B cell production and expansion

Abstract

The present disclosure provides a method for producing and expanding a population of B cells (e.g., human B cells for clinical application). This expanded population can be used to produce a B cell vaccine containing B cells that express 4-1BBL and can promote tumor cell death.

Description

Technology Field

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 590,202 filed on October 13, 2023, under 35 USC § 119(e), the entire contents of which are incorporated herein by reference.

[0003] Instructions regarding federally funded research

[0004] The present invention was made with government support under approval numbers R37CA258426 and 2P50CA221747 granted by the National Institutes of Health. The government holds specific rights to the present invention. Background Technology

[0005] There are many types of antigen-presenting cells (APCs) that can be used in clinical applications. Although classical APCs are dendritic cells and macrophages, the role and utility of activated B cells as APCs have been increasingly recognized. Under steady-state conditions, naive B cells are poor APCs. Furthermore, their numbers are too small to be of any practical clinical use. However, upon the acquisition and upregulation of co-receptors through activation, B cells become effective APCs.

[0006] Furthermore, B cells are important role factors in brain tumor immunity, capable of providing both regulatory and anti-tumor functions. B cells start in a naive state, and upon BCR and cytokine stimulation, non-switched IgM within the embryonic center +They undergo differentiation into memory B-cells. Further activation leads to isomorphic switching and development into switched memory B-cells, which can subsequently mature into plasmablasts and plasma cells (the most activated B-cell state). The functions of anti-tumor B-cells involved in immune checkpoint blocking efficacy include differentiation into plasmablasts and the subsequent production of tumor-reactive antibodies, as well as T-cell activation via antigen presentation or complement.

[0007] The present disclosure provides a method for producing and expanding a population of B cells that can be used to produce a B cell vaccine containing B cells that express 4-1BBL and can promote tumor cell death, at least in part, and a composition thereof.

[0008] In some embodiments, 4-1BBL + A method for producing an expanded population of B cells is provided.

[0009] In some embodiments, 4-1BBL + A method for producing an expanded population of B cells is (i) 4-1 BBL in a bioreactor + A population of B cells was cultured for a certain period in the presence of interleukin-4 (IL-4), 5-15% v / v serum, and CD40 ligand (CD40L) to produce 4-1 BBL + It includes a step of producing an expanded population of B cells.

[0010] In some embodiments, 4-1BBL + A population of B cells is not cultured in the presence of BAFF.

[0011] In some embodiments, 4-1BBL cultured in step (i) + B cell population CD19 in bioreactor + A population of B cells is prepared by culturing them in the presence of B-cell activating factor (BAFF) and CD40L.

[0012] In some embodiments, CD19 +A population of B cells is cultured in the presence of 50-200 nM, 50-150 nM, or 50-100 nM BAFF.

[0013] In some embodiments, CD19 + A population of B cells is cultured in the presence of 100 nM BAFF.

[0014] In some embodiments, CD19 + A population of B cells is cultured in the presence of 100 nM recombinant human BAFF.

[0015] In some embodiments, CD19 + B cell populations are not cultured in the presence of IL-4.

[0016] In some embodiments, CD19 + B cell populations and 4-1BBL + Populations of B cells are cultured in the presence of different amounts of CD40L.

[0017] In some embodiments, CD19 + B cell populations and 4-1BBL + A population of B cells is cultured in the presence of an equal amount of CD40L.

[0018] In some embodiments, CD19 + B cell populations and 4-1BBL + A population of B cells is cultured in the presence of 5-20 U / mL CD40L or 5-15 U / mL CD40L.

[0019] In some embodiments, CD19 + B cell populations and 4-1BBL + A population of B cells is cultured in the presence of 8-12 U / mL CD40L.

[0020] In some embodiments, CD19 + A population of B cells is cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L.

[0021] In some embodiments, 4-1BBL+ A population of B cells is cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L.

[0022] In some embodiments, CD19 + A population of B cells is cultured in the presence of 8 U / mL CD40L or approximately 8 U / mL CD40L, and 4-1BBL + A population of B cells is cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L. In some embodiments, 4-1BBL + A population of B cells is cultured in the presence of 20-400 U / mL IL-4, 20-300 U / mL IL-4, 20-200 U / mL IL-4, 40-400 U / mL IL-4, 40-300 U / mL IL-4, 40-200 U / mL IL-4, 40-100 U / mL IL-4, 40-60 IU / mL IL-4, 20-100 U / mL IL-4, 20-60 U / mL IL-4, 50 U / mL IL-4, 30-60 ng / mL IL-4, or 40-60 ng / mL IL-4.

[0023] In some embodiments, the method is CD19 + 4-1BBL from a population of B cells + It additionally includes isolating B cells.

[0024] In some embodiments, the duration is at least 4 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 21 days.

[0025] In some embodiments, the duration is 12 to 14 days.

[0026] In some embodiments, 4-1BBL in (i) + The expanded population of B cells is at least 5x10 6 Dogs or at least 10x10 6 It includes dog B cells.

[0027] In some embodiments, at least 90%, at least 95%, or at least 98% of the cells of the expanded population are 4-1BBL + It is a B cell.

[0028] In some embodiments, 4-1BBL + The population of B cells is at least 5x10 6 Dogs or at least 10x10 6 It includes dog B cells.

[0029] In some embodiments, 4-1BBL + The extended population of B cells does not contain detectable amounts of non-B cells, and optionally here 4-1BBL + The expanded population of B cells does not contain a detectable amount of T cells.

[0030] In some embodiments, the serum concentration is maintained at 5-15% v / v for a certain period.

[0031] In some embodiments, CD19 + A population of B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum.

[0032] In some embodiments, the serum is AB serum, optionally human AB serum.

[0033] In some embodiments, the serum is heat-inactivated serum.

[0034] In some embodiments, CD40L is a multimeric CD40L.

[0035] In some embodiments, the polymeric CD40L is a soluble 4-trimeric CD40L, optionally where the trimer is linked by surfactant protein D.

[0036] In some embodiments, CD19 + A population of B cells is stimulated with CD40L at least once on days 0–4 of a certain period and / or 4–1 BBL + A population of B cells is stimulated with CD40L at least once on days 4–11 of a certain period.

[0037] In some embodiments, the method is CD19 before the culture step. + It includes seeding a population of B cells at a density of 400,000 to 800,000 cells / mL.

[0038] In some embodiments, the method is CD19 before the culture step. + It involves seeding a population of B cells at a density of about 750,000 cells / mL.

[0039] In some embodiments, the method is 4-1BBL before the culture step. + It includes seeding a population of B cells at a density of 100,000 to 800,000 cells / mL.

[0040] In some embodiments, the method is 4-1BBL before the culture step. + It includes seeding a population of B cells at a density of about 250,000 to 500,000 cells / mL.

[0041] In some embodiments, CD19 + The population of B cells is isolated and expanded from the population of B cells obtained from human subjects.

[0042] In some embodiments, CD19 + B cell populations and 4-1BBL + A population of B cells is cultured in the presence of cell medium, optionally Good Manufacturing Practice (GMP) grade cell medium. In some embodiments, the same cell medium is used throughout the culture steps. In some embodiments, the cell medium is HSC-Brew GMP medium containing 10% human AB serum.

[0043] In some embodiments, CD19 + Culture of B cell populations and / or 4-1 BBL + 20%, 10%, 5%, or 3% or less of the cell medium is removed from the culture of a population of B cells.

