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).

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 / 590187, filed on October 13, 2023, the entire contents of which is incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] 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.

[0006] 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.

[0007] For these reasons among others, methods of producing B cells at a large-scale are desired. There are several commercially available kits for B cell expansion. However, these commercially available kits are known to have limitations (e.g., low scale or yield, useful for basic research only, and / or high variability from one expansion to the next).

[0008] SUMMARY

[0009] The present disclosure is based, at least in part, on methods related to expansion of populations of B cells (e.g., human B cells).

[0010] In some embodiments, a method of producing an expanded population of B cells comprises seeding a population of B cells at a density of 400,000 to 1,000,000 cells / mE, and culturing in a bioreactor a population of B cells in the presence of interleukin (IE-4), 5-15% v / v serum, and CD40 ligand (CD40L) for a period of time to produce an expanded population of B cells.

[0011] In some embodiments, a method of producing an expanded population of B cells comprises culturing in a bioreactor a population of B cells in the presence of 20 - 60 lU / mL Interleukin-4 (IL-4), 5-15% v / v serum, and 10 - 30 lU / mL CD40 ligand (CD40L) for a period of time to produce an expanded population of B cells.

[0012] In some embodiments, the population of B cells is cultured in the presence of 50 lU / mL IL-4.

[0013] In some embodiments, the period of time is 12-25 days, optionally 12-21 days or 12- 16 days.

[0014] In some embodiments, the period of time is at least 12 days, at least 13 days, at least 14 days, or at least 21 days.

[0015] In some embodiments, the period of time is 14 days or 21 days.

[0016] In some embodiments, the population of B cells comprises at least 5xl06or at least 10xl06B cells.

[0017] In some embodiments, at least 90%, at least 95%, or at least 98% of the expanded population of B cells are B cells.

[0018] In some embodiments, 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.

[0019] In some embodiments, IL-4 is maintained at a concentration of 50 lU / mL. In some embodiments, IL-4 is added on days 0, 5, 7, 9, and 12; optionally, in some embodiments the expanded population of B cells is harvested on day 14. If the population of B cells is cultured until day 21, in some embodiments, IL-4 is added every 2-3 days.

[0020] In some embodiments, the population of B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum.

[0021] In some embodiments, the serum is AB serum, optionally human AB serum.

[0022] In some embodiments, the serum is heat-inactivated serum.

[0023] In some embodiments, the population of B cells is cultured in the presence of 10 - 40 U / mL CD40L.

[0024] In some embodiments, the concentration of CD40L is maintained at 10 - 40 U / mL.

[0025] In some embodiments, the population of B cells is cultured in the presence of 20 - 30 U / mL CD40L. In some embodiments, the concentration of CD40L is maintained at 20 - 30 U / mL.

[0026] In some embodiments, the population of B cells is cultured in the presence of 12 U / mL or about 12 U / mL CD40L.In some embodiments, the population of B cells is cultured in the presence of 24 U / mL or about 24 U / mL CD40L.

[0027] In some embodiments, the concentration of CD40L is maintained at 24 U / mL or about 24 U / mL.

[0028] In some embodiments, the CD40L is a multimeric CD40L.

[0029] In some embodiments, the multimeric CD40L is soluble 4-trimer CD40L.

[0030] In some embodiments, the population of B cells is stimulated with CD40L on days 0, 7, and 9 of the period of time; optionally, wherein the period of time is 14 days or 21 days.

[0031] In some embodiments, the population of B cells is stimulated with IL-4 on days 0, 5, 7, 9; and optionally, day 12 of the period of time; optionally, wherein the period of time is 14 days or 21 days.

[0032] In some embodiments, 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.

[0033] In some embodiments, the method comprises seeding the population of B cells at a density of about 500,000 cells / mL prior to the culturing step.

[0034] In some embodiments, the population of B cells is obtained from a human subject.

[0035] In some embodiments, the population of B cells is cultured in the presence of a cell media, optionally a Good Manufacturing Practice (GMP) grade cell media.

[0036] In some embodiments, the cell media is not removed from the population of B cells for the entire duration of the period of time.

[0037] In some embodiments, 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.

