disclosure further provide methods of using expanded populations of B cells (e.g., for clinical applications). In some embodiments, expanded populations of B cells (e.g., expanded population of 4-lBBL+B cells) are used for treatment of cancer patients to mount an effect immune response. In some embodiments, the populations of B cells are expanded from cells obtained from a cancer patient to be treated. In some embodiments, methods of using expanded B cells comprise administering (e.g., delivering) the population of expanded cells to a subject. In some embodiments, the B cells were expanded from cells obtained from the subject to be treated. In some embodiments, methods of using expanded B cells comprise using the expanded B cell population to treat a subject with cancer. In some embodiments, the B cells were expanded from cells obtained from the subject with cancer.

[0158] In some embodiments, expanded populations of B cells (e.g., expanded population of CD19+B cells) are purified to isolate 4-lBBL+B cells, which can be used to promote antitumor immunity. In some embodiments, expanded populations of B cells are used as a vaccine to treat tumors, including glioblastoma. In some embodiments, expanded populations of B cells (e.g., expanded population of CD19+B cells) are purified to isolate 4-lBBL+B cells and contacted with tumor-derived antigens. In some embodiments, expanded populations of B cells (e.g., expanded population of CD19+B cells) are purified to isolate 4- 1BBL+B cells, incubated with a CD40 agonist; and contacted with tumor-derived antigens.

[0159] As used herein, the term "anti-cancer composition" refers to any substance that, when administered in a therapeutically effective amount to a subject suffering from cancer, provides a therapeutic benefit such as (1) curing the cancer; (2) slowing the progress of the cancer; (3) causing the tumor to regress; or (4) alleviating one or more symptoms of the cancer.

[0160] In some embodiments, expanded populations of B cells (e.g., expanded population of CD19+ B cells, expanded population of 4-1BBL+ B cells) are used for (i) ex vivo expansion of donor- specific Tregs (which can be used for induction of tolerance in clinical organ, tissue and cellular transplantations), (ii) the amelioration of autoimmune diseases in humans where the immunogenic antigen is known through the use of antigen- specific Tregs; or (iii) the prevention and / or cure of allergies in humans where the specific protein allergen is known through the use of antigen- specific Tregs. In some embodiments, in a transplant situation, B cells from either the donor or the recipient of the transplant can be used without antigen incubation. For example, expanded B cells can be used as stimulators for the expansion of antigen- specific regulatory T cells (Tregs) to be used clinically.

[0161] The term “cancer” is meant to encompass any cancer, neoplastic and preneoplastic disease that is characterized by abnormal growth of cells. The cancer may be selected from the group consisting of colon carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, Hodgkin’s Disease, non-Hodgkin’s lymphomas, rectum cancer, urinary cancers, uterine cancers, oral cancers, skin cancers, stomach cancer, brain tumors, liver cancer, laryngeal cancer, esophageal cancer, mammary tumors, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelio sarcoma, Ewing’s sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystandeocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, endometrial cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioblastomas, neuronomas, craniopharingiomas, schwannomas, glioma, astrocytoma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias and lymphomas, acute lymphocytic leukemia and acute myelocytic polycythemia vera, multiple myeloma, Waldenstrom’s macroglobulinemia, and heavy chain disease, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, childhood-null acute lymphoid leukemia (ALL), thymic ALL, B-cell ALL, acute megakaryocytic leukemia, Burkitt’s lymphoma, and T cell leukemia, small and large nonsmall cell lung carcinoma, acute granulocytic leukemia, germ cell tumors, endometrial cancer, gastric cancer, hairy cell leukemia, thyroid cancer and other cancers known in the art. In some embodiments, the cancer is selected from the group consisting of glioblastoma, melanoma, breast cancer and pancreatic cancer. In a preferred embodiment, the cancer is a glioblastoma.

[0162] Methods of administering a population of expanded B cells (e.g., expanded population of CD19+B cells, expanded population of 4-lBBL+B cells) comprise administering an effective amount of the composition to the subject. The B cells compositions may be coadministered with other cancer treatments including radiation, other chemotherapeutic s, or other lymphocytes, including T cells or NK cells. Co-administration is used to indicate that the same subject may have received an additional therapeutic in addition to the B cells compositions described here. The therapies may be administered to the subject simultaneously as separate treatments, as part of a unitary composition or in any order. The administration of the therapies may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more.

[0163] In some embodiments of the disclosure, provided herein are methods of treating a subject having a disease or disorder comprising administering an effective amount of a B cell vaccine (referred to, in some embodiments, as “Bvax”) expanded B cell population (e.g., expanded population of 4-lBBL+B cells) described herein to the subject. In some embodiments, the disease or disorder relates to the immune system. The disease or disorder may be cancer. Thus, in some embodiments, the subject has cancer. In some embodiments, the cancer is a brain cancer, lung cancer, liver cancer, skin cancer, prostate cancer, breast cancer, blood cancer, or any other cancer.

[0164] A brain cancer may be a slower growing, less aggressive glioma (referred to as low grade or benign glioma) or a high grade gliomas. A high grade glioma may include high grade astrocytomas (36% of gliomas) and glioblastoma (GBM; 47% of gliomas and 22% of all primary brain tumors). Many high grade gliomas are located within the corpus callosum, basal ganglia, brainstem and areas of eloquent cortex (speech, motor or visual areas). Gliomas are the most common type of primary brain tumor, accounting for about 80% of all malignant brain tumors. These tumors arise from glial cells, which are supportive cells that surround and support the neurons in the brain. Gliomas can develop in any part of the brain or spinal cord, and they can be classified based on their location, cell type, and degree of malignancy. The three main types of glial cells are astrocytes, oligodendrocytes, and ependymal cells. Astrocytomas are the most common type of glioma, followed by oligodendrogliomas and ependymomas. In some embodiments, a patient of the present disclosure has been diagnosed with a glioma, for example, that has developed in the brain. In some embodiments, the glioma is an astrocytoma. In other embodiments, the glioma is an oligodendrogliomas. In yet other embodiments, the glioma is an ependymomas.

[0165] In some embodiments, the Bvax is produced by isolating 4-lBBL+B cells from a population of expanded B cells (e.g., expanded population of CD19+B cells). In some embodiments, the Bvax is produced by combining a population of expanded B cells (optionally wherein the population of expanded B cell has been selected for 4-lBBL+B cells) with a CD40 agonist. In some embodiments, the Bvax is produced by combining a population of expanded B cells (optionally wherein the population of expanded B cell has been selected for 4-lBBL+B cells) with a CD40 agonist and IFN-y. In some embodiments, the Bvax is produced by combining a population of expanded B cells (optionally wherein the population of expanded B cell has been selected for 4-lBBL+B cells) with IFN-y. In some embodiments, the Bvax is produced without using IFN-y.

[0166] In some embodiments, the Bvax is produced by isolating 4-lBBL+B cells from a population of expanded B cells. In some embodiments, the Bvax is produced by combining a population of expanded B cells (optionally wherein the population of expanded B cell has been selected for 4-lBBL+B cells) with a tumor lysate. In some embodiments, the Bvax is produced by isolating 4-lBBL+B cells from a population of expanded B cells. In some embodiments, the Bvax is produced by combining a population of expanded B cells (optionally wherein the population of expanded B cell has been selected for 4-lBBL+B cells) with a tumor lysate and a CD40 agonist.

[0167] In some embodiments that involve combining a population of expanded B cells with a CD40 agonist and IFN-y, the B cells are first incubated the CD40 agonist before addition of IFN-y (e.g., 18-24 hours after addition of the CD40 agonist.

[0168] As used herein, the term "CD40 agonist" refers to a reagent that specifically binds to a CD40 molecule and induces CD40 signaling. CD40 is a transmembrane protein receptor that is expressed by antigen-presenting cells (APCs) and is involved in co-stimulation of immune cells. Current strategies to induce CD40 signaling include both antibody -based and CD40 ligand-based approaches. Thus, in some embodiments, the CD40 agonist is selected from CD154 (i.e., the cognate ligand for CD40) and a CD40 antibody or portion thereof capable of agonizing CD40. The B cells may be incubated with the CD40 agonist for up to 48 hours prior to addition of tumor-derived antigen. In certain embodiments, the B cells are incubated with the CD40 agonist for at least 12 hours prior to the addition of IFN-y and as long as 48 hours.

[0169] In some embodiments, the Bvax further comprises tumor-derived antigens. In such embodiments, the Bvax is produced by incubating the expanded population of B cells with the tumor-derived antigens. Any sample containing cancer cells may be used as a source of tumor-derived antigens for the present disclosure. Suitable samples include, for example, tissue samples, tumors, tumor lysates, biopsies, and bodily fluids (e.g., blood, serum, plasma, sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears, saliva). Alternatively, the sample could comprise an organoid that was generated from a cancer specimen (i.e., a "tumor organoid"). In some embodiments, the tumor-derived antigen is a tumor cell lysate. In some embodiments, the tumor-derived antigen may be a single or small number of polypeptides identified as comprising tumor antigens. In certain embodiments, the tumor cell lysate is derived from a subject with cancer. The term “antigen” refers to any molecule that is recognized by the immune system and that can stimulate an immune response. A "tumor- derived antigen" is an antigen that is preferentially or differentially expressed by a tumor cell and not expressed or differentially expressed on normal, healthy cells. Thus, by incubating the B cells with tumor-derived antigens, the B cells are activated to act as more effective antigen presenting cells and stimulate CD8+ T cells to recognize these antigens. These B cells may also be able to produce tumor- specific antibodies that may recognize the tumor.