[0044] In some embodiments, CD19+ B cell population and / or 4-1BBL + The population of B cells is supplemented with growth medium for a certain period, at any time on the 0th, 4th, and / or 7th day of the period.

[0045] In some embodiments, CD19 + B cell population and / or 4-1BBL + A population of B cells is further cultured in the presence of cyclosporine.

[0046] In some embodiments, CD19 + B cells and / or 4-1BBL + A population of B cells is supplemented with IL-4 for a set period, at any time every 2-4 days and / or on the 5th, 7th, 9th and / or 12th days of the set period.

[0047] In some embodiments, a population of 4-1BBL cells is supplemented with IL-4 on the 5th, 7th, 9th and / or 12th days of a period.

[0048] In some embodiments, the bioreactor includes a gas-permeable rapid-expanding cell culture membrane.

[0049] In some embodiments, the CD19+ B cell population is a population of human B cells.

[0050] In some embodiments, a population of human B cells is isolated from whole blood, optionally, the population of human B cells is isolated using micro beads, wherein the micro beads comprise antibodies, optionally anti-CD19 antibodies.

[0051] In some embodiments, the B cell population is not treated with IFN-γ.

[0052] In some embodiments, 4-1BBL + A composition comprising an extended population of B cells is provided.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0066] In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the population express CD86.

[0067] In some embodiments, 2% or less, 1% or less, or 0.5% or less of the cells in the population are T cells.

[0068] In some embodiments, the composition does not contain feeder cells.

[0069] In some embodiments, the cell population was expanded from a cell population obtained from a patient, optionally where the patient has cancer and the cancer is a glioma.

[0070] In some embodiments, a method for producing a B cell vaccine is provided.

[0071] In some embodiments, a method for producing a B cell vaccine comprises the following steps:

[0072] (i) CD19 in the bioreactor +A step of producing an expanded population of CD19+ B cells by culturing a population of B cells in the presence of B-cell activating factor (BAFF), serum, and CD40 ligand (CD40L);

[0073] (ii) CD19 in (i) + 4-1BBL from an expanded population of B cells + Step of isolating B cells;

[0074] (iii) 4-1BBL isolated from (ii) in the bioreactor + B cells were cultured in the presence of interleukin-4 (IL-4) and CD40L to produce 4-1BBL + A step of producing an expanded population of B cells, and

[0075] (iv) 4-1BBL from (iii) + An expanded population of B cells is incubated with a tumor lysate containing at least one tumor antigen to produce 4-1BBL containing at least one tumor antigen. + A step of producing an expanded population of B cells and thereby producing a B cell vaccine.

[0076] In some embodiments, CD19 in (i) + A population of B cells is cultured for 0 to 4 days.

[0077] In some embodiments, the isolated 4-1BBL in (iii) + B cells are cultured for 4 to 11 days.

[0078] In some embodiments, CD19 in (i) + A population of B cells is cultured in the presence of 100 nM BAFF or 100 ng / mL BAFF and 8 U / mL CD40L or about 8 U / mL CD40L.

[0079] In some embodiments, the isolated 4-1BBL in (iii) +B cells are cultured in the presence of 50 U / mL or about 50 U / mL IL-4 and 12 U / mL or about 12 U / mL CD40L.

[0080] In some embodiments, CD19 in (i) + A population of B cells is isolated from a population of B cells using micro beads, wherein the micro beads contain antibodies, optionally anti-CD19 antibodies.

[0081] In some embodiments, the isolated 4-1BBL in (ii) + B cells are isolated from an expanded population of CD19+ B cells using a labeled anti-4-1BBL antibody.

[0082] In some embodiments, at least 25% growable 4-1BBL + A B cell vaccine containing an expanded population of B cells is provided.

[0083] In some embodiments, the B cell vaccine is 25%–75% viable 4-1BBL + It includes an expanded population of B cells, which are B cells. In some embodiments, the B cell vaccine contains 40%–60% viable 4-1BBL + It includes an extended population of B cells, which are B cells.

[0084] In some embodiments, the B cell vaccine includes an antigen, optionally additionally a tumor antigen.

[0085] In some embodiments, the tumor antigen is from a tumor lysate containing the tumor antigen, optionally, where the tumor antigen is associated with glioblastoma.

[0086] In some embodiments, CD40 agonists are additionally included.

[0087] In some embodiments, a method for treating a disease or disorder in a human subject is provided, comprising administering a B cell vaccine.

[0088] In some embodiments, a method for treating a disease or disorder in a human subject includes administering the B cell vaccine disclosed herein.

[0089] In some embodiments, the disease or disorder is cancer.

[0090] In some embodiments, the cancer is brain cancer; optionally, the cancer is glioblastoma.

[0091] In some embodiments, a method comprising administering a B cell vaccine to a human subject is disclosed.

[0092] In some embodiments, the method comprises administering the B cell vaccine disclosed herein to a human subject.

[0093] In some embodiments, the human subject has cancer.

[0094] In some embodiments, the human subject has brain cancer, optionally glioblastoma.

[0095] In some embodiments, the method further comprises administering a therapeutic agent, optionally wherein the therapeutic agent is an immune checkpoint inhibitor.

[0096] In some embodiments, a method for producing a B cell vaccine is provided.

[0097] In some embodiments, a method for producing a B cell vaccine comprises the following steps:

[0098] (i) 4-1BBL according to the method of any one of paragraphs 1 through 38 + A step of producing an expanded population of B cells; and

[0099] (ii) 4-1BBL in (i) + A step of incubating an expanded population of B cells with a tumor lysate containing at least one tumor antigen.

[0100] In some embodiments, the tumor antigen in (ii) is associated with glioblastoma.

[0101] Each limitation of the present invention may encompass various embodiments of the present invention. Accordingly, it is expected that each limitation of the present invention involving any one element or combination of elements may be included in each aspect of the present invention. The present invention is not limited to the details of the composition and arrangement of components presented in the following description or illustrated in the drawings in its application. Other embodiments of the present invention are possible and may be practiced or performed in various ways. Furthermore, phrases and terms used herein are for illustrative purposes only and should not be construed as limiting. The use of "including," "comprising," "having," "containing," "accompanying," and variations thereof is intended to encompass additional items as well as the items listed thereafter and their equivalents. Brief explanation of the drawing