[0038] In some embodiments, the population of B cells is supplemented with growth media during the period of time, optionally every 2-4 days during the period of time.

[0039] In some embodiments, the population of B cells is supplemented with growth media on days 0, 5, 7, 9 and 12 of the period of time.

[0040] In some embodiments, the population of B cells are further cultured in the presence of cyclosporine.

[0041] In some embodiments, the bioreactor comprises a gas permeable rapid expansion cell culture membrane.

[0042] In some embodiments, the population of B cells is a population of human B cells.

[0043] In some embodiments, the population of human B cells is isolated from whole blood. In some embodiments, the population of human B cells is isolated using a microbead.

[0044] In some embodiments, the microbead comprises an antibody, optionally an anti-CD19 antibody.

[0045] In some embodiments, a composition comprising an expanded population of B cells is comprising at least 50 million cells disclosed.

[0046] In some embodiments, at least 95% of the expanded population of B cells are CD19+B cells, at least 90% of the expanded population of B cells express HLA-DR, at least 75% of the expanded population of B cells express CD80; and at least 75% of the expanded population of B cells express CD86.

[0047] In some embodiments, the expanded population of B cells comprises at least 15 million cells, at least 20 million cells, at least 25 million cells, at least 30 million cells, at least 75 million cells, at least 100 million cells, at least 150 million cells, at least 200 million cells, or 20 million - 35 million cells..

[0048] In some embodiments, the expanded population of B cells comprises no more than 1,000 million cells, no more than 750 million cells, or no more than 500 million cells.

[0049] In some embodiments, a composition comprising an expanded population of B cells comprising at least 25 million cells is disclosed.

[0050] In some embodiments, at least 95% of the expanded population of B cells are CD19+B cells, at least 90% of the expanded population of B cells express HLA-DR, at least 75% of the expanded population of B cells express CD80, and at least 75% of the expanded population of B cells express CD86.

[0051] In some embodiments, the population of cells is capable of expanding CD4+T cells.

[0052] In some embodiments, the expanded population of B cells comprises at least 97%, at least 98%, or at least 99% CD19+B cells.

[0053] In some embodiments, the expanded population of B cells comprises at least 95%, at least 97%, at least 98%, or at least 99% HLA-DR+B cells.

[0054] In some embodiments, the expanded population of B cells comprises 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% CD80+B cells.

[0055] In some embodiments, the expanded population of B cells comprises 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% CD86+B cells.

[0056] In some embodiments, the expanded population of B cells comprises no more than 2%, no more than 1%, or no more than 0.5% of T cells. In some embodiments, the expanded population of B cells are human B cells.

[0057] In some embodiments, the expanded population of B cells was expanded from a population of B cells obtained from a patient.

[0058] 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.

[0059] BRIEF DESCRIPTION OF THE DRAWINGS

[0060] 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.

[0061] FIG. 1 provides a graph showing the total number of expanded B cells in the presence of varying concentrations of IE-4.

[0062] FIGs. 2A-2D provide depictions showing total number of expanded B cells and expression of relevant phenotypic markers following different B cell isolation protocols.

[0063] FIG. 3 provides a graph showing expansion of B cell populations obtained using different isolation and culture protocols.

[0064] FIG. 4 provides a graph showing the ability of B cell populations obtained from different isolation and culture protocols to express phenotypic markers.

[0065] FIGs. 5A-5B provide graphs showing the ability of B cell populations to expand in a bioreactor culture condition.

[0066] FIGs. 6A-6C provide depictions showing the ability of B cell populations expanded in a bioreactor culture condition to express phenotypic markers. FIGs. 7A-7B provide representative flow cytometry data from a single B cell expansion experiment.

[0067] FIG. 8 provides an exemplary method of B cell expansion of the disclosure.

[0068] FIG. 9provides donor B cell isolation and expansion process.

[0069] FIG. 10 provides manufacturing activities at different timepoints.

[0070] DETAILED DESCRIPTION

[0071] 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.

[0072] 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).

[0073] 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.

[0074] 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).

[0075] The disclosure described herein further provides populations of cells produced using the methods provided herein.