[0170] In some method of treatment embodiments, the Bvax is used to treat a subject with cancer. The methods comprise administering an effective amount of the composition to the subject. Bvax compositions may be co-administered with other cancer treatments including radiation, other chemotherapeutic s, or other lymphocytes, including T cells or NK cells. Coadministration is used to indicate that the same subject may have received an additional therapeutic in addition to the Bvax compositions described here. The therapies may be administered to the subject simultaneously as separate treatments, as part of a unitary composition or in any order. The administration of the therapies may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more.

[0171] The methods disclosed herein can further include a conventional cancer treatment regimen. In some embodiments, the methods further comprise administering radiation therapy to the subject. As used herein, the term “radiation therapy” refers to any manner of treatment of solid tumors and cancers with ionizing radiation and includes, without limitation, external beam radiotherapy, stereotatic radiotherapy, virtual simulation, 3-dimensional conformal radiotherapy, intensity-modulated radiotherapy, ionizing particle therapy, and radioisotope therapy.

[0172] In some embodiments, the method further comprises administering a therapeutic agent to the subject (e.g., coadministratation of a therapeutic agent and the population of expanded B cells).

[0173] In some embodiments, the methods further comprise administering a chemotherapeutic to the subject. Suitable chemotherapeutic s for use with the present methods include, without limitation, platinum-based agents, such as cisplatin, gemcitabine, and carboplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU) and other alkylating agents; antimetabolites, such as methotrexate; purine analog antimetabolites; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as taxanes (e.g., docetaxel and paclitaxel), aldesleukin, interleukin-2, etoposide (VP-16), interferon .alpha., and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vinca alkaloid natural antineoplastics, such as vinblastine and vincristine. In some embodiments, the chemotherapeutic is a triazene such as dacarbazine, mitozolomide or temozolomide. In some embodiments, the chemotherapeutic agent is temozolomide.

[0174] In some embodiments, the methods further comprise administering a checkpoint inhibitor. As used herein, the term “checkpoint inhibitor" refers to a molecule that blocks or inhibits immunosuppressive activities of checkpoint proteins. In another embodiment, the checkpoint inhibitor inhibits PD1, PD-L1, CTLA4, or a combination thereof. In some embodiments, the checkpoint inhibitor polypeptide is an antibody. In other embodiments, the checkpoint inhibitor is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti- PD 1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof. In some embodiments, the methods further comprise administering T cells to the subject. The T cells used in these methods may be obtained from a subject, and then expanded and activated ex vivo to enhance their immuno stimulatory capabilities. In certain embodiments, the T cells are CD8+ T cells. The T cells may be selected or engineered with antigen receptors directed against tumor antigens. As used herein, the term "chimeric antigen receptor T cell" refers to a genetically engineered antibody-T cell chimera that comprises a chimeric antigen receptor (CAR). Techniques for chimeric antigen receptor T cell therapies are known and available in the art. See, e.g., Kenderian et al., Cancer Res. 74(22):6383-9 (2014).

[0175] The terms “subject” and “patient” are used interchangeably and refer to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. For example, a suitable subject includes a subject in need of cancer treatment.

[0176] As used herein, “treating” or “treatment” describes the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treating includes the administration of a composition of present invention to prevent the onset of the symptoms or complications, to alleviate the symptoms or complications, or to eliminate the disease, condition, or disorder. Specifically, compositions disclosed herein can be used to treat a cancer. Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor in the subject, reducing progression of a cancer to a more aggressive form, reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with a disease or at risk of developing the disease, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of symptoms or slow the progression of symptoms, etc.

[0177] As used herein, the term “administering” refers to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra- arterial administration, intramuscular administration, intradermal administration, intrathecal administration, intra-lymphatic and subcutaneous administration. Administration can be continuous or intermittent. In some embodiments, the composition is administered intravenously or intracranially.

[0178] The term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological or clinical results. That result can be reducing, alleviating, inhibiting or preventing one or more symptoms of a disease or condition, reducing, inhibiting or preventing the growth of cancer cells, reducing, inhibiting or preventing metastasis of the cancer cells or invasiveness of the cancer cells or metastasis, or reducing, alleviating, inhibiting or preventing one or more symptoms of the cancer or metastasis thereof, or any other desired alteration of a biological system. In some embodiments, the effective amount is an amount suitable to provide the desired effect, e.g., produce an anti-tumor response.

[0179] Also provided herein are compositions comprising a population of expanded B cells. In some embodiments, the population of expanded B cells in the composition can be provided using any method of expanding B cells provided herein.

[0180] In some embodiments, a composition comprising a population of at least 1 million cells (e.g., population of B cells, expanded population of CD19+B cells, expanded population of 4-lBBL+B cells, etc.) is provided herein, wherein at least 95% of the cells in the population are CD19+B cells, at least 95% of the cells in the population express HLA-DR, at least 85% of the cells in the population express CD80, and at least 85% of the cells in the population express CD86. In some embodiments, the population (e.g., population of B cells, expanded population of B cells, expanded population of CD19+B cells, expanded population of 4-1BBL+ B cells, etc.) comprises at least 100,000 cells or at least about 100,000 cells, at least 150,000 cells or at least about 150,000 cells, at least 200,000 cells or at least about 200,000 cells, at least 250,000 cells or at least about 250,000 cells, at least 300,000 cells or at least about 300,000 cells, at least 350,000 cells or at least about 350,000 cells, at least 400,000 cells or at least about 400,000 cells, at least 450,000 cells or at least about 450,000 cells, at least 500,000 cells or at least about 500,000 cells, at least 1 million cells or at least about 1 million cells, at least 2 million cells or at least about 2 million cells, at least 3 million cells or at least about 3 million cells, at least 4 million cells or at least about 4 million cells, at least 5 million cells or at least about 5 million cells.

[0181] In some embodiments, the population (e.g., population of B cells, expanded population of B cells, expanded population of CD19+B cells, expanded population of 4- 1BBL+B cells, etc.) comprises at least 10 million cells, at least 20 million cells, at least 30 million cells, at least 40 million cells, at least 50 million cells, at least 60 million cells, at least 70 million cells, at least 75 million cells, at least 100 million cells, at least 150 million cells or at least 200 million cells.

[0182] In some embodiments, the population (e.g., population of B cells, expanded population of B cells, expanded population of CD19+B cells, expanded population of 4- 1BBL+B cells, etc.) comprises 10 million to 1,000 million cells, 20 million to 1,000 million cells, 30 million to 1,000 million cells, 40 million to 1,000 million cells, 50 million to 1,000 million cells, 60 million to 1,000 million cells, 70 million to 1,000 million cells, 75 million to 1,000 million cells, 100 million to 1,000 million cells, 150 million cells to 1,000 million cells, or 200 million to 1,000 million cells. In some embodiments, the population comprises 10 million to 500 million cells, 20 million to 500 million cells, 30 million to 500 million cells, 40 million to 500 million cells, 50 million to 500 million cells, 60 million to 500 million cells, 70 million to 500 million cells, 75 million to 500 million cells, 100 million to 500 million cells, 150 million cells to 500 million cells, or 200 million to 500 million cells. Such a population of cells can include 4-1BBL+ cells. In some embodiments of such a population of cells, at least 50% of B cells in the cell population express 4-1BBL (4-lBBL+). In some embodiments of such a population of cells, 50% to 60%, 50% to 70%, 50% to 80%, or 50% to 90% of B cells in the cell population express 4-1BBL.

[0183] As described herein, such a population of cells can be subject to selection for 4- 1BBL+cells. In some embodiments, such population of cells subjected to selection for 4- 1BBL+cells can be in a composition. Such composition can comprise, e.g., at least 1 million cells, at least 2 million cells, at least 3 million cells, at least 4 million cells, at least 5 million cells, at least 10 million cells, at least 15 million cells, at least 20 million cells, or at least 25 million cells. In some embodiments, the population comprises 1 million to 500 million cells, 2 million to 500 million cells, 3 million to 500 million cells, 4 million to 500 million cells, 5 million to 500 million cells, 10 million to 500 million cells, 15 million to 500 million cells, 20 million to 500 million cells, or 25 million to 500 million cells.

[0184] In some aspects a composition comprising a population of cells, e.g., expanded B cells that has been further selected for 4-lBBL+cells (e.g., expanded population of 4-lBBL+B cells) can be a composition wherein at least 95% of the cells in the population are CD19+B cells, at least 95% of the cells in the population express HLA-DR, at least 85% of the cells in the population express CD80, at least 85% of the cells in the population express CD86, and at least 80% of B cells in the cell population express 4-1BBL. In some aspects, at least 85% of B cells in the cell population express 4-1BBL. In some aspects, at least 90% of B cells in the cell population express 4-1BBL. In some aspects, at least 95% of B cells in the cell population express 4-1BBL.