[0102] The following drawings form part of this specification and are included to further demonstrate specific aspects of the disclosure, which may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. For clarity, not all components may be indicated in all drawings. It should be understood that the data illustrated in the drawings does not limit the scope of the disclosure in any way. Figure 1 provides a graph showing the total number of B cells expanded in the presence of various concentrations of IL-4. Figures 2a-2d provide figures showing the total number of expanded B cells and the expression of associated phenotypic markers according to different B cell isolation protocols. Figure 3 provides a graph showing the expansion of B cell populations obtained using different isolation and culture protocols. Figure 4 provides a graph showing the ability of B cell populations obtained from different isolation and culture protocols to express phenotypic markers. Figures 5a-5b provide graphs showing the ability of a B cell population to expand under bioreactor culture conditions. Figures 6a-6c provide a diagram showing the ability of an expanded B cell population to express a phenotypic marker under bioreactor culture conditions. Figures 7a-7b provide representative flow cytometry data from a single B cell expansion experiment. FIGS. 8-9 provides an exemplary method of B cell expansion of the present disclosure. Figure 10 provides an overview of the development of a tumor lysate protocol and the optimization of tumor lysate production under GMP conditions. Figures 11a and 11b provide the analysis of the cellular components of BVax and the limitations identified in the original protocol. Figure 11a illustrates the cellular composition of BVax. Figure 11b addresses the limitations faced in the original protocol, including low cell counts. Factors contributing to low cell counts include the downregulation of 4-1BBL during the storage and transport of Leucopac samples at 4°C, as well as immunodeficient patients presenting with low B cell counts. Figures 12a–12f provide an overview of BVax components, CD40L titration, IL4 and cell density optimization, and B cell expansion. Figure 12a shows the components of BVax, while Figures 12b and 12c exemplify small-scale and large-scale CD40L titrations, respectively. Figure 12d provides data on IL4 optimization, and Figure 12e focuses on cell density optimization. The conclusions of these optimizations suggest that reducing CD40L from 24U per mL to 8U–12U and lowering IL4 from 360U per mL to 50U results in cost savings exceeding 80%. Additionally, cell density was 0.25 x 10⁶ per mL throughout the expansion. 6 It can be recalibrated, and G-Rex products can be utilized while increasing the frequency of fresh medium replacement. Figures 13a–13c provide details regarding the components of BVax and issues concerning the isolation of 4-1BBL+ cells. Figure 13a shows the components of BVax, while Figure 13b presents the original objective for 4-1BBL+ isolation. Figure 13c details unsuccessful attempts at 4-1BBL+ isolation, and Figure 13d provides cell viability staining using 0.4% trypan blue solution from Countess, illustrating the problems encountered during 4-1BBL+ isolation. These data suggest that while biotinylated antibodies successfully select cells, the viability of 4-1BBL+ cells after selection is less than 20%. Additionally, CD19+ B cells expand and become vulnerable after a 7–12 day expansion period, and high pressure in the magnetic column contributes to membrane rupture. Figure 14 provides a schematic diagram of the principle of B cell isolation. Figure 15 provides a schematic diagram of daily monitoring of microbead attachment to B cells. Figure 16 provides insights into limiting B cell growth and vulnerability during the expansion process. Figure 17 provides a schematic diagram of a 4-day priming culture using IL-4 or BAFF. FIG. 18 is the 4-1BBL of the 4th day using BAFF versus IL-4. + Provides a comparative analysis of B cell isolation. Fig. 19 is 4-1BBL + Provides data showing the effect of IL-4 on cell recovery. Fig. 20 shows a 4-1BBL using BAFF. + Provides data showing increased cell recovery. Fig. 21 is 4-1BBL + Provides results of B cell expansion. Figure 22 provides a schematic diagram illustrating a newly developed protocol. Figure 23 provides a summary of the challenges faced during the GMP optimization of BVax. Specific details for implementing the invention

[0103] Previous studies have demonstrated that B cells can be isolated and efficiently expanded. The inventors of this disclosure recognized that currently available methods and commercially available kits for B cell expansion are limited by their low scale (or impracticality for large-scale use), 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, which can limit their clinical use. Accordingly, the inventors of this disclosure identified a novel method for B cell expansion capable of providing a higher yield of large-scale B cells suitable for clinical application with high reproducibility. The novel approach described herein, in some embodiments, further provides the ability for B cells to grow more easily and reliably in culture (compared to other APCs), the ability for B cells to expand to the number required for clinical application, and the ability to be compatible with Good Manufacturing Practice (GMP), and does not require the use of transfected cells (e.g., which raise regulatory concerns under the Federal Drug Administration). The present disclosure provides that, in some embodiments, the disclosed method can be used to produce a B cell vaccine (e.g., a cancer vaccine) derived from an expanded B cell population produced using the expansion and culture protocol of the present disclosure. Such a B cell vaccine may be useful for treating cancer in human patients requiring treatment for cancer. The disclosure described herein further provides a population of cells produced using the method provided herein.

[0104] In some embodiments, the method described herein may be used to stimulate B cells for expansion by utilizing multimerized CD40L in the presence of B cell growth factor, utilizing GMP reagents to ensure that a desired B cell surface marker is expressed on the B cells, and subsequently to expand B cells incubated with an antigen for which an immune response is desired (e.g., proteins and protein fragments from cancer cells, autoantigens, infectious agents, etc.).

[0105] The methods described herein involve, in some embodiments, specific variations in the number, concentration, or purity of starting cells (e.g., purified B cells versus whole peripheral blood mononuclear cells (PMBCs)), culture media and supplements, stimulants, restimulation requirements, cytokine combinations, culture vessels, and duration of extended culture. In some embodiments, PMBCs are isolated from whole blood (leukocyte apheresis products may also be used for further extended culture). In some embodiments, B cells are isolated using a pull-down reagent (e.g., anti-CD19 micro beads, e.g., GMP-grade anti-CD19 micro beads). In some embodiments, B cells are purified (e.g., from PMBCs) using a cell sorting platform (e.g., CliniMACS Plus closed system).

[0106] Method of the present disclosure

[0107] Some aspects of the present disclosure relate to populations of B cells (e.g., CD19 + Population of B cells, 4-1BBL + Method for producing a population of B cells) and / or an expanded population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + Provides a method for producing an expanded population of B cells.

[0108] In some embodiments, an extended population of B cells (e.g., CD19+ Extended population of B cells, 4-1BBL + A method for producing an expanded population of B cells, etc., is (i) 4-1 BBL in a bioreactor + A population of B cells was cultured for a certain period in the presence of interleukin-4 (IL-4), 5-15% v / v serum, and CD40L to produce 4-1 BBL + It includes the step of producing an expanded population of B cells. In some embodiments, an expanded population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + Methods for producing an expanded population of B cells include a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + It comprises culturing a population of B cells in the presence of cell medium, one or more cytokines, CD40 ligands (CD40L), vitamins, and / or growth factors. In some embodiments, culturing a population of B cells in the presence of cell medium or any molecule (e.g., one or more cytokines, CD40 ligands (CD40L), vitamins, and / or growth factors) involves adding said cell medium or any molecule to the population of B cells at a given time. In some embodiments, a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + Culturing an expanded population of B cells in cell medium or in the presence of any molecule (e.g., one or more cytokines, CD40 ligand (CD40L), vitamins and / or growth factors) results in a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL +It involves maintaining any molecule or a specific amount or concentration of the cell medium for a certain period of time in a population of B cells.

[0109] An extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + A method for producing an expanded population of B cells comprises, in some embodiments, the following steps: (a) adding cell medium, 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) adding IL-4 (and optionally a growth medium) to the B cell population on day 5; (c) adding CD40L and IL-4 (and optionally a growth medium) to the B cell population on day 7; (d) adding CD40L and IL-4 (and optionally a growth medium) to the B cell population on day 9; (e) adding IL-4 (and optionally a growth medium) to the B cell population on day 12; and (f) collecting the B cell population on day 14, thereby producing an expanded B cell population (e.g., CD19 + Extended population of B cells, 4-1BBL + The stage of producing an expanded population of B cells.