[0076] Methods of the disclosure

[0077] Some aspects of the disclosure provide a method of producing an expanded population of B cells. In some embodiments, a method of producing an expanded population of B cells comprises culturing a population of 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 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 for a period of time.

[0078] A method of producing an expanded population of 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.

[0079] In some embodiments, a method of producing an expanded population of B cells comprises culturing in a bioreactor a population of B cells in the presence of IL-4, serum, and CD40L for a period of time to produce an expanded population of B cells.

[0080] 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 Interieukin-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.

[0081] In some embodiments, the one or more cytokines are added to the population of 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 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 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 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 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 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 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.

[0082] In some embodiments, IL-4 is added to the population of B cells at a concentration of 5 IU / 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, 120 lU / mL or about 120 lU / mL, 140 lU / mL or about 140 lU / mL, 160 lU / mL or about 160 lU / mL, 180 lU / mL or about 180 lU / mL, 200 lU / mL or about 200 lU / mL, 220 lU / mL or about 220 lU / mL, 240 lU / mL or about 240 lU / mL, 260 lU / mL or about 260 lU / mL, 280 lU / mL or about 280 lU / mL, 300 lU / mL or about 300 lU / mL, 320 lU / mL or about 320 lU / mL, 340 lU / mL or about 340 lU / mL, 360 lU / mL or about 360 lU / mL, 380 lU / mL or about 380 lU / mL, 400 lU / mL or about 400 lU / mL, 420 lU / mL or about 420 lU / mL, 440 lU / mL or about 440 lU / mL, 460 lU / mL or about 460 lU / mL, 480 lU / mL or about 480 lU / mL or 500 lU / mL or about 500 lU / mL. In some embodiments, IL-4 is added to the is added to a population of B cells at a concentration of 30 - 70 lU / mL, 40 - 60 lU / mL, 100 - 1000 lU / mL, 100 - 750 lU / mL, 200 - 600 lU / mL, 200 - 500 lU / mL, 250 - 400 lU / mL, or 300 - 400 lU / mL.

[0083] In some embodiments, IL-4 is added to a population of 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. In some embodiments, IL-4 is added to a population of B cells at a concentration of 20 - 200 U / mL. In some embodiments, IL-4 is added to a population of B cells at a concentration of 20 - 100 U / mL. In some embodiments, IL-4 is added to a population of B cells at a concentration of 20 - 60 U / mL. In some embodiments, IL-4 is added to a population of B cells at a concentration of 50 lU / mL or about 50 lU / mL.

[0084] In some embodiments, the concentration of IL-4 is maintained within the population of B cells at a concentration of 5 IU / 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, 100 lU / mL, 120 lU / mL, 140 lU / mL, 160 lU / mL, 180 lU / mL, 200 lU / mL, 220 lU / mL, 240 lU / mL, 260 lU / mL, 280 lU / mL, 300 lU / mL, 320 lU / mL, 340 lU / mL, 360 lU / mL, 380 lU / mL, 400 lU / mL, 420 lU / mL, 440 lU / mL, 460 lU / mL, 480 lU / mL or 500 lU / mL. In some embodiments, the concentration of IL-4 is maintained within the population of B cells at a concentration of 100-1000 lU / mL, 100-750 lU / mL, 200-600 lU / mL, 200-500 lU / mL, 250-400 lU / mL, or 300 - 400 lU / mL for a period of time (e.g., culture period).

[0085] In some embodiments, IL-4 is added to the population of 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. In some embodiments, the one or more cytokines (e.g., IL-4) are added to the population of 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 (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 on days 0, 6, 7, 13 and / or 14 of the culture period (e.g., period of time). 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.

[0086] 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.

[0087] 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). In some embodimentsCD40L is added to the population of B cells on days 0, 6, 7, 13 and / or 14 of the culture period (e.g., period of time).

[0088] In some embodiments, CD40L (e.g., multimeric CD40L) is added to the population of 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 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, 20 - 30 lU / mL, or 20 - 25 lU / mL. In some embodiments, CD40L (e.g., multimeric CD40L) is added to the population of B cells at a concentration of 12 lU / mL or about 12 lU / mL.