[0185] As described herein, a population of cells (e.g., expanded B cells that have been selected for 4-1BBL+ cells) can be exposed to tumor cells. In some aspects, in the resulting population of cells at least 98% or at least 99% of B cells in the population express MHC- class I and / or at least 98% or at least 99% of B cells in the population express MHC-class II. In some aspects, a composition comprising the resulting population of tumor cells comprises one or more antigens from one or more tumor cells. In some aspects, the resulting population of cells is capable of expanding CD8+T cells, promoting GzmB expression in CD8+T cells, and / or killing tumor cells.

[0186] As a result of the fact that the methods of expanding B cells provided herein can be performed in the absence of feeder cells, in some aspects, a composition comprising a population of cells provided herein does not contain feeder cells.

[0187] EXAMPLES

[0188] Example 1.

[0189] Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density centrifugation and cultured in RPMI media containing 15% v / v heat-inactivated human AB serum (Sigma), HEPES buffer (Invitrogen), Penicillin / streptomycin / glutamine (Invitrogen) with the addition of 20% soluble 4-trimer CD40L (SPD-CD40L fusion protein, UltraCD40L; Multimeric Bio therapeutics), and 400 ng / ml cyclosporine-A (Novartis) at 37°C with 5% CO2. Varying concentrations of IL-4 (R&D Systems) were used. Cells were cultured over a 14 day period, and the total cell count and B cell proportion were assessed.

[0190] As shown in FIG. 1, 40ng / mL of IL-4 exhibited the greatest fold expansion of B cells in 14 day culture (>10 fold), indicating that this concentration of IL-4 may be desirable for future B cell expansion.

[0191] Example 2.

[0192] Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density centrifugation and cultured in RPMI media containing 15% v / v heat-inactivated human AB serum (Sigma), HEPES buffer (Invitrogen), Penicillin / streptomycin / glutamine (Invitrogen) with the addition of 40 ng / mL IL-4 (R&D Systems), 20% soluble 4-trimer CD40L (SPD-CD40L fusion protein, UltraCD40L; Multimeric Bio therapeutics), and 400ng / ml cyclosporine-A (Novartis) at 37°C with 5% CO2. Cells were cultured in T75 flasks (Coming), counted every 3-4 days, and maintained at a cell density of 0.5-1 x 106cells / mL. After 14 days in culture, the expanded B cells were phenotypically characterized by flow cytometry.

[0193] As shown in FIG. 2, there was ~ 10-fold increase in the absolute number of cells after 14 days in culture when PBMC was the starting population with increasing proportion of CD 19+ cells (i.e., B cells) (FIG. 2A-2B). The B cell cultures also showed an increased percentage of cells that expressed CD86 and CD80 with the majority expressing HLA-DR throughout the culture (FIG. 2C-2D).

[0194] These data demonstrate that B cells can be successfully expanded from PBMCs and that these expanded “donor” B cells demonstrated the phenotype of mature APCs.

[0195] Example 3.

[0196] B cell expansion cultures were performed as described above. Briefly, peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density centrifugation. All cells culture combinations were expanded in RPMI media containing 15% v / v heat- inactivated human AB serum (Sigma), HEPES buffer (Invitrogen), Penicillin / streptomycin / glutamine (Invitrogen) with the addition of 40 ng / mL IL-4 (R&D Systems) at 37°C with 5% CO2. Cells were cultured in T75 flasks (Corning), counted every 3-4 days, and maintained at a cell density of 0.5-1 x 106cells / mL. After 14 days in culture, the expanded B cells were characterized phenotypically by flow cytometry.

[0197] Three culture conditions were tested:

[0198] (1) Ultra PBMCs: PBMCs were cultured in the presence of 20% soluble 4-trimer CD40L, and 400ng / ml cyclosporine-A (Novartis)

[0199] (2) Ultra CD19+: B cells were isolated with a CD 19 microbead kit and were cultured in the presence of 20% soluble 4-trimer CD40L

[0200] (3) MB CD19+: B cells were isolated with a CD19 microbead kit and were cultured in the presence of CD40L Multimer.

[0201] Culture results are shown in Figure 3. A B cell isolation step followed by culture in the presence of CD40L Multimer exhibited the highest fold expansion (3.2 fold). Ultra CD40L (Multimeric) in both the isolated B cell and PBMC culture exhibited <2 fold expansion. These data suggest that optimal B cell expansion occurs with upfront B cell isolation followed by subsequent culture in the presence of CD40L multimer. The phenotypic nature of the expanded cells were also monitored by 5-color flow cytometric analyses performed on a Beckman-Coulter FC500 flow cytometer. The percentage of CD19+ cells was >80% on day 0 in both the Ultra CD19+ and MB CD19+ cultures, but approximately 10% in the Ultra PBMCs culture (FIG. 4, Top left panel). All culture conditions exhibited increased expression of co-activation markers (CD80 and CD86) over the 14 day culture (FIG. 4, bottom panels). Contaminating CD3+ T cells were highest in the Ultra PBMC culture and lowest in the MB CD 19+ culture (FIG. 4, Top right panel).

[0202] As shown in FIG. 4, MB CD 19+ culture conditions provided substantial activation of B cells. Using an isolated B cell population for culture rather than a starting population of PBMCs (with cyclosporine supplemented in the media to limit T cell growth) provided a purer B cell culture on Day 14 with minimal contaminating CD3+ T cells. Phenotypically, these B cells are activated APCs.

[0203] Example 4.

[0204] PMBCs were isolated from whole blood as above, and B cells were isolated using the CD 19 microbead kit (Miltenyi Biotec). Isolated B cells were cultured in StemMACS HSC Expansion Media (Miltenyi Biotec) supplemented with 10% v / v heat inactivated human AB Serum (Valley Biomedical) at 37 °C with 5% CO2 and stimulated with IL-4 (240 lU / mL media; Miltenyi Biotec) and CD40L multimer (24U / mL media; Miltenyi) (N=3). These concentrations were determined based on the results of previous expansions performed, and the biological activities of the specific lots of IL-4 and CD40L used in those experiments. The seeding concentration was 500,000 cells / mL media. CD40L stimulations were performed on Days 0, 7, and 9 of culture, and fresh IL-4 and media were supplemented every 2-4 days of culture.

[0205] In one culture replicate, StemMACS HSC Expansion media was replaced with HSC- Brew GMP Medium (Miltenyi) to ensure GMP compliance, and a G-Rex®10M bioreactor was used (Wilson Wolf) instead of a T25 or T75 flask. In this GMP compliant replicate, CD 19 selection was performed with GMP grade CD 19 microbeads (Miltenyi Biotec) on the CliniMACS Plus closed system (Miltenyi Biotec).

[0206] As shown in PIG. 5, B cells expanded from a starting population of (PIG. 5A) 14 million cells into 111 million cells over 14 days, which equated to (PIG. 5B) an 8 fold expansion over 14 days. In the GMP compliant culture, 30 million B cells expanded into 213 million cells after 14 days (~7 fold expansion). Notably, the use of the bioreactor and the GMP compliant reagents provided a significant expansion of B cells, suggesting that this protocol can be translated to a clinical setting.

[0207] Flow cytometric analyses were performed on cell culture samples prior to culture initiation (Day 0) and also on Day 14 of culture. The following antibodies were used: PE-Cy 7 CD20 (Day 0) (Biolegend), PE Cy-7 CD19 (Day 14) (Biolegend), APC-Cy7 HLA-DR (Biolegend), FITC CD80 (Biolegend), PE CD86 (Biolegend), and efluor405 Live / Dead fixable viability dye (Invitrogen). Briefly, 5xl05cells were stained with the above antibodies for 30 min., washed with MACS buffer supplemented with 0.5% BSA and fixed with 1% paraformaldehyde. Data was acquired on a Beckman Coulter Cytoflex. At least 50,000 cells were analyzed per sample. Data analyses was performed with FlowJo software after gating out doublets and dead cells. Descriptive statistics were performed in Prism.

[0208] Flow cytometry data is shown in FIG. 6. Descriptive statistics from (FIG. 6A) Day 0 and (FIG. 6B) Day 14 of culture are provided. The cell population used for culture after B cell isolation highly expressed CD20 (89.2% + / - 4.48) and HLA-DR (99.4 % + / - 0.418) but had low expression of CD80 (1.22% + / - 0.836) and CD86 (2.66% + / - 1.86) (FIG. 6C). On day 14, samples showed high expression of CD 19 (98.5% + / - 0.985), HLA-DR (99.9% + / - 0.117), CD80 (94.5% + / - 4.05) and CD86 (99.7% + / - 0.382).