[0110] In some embodiments, one or more cytokines include 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, one or more cytokines include interleukin-4 (IL-4). In some embodiments, one or more cytokines include interleukin-4 (IL-4) and at least one of IL-2, IL-6, IL-7, IL-12, and / or IL-15. In some embodiments, one or more cytokines are added to a population of B cells at the start of their culture and / or at intervals during the culture (e.g., on days 0, 5, 7, 9, 12, 14, 16, and / or 18 of the culture period). In some embodiments, one or more cytokines are useful for stimulating, promoting, and / or activating B cell growth or survival.

[0111] In some embodiments, one or more cytokines are B cell populations (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 10-100 ng / mL. In some embodiments, one or more cytokines are added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells at concentrations 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 one or more cytokines is applied to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + Within a population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + In an expanded population of B cells) it is maintained 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 certain period (e.g., period of cell culture and expansion). In some embodiments, one or more cytokines are present in a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at concentrations 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 a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells at concentrations 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 applied to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + Within a population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + Within an expanded population of B cells) it is maintained 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 certain period (e.g., culture period). In some embodiments, IL-4 is maintained in a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at concentrations 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.

[0112] In some embodiments, IL-4 is a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +5 IU / mL or about 5 IU / mL, 10 IU / mL or about 10 IU / mL, 15 IU / mL or about 15 IU / mL, 20 IU / mL or about 20 IU / mL, 25 IU / mL or about 25 IU / mL, 30 IU / mL or about 30 IU / mL, 35 IU / mL or about 35 IU / mL, 40 IU / mL or about 40 IU / mL, 45 IU / mL or about 45 IU / mL, 50 IU / mL or about 50 IU / mL, 60 IU / mL or about 60 IU / mL, 70 IU / mL or about 70 IU / mL, 80 IU / mL or about 80 IU / mL, 90 IU / mL or about 90 IU / mL, in an expanded population of B cells It is added at a concentration of 100 IU / mL or about 100 IU / mL.

[0113] In some embodiments, IL-4 is a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +expanded population of B cells) at 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 IU / mL, 40 - 400 U / mL, 40 - 300 U / mL, 40 - 200 U / mL, 40 - 100 U / mL, 40 - 60 IU / mL, 20 - 50 U / mL, 100 - 1000 IU / mL, It is added at concentrations of 100 - 750 IU / mL, 100 - 500 IU / mL, 100 - 250 IU / mL, 200 - 1000 IU / mL, 200 - 750 IU / mL, 200 - 600 IU / mL, 200 - 500 IU / mL, or 250 - 400 IU / mL.

[0114] In some embodiments, IL-4 is used for a population of B cells (e.g., a population of CD19+ B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 20–400 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 20–300 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded 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 (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded 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 (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded 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 (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 20–50 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 30–400 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 30–300 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 +A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 30–200 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 30–100 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., a population of CD19+ B cells) and / or an expanded population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 30–60 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 30–50 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 30–70 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells at a concentration of 40–400 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 40–300 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 40–200 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 40–100 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 40–60 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 40–50 U / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 +A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 100 to 1,000 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 100–750 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 100–500 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 100–250 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 200–1000 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells at a concentration of 200–750 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 200–600 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 200–500 IU / mL. In some embodiments, IL-4 is added to a population of B cells (e.g., CD19 + In a population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 250–400 IU / mL. In some embodiments, the concentration of IL-4 is such that the population of B cells (e.g., CD19) + Population of B cells, 4-1BBL + Within a population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + An expanded population of B cells) is maintained at a concentration of 5 IU / mL, 10 IU / mL, 15 IU / mL, 20 IU / mL, 25 IU / mL, 30 IU / mL, 35 IU / mL, 40 IU / mL, 45 IU / mL, 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, or 100 IU / mL. In some embodiments, the concentration of IL-4 is maintained in a population of B cells (e.g., CD19 +Within a population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is maintained at a concentration of 100-1000 IU / mL, 100-750 IU / mL, 200-600 IU / mL, 200-500 IU / mL, or 250-400 IU / mL for a certain period (e.g., culture period) within an expanded population of B cells.

[0115] In some embodiments, IL-4 is a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + An expanded population of B cells is added to the population at a concentration of 100 IU / mL, 200 IU / mL, 300 IU / mL, 400 IU / mL, 500 IU / mL, 600 IU / mL, 700 IU / mL, 800 IU / mL, 900 IU / mL, or 1000 IU / mL. In some embodiments, one or more cytokines (e.g., IL-4) are added to the population of B cells on day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, or day 20 of the culture period (e.g., a certain period). In some embodiments, one or more cytokines (e.g., IL-4) are used for a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells every 2 to 4 days. In some embodiments, one or more cytokines (e.g., IL-4) are added to a population of B cells on the 5th, 7th, 9th, and / or 12th days of the culture period.

[0116] In some embodiments, CD40L (e.g., multimeric CD40L) is added to a population of B cells at a concentration of 0.1-1 mg / mL. In some embodiments, CD40L is added to a 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 a 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 certain period (e.g., culture period). In some embodiments, CD40L is added to a 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.

[0117] In some embodiments, the concentration of CD40L is in a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +In an expanded population of B cells, it is maintained 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 certain period (e.g., culture period). In some embodiments, CD40L is used in a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at concentrations 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.

[0118] In some embodiments, CD40L is useful for stimulating, promoting, and / or activating B cell growth or survival. In some embodiments, CD40L is useful for B cell populations (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + CD40L is added to an expanded 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 a population of B cells on days 0, 7, 9, and / or 14 of the culture period (e.g., a certain period).

[0119] In some embodiments, CD40L (e.g., multimeric CD40L) is a population of B cells (e.g., CD19 + A population of B cells) and / or an extended population of B cells (e.g., CD19+ Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of at least 5 IU / mL. In some embodiments, CD40L is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at concentrations of 6 IU / mL, 7 IU / mL, 8 IU / mL, 9 IU / mL, 10 IU / mL, 11 IU / mL, 12 IU / mL, 13 IU / mL, 14 IU / mL, 15 IU / mL, 16 IU / mL, 17 IU / mL, 18 IU / mL, 19 IU / mL, 20 IU / mL, 25 IU / mL, 30 IU / mL, 35 IU / mL, 40 IU / mL, 45 IU / mL, or 50 IU / mL, or any range or combination thereof. In some embodiments, CD40L is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +expanded population of B cells): 5 - 100 IU / mL, 5 - 90 IU / mL, 5 - 80 IU / mL, 5 - 70 IU / mL, 5 - 60 IU / mL, 5 - 50 IU / mL, 5 - 40 IU / mL, 5 - 30 IU / mL, 5 - 20 IU / mL, 5 - 15 IU / mL, 5 - 10 IU / mL, 8 - 100 IU / mL, 8 - 90 IU / mL, 8 - 80 IU / mL, 8 - 70 IU / mL, 8 - 60 IU / mL, 8 - 50 IU / mL, 8 - 40 IU / mL, 8 - 30 IU / mL, 8 - 20 IU / mL, 8 - 15 IU / mL, 8 - 10 It is added at concentrations of IU / mL, 8 - 12 IU / mL, 8 - 100 IU / mL, 10 - 100 IU / mL, 10 - 90 IU / mL, 10 - 80 IU / mL, 10 - 70 IU / mL, 10 - 60 IU / mL, 10 - 50 IU / mL, 10 - 40 IU / mL, 10 - 30 IU / mL, 10 - 20 IU / mL, or 10 - 15 IU / mL.