[0089] In some embodiments, the concentration of CD40L is maintained in the population of 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, 21 lU / mL, 22 lU / mL, 23 lU / mL, 24 lU / mL, 25 lU / mL, 26 lU / mL, 27 lU / mL, 28 lU / mL, 29 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 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, 20 - 30 lU / mL, or 20 - 25 lU / mL.

[0090] In some embodiments, the population of B cells are cultured in the presence of 24 U / mL CD40L or about 24 U / mL CD40L.

[0091] 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). In some embodiments, CD40L is added to the population of B cells on days 0, 6, 7, 13 and / or 14 of the culture period (e.g., period of time).

[0092] 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 trimers. 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 trimers. 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.

[0093] In some embodiments, serum (e.g., human serum, e.g., human AB serum) is added to the population of 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.

[0094] In some embodiments, vitamins are added to the population of 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).

[0095] 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.

[0096] 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.

[0097] The population of 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 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 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 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.

[0098] The 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.

[0099] 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. 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., CD 19) 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.

[0100] In some embodiments, a population of B cells or expanded population of 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 or expanded population of 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 or expanded population of B cells are B cells. In some embodiments, a population of B cells or expanded population of 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 or expanded 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 or expanded population of B cells does not include detectable amounts of non-B cells.

[0101] 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.

[0102] In some embodiments, a population of B cells (e.g., a population of expanded 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.

[0103] 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 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.

[0104] In some embodiments, expanded populations of B cells are purified to isolate 4- 1BBL+B cells, which can be used to promote anti-tumor 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 are purified to isolate 4-lBBL+B cells and contacted with tumor-derived antigens. In some embodiments, expanded populations of B cells are purified to isolate 4-lBBL+B cells, incubated with a CD40 agonist; and contacted with tumor-derived antigens.

[0105] 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.

[0106] In some embodiments, expanded populations of 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.

[0107] 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.

[0108] Methods of administering a population of expanded 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.

[0109] 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.

[0110] 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).

[0111] 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.

[0112] 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.

[0113] 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).

[0114] 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.

[0115] 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.

[0116] 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, intraarterial 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.

[0117] 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.

[0118] 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.

[0119] In some embodiments, a composition comprising a population (e.g., population of B cells or expanded population of B cells) of at least 1 million cells is provided herein, wherein at least 95% of the cells in the population are CD 19+ 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.

[0120] In some embodiments, the population of B cells or expanded population of B cells 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.

[0121] In some embodiments, the population of B cells or expanded population of B cells 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. In some embodiments, the population 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. 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.

[0122] As described herein, such a population of cells (e.g., population of B cells or expanded population of B cells) can be subject to selection for 4-1BBL+ cells. 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 of cells (e.g., population of B cells or expanded population of B cells) 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. In some aspects a composition comprising a population of cells, e.g., expanded B cells that has been further selected for 4-1BBL+ cells can be a composition wherein at least 97% of the cells in the population are CD 19+ 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.

[0123] 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.

[0124] EXAMPLES

[0125] Example 1.

[0126] 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.

[0127] As shown in FIG. 1, 40 ng / 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.

[0128] Example 2.

[0129] 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 400 ng / 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.

[0130] 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).

[0131] 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.

[0132] Example 3.

[0133] 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.

[0134] Three culture conditions were tested:

[0135] (1) Ultra PBMCs: PBMCs were cultured in the presence of 20% soluble 4-trimer CD40L, and 400 ng / ml cyclosporine-A (Novartis)

[0136] (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

[0137] (3) MB CD19+: B cells were isolated with a CD19 microbead kit and were cultured in the presence of CD40L Multimer.

[0138] 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.

[0139] 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).

[0140] 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.

[0141] Example 4.

[0142] 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.

[0143] 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).

[0144] 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.

[0145] Plow 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.

[0146] 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 HEA-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 CD19 (98.5% + / - 0.985), HEA-DR (99.9% + / - 0.117), CD80 (94.5% + / - 4.05) and CD86 (99.7% + / - 0.382).