[0209] FIG. 7 provides representative flow cytometry data from a single B cell expansion experiment (this single set of data is included within the statistics provided in Figure 6). (FIG. 7 A) Low expression of CD80 and CD86 on Day 0 (Top panel). High expression of CD80, CD86 and HLA-DR is observed after 14 days in culture (Bottom panel). (FIG. 7B) In addition, expression of 4-1BBL was assessed in this experiment as it is an activation marker and is present on activated APCs. (Top Panel): Low expression of 4-1BBL on Day 0 of culture. (Bottom Panel): After 14 days, over 50% of B cells express 4-1BBL.

[0210] These data show that expanded B cells uniformly expressed high levels of CD80, CD86, and HLA-DR suggesting these cells are mature, APCs and can be used for downstream applications.

[0211] Example 5.

[0212] An exemplary protocol is as described in FIG. 8. The exemplary protocol in FIG. 8 is a representative embodiment of the methods described herein. This exemplary protocol utilizes the G-Rex®10M bioreactor and provides for a 21 -day culture period (final volume of 50 mL). Notably, the bioreactor does not allow media to be removed, but does allow for media components (e.g., IL-4) to be added. Cells obtained from PMBCs (and purified using anti-CD19 GMP microbeads) are cultured in HSC-Brew GMP Medium supplemented with 10% v / v heat inactivated human AB Serum (“Growth Media”) at 37 °C with 5% CO2. CD40L multimer stimulations are performed on Days 0, 7, 9, and 14 of culture, and IL-4 is supplemented throughout the culture duration at defined intervals (Days 0, 5, 7, 9, 12, 14, 16, and 18). The concentrations of IL-4 and CD40L are expressed in standardized units (IU and U) to provide consistency across experiments given variability in biological activity lot to lot. Seeding concentration of cells is 500,000 cells / mL with a starting cell number of 10 million cells per G-Rex® 10M.

[0213] Example 6.

[0214] An exemplary protocol is as described in FIG. 9. The exemplary protocol in FIG. 9 is a representative embodiment of the methods described herein. This exemplary protocol utilizes the G-Rex® 10M bioreactor and provides for a 12-day culture period (final volume of 100 mL).

[0215] Day 0: CD8+T cells are isolated using anti-CD8 GMP microbeads (e.g., using a CiiniMacs® system). The negative fraction is then collected to isolate (e.g., using a CiiniMacs® system) CD19+B cells. CD19+B cells are counted for viability and then plated in the bioreactor at 500,000 cells / mL with a starting cell number of 10 million cells. CD19+B cells are cultured in HSC-Brew GMP Medium (20 mL) supplemented with 10% v / v heat inactivated human AB Serum, CD40L multimer (480 IU), and IL-4 (4800 IU) (37 °C with 5% CO2).

[0216] Day 5 : Additional growth media (up to 30 mL total volume) and IL-4 (7200 IU) are added to the CD19+B cells in culture (37 °C with 5% CO2).

[0217] Day 7: Additional growth media (up to 50 mL total volume), IL-4 (12000 IU), and CD40L multimer (1200 UI) are added to the CD19+B cells in culture (37°C with 5% CO2). Cells are counted for viability.

[0218] Day 9: Additional growth media (up to 50 mL total volume), IL-4 (18000 IU), and CD40L multimer (1800 UI) are added to the CD19+B cells in culture (37°C with 5% CO2).

[0219] Day 12: CD19+B Cells are harvested and checked for viability. Cells are further isolated to select 4-lBBL+B cells. After isolation, 4-lBBL+B cells are checked for viability, quality testing, and are preserved via cryopreservation to produce an expanded population of 4- 1BBL+B cells. In some embodiments, the expanded population of 4-lBBL+B cells can be useful as a vaccine. In some embodiments, the expanded population of 4-lBBL+B cells can be incubated with one or more tumor antigens (e.g., to produce a cancer vaccine). Example 7.

[0220] The peripheral blood samples of glioblastoma patients are collected in EDTA-treated tubes and PBMC is isolated by Ficoll gradient. B cells are isolated from PMBC and cultured using the exemplary method described in Example 5 or 6.

[0221] 4-lBBL+B cells are resuspended at 2xl06cells / ml in complete RPMI media and stimulated with 5 pg / ml human anti-CD40. After 24 hours, 1000 Ul / ml recombinant human IFNymay be added, in some embodiments, and incubated for additional 24-48 hours. In some embodiments, IFNyis not added. B cells may be supplemented with 100 nM recombinant human BAFF throughout the entire in-vitro activation process.

[0222] The final product B cell population can be referred to as “Bvax.” B cells are cocultured with cancer tumor lysate (or a tumor antigen) (e.g., at a 1:1 ratio) for 5-72 hours, in some embodiments.

[0223] Example 8.

[0224] The exemplary protocol in Table 1 is a representative embodiment of the methods described herein. This exemplary protocol utilizes the G-Rex®10M, 50M, or 100M bioreactor based on seeded cell concentration and provides for a 11 -day culture period.

[0225] Table 1.

[0226] Table 2. Table 3. Day 0:

[0227] 1. CD19+B cells are isolated using anti-CD19 GMP microbeads (e.g., using a CiiniMacs® system). CD19+ B Cells are counted for viability and then plated in a G- Rex®100M bioreactor at 500,000 cells / mL according to Table 2. CD19+B cells are cultured in HSC-Brew GMP Medium supplemented with 10% v / v heat inactivated human AB Serum, CD40L multimer (8 lU / mL), and BAFF (lOOng / mL) (37°C with 5% CO2). a. A lower dose of CD40L multimer and BAFF are used during this step to promote healthy B Cell culture, while preventing over activation and proliferation of the cells. Our research has shown that 8 lU / mL of CD40L multimer is sufficient to promote survival of CD19+B Cells with the addition of BAFF. We have chosen to utilize a set dose of lOOng / mL of BAFF in place of IL-4 to avoid unnecessary proliferation and expansion of cells that are observed with an IL-4 + CD40L multimer regimen. The goal of the 4-day culture is to promote healthy culture but limit the fragility of the cells when harvested on day 4 to avoid cell membrane rupture during magnetic selection as observed when isolating cells from a 4-day culture with IL-4 and CD40L multimer.

[0228] 2. The negative fraction of the original leukopak product that is now CD19+depleted is then collected to isolate (e.g., using a CiiniMacs® system) CD8+T cells. CD8+T Cells are counted for viability and cryopreserved for future use in GMP cryopreservant with 10% DMSO.

[0229] Day 4:

[0230] 1. The CD19+B-cell population is harvested and 4-lBBL+cells are magnetically labeled using Miltenyi Biotec CD137L (4-1BBL) Biotinylated Antibody, and CliniMACS® Anti-Biotin cGMP MicroBeads (e.g., using a CiiniMacs® system).

[0231] 2. Freshly isolated 4-lBBL+B Cells are counted for viability and then plated in a G- Rex® bioreactor at 250,000 cells / mL according to seeding conditions in Table 2. 4- 1BBL+B cells are cultured in HSC-Brew GMP Medium supplemented with 10% v / v heat inactivated human AB Serum, CD40L multimer (12 lU / mL), and IL-4 (50 lU / mL) (37°C with 5% CO2). a. The goal of this stage of the process is to promote a robust proliferative and activation stage of 4-lBBL+B Cells which is achieved with a co-regime of IL- 4 and CD40L multimer. The CD40L multimer dose is raised at this stage to influence a greater expansion and proliferation of cells, based on data collected in an optimization stage.

[0232] Day 7:

[0233] 1. Exhausted GM is carefully removed until the flask minimum is reached without disturbing cells at the bottom of the bioreactor according to guidelines in Table 3. Cells are resuspended in remaining media and counted for viability, with cells removed for flow analysis. Based on counted viability, 4-1BBL+ cells are resuspended to reestablish 0.25 xlO6cells / mL in HSC-Brew GMP Medium supplemented with 10% v / v heat inactivated human AB Serum, CD40L multimer (12 lU / mL), and IL-4 (50 lU / mL) (37°C with 5% CO2). Seeding conditions are detailed in Table 2. If expansion is greater than the maximum seeding abilities, split cells into multiple flasks according to cell range specifications in Table 2. a. During culture, clustered 4-lBBL+B cells will settle on the floor of the G- Rex® bioreactor within the gas permeable membrane. This enables removal of excess or exhausted media during culture. To promote the proliferation and expansion of 4-1BBL+ B Cells, exhausted culture media will be removed, and cells resuspended in fresh GM to reestablish a consistent cell concentration (0.25 xlO6 / mL). This fresh GM will contain fresh supplementation of CD40L multimer (12 lU / mL) and IL-4 (50 lU / mL) to promote additional activation and proliferation. The removal of excess media also enables easier resuspension in the G-Rex® bioreactor, as less media allows for more displacement from a pipette to break up cell clumps. This technique is used to avoid cell clumps, which can interfere with obtaining an accurate and reliable cell count.