[0120] In some embodiments, the concentration of CD40L is in a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +In an expanded population of B cells, it is maintained at a concentration of at least 5 IU / mL. In some embodiments, CD40L is added to a population of B cells at a concentration of 6 IU / mL, 7 IU / mL, 8 IU / mL, 9 IU / mL, 10 IU / mL, 11 IU / mL, 12 IU / mL, 13 IU / mL, 14 IU / mL, 15 IU / mL, 16 IU / mL, 17 IU / mL, 18 IU / mL, 19 IU / mL, 20 IU / mL, 25 IU / mL, 30 IU / mL, 35 IU / mL, 40 IU / mL, 45 IU / mL, or 50 IU / mL, or any range or combination thereof. In some embodiments, CD40L is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + expanded population of B cells): 5 - 100 IU / mL, 5 - 90 IU / mL, 5 - 80 IU / mL, 5 - 70 IU / mL, 5 - 60 IU / mL, 5 - 50 IU / mL, 5 - 40 IU / mL, 5 - 30 IU / mL, 5 - 20 IU / mL, 5 - 15 IU / mL, 5 - 10 IU / mL, 8 - 100 IU / mL, 8 - 90 IU / mL, 8 - 80 IU / mL, 8 - 70 IU / mL, 8 - 60 IU / mL, 8 - 50 IU / mL, 8 - 40 IU / mL, 8 - 30 IU / mL, 8 - 20 IU / mL, 8 - 15 IU / mL, 8 - 10 It is added at concentrations of IU / mL, 8 - 12 IU / mL, 8 - 100 IU / mL, 10 - 100 IU / mL, 10 - 90 IU / mL, 10 - 80 IU / mL, 10 - 70 IU / mL, 10 - 60 IU / mL, 10 - 50 IU / mL, 10 - 40 IU / mL, 10 - 30 IU / mL, 10 - 20 IU / mL, or 10 - 15 IU / mL.

[0121] In some embodiments, CD19 + B cell populations and 4-1BBL + A population of B cells is cultured in the presence of different amounts of CD40L disclosed herein. In some embodiments, CD19 + B cell populations and 4-1BBL + A population of B cells is cultured in the presence of an equal amount of CD40L as disclosed herein.

[0122] In some embodiments, CD19 + A population of B cells is cultured in the presence of 8 U / mL CD40L or approximately 8 U / mL CD40L, and 4-1BBL + A population of B cells is cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L. In some embodiments, CD19 + A population of B cells was cultured for 4 days in the presence of 8 U / mL CD40L or approximately 8 U / mL CD40L, and 4-1BBL + A population of B cells is cultured for 7 days in the presence of 12 U / mL CD40L or about 12 U / mL CD40L. In some embodiments, CD19 + B cell populations were cultured for 4 days using a low dose of CD40L (e.g., 8 U / mL) to prevent CD19+ B cell activation / expansion, and isolated 4-1BBL +B cells are cultured at a higher dose of CD40L (12 U / mL) to induce higher proliferation and activation. In some embodiments, CD40L is useful for stimulating, promoting, and / or activating B cell growth or survival. In some embodiments, CD40L is added to a 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 a population of B cells on days 0, 7, 9, and / or 14 of the culture period (e.g., a certain period).

[0123] In some embodiments, CD40L is a polymeric CD40L. In some embodiments, the polymeric CD40L comprises CD40L trimers. In some embodiments, the polymeric CD40L comprises at least two CD40L trimers. The at least two trimers may be linked, for example, by a surfactant. In some embodiments, the at least two trimers are linked by surfactant protein D. In some embodiments, the polymeric CD40L comprises at least three CD40L trimers. The at least three trimers may be linked, for example, by a surfactant. In some embodiments, the at least three trimers are linked by surfactant protein D. In some embodiments, the polymeric CD40L comprises at least four CD40L trimers. The at least four trimers may be linked, for example, by a surfactant. In some embodiments, the at least four trimers are linked by surfactant protein D. In some embodiments, the polymeric CD40L comprises two to five CD40L trimers. The two to five trimers may be linked, for example, by a surfactant. In some embodiments, the two to five trimers are linked by surfactant protein D. In some embodiments, the polymeric CD40L comprises four CD40L trimers. The four trimers may be linked, for example, by a surfactant. In some embodiments, the four trimers are linked by surfactant protein D. In some embodiments, the polymeric CD40L is a soluble 4-trimeric CD40L.

[0124] In some embodiments, serum (e.g., human serum, e.g., human AB serum) is a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells. In some embodiments, serum (e.g., human serum, e.g., human AB serum) is added to a 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 a 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.

[0125] In some embodiments, B-cell activating factor (BAFF) is a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells. In some embodiments, BAFF is recombinant human BAFF.

[0126] In some embodiments, BAFF (e.g., recombinant human BAFF) is a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +BAFF (e.g., recombinant human BAFF) is added to an expanded population of B cells at a concentration of 100 nM or about 100 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is 10 nM, about 10 nM, or at least 10 nM; 20 nM, about 20 nM, or at least 20 nM; 30 nM, about 30 nM, or at least 30 nM; 40 nM, about 40 nM, or at least 40 nM; 50 nM, about 50 nM, or at least 50 nM; 60 nM, about 60 nM, or at least 60 nM; 70 nM, about 70 nM, or at least 70 nM; 80 nM, about 80 nM, or at least 80 nM; 90 nM, about 90 nM, or at least 90 nM; 100 nM, about 100 nM or at least 100 nM; 120 nM, about 120 nM or at least 120 nM; 140 nM, about 140 nM or at least 140 nM; 160 nM, about 160 nM or at least 160 nM; 180 nM, about 180 nM or at least 180 nM; 200 nM, about 200 nM or at least 200 nM; 300 nM, about 300 nM or at least 300 nM; or 400 nM, about 400 nM or at least 400 nM; or added at a concentration of any range or combination thereof.

[0127] In some embodiments, BAFF (e.g., recombinant human BAFF) is a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells at concentrations of 10–400 nM, 50–400 nM, 10–300 nM, 50–300 nM, 50–200 nM, 50–150 nM, or 50–100 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 10–400 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 50–400 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 10–300 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +It is added to an expanded population of B cells at a concentration of 50–300 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 50–200 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 50–150 nM. In some embodiments, BAFF (e.g., recombinant human BAFF) is added to a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It is added to an expanded population of B cells at a concentration of 50-100 nM.

[0128] In some embodiments, the vitamin is a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +Vitamins are added to an expanded population of B cells. Vitamins may function as enzyme cofactors (e.g., vitamin K, selected B vitamin), 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. Vitamins to be added to a 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 B1), pyridoxine (vitamin B6), biotin (vitamin B7), pantothenate (vitamin B5), or nicotinamide (vitamin B3).

[0129] In some embodiments, cyclosporine is added to a population of cells. In some embodiments, the method comprises culturing a population of cells in the presence of cyclosporine. In some embodiments, cyclosporine functions to limit T cell growth. In some embodiments, the concentration of cyclosporine in a population of cells is maintained 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.

[0130] A population of B cells may be cultured for any reasonable period of time (e.g., any reasonable culture period). In some embodiments, a population of B cells is cultured for 10 to 25 days. In some embodiments, a 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. A population of B cells may be cultured at any reasonable temperature as known to a person skilled in the art. In some embodiments, a population of B cells is cultured at a temperature of 30 to 40°C. In some embodiments, a population of B cells is cultured at a temperature of 37°C.