[0147] 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) Eow expression of CD80 and CD86 on Day 0 (Top panel). High expression of CD80, CD86 and HEA-DR is observed after 14 days in culture (Bottom panel). (FIG. 7B) In addition, expression of 4-1BBE was assessed in this experiment as it is an activation marker and is present on activated APCs. (Top Panel): Eow expression of 4-1BBE on Day 0 of culture. (Bottom Panel): After 14 days, over 50% of B cells express 4-1BBE.

[0148] These data show that expanded B cells uniformly expressed high levels of CD80, CD86, and HEA-DR suggesting these cells are mature, APCs and can be used for downstream applications.

[0149] Example 5.

[0150] 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 mF). Notably, the bioreactor does not allow media to be removed, but does allow for media components (e.g., IE-4) to be added.

[0151] 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. CD40E multimer stimulations are performed on Days 0, 7, 9, and 14 of culture, and IE-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.

[0152] Example 6.

[0153] Allogenic Peripheral Blood. Collection from the Kidney Donor

[0154] B cells (e.g., donor B cells) will come from whole blood (e.g., donor whole blood). About 300 mL of whole blood will be collected. PBMC will be isolated from the blood followed by B cell separation from the PBMC. The isolated B cells will be cultured and expanded (FIG. 9).

[0155] A detailed description of the procedure in FIG. 9 is provided in the sections below. A procedural timetable, FIG. 10, depicts the manufacturing activities at different time points in chronological order. Manufacturing reagents and ancillary materials are described in the table below.

[0156] B Cell Isolation and Expansion

[0157] After overnight storage or immediately after PBMC isolation, B cells (e.g. donor B cells) are labeled with CliniMACS CD 19 Reagent and the labeled B cells are separated by positive selection using Miltenyi CliniMACS plus. Flow cytometry analysis of the pre and post isolation samples is performed to assess the purity and recovery of the B cells. The resultant B cells are washed and resuspended in culture medium containing HSC Brew GMP Medium (Miltenyi Biotec), 10% human AB serum, 360 lU / mL IL-4 and 24 U / mL soluble CD40L. The B cells are cultured in Wilson-Wolf G-Rex® 10M device and incubated at 37°C and 5% CO2.

[0158] B cell stimulation is provided by addition of soluble CD40L on Days 0, 6 / 7, and 13 / 14 of B cell culture. Fresh IL-4 and additional medium is added throughout culture according to the cell maintenance schedule. Cultured and expanded B cells. In some embodiments, B cells to be used have: 7-AAD viability >70%, CD19+ >70%, CD80+ >70%, CD86+ >70%, CD3+ <10%. At day 13 / 14 of B cell culture and expansion, in some embodiments, a part of B cells will be restimulated with CD40L and continue to be cultured for an additional 7 days. In some embodiments, B cells are then cryopreserved and / or may be used if, for instance, an ongoing B cell culture is compromised.

[0159] Example 7.

[0160] Additional Optimization of B Cell Expansion

[0161] Additional expansions were performed using the established methodology. This included modification to the concentrations of both IL-4 and CD40L, and also the seeding density of the initial Grex 10M.

[0162] Methods

[0163] B cells were isolated from a fresh leukapheresis product using the CD 19 microbead kit (Miltenyi Biotec). Isolated B cells were cultured in HSC-Brew GMP Medium (Miltenyi Biotec) supplemented with 10% heat inactivated human AB Serum (Valley Biomedical) at 37°C with 5% CO2 and stimulated with IL-4 (50IU / mL; Miltenyi Biotec) and varying concentration of CD40L multimer (Miltenyi Biotec) as detailed below. 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 for experimental cultures and were compared to a Miltenyi Biotec protocol. 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. Notably, in one of these cultures, expanded B cells were resuspended on Day 9 to determine if additional manipulation and disruption of cell clumps promoted additional cell expansion.

[0164] In some embodiments, the concentration of CD40L is 12 U / mL (or 240 U / 20 mL) and of IL- 4 is 50 lU / mL. In some embodiments, when CD40L and / or IL-4 are added to a B cell culture or a B cell culture is restimulated, it is considered as though all of the previous additions of CD40L and IL-4 are used up and none remains in the system.