[0234] Day 11:

[0235] 1. 4-lBBL+B cells are harvested, counted for viability, and cells removed for flow analysis. a. In some embodiments, the expanded population of 4-lBBL+B cells can be useful as a vaccine. In some embodiments, the expanded population of 4- 1BBL+B cells can be incubated with one or more tumor antigens (e.g., to produce a cancer vaccine). Example 9. CD40L Multimer Optimization (Figures 12B-C)

[0236] Purpose: This study aimed to understand how different concentrations of the CD40L multimer affect the proliferation, activation, and expansion of CD 19+ B cells. The only variable tested in this study was different concentrations of CD40L multimer in culture.

[0237] Methods and Materials: Cryopreserved CD19+ B cells (FIG. 12A) and freshly isolated CD19+ B cells (FIG. 12B) were used to test different concentrations of the CD40L multimer in culture with IL4 to promote cell expansion and proliferation. CD 19+ B cells were plated at 0.5 xlO6cells / mL of HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum. The concentration of IL-4 was kept constant at 360 lU / mL as previously described before the time point of this experiment. In Figure 12A, a range of 8-12 lU / mL CD40L multimer was tested in comparison to the previously developed dose of 24 lU / mL. In Figure 12B, a range of 6-12 lU / mL CD40L multimer was tested to further validate previous findings. On day 7, the cells were resuspended and counted, and HSC-Brew GMP medium supplemented with 10% v / v heat- inactivated human AB serum was added with new IL-4 and new CD40L multimer according to the specific well conditions. Cells were harvested on day 12, and cells were counted to measure population viability.

[0238] Results and Conclusions: The original results from cryopreserved CD 19+ B cells revealed that a high concentration of 24 lU / mL was not necessary and could be decreased. The results of freshly isolated CD19+ B cells, similar to those of a clinical protocol, demonstrated that CD19+ B cells were healthy and proliferative in all groups regardless of the decrease in protein concentration. However, the cells expanded at a greater rate when 12 lU / mL of CD40L multimer was added to culture. This newly developed lower concentration of the CD40L multimer reduces the cost of each expansion and decreases the amount of reagents needed.

[0239] Example 10. IL-4 Optimization (Figure 12D)

[0240] Purpose: This study aimed to understand how different concentrations of IL-4 affect the proliferation, activation, and expansion of CD 19+ B cells. The only variable tested in this study was different IL-4 concentrations in culture. Methods and Materials: Cryopreserved CD 19+ B cells were used to test different concentrations of IL-4 in culture with a set concentration of the CD40L multimer to promote cell expansion and proliferation. CD19+ B cells were plated at 0.5 xlO6cells / mL of HSC- Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum. The concentration of the CD40L multimer was kept consistent at 12 lU / mL on the basis of previous optimization. In FIG. 12D, a range of 50-275 lU / mL of IL-4 was used and compared to a well that utilized the previously developed dose of 360 lU / mL. CD 19+ B cells were expanded for 7 days in culture. On day 5, the cells were resuspended and counted, and new IL-4 was added to HSC-Brew GMP medium supplemented with 10% v / v heat- inactivated human AB serum according to the specific well conditions. No additional CD40L multimer was added on day 5 as the current 12-day protocol did not account for it.

[0241] Results and Conclusions: The results from this experiment were consistent across all groups, with a slight increase in expansion at lower concentrations of IL-4. This experiment revealed that the original concentration of IL-4 in culture was too high and could be decreased to 50 lU / mL across the course of the expansion period. This newly developed lower concentration of the IL-4 reduces the cost of each expansion and decreases the amount of reagents needed.

[0242] Example 11. Cell Density Optimization (Figure 12E)

[0243] Purpose: This study aimed to understand how different cell densities used during culture affect the proliferation, activation, and expansion of CD 19+ B cells.

[0244] Methods and Materials: Cryopreserved CD 19+ B cells were used to test different densities of plated cells for different durations of expansion. CD 19+ B cells were plated at a density ranging from 0.15 xlO6- 0.5 xlO6cells / mL HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, 50 lU / mL IL-4, and 12 lU / mL CD40L multimer. The concentrations of the CD40L multimer and IL-4 were newly developed from previous optimization experiments. CD19+ B cells were expanded for 7 days of culture. On day 5, the cells were resuspended and counted, and new IL-4 was added to HSC-Brew GMP medium supplemented with 10% v / v heat- inactivated human AB serum according to the specific well conditions. No additional CD40L multimer was added on day 5 as the current 12-day protocol did not account for it. Results and Conclusions: The expansion of each group was consistent across all conditions; however, there was a trend toward higher viability as the cell density decreased across all groups. This experiment revealed that plating the cells at a lower density of 0.15 xlO6- 0.25 xlO6cells / mL resulted in healthier expansion. A density of 0.25 xlO6cells / mL was recommended to help reduce the extraneous costs of plating at a lower density of 0.15 xlO6cells / mL.

[0245] Example 12. Isolation Background (Figures 13C-D)

[0246] Purpose: This study aimed to understand how optimized doses of IL-4 and the CD40L multimer could help alleviate previous issues associated with 4-1BBL+ isolation from an expanded CD 19+ B-cell population.

[0247] Methods and Materials: Freshly isolated CD19+ B cells were expanded with updated doses of IL-4 and CD40L, and attempts were made to isolate 4-1BBL+ B cells on different days during expansion. Both magnetic isolation and flow-based sorting were tested, as shown in Figure 13C. All the results were unsuccessful and confirmed that fewer than 20% of the cells were viable after isolation.

[0248] Results and Conclusions: The data collected from this experiment help illustrate the need to restructure our expansion and isolation process. Even after optimization of the concentration of supplemented IL-4 and CD40L multimer, the cells remained in a highly fragile state and cell membranes could be easily ruptured while passing through a magnetic field.

[0249] Example 13. Microbead Isolation Optimization (Figures 14 and 15)

[0250] Purpose: This experiment aimed to understand how long the original CD 19 microbead remained on the surface of CD19+ B cells after magnetic selection (CliniMACS® system). This information helped us design an optimized method for the isolation of 4-1BBL+.

[0251] Methods and Materials: CD 19+ B cells were isolated from a freshly mobilized leukopak via a CD 19 microbead. Every 24 hours, starting from day 0 to day 4, the cells were harvested and stained with a fluorochrome-conjugated anti-CD19 antibody. The assay was performed when the CD 19 marker became available and could be recognized by a flow cytometer. The re- emergence of the CD 19 population marked by flow was only possible once the CD 19 microbead had fallen off the cell surface before staining. The cells were plated at a density of 0.25 xlO6cells / mL throughout the entire experiment in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, 50 lU / mL IL-4, and 12 lU / mL CD40L multimer.

[0252] Results and Conclusions: The CD 19 population had completely remerged by day 4 (96 hours in culture). On the basis of these data, we concluded that the original CD 19 microbeads had fallen off the entirety of the CD 19+ B-cell population by this time. These data revealed that 4 days after the original CD19+ B-cell isolation was the earliest that a 4-1BBL+ B-cell isolation could be performed. This was because the CliniMACS® system cannot distinguish between different microbeads (4-1BBL or CD 19); therefore, if both populations remained with microbeads, the isolated population would not be purely 4-1BBL+ B cells.

[0253] Example 14. 4-Day CD19+ Culture Optimization (Figures 17-20)

[0254] Purpose: This study was designed to assess how the use of IL-4 or BAFF in combination with a CD40L multimer could promote the viability of CD 19+ B cells in a 4-day long culture period while limiting their activation and expansion. The goal was to preserve cells that could undergo an additional round of magnetic selection.

[0255] Methods and Materials: CD 19+ B cells were isolated from a freshly mobilized leukopak via a CD 19 microbead positive selection and cryopreserved CD 19+ B cells were used. In the figures CD19+ B cells were cultured at a density of 0.50 xl06cells / mL in HSC-Brew GMP medium supplemented with 10% v / v heat- inactivated human AB serum and 8-10 lU / mL of the CD40L multimer. One group of plated cells received an additional supplementation of 50 lU / mL IL-4, whereas the other group received an additional supplementation of 100 ng / mL BAFF instead. After 4 days of culture, the CD 19+ B cells were harvested and counted for cell viability. 4-1BBL+ B cells were then magnetically labeled from the harvested CD19+ B-cell population and isolated. The isolated 4-1BBL+ B-cell population was collected and counted for cell viability. Results and Conclusions: The viability of CD19+ B cells was high across both the IL-4 and CD40L multimer conditions at the time of harvest on day 4. However, the CD 19+ B cells from the BAFF + CD40L condition maintained much greater viability throughout the magnetic labeling process of the 4-1BBL+ cells. Additionally, the isolated 4-1BBL+ cells maintained significantly greater viability only in the group originating from CD 19+ B cells previously cultured with BAFF and CD40L. These data illustrated that the isolation of 4- 1BBL+ B cells was now possible via a magnetic separation technique (CliniMACS® system) from CD 19+ B cells that were cultured with BAFF + CD40L over a 4-day period. During these 4 days the cells remained healthy and the original CD 19 microbead had been shed from the cell surface.