[0131] Populations of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + An expanded population of B cells can be seeded at any reasonable density depending on the culture technique being used (e.g., culture on plates, suspension culture, culture in a bioreactor). In some embodiments, the method involves a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + The method comprises seeding an expanded 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 a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL+ The method comprises seeding an expanded 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 a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + It includes seeding an expanded 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.

[0132] Populations of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +The expanded population of B cells is further cultured in the presence of cell medium (also referred to as "growth medium," "expansion medium," or "culture medium"). Any reasonable cell medium known to a person skilled in the art may be used in the method described herein. In some embodiments, the method of expanding the population of B cells may be performed without replacing or removing the medium during the culture period. In some embodiments, the method of expanding the population of B cells may be performed without replacing or removing the medium during the culture period, but instead by replenishing with fresh or "new" medium during the culture period. In some embodiments, the cell medium is replaced or replenished when conditions are required (e.g., when the glucose level in the medium falls below a threshold level). In some embodiments, the cell medium is RPMI medium.

[0133] In some embodiments, the method is carried out using a bioreactor. In some embodiments, the bioreactor comprises a gas-permeable rapid-expanding cell culture membrane. The inventors have found that, in some embodiments, the use of the bioreactor enables large-scale production of expanded B cell populations. In some embodiments, the bioreactor is a GRex® 10M bioreactor (Wilson Wolf). In some embodiments, the bioreactor is a oscillating 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 the literature ["Recent Advances in the Development of Bioreactors for Manufacturing of Adoptive Cell Immunotherapies" Bioengineering 2022, 9(12), 808] (the contents of which are incorporated herein by reference).

[0134] A population of B cells (e.g., CD19) for use in the method described herein + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + An expanded population of B cells is generally human B cells. In some embodiments, the population of human B cells is isolated from whole blood. In these embodiments, whole blood contains whole peripheral blood mononuclear cells (PMBCs). In some embodiments, PMBCs are obtained from whole blood or apheresis products (e.g., obtained using a Ficoll density gradient separation). In some embodiments, B cells are obtained or isolated using micro beads containing anti-CD19 antibodies. In some embodiments, B cells are obtained or isolated using cell separation, e.g., magnetic cell separation. For magnetic cell separation, a monoclonal antibody that binds to a target (e.g., CD19) on B cells may be conjugated to magnetic particles and then separated using a magnetic matrix. In some embodiments, a monoclonal antibody that binds to a target (e.g., CD19) on B cells may be conjugated to paramagnetic iron dextran particles (e.g., MACS micro beads) and then separated using a matrix of paramagnetic spheres. Exemplary cell separation systems include, for example, the Clinimax® system.

[0135] In some embodiments, 4-1BBL + B cells use antibodies against CD19 + It is isolated from a population of B cells. In some embodiments, the antibody is labeled. In some embodiments, the label is biotin. In some embodiments, 4-1BBL +B cells are isolated using a labeling process comprising an anti-4-1BBL biotinylated antibody and anti-biotin micro beads. In some embodiments, GMP-grade Clinimax® anti-biotin micro beads are used to further label the cells or cell populations disclosed herein with magnetic beads to be detected during isolation. In some embodiments, 4-1BBL + B cells are obtained or isolated using cell separation, e.g., magnetic cell separation. For magnetic cell separation, a monoclonal antibody binding to a target on B cells (e.g., 4-1BBL) can be conjugated to magnetic particles and then separated using a magnetic matrix. In some embodiments, a monoclonal antibody binding to a target on B cells (e.g., 4-1BBL) can be conjugated to paramagnetic iron dextran particles (e.g., MACS micro beads) and then separated using a matrix of paramagnetic spheres. Exemplary cell separation systems include, for example, the Clinimax® system.

[0136] In some embodiments, a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + An extended population of B cells includes B cells, T cells, NK cells, monocytes, dendritic cells, red blood cells, white blood cells, and / or platelets. In some embodiments, a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +An extended population of B cells comprises B cells, epithelial cells, neurons, hormone-secreting cells, immune cells, secretory cells, blood cells, stromal 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 within the population of B cells are B cells. In some embodiments, the population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + An 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, the 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, the population of B cells does not contain a detectable amount of non-B cells.

[0137] In some embodiments, the method described herein produces a 2- to 20-fold expansion of B cells, which provides a sufficient number of cells for use in a clinical setting. In some embodiments, the method described herein produces 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 expansion level is determined by counting viable B cells. In some embodiments, viable B cells may be counted by cell staining with, for example, trypan blue (and optical microscopy) or 7-amino-actinomycin D, a biodye emitting at 670 nm (or a commercially available solution of 7AAD via a probe), and by flow cytometry using techniques known to those skilled in the art.

[0138] In some embodiments, a population of B cells (e.g., CD19 + Population of B cells, 4-1BBL + A population of B cells) and / or an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + A method for producing an expanded population of B cells is applied to a cryopreservation procedure. In some embodiments, the cell population is frozen after expansion. In some embodiments, the cell population is maintained in a frozen state for a certain period (e.g., at least 1, 2, 4, 8, 12 weeks or more). In some embodiments, the cell population has been previously frozen and thawed (e.g., 1, 2, 3, 4, 5 or more freeze / thaw cycles). In some embodiments, the cell population is maintained in a liquid culture medium. In some embodiments, the cell population was subcultured 1, 2, 3, 4, 5 or more times using any known method.

[0139] Aspects of the present disclosure further provide a method of using an expanded population of B cells (e.g., for clinical application). In some embodiments, an expanded population of B cells (e.g., 4-1BBL + An expanded population of B cells is used in the treatment of cancer patients to induce an effective immune response. In some embodiments, the population of B cells is expanded from cells obtained from the cancer patient to be treated. In some embodiments, the method of using expanded B cells includes 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, the method of using expanded B cells includes treating a subject with cancer using the expanded B cell population. In some embodiments, the B cells were expanded from cells obtained from a subject with cancer.

[0140] In some embodiments, an extended population of B cells (e.g., CD19 + An expanded population of B cells is 4-1BBL that can be used to promote anti-tumor immunity. + B cells are purified to isolate them. In some embodiments, an expanded population of B cells is used as a vaccine to treat tumors, including glioblastoma. In some embodiments, an expanded population of B cells (e.g., CD19 + Purify the expanded population of B cells) to obtain 4-1BBL + B cells are isolated and contacted with tumor-derived antigens. In some embodiments, an expanded population of B cells (e.g., CD19 + Purify the expanded population of B cells) to obtain 4-1BBL + B cells are isolated, incubated with a CD40 agonist, and exposed to tumor-derived antigens.

[0141] The term “anticancer composition” as used herein 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 progression of the cancer; (3) causing the tumor to regress; or (4) alleviating one or more symptoms of the cancer.

[0142] In some embodiments, an extended population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL + An expanded population of B cells is used for (i) the in vitro expansion of donor-specific Tregs (which may be used to induce tolerance in clinical organs, tissues, and cell transplants), (ii) the improvement of autoimmune diseases in humans where immunogenic antigens are known through the use of antigen-specific Tregs; or (iii) the prevention and / or cure of allergies in humans where specific protein allergens are known through the use of antigen-specific Tregs. In some embodiments, in transplant contexts, B cells from the donor or recipient of the transplant may be used without antigen incubation. For example, expanded B cells may be used as stimulants for the expansion of antigen-specific regulatory T cells (Tregs) for clinical use.