[0165] 5 Conditions:

[0166] • 6u CD40L: 4.2 million cells, seeding density 500,000 / mL

[0167] • 12u CD40L: 4.2 million cells, seeding density 500,000 / mL • 12u CD40L: 4.2 million cells, seeding density 500,000 / mL (resuspend Day 9)

[0168] • 24u CD40L: 4.2 million cells, seeding density 500,000 / mL

[0169] • Miltenyi Biotec (MB) Protocol: 1.5 million cells, seeding density 150,000 / mL (40uL CD40L, 50u / mL IL-4) o No feeding on Day 5. Fully harvest, count, and reculture in fresh media Day 7 / 10. Final manipulation on Day 14.

[0170] Results: Results of these expansions are shown in the below table. As shown, among the experimental groups, the 12 U / mL CD40L with 50 lU / mL IL-4 provided the greatest fold expansion with preserved phenotype of expanded B cells. Additionally, the MB protocol did have the greatest fold expansion; however, in terms of absolute cells expanded all of our protocols provided a greater number of absolute cells expanded. This was likely a result of the higher seeding density utilized at the initiation of the culture, evidenced by the greater fold expansion of our experimental groups as compared to the MB protocol at Day 7. Importantly, viability and phenotype were maintained in these higher concentrated cultures.

[0171] Gated on lymphocytes, L / D, CD 19+

[0172] Conclusion: Based on these results, in order to cultivate large numbers of expanded B cells a higher seeding density is required at the initiation of culture as it promotes greater expansion in the first week of culture. Most importantly, the higher seeding density did not impact phenotype or viability of the final product. Final Optimized Protocol (14 Day)

[0173] OTHER EMBODIMENTS 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. 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. 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. 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.

[0174] Embodiment 5. The method of any preceding embodiment, wherein the period of time is 14 days.

[0175] Embodiment 6. The method of any preceding embodiment, wherein the expanded population of B cells comprises at least 5xl06or at least 10xl06B cells.

[0176] 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.

[0177] 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.

[0178] Embodiment 9. The method of any preceding embodiment, wherein the concentration of IL-4 is maintained at 20-60 ng / mL or 200-400 lU / mL.

[0179] 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.

[0180] 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.

[0181] Embodiment 12. The method of any preceding embodiment, wherein the concentration of serum is maintained at 5-15% v / v.

[0182] 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. Embodiment 14. The method of any preceding embodiment, wherein the serum is AB serum, optionally human AB serum.

[0183] Embodiment 15. The method of any preceding embodiment, wherein the serum is heat-inactivated serum.

[0184] Embodiment 16. The method of any preceding embodiment, wherein the concentration of CD40L is maintained at 0.1-1 mg / mL.

[0185] Embodiment 17. The method of any preceding embodiment, wherein the CD40L is a multimeric CD40L.

[0186] Embodiment 18. The method of embodiment 17, wherein the multimeric CD40L is soluble 4-trimer CD40L.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] Embodiment 22. The method of any preceding embodiment, wherein the population of B cells was obtained from a human subject.

[0191] 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. 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.

[0192] 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.

[0193] 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.

[0194] Embodiment 27. The method of any preceding embodiment, wherein the population of cells are further cultured in the presence of cyclosporine.

[0195] 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.

[0196] 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.

[0197] Embodiment 30. The method of any preceding embodiment, wherein the bioreactor comprises a gas permeable rapid expansion cell culture membrane.

[0198] Embodiment 31. The method of any preceding embodiment, wherein the population of B cells is a population of human B cells.

[0199] Embodiment 32. The method of embodiment 31, wherein the population of human B cells was isolated from whole blood.

[0200] Embodiment 33. The method of embodiment 32, wherein the population of human B cells was isolated using a microbead. Embodiment 34. The method of embodiment 33, wherein the microbead comprises an antibody, optionally an anti-CD19 antibody.

[0201] Embodiment 35. A composition comprising a population of expanded B cells produced according to the method of any one of embodiments 1-34.

[0202] Embodiment 36. 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.

[0203] Embodiment 37. The composition of embodiment 35 or 36, wherein at least 20% of B cells in the cell population express 4-1BBL.