[0256] Example 15. 4-1BBL+ Expansion Culture (Figure 21)

[0257] Purpose: This experiment was designed to assess how isolated 4-1BBL+ cells from the newly developed isolation protocol can expand, proliferate, become activated and retain their surface 4- IBB Ligand.

[0258] Methods and Materials: CD 19+ B cells were isolated from a freshly mobilized leukopak via a CD19 microbead positive selection. CD19+ B cells were cultured at a density of 0.50 x 106cells / mL in HSC-Brew GMP medium supplemented with 10% v / v heat- inactivated human AB serum, 8 lU / mL CD40L multimer, and 100 ng / mL BAFF. On day 4, CD19+ B cells were harvested, and 4-1BBL+ B cells were subsequently isolated. 4-1BBL+ B cells were then cultured at either 0.25 xlO6cells / mL or 0.50 xlO6cells / mL over a range of different time periods to determine how they expanded. On day 3 of the groups that were expanded for up to 6 days, the cells were resuspended, counted and readjusted to their original plated density in HSC-Brew GMP medium supplemented with 10% v / v heat- inactivated human AB serum, IL-4, and CD40L. The condition that expanded over the course of 5 days did not include a second restimulation. CD40L multimer dosing was kept consistent at 12 lU / mL, and IL-4 was kept consistent at 50 lU / mL on the basis of previous optimization. 4-1BBL+ B cells were then harvested after expansion, and 4-1BBL was stained with a fluorochrome-conjugated anti-4- 1BBL antibody to measure purity.

[0259] Results and Conclusions: All 4-1BBL+ B cells expanded; however, the greatest expansion was observed under lower density conditions, further supporting the claim that the 0.25 xlO6 cells / mL density was the most proficient and cost effective at maintaining the most proliferative environment. The best results were obtained from the group that was expanded over the course of 6 days with restimulation halfway through. Flow results from this group demonstrated a 58% recognition of the 4-1BBL+ B cell population. Our most updated protocol utilizes this methodology; however, we have made a slight adjustment to add an additional day of expansion to alleviate concerns with the 4-1BBL+ B cell culture needing to be manipulated on weekend days.

[0260] OTHER EMBODIMENTS

[0261] Embodiment 1. A method of producing an expanded population of B cells comprising: culturing a population of B cells in the presence of 20 - 60 ng / mL Interleukin-4 (IL- 4), 5-15% v / v serum and CD40 ligand (CD40L) for a period of time, wherein the population of B cells is cultured in a bioreactor.

[0262] Embodiment 2. A method of producing an expanded population of B cells comprising culturing a population of B cells in the presence of 200-400 lU / mL Interleukin-4 (IL-4), 5-15% v / v serum, and CD40 ligand (CD40L) for a period of time, wherein the population of B cells is cultured in a bioreactor.

[0263] Embodiment 3. The method of embodiment 1 or 2, wherein the period of time is 12-25 days, optionally 12-21 days or 12-16 days.

[0264] Embodiment 4. The method of any preceding embodiment, wherein the period of time is at least 12 days, at least 13 days, at least 14 days, or at least 21 days.

[0265] Embodiment 5. The method of any preceding embodiment, wherein the period of time is 14 days.

[0266] Embodiment 6. The method of any preceding embodiment, wherein the expanded population of B cells comprises at least 5xl06or at least 10xl06B cells. Embodiment 7. The method of any preceding embodiment, wherein at least 90%, at least 95%, or at least 98% of the cells of the expanded population are B cells.

[0267] Embodiment 8. The method of any preceding embodiment, wherein the expanded population of B cells does not comprise a detectable amount of non-B cells, optionally wherein the expanded population of B cells does not comprise a detectable amount of T cells.

[0268] Embodiment 9. The method of any preceding embodiment, wherein the concentration of IL-4 is maintained at 20-60 ng / mL, 40 ng / mL, or 200-400 lU / mL for the period of time.

[0269] Embodiment 10. The method of any preceding embodiment, wherein the population of B cells is cultured in the presence of 30-60 ng / mL IL-4 or 40-60 ng / mL IL-4.

[0270] Embodiment 11. The method of any preceding embodiment, wherein the population of B cells is cultured in the presence of 40 ng / mL IL-4 or 240 lU / mL IL-4.

[0271] Embodiment 12. The method of any preceding embodiment, wherein the concentration of serum is maintained at 5-15% v / v for the period of time.

[0272] Embodiment 13. The method of any preceding embodiment, wherein the population of B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum.

[0273] Embodiment 14. The method of any preceding embodiment, wherein the serum is AB serum, optionally human AB serum.

[0274] Embodiment 15. The method of any preceding embodiment, wherein the serum is heat-inactivated serum.

[0275] Embodiment 16. The method of any preceding embodiment, wherein the concentration of CD40L is maintained at 0.1-1 mg / mL. Embodiment 17. The method of any preceding embodiment, wherein the CD40L is a multimeric CD40L.

[0276] Embodiment 18. The method of embodiment 17, wherein the multimeric CD40L is soluble 4-trimer CD40L, optionally wherein the trimers are linked by surfactant protein D.

[0277] Embodiment 19. The method of any preceding embodiment, wherein the population of B cells is stimulated with CD40L on days 0, 7, and 9 of the period of time.

[0278] Embodiment 20. The method of any preceding embodiment, wherein the method comprises seeding the population of B cells at a density of 400,000 to 600,000 cells / mL prior to the culturing step.

[0279] Embodiment 21. The method of any preceding embodiment, wherein the method comprises seeding the population of B cells at a density of about 500,000 cells / mL prior to the culturing step.

[0280] Embodiment 22. The method of any preceding embodiment, wherein the population of B cells was obtained from a human subject.

[0281] Embodiment 23. The method of any preceding embodiment, wherein the population of B cells are cultured in the presence of a cell media, optionally a GMP grade cell media.

[0282] Embodiment 24. The method of embodiment 23, wherein the cell media is not removed from the population of B cells for the entire duration of the period of time.

[0283] Embodiment 25. The method of embodiment 23, wherein no more than 20%, 10%, 5%, or 3% of the cell media is removed from the of B cells for the entire duration of the period of time.

[0284] Embodiment 26. The method of any one of embodiments 23-25, wherein the population of B cells is supplemented with growth media during the period of time, optionally on days 5, 7, 9, and / or 12 of the period of time. Embodiment 27. The method of any preceding embodiment, wherein the population of cells are further cultured in the presence of cyclosporine.

[0285] Embodiment 28. The method of any preceding embodiment, wherein the population of B cells is supplemented with IL-4 during the period of time, optionally every 2- 4 days and / or on days 5, 7, 9, and / or 12 of the period of time.

[0286] Embodiment 29. The method of any preceding embodiment, wherein the population of B cells is supplemented with IL-4 on days 5, 7, 9, and / or 12 of the period of time.

[0287] Embodiment 30. The method of any preceding embodiment, wherein the bioreactor comprises a gas permeable rapid expansion cell culture membrane.

[0288] Embodiment 31. The method of any preceding embodiment, wherein the population of B cells is a population of human B cells.

[0289] Embodiment 32. The method of embodiment 31, wherein the population of human B cells was isolated from whole blood.

[0290] Embodiment 33. The method of embodiment 32, wherein the population of human B cells was isolated using a microbead.

[0291] Embodiment 34. The method of embodiment 33, wherein the microbead comprises an antibody, optionally an anti-CD19 antibody.

[0292] Embodiment 35. The method of any preceding embodiment further comprising isolating 4-lBBL+B cells from the expanded population of B cells.

[0293] Embodiment 36. The method of any preceding embodiment wherein the population of B cells is not treated with IFN-y. Embodiment 37. A composition comprising a population of expanded B cells produced according to the method of any one of embodiments 1-36.

[0294] Embodiment 38. A composition comprising a population of at least 50 million cells, wherein at least 95% of the cells in the population are CD19+B cells, at least 90% of the cells in the population express HLA-DR, at least 75% of the cells in the population express CD80; and at least 75% of the cells in the population express CD86.

[0295] Embodiment 39. The composition of embodiment 37 or 38, wherein at least 20% of B cells in the cell population express 4-1BBL.

[0296] Embodiment 40. The composition of embodiment 37 or 38, wherein 20% to 95% of B cells in the cell population express 4-1BBL.

[0297] Embodiment 41. The composition of any one of embodiments 37-40, wherein the population of cells comprises at least 75 million cells.

[0298] Embodiment 42. The composition of embodiment 41, wherein the population of cells comprises at least 100 million cells.

[0299] Embodiment 43. The composition of embodiment 42, wherein the population of cells comprises at least 150 million cells.

[0300] Embodiment 44. The composition of embodiment 43, wherein the population of cells comprises at least 200 million cells.

[0301] Embodiment 45. The composition of any one of embodiments 37-44, wherein the population of cells comprises no more than 1,000 million cells.

[0302] Embodiment 46. The composition of embodiment 45, wherein the population of cells comprises no more than 750 million cells.