[0143] The term "cancer" is intended to encompass any cancer, neoplastic, and systemic disease characterized by the abnormal growth of cells. Cancer includes colorectal carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, Hodgkin's disease, non-Hodgkin lymphoma, rectal cancer, urological cancer, uterine cancer, oral cancer, skin cancer, gastric cancer, brain tumor, liver cancer, laryngeal cancer, esophageal cancer, breast tumor, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, Ewing sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, cervical cancer, testicular tumor, endometrial cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelium It may be selected from the group consisting of carcinoma, glioblastoma, neuroma, craniopharyngioma, Schwann cell tumor, glioma, astrocytoma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia and lymphoma, acute lymphoblastic leukemia and acute myeloid polycythemia vera, multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease, acute non-lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, childhood-aplastic acute lymphoblastic leukemia (ALL), thymic ALL, B-cell ALL, acute megakaryocytic leukemia, Burkitt lymphoma, and T-cell leukemia, small cell and large cell non-small 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 glioblastoma.

[0144] Expanded population of B cells (e.g., CD19 + Extended population of B cells, 4-1BBL +A method of administering an expanded population of B cells comprises administering an effective amount of the composition to a subject. The B cell composition may be co-administered with radiation, other chemotherapy agents, or other cancer treatments including other lymphocytes such as T cells or NK cells. Co-administration is used to indicate that the same subject may have received additional therapeutic agents in addition to the B cell composition described herein. The regimen may be administered to the subject simultaneously as separate treatments, as part of a single composition, or in any order. The regimen may be administered such that one is administered before the other with a time difference 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.

[0145] In some embodiments of the present disclosure, as a method for treating a subject having a disease or disorder, an expanded B cell population (e.g., 4-1BBL) of the B cell vaccine described herein (referred to as “Bvax” in some embodiments). + A method comprising administering an effective amount of an expanded population of B cells to a subject is provided herein. In some embodiments, the disease or disorder relates to the immune system. The disease or disorder may be cancer. Thus, in some embodiments, the subject has cancer. In some embodiments, the cancer is brain cancer, lung cancer, liver cancer, skin cancer, prostate cancer, breast cancer, blood cancer, or any other cancer.

[0146] Brain cancer can be a slower-growing, less aggressive glioma (referred to as low-grade or benign glioma) or a high-grade glioma. High-grade gliomas can include high-grade astrocytomas (36% of gliomas) and glioblastomas (GBMs; 47% of gliomas and 22% of all primary brain tumors). Many high-grade gliomas are located within the corpus callosum, basal ganglia, brainstem, and major functional cortical regions (language, motor, or visual areas). Gliomas are the most common type of primary brain tumor, accounting for approximately 80% of all malignant brain tumors. These tumors originate from glial cells, which are supporting cells that surround and support neurons in the brain. Gliomas can occur in any part of the brain or spinal cord and can be classified based on their location, cell type, and degree of malignancy. The three main types of glial cells are astrocytes, oligodendrons, and ependymocytes. Astrocytomas are the most common type of glioma, followed by oligodendrogliomas and ependymomas. In some embodiments, the patient of the present disclosure was diagnosed with a glioma occurring, for example, in the brain. In some embodiments, the glioma is an astrocytomas. In other embodiments, the glioma is oligodendrogliomas. In yet another embodiment, the glioma is ependymomas.

[0147] In some embodiments, Bvax is an expanded population of B cells (e.g., CD19 + 4-1BBL from an expanded population of B cells + It is produced by isolating B cells. In some embodiments, Bvax is produced from a population of expanded B cells (optionally, where the population of expanded B cells is 4-1 BBL). + Bvax is produced by combining a CD40 agonist with a population of expanded B cells (selected for B cells). In some embodiments, Bvax is produced by combining a population of expanded B cells (optionally, where the population of expanded B cells is 4-1 BBL). +Bvax is produced by combining a CD40 agonist and IFN-γ with a population of expanded B cells (selected for B cells). In some embodiments, Bvax is produced by combining a population of expanded B cells (optional, wherein the population of expanded B cells is 4-1 BBL). + It is produced by combining (selected for B cells) with IFN-γ. In some embodiments, Bvax is produced without using IFN-γ.

[0148] In some embodiments, Bvax is 4-1 BBL from an expanded population of B cells + It is produced by isolating B cells. In some embodiments, Bvax is produced from a population of expanded B cells (optional, wherein the population of expanded B cells is 4-1 BBL). + It is produced by combining (selected for B cells) with a tumor lysate. In some embodiments, Bvax is produced from a population of expanded B cells, 4-1 BBL + It is produced by isolating B cells. In some embodiments, Bvax is produced from a population of expanded B cells (optional, wherein the population of expanded B cells is 4-1 BBL). + It is produced by combining (selected for B cells) with a tumor lysate and a CD40 agonist.

[0149] In some embodiments involving the combination of an expanded population of B cells with a CD40 agonist and IFN-γ, B cells are first incubated with the CD40 agonist and then IFN-γ is added (e.g., 18-24 hours after the addition of the CD40 agonist).

[0150] The term “CD40 agonist” as used herein refers to a reagent that specifically binds to a CD40 molecule and induces CD40 signaling. CD40 is a transmembrane protein receptor expressed by antigen-presenting cells (APCs) and involved in the co-stimulation of immune cells. Current strategies for inducing CD40 signaling include both antibody-based and CD40 ligand-based approaches. Accordingly, in some embodiments, the CD40 agonist is selected from CD154 (i.e., a homologous ligand for CD40) and a CD40 antibody or a portion thereof capable of acting agonistly against CD40. B cells may be incubated with the CD40 agonist for 48 hours or less prior to the addition of the tumor-derived antigen. In certain embodiments, B cells are incubated with the CD40 agonist for at least 12 hours prior to the addition of IFN-γ and for as long as 48 hours.

[0151] In some embodiments, Bvax further comprises a tumor-derived antigen. In these embodiments, Bvax is produced by incubating an expanded population of B cells with the tumor-derived antigen. Any sample containing cancer cells may be used as a source of the tumor-derived antigen for the present disclosure. Suitable samples include, for example, tissue samples, tumors, tumor lysates, biopsies, and body fluids (e.g., blood, serum, plasma, sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears, saliva). Alternatively, samples may include organoids produced from cancer specimens (i.e., “tumor organoids”). In some embodiments, the tumor-derived antigen is a tumor cell lysate. In some embodiments, the tumor-derived antigen may be a single or a few polypeptides identified as containing the tumor antigen. In certain embodiments, the tumor cell lysate is derived from a subject having cancer. The term “antigen” refers to any molecule that is recognized by the immune system and capable of stimulating an immune response. "Tumor-derived antigens" are antigens that are preferentially or differentially expressed by tumor cells but are not expressed or are differentially expressed on normal, healthy cells. Therefore, by incubating B cells with tumor-derived antigens, the B cells are activated to act as more effective antigen-presenting cells and stimulate CD8+ T cells to recognize these antigens. These B cells can also produce tumor-specific antibodies capable of recognizing the tumor.