[0204] Embodiment 38. The composition of embodiment 35 or 36, wherein 20% to 95% of B cells in the cell population express 4-1BBL.

[0205] Embodiment 39. The composition of any one of embodiments 35-38, wherein the population of cells comprises at least 75 million cells, at least 100 million cells, at least 150 million cells, or at least 200 million cells.

[0206] Embodiment 40. The composition of any one of embodiments 35-39, wherein the population of cells comprises no more than 1,000 million cells, no more than 750 million cells, or no more than 500 million cells.

[0207] Embodiment 41. 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 20% of B cells in the cell population express 4-1BBL. Embodiment 42. The composition of embodiment 41, wherein the population of cells is capable of expanding CD8+ T cells.

[0208] Embodiment 43. The composition of embodiment 41 or 42, wherein the population of cells is capable of promoting GzmB expression in CD8+ T cells.

[0209] Embodiment 44. The composition of any one of embodiments 35-43, wherein at least 97%, at least 98%, or at least 99% of the cells in the population are CD19+B cells.

[0210] Embodiment 45. The composition of any one of embodiments 35-44, wherein at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population express HLA-DR.

[0211] Embodiment 46. The composition of any one of embodiments 35-45, 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.

[0212] Embodiment 47. The composition of any one of embodiments 35-46, 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.

[0213] Embodiment 48. The composition of any one of embodiments 35-47, wherein no more than 2%, no more than 1%, or no more than 0.5% of the cells in the population are T cells.

[0214] Embodiment 49. The composition of any one of embodiments 35-48, wherein the cells in the population are human cells.

[0215] Embodiment 50. The composition of any one of embodiments 35-49, wherein the population of cells was expanded from a cell population obtained from a patient.

[0216] Equivalents and Scope 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 configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations 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.

[0217] 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.

[0218] 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.

[0219] 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.”

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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 B cells comprising: seeding a population of B cells at a density of 400,000 to 1,000,000 cells / mL, and culturing in a bioreactor a population of B cells in the presence of interleukin (IL-4), 5-15% v / v serum, and CD40 ligand (CD40L) for a period of time to produce an expanded population of B cells.

2. A method of producing an expanded population of B cells comprising: culturing in a bioreactor a population of B cells in the presence of 20 - 60 lU / mLInterleukin-4 (IL-4), 5-15% v / v serum, and 10 - 30 lU / mL CD40 ligand (CD40L) for a period of time to produce an expanded population of B cells.

3. The method of claim 1, wherein the period of time is 12-25 days, optionally 12-21 days or 12-16 days.

4. The method of any claim 1 or claim 3, wherein the period of time is at least 12 days, at least 13 days, at least 14 days, or at least 21 days.

5. The method of any one of claims 1-4, wherein the period of time is 14 days or 21 days.

6. The method of any one of claims 1-5, wherein the population of B cells comprises at least 5xl06or at least 10xl06B cells.

7. The method of any one of claims 1-6, wherein at least 90%, at least 95%, or at least 98% of the expanded population of B cells are B cells.

8. The method of any one of claims 1-7, 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.

9. The method of any one of claims 1 or 7-8, the population of B cells is cultured in the presence of 50 lU / mL IL-4.

10. The method of any one of claims 1-9, wherein the population of B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum.

11. The method of any one of claims 1-10, wherein the serum is AB serum, optionally human AB serum.

12. The method of any one of claims 1-11, wherein the serum is heat-inactivated serum.

13. The method of any one of claims 1-12, wherein the population of B cells is cultured in the presence of 10 - 40 U / mL CD40L.

14. The method of any one of claims 1-12, wherein the concentration of CD40L is maintained at 10 - 40 U / mL.

15. The method of any one of claims 1-12, wherein the population of B cells is cultured in the presence of 20 - 30 U / mL CD40L; optionally, the population of B cells is cultured in the presence of 12 U / mL or about 12 U / mL CD40L.

16. The method of any one of claims 1-12, wherein the population of B cells is cultured in the presence of 24 U / mL or about 24 U / mL CD40L.