[0303] Embodiment 47. The composition of embodiment 46, wherein the population of cells comprises no more than 500 million cells. Embodiment 48. A composition comprising a population of at least 25 million cells, wherein at least 95% of the cells in the population are CD19+B cells, at least 90% of the cells in the population express HLA-DR, at least 75% of the cells in the population express CD80; at least 75% of the cells in the population express CD86; and at least 80% of B cells in the cell population express 4-1BBL.

[0304] Embodiment 49. The composition of 48, wherein at least 98% of B cells in the population express MHC-class I.

[0305] Embodiment 50. The composition of embodiment 49, wherein at least 99% of B cells in the population express MHC-class I.

[0306] Embodiment 51. The composition of any one of embodiments 48-50, wherein at least 98% of B cells in the population express MHC-class II.

[0307] Embodiment 52. The composition of embodiment 51, wherein at least 99% of B cells in the population express MHC-class II.

[0308] Embodiment 53. The composition of any one of embodiments 48-52 further comprising one or more tumor antigens.

[0309] Embodiment 54. The composition of any one of embodiments 48-53, wherein the population of cells is capable of expanding CD8+ T cells.

[0310] Embodiment 55. The composition of any one of embodiments 48-54, wherein the population of cells is capable of promoting GzmB expression in CD8+ T cells.

[0311] Embodiment 56. The composition of any one of embodiments 48-55, wherein the population of cells is capable of killing tumor cells.

[0312] Embodiment 57. The composition of any one of embodiments 37-56, wherein at least 98% of the cells in the population are CD19+ B cells.

[0313] Embodiment 58. The composition of embodiment 57, wherein at least 99% of the cells in the population are CD 19+ B cells. Embodiment 59. The composition of any one of embodiments 37-58, wherein at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population express HLA-DR.

[0314] Embodiment 60. The composition of any one of embodiments 37-59, wherein at least 80%, at least 85%, at least 90%, or at least 95% of the cells in the population express CD80.

[0315] Embodiment 61. The composition of embodiment 60, wherein at least 96% of the cells in the population express CD80.

[0316] Embodiment 62. The composition of embodiment 61, wherein at least 97% of the cells in the population express CD80.

[0317] Embodiment 63. The composition of embodiment 62, wherein at least 98% of the cells in the population express CD80.

[0318] Embodiment 64. The composition of embodiment 63, wherein at least 99% of the cells in the population express CD80.

[0319] Embodiment 65. The composition of any one of embodiments 37-64, wherein at least 80%, at least 85%, or at least 90% of the cells in the population express CD86.

[0320] Embodiment 66. The composition of embodiment 68, wherein at least 95% of the cells in the population express CD86.

[0321] Embodiment 67. The composition of embodiment 66, wherein at least 96% of the cells in the population express CD86.

[0322] Embodiment 68. The composition of embodiment 67, wherein at least 97% of the cells in the population express CD86.

[0323] Embodiment 69. The composition of embodiment 68, wherein at least 98% of the cells in the population express CD86.

[0324] Embodiment 70. The composition of embodiment 69, wherein at least 99% of the cells in the population express CD86. Embodiment 71. The composition of any one of embodiments 37-70, wherein no more than 2% of the cells in the population are T cells.

[0325] Embodiment 72. The composition of embodiment 71, wherein no more than 1% of the cells in the population are T cells.

[0326] Embodiment 73. The composition of embodiment 72, wherein no more than 0.5% of the cells in the population are T cells.

[0327] Embodiment 74. The composition of any one of embodiments 37-73, wherein the cells in the population are human cells.

[0328] Embodiment 75. The composition of any one of embodiments 37-74, wherein the population of cells does not contain feeder cells.

[0329] Embodiment 76. The composition of any one of embodiments 37-75, wherein the population of cells was expanded from a cell population obtained from a patient.

[0330] Embodiment 77. The composition of embodiment 76, wherein the patient has cancer.

[0331] Embodiment 78. The composition of embodiment 77, wherein the cancer is a glioma.

[0332] Embodiment 79. A B cell vaccine comprising at least a subset of the B cells from the expanded population of B cells of any one of embodiments 1-36.

[0333] Embodiment 80. The B cell vaccine of embodiment 79, wherein at least 50%, 60%, 70%, 80%, 90%, or 95% of the total cells of the subset of B cells are 4-lBBL+B cells.

[0334] Embodiment 81. The B cell vaccine of embodiment 79 or 80 further comprising an antigen, optionally a tumor antigen.

[0335] Embodiment 82. The B cell vaccine of embodiment 81, wherein the subset of B cells was incubated with a tumor lysate comprising the tumor antigen. Embodiment 83. The B cell vaccine of any one of embodiments 79-82 further comprising a CD40 agonist.

[0336] Embodiment 84. A method of treating a disease or disorder in a human subject comprising administering the B cell vaccine of any one of embodiments 79-83.

[0337] Embodiment 85. The method of embodiment 84, wherein the disease or disorder is a cancer.

[0338] Embodiment 86. The method of embodiment 85, wherein the cancer is a brain cancer.

[0339] Embodiment 87. The method of embodiment 86, wherein the cancer is glioblastoma.

[0340] Embodiment 88. A method comprising administering the B cell vaccine of any one of embodiments 79-83 to a human subject.

[0341] Embodiment 89. The method of any one of embodiments 84-88, wherein the human subject has cancer.

[0342] Embodiment 90. The method of 89, wherein the human subject has brain cancer, optionally glioblastoma.

[0343] Embodiment 91. The method of any one of embodiments 84-90, wherein the method further comprises administering a therapeutic agent, optionally wherein the therapeutic agent is an immune checkpoint inhibitor.

[0344] Equivalents and Scope

[0345] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configfigurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configfigurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0346] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0347] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0348] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0349] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0350] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

CLAIMSWhat is claimed is:

1. A method of producing an expanded population of 4-lBBL+B cells comprising:(i) culturing in a bioreactor a population of 4-lBBL+B cells in the presence of Interleukin-4 (IL-4), 5-15% v / v serum and CD40 ligand (CD40L) for a period of time to produce an expanded population of 4-lBBL+B cells.

2. The method of claim 1, wherein the population of 4-lBBL+B cells cultured in step (i) is prepared by culturing in a bioreactor a population of CD19+B cells in the presence of B-cell activating factor (BAFF) and CD40L.

3. The method of claim 2, wherein the population of CD19+B cells is cultured in the presence of 50 - 200 nM, 50 - 150 nM, or 50 - 100 nM BAFF.

4. The method of claim 1 or claim 2, wherein the population of 4-lBBL+B cells is not cultured in the presence of BAFF.

5. The method of claim 2, wherein the population of CD19+B cells is cultured in the presence of 100 nM BAFF.

6. The method of claim 2, wherein the population of CD19+B cells is cultured in the presence of 100 nM recombinant human BAFF.

7. The method of any one of claims 2-6, wherein the population of CD19+B cells is not cultured in the presence of IL-4.

8. The method of any one of claims 2-5, wherein the population of CD19+B cells and the population of 4-lBBL+B cells are cultured in the presence of different amounts of CD40L.

9. The method of any one of claims 2-5, wherein the population of CD19+B cells and the population of 4-lBBL+B cells are cultured in the presence of same amounts of CD40L.

10. The method of any one of claims 2-5, wherein the population of CD19+B cells and the population of 4-lBBL+B cells are cultured in the presence of 5 - 20 U / mL CD40L or 5 - 15 U / mL CD40L.

11. The method of any one of claims 2-5, wherein the population of CD19+B cells and the population of 4-lBBL+B cells are cultured in the presence of 8 - 12 U / mL CD40L.

12. The method of any one of claims 2-5, wherein the population of CD19+B cells are cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L.

13. The method of any one of claims 1-5, wherein the population of 4-lBBL+B cells are cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L.

14. The method of any one of claims 2-13, wherein the population of CD19+B cells are cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L and the population of 4-lBBL+B cells are cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L.

15. The method of any one of claims 1-13, wherein the population of 4-lBBL+B cells are cultured in the presence of 20 - 400 U / mL IL-4, 20 - 300 U / mL IL-4, 20 - 200 U / mL IL-4, 40 - 400 U / mL IL-4, 40 - 300 U / mL IL-4, 40 - 200 U / mL IL-4, 40 - 100 U / mL IL-4, 40 - 60 lU / mL IL-4, 20 - 100 U / mL IL-4, 20 - 60 U / mL IL-4, 50 U / mL IL-4, 30-60 ng / mL IL-4, or 40-60 ng / mL IL-4.

16. The method of any one of claims 2-15 further comprising isolating 4-lBBL+B cells from the population of CD19+B cells.

17. The method of any one of claims 1-16, wherein the period of time is at least 4 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 21 days.

18. The method of claim 1, wherein the period of time is 12 - 14 days.

19. The method of any one of claims 1-18, wherein the expanded population of 4- 1BBL+B cells in (i) comprises at least 5xl06or at least 10xl06B cells.

20. The method of any one of claims 2-19, wherein at least 90%, at least 95%, or at least 98% of the cells of the expanded population are 4-lBBL+B cells.