17. The method of any one of claims 1-16, wherein the CD40L is a multimeric CD40L.

18. The method of claim 17, wherein the multimeric CD40L is soluble 4-trimer CD40L.

19. The method of any one of claims 1-18, wherein the population of B cells is stimulated with CD40L on days 0, 7, and 9 of the period of time; optionally, wherein the period of time is 14 days.

20. The method of any one of claims 1-19, wherein the population of B cells is stimulated with IL-4 on days 0, 5, 7, 9; optionally, day 12 of the period of time; optionally, wherein the period of time is 14 days or 21 days.

21. The method of any one of claims 1-19, 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.

22. The method of any one of claims 1-21, wherein the method comprises seeding the population of B cells at a density of about 500,000 cells / mL prior to the culturing step.

23. The method of any one of claims 1-22, wherein the population of B cells is obtained from a human subject.

24. The method of any one of claims 1-23, wherein the population of B cells is cultured in the presence of a cell media, optionally a Good Manufacturing Practice (GMP) grade cell media.

25. The method of claim 24, wherein the cell media is not removed from the population of B cells for the entire duration of the period of time.

26. The method of claim 24, 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.

27. The method of any one of claims 24-26, wherein the population of B cells is supplemented with growth media during the period of time, optionally every 2-4 days during the period of time.

28. The method of claim 27, wherein the population of B cells is supplemented with growth media on days 0, 5, 7; optionally, days 9 and 12 of the period of time.

29. The method of any one of claims 1-27, wherein the population of B cells are further cultured in the presence of cyclosporine.

30. The method of any one of claims 1-29, wherein the bioreactor comprises a gas permeable rapid expansion cell culture membrane.

31. The method of any one of claims 1-30, wherein the population of B cells is a population of human B cells.

32. The method of claim 31, wherein the population of human B cells is isolated from whole blood.

33. The method of claim 32, wherein the population of human B cells is isolated using a microbead.

34. The method of claim 33, wherein the microbead comprises an antibody, optionally an anti-CD19 antibody.

35. A composition comprising an expanded population of B cells comprising at least 50 million cells, wherein: at least 95% of the expanded population of B cells are CD19+B cells, at least 90% of the expanded population of B cells express HLA-DR, at least 75% of the expanded population of B cells express CD80; and at least 75% of the expanded population of B cells express CD86.

36. The composition of claim 35, wherein the expanded population of B cells comprises at least 15 million cells, at least 20 million cells, at least 25 million cells, at least 30 million cells, at least 75 million cells, at least 100 million cells, at least 150 million cells, at least 200 million cells, or 20 million - 35 million cells.

37. The composition of any one of claims 35-36, wherein the expanded population of B cells comprises no more than 1,000 million cells, no more than 750 million cells, or no more than 500 million cells.

38. A composition comprising an expanded population of B cells comprising at least 25 million cells, wherein at least 95% of the expanded population of B cells are CD19+B cells, at least 90% of the expanded population of B cells express HLA-DR, at least 75% of the expanded population of B cells express CD80; at least 75% of the expanded population of B cells express CD86; and at least 20% of the expanded population of B cells express 4-1BBL.

39. The composition of claim 38, wherein the population of cells is capable of expanding CD4+T cells.

40. The composition of any one of claims 35-39, wherein the expanded population of B cells comprises at least 97%, at least 98%, or at least 99% CD19+B cells.

41. The composition of any one of claims 35-40, wherein the expanded population of B cells comprises at least 95%, at least 97%, at least 98%, or at least 99% HLA-DR+B cells.

42. The composition of any one of claims 35-41, wherein the expanded population of B cells comprises 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% CD80+B cells.

43. The composition of any one of claims 35-42, wherein the expanded population of B cells comprises 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% CD86+B cells.

44. The composition of any one of claims 35-43, wherein the expanded population of B cells comprises no more than 2%, no more than 1%, or no more than 0.5% of T cells.

45. The composition of any one of claims 35-44, wherein the expanded population of B cells are human B cells.

46. The composition of any one of claims 35-45, wherein the expanded population of B cells was expanded from a population of B cells obtained from a patient.

Images