21. The method of any one of claims 1-12, wherein the population of 4-lBBL+B cells comprise at least 5xl06or at least 10xl06B cells.

22. The method of any one of claims 1-21, wherein the expanded population of 4- 1BBL+B cells does not comprise a detectable amount of non-B cells, optionally wherein the expanded population of 4-lBBL+B cells does not comprise a detectable amount of T cells.

23. The method of any one of claims 1-22, wherein the concentration of serum is maintained at 5-15% v / v for the period of time.

24. The method of any one of claims 1-23, wherein the population of CD19+B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum.

25. The method of any one of claims 1-24, wherein the serum is AB serum, optionally human AB serum.

26. The method of any one of claims 1-25, wherein the serum is heat-inactivated serum.

27. The method of any one of claims 1-26, wherein the CD40L is a multimeric CD40L.

28. The method of claim 27, wherein the multimeric CD40L is soluble 4-trimer CD40L, optionally wherein the trimers are linked by surfactant protein D.

29. The method of any one of claims 1-28, wherein the population of CD19+B cells is stimulated with CD40L at least one time on days 0-4 of the period of time and / or wherein the population of 4-lBBL+B cells are stimulated with CD40L at least once on days 4-11 of the period of time.

30. The method of any one of claims 2-29, wherein the method comprises seeding the population of CD19+B cells at a density of 400,000 to 800,000 cells / mL prior to the culturing step.

31. The method of any one of claims 2-29, wherein the method comprises seeding the population of CD19+B cells at a density of about 750,000 cells / mL prior to the culturing step.

32. The method of any one of claims 1-31, wherein the method comprises seeding the population of 4-lBBL+B cells at a density of 100,000 to 800,000 cells / mL prior to the culturing step.

33. The method of any one of claims 1-31, wherein the method comprises seeding the population of 4-lBBL+B cells at a density of about 250,000 to 500,000 cells / mL prior to the culturing step.

34. The method of any one of claims 2-33, wherein the population of CD19+B cells is isolated and expanded from a population of B cells obtained from a human subject.

35. The method of any one of claims 2-34, wherein the population of CD19+B cells and the population of 4-lBBL+B cells are cultured in the presence of a cell media, optionally a good manufacturing practice (GMP) grade cell media.

36. The method of claim 35, wherein no more than 20%, 10%, 5%, or 3% of the cell media is removed from culturing the population of CD19+B cells and / or culturing the population of 4-lBBL+B cells.

37. The method of claim 35 or claim 36, wherein the population of CD19+B cells and / or the population of 4-lBBL+B cells is supplemented with growth media during the period of time, optionally on days 0, 4, and / or 7 of the period of time.

38. The method of any one of claims 2-37, wherein the population of CD19+B cells and / or the population of 4-lBBL+B cells are further cultured in the presence of cyclosporine.

39. The method of any one of claims 2-38, wherein CD19+B cells and / or the population of 4-lBBL+B cells are supplemented with IL-4 during the period of time, optionally every 2-4 days and / or on days 5, 7, 9, and / or 12 of the period of time.

40. The method of any one of claims 2-39, wherein the population of 4-1BBL cells are supplemented with IL-4 on days 5, 7, 9, and / or 12 of the period of time.

41. The method of any one of claims 2-40, wherein the bioreactor comprises a gas permeable rapid expansion cell culture membrane.

42. The method of any one of claims 2-41, wherein the population of CD19+B cells is a population of human B cells.

43. The method of claim 42, wherein the population of human B cells is isolated from whole blood, and optionally wherein the population of human B cells is isolated using a microbead, wherein the microbead comprises an antibody, optionally an anti-CD19 antibody.

44. The method of any one of claims 1-43, wherein the population of CD19+B cells and / or the population of 4-lBBL+B cells is not treated with IFN-y.

45. A composition comprising an expanded population of 4-lBBL+B cells produced according to the method of any one of claims 1-44.

46. A composition comprising a population of at least 50 million cells, wherein at least 95% of the cells in the population are CD19+B cells, at least 90% of the cells in the population express HLA-DR,at least 75% of the cells in the population express CD80, and at least 75% of the cells in the population express CD86.

47. The composition of claim 46, wherein 20% to 95% of B cells in the cell population express 4-1BBL.

48. The composition of claim 46 or claim 47, wherein the population of cells comprises at least 75 million cells, at least 100 million cells, at least 150 million cells, at least 200 million cells, at least 500 million cells, at least 750 million cells or at least 1 billion cells.

49. A composition comprising a population of at least 25 million cells, wherein at least 95% of the cells in the population are CD19+B cells, at least 90% of the cells in the population express HLA-DR, at least 75% of the cells in the population express CD80, at least 75% of the cells in the population express CD86, and at least 80% of B cells in the cell population express 4-1BBL.

50. The composition of 49, wherein at least 98% or 99% of B cells in the population express MHC-class I.

51. The composition of claim 49 or claim 50, wherein at least 98% or 99% of B cells in the population express MHC-class II.

52. The composition of any one of claims 49-51 further comprising one or more tumor antigens.

53. The composition of any one of claims 46-52, wherein at least 98% or 99% of the cells in the population are CD19+B cells.

54. The composition of any one of claims 46-53, wherein at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population express HLA-DR and optionally wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the population express CD80.

55. The composition of any one of claims 46-54, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the population express CD86.

56. The composition of any one of claims 46-55, wherein no more than 2%, no more than 1%, or no more than 05% of the cells in the population are T cells.

57. The composition of any one of claims 46-56, wherein the composition does not contain feeder cells.

58. The composition of any one of claims 46-57, wherein the population of cells was expanded from a cell population obtained from a patient, optionally wherein the patient has cancer and the cancer is glioma.

59. A method of producing a B cell vaccine comprising:(i) culturing in a bioreactor a population of CD19+B cells in the presence of B-cell activating factor (BAFF), serum, and CD40 ligand (CD40L) to produce an expanded population of CD19+B cells;(ii) isolating 4-lBBL+B cells from the expanded population of CD19+B cells in (i);(iii) culturing in a bioreactor the isolated 4-lBBL+B cells from (ii) in the presence of Interleukin-4 (IL-4) and CD40L to produce an expanded population of 4-lBBL+B cells, and(iv) incubating the expanded population of 4-lBBL+B cells from (iii) with a tumor lysate comprising at least one tumor antigen to produce an expanded population of 4-lBBL+B cells comprising at least one tumor antigen thereby producing a B cell vaccine.

60. The method of claim 59, wherein the population of CD19+B cells in (i) are cultured for 0 - 4 days.

61. The method of claim 59 or claim 60, wherein the isolated 4-lBBL+B cells in (iii) are cultured for 4 - 11 days.

62. The method of any one of claims 59-61, wherein the population of CD19+B cells in (i) is cultured in the presence of 100 nM BAFF or 100 ng / mL BAFF and 8 U / mL CD40L or about 8 U / mL CD40L.

63. The method of any one of claims 59-62, wherein the isolated 4-lBBL+B cells in (iii) are cultured in the presence of 50 U / mL or about 50 U / mL IL-4 and 12 U / mL or about 12 U / mL CD40L.

64. The method of any one of claims 59-63, wherein the population of CD19+B cells in (i) is isolated from a population of B cells using a microbead, wherein the microbead comprises an antibody, optionally an anti-CD19 antibody.

65. The method of any one of claims 59-63, wherein the isolated 4-lBBL+B cells in (ii) are isolated from the expanded population of CD19+B cells using a labeled anti-4- 1BBL antibody.

66. A B cell vaccine comprising an expanded population of B cells which are at least 25% viable 4-lBBL+B cells.

67. The B cell vaccine of claim 66 comprising an expanded population of B cells which are 25% - 75% viable 4-lBBL+B cells.

68. The B cell vaccine of claim 66 comprising an expanded population of B cells which are 40% - 60%69. The B cell vaccine of any one of claims 59-66, wherein the tumor antigen is associated with glioblastoma.

70. The B cell vaccine of any one of claims 59-66 further comprising a CD40 agonist.

71. A method of treating a disease or disorder in a human subject comprising administering the B cell vaccine of any one of claims 66-70.

72. The method of claim 71, wherein the disease or disorder is a cancer.

73. The method of claim 72, wherein the cancer is a brain cancer; optionally, wherein the cancer is glioblastoma.

74. A method comprising administering the B cell vaccine of any one of claims 66-70 to a human subject.

75. The method of any one of claims 72-74, wherein the human subject has cancer.

76. The method of claim 75, wherein the human subject has brain cancer, optionally glioblastoma.

77. The method of any one of claims 71-76, wherein the method further comprises administering a therapeutic agent, optionally wherein the therapeutic agent is an immune checkpoint inhibitor.

78. A method of producing a B cell vaccine comprising:(i) producing an expanded population of 4-lBBL+B cells according to the method of any one of claims 1-44; and(ii) incubating the expanded population of 4-1 BBL+B cells in (i) with a tumor lysate comprising at least one tumor antigen.

79. The method of claim 78, wherein the tumor antigen in (ii) is associated with glioblastoma.

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