[0152] In some therapeutic method embodiments, Bvax is used to treat a subject having cancer. The method comprises administering an effective amount of the composition to the subject. The Bvax composition may be co-administered with other cancer treatments, including radiation, other chemotherapy agents, or other lymphocytes, including T cells or NK cells. Co-administration is used to indicate that the same subject may have received additional therapeutic agents in addition to the Bvax composition described herein. The regimen may be administered to the subject simultaneously as separate treatments, as part of a single composition, or in any order. The regimen 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.

[0153] The method disclosed herein may further include conventional cancer treatment regimens. In some embodiments, the method further includes administering radiation therapy to a subject. As used herein, the term “radiation therapy” refers to any method of treating solid tumors and cancers using ionizing radiation, and includes, but is not limited to, external beam radiation therapy, stereotactic radiation therapy, virtual simulation, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy, ionizing particle therapy, and radioisotope therapy.

[0154] In some embodiments, the method further comprises administering a therapeutic agent to a subject (e.g., co-administration of the therapeutic agent and a population of expanded B cells).

[0155] In some embodiments, the method further comprises administering a chemotherapeutic agent to a subject. Chemotherapeutic agents suitable for use in the method of the present invention include, but are not limited to, platinum-based agents, e.g., cisplatin, gemcitabine, and carboplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, e.g., carmustine (BCNU) and other alkylating agents; antimetabolites, e.g., methotrexate; purine analogue antimetabolites; pyrimidine analogue antimetabolites, e.g., fluorouracil (5-FU) and gemcitabine; hormonal antineoplastic agents, e.g., goserelin, leuprolide, and tamoxifen; natural antineoplastic agents, e.g., taxanes (e.g., docetaxel and paclitaxel), aldesleukin, interleukin-2, etoposide (VP-16), interferon alpha, and tretinoin (ATRA); It includes antibiotic natural antineoplastic agents, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vinca alkaloid natural antineoplastic agents, such as vinblastine and vincristine. In some embodiments, the chemotherapy agent is a triagen, such as dacarbazine, mitozolomide, or temozolomide. In some embodiments, the chemotherapy agent is temozolomide.

[0156] In some embodiments, the method further comprises administering a checkpoint inhibitor. As used herein, the term “checkpoint inhibitor” refers to a molecule that blocks or inhibits the immunosuppressive activity of a checkpoint protein. 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 its antigen-binding fragment that specifically binds to CTLA4, an anti-PD1 antibody or its antigen-binding fragment that specifically binds to PD1, an anti-PD-L1 antibody or its antigen-binding fragment that specifically binds to PD-L1, and a combination thereof.

[0157] In some embodiments, the method further comprises administering T cells to a subject. The T cells used in these methods may be obtained from a subject and then expanded and activated in vitro to enhance their immunostimulatory capacity. In certain embodiments, the T cells are CD8+ T cells. The T cells may be selected or engineered to act as an indicated antigen receptor against tumor antigens. As used herein, the term "chimeric antigen receptor T cell" refers to a genetically engineered antibody-T cell chimera comprising a chimeric antigen receptor (CAR). Techniques for chimeric antigen receptor T cell therapy are known and available in the art. For example, refer to the literature [Kenderian et al., Cancer Res. 74(22):6383-9 (2014)].

[0158] The terms "subject" and "patient" are used interchangeably and refer to any animal (e.g., mammals) that is a recipient of a specific treatment, including but not limited to humans, non-human primates, rodents, etc. For example, suitable subjects include subjects requiring cancer treatment.

[0159] As used herein, “treating” or “treatment” describes managing and caring for a subject for the purpose of combating a disease, pathological condition, or disorder. Treating includes the administration of a composition of the present invention to prevent the onset of symptoms or complications, to alleviate symptoms or complications, or to eliminate a disease, pathological condition, or disorder. Specifically, the composition disclosed herein may be used to treat cancer. Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor in a subject, reducing the progression of cancer to a more aggressive form, reducing the proliferation of cancer cells or the rate of tumor growth, killing cancer cells, reducing the metastasis of cancer cells, or reducing the likelihood of cancer recurrence in a subject. As used herein, treating a subject refers to any type of treatment that provides benefit to a subject suffering from a disease or at risk of developing a disease, including improvement of the subject’s pathological condition (e.g., one or more symptoms), delay of disease progression, delay of symptom onset, or slowing of symptom progression.

[0160] As used herein, the term “administering” refers to any method of providing a pharmaceutical formulation to a subject. Such methods are widely 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, vaginal administration, ophthalmic administration, auricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, intrathecal administration, intralymphatic and subcutaneous administration. Administration may be continuous or intermittent. In some embodiments, the composition is administered intravenously or intracranially.

[0161] The term "effective dose" refers to an amount sufficient to achieve a beneficial or desirable biological or clinical result. The result may be a reduction, alleviation, inhibition, or prevention of one or more symptoms of a disease or condition; a reduction, inhibition, or prevention of the growth of cancer cells; a reduction, inhibition, or prevention of the metastasis of cancer cells or the invasion or spread of cancer cells; a reduction, alleviation, inhibition, or prevention of one or more symptoms of cancer or its metastasis; or any other intended alteration of a biological system. In some embodiments, the effective dose is an amount suitable for providing an intended effect, for example, to induce an antitumor response.

[0162] Additionally, a composition comprising a population of expanded B cells is provided herein. In some embodiments, the population of expanded B cells in the composition may be provided using any method of expanding B cells provided herein.

[0163] In some embodiments, a population of at least one million cells (e.g., a population of B cells, CD19 + Extended population of B cells, 4-1BBL + A composition comprising an extended population of B cells, etc.) is provided herein, wherein at least 95% of the cells in the population are CD19 + It is B cells, and at least 95% of the cells in the population express HLA-DR, at least 85% of the cells in the population express CD80, and at least 85% of the cells in the population express CD86. In some embodiments, the population (e.g., a population of B cells, an extended population of B cells, CD19 + Extended population of B cells, 4-1BBL +An expanded population of B cells, etc.) comprises at least 100,000 cells or at least about 100,000 cells, at least 150,000 cells or at least about 150,000 cells, at least 200,000 cells or at least about 200,000 cells, at least 250,000 cells or at least about 250,000 cells, at least 300,000 cells or at least about 300,000 cells, at least 350,000 cells or at least about 350,000 cells, at least 400,000 cells or at least about 400,000 cells, at least 450,000 cells or at least about 450,000 cells, at least 500,000 cells or at least about 500,000 cells, at least 1 million cells or at least about 1 million It includes 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.

[0164] In some embodiments, populations (e.g., populations of B cells, extended populations of B cells, CD19) + Extended population of B cells, 4-1BBL + An expanded population of B cells, etc.) comprises at least 10 million cells, at least 20 million cells, at least 30 million cells, at least 40 million cells, at least 50 million cells, at least 60 million cells, at least 70 million cells, at least 75 million cells, at least 100 million cells, at least 150 million cells, or at least 200 million cells.

[0165] In some embodiments, populations (e.g., populations of B cells, extended populations of B cells, CD19) + Extended population of B cells, 4-1BBL +An expanded population of B cells, etc., comprises 10 million to 1,000 million cells, 20 million to 1,000 million cells, 30 million to 1,000 million cells, 40 million to 1,000 million cells, 50 million to 1,000 million cells, 60 million to 1,000 million cells, 70 million to 1,000 million cells, 75 million to 1,000 million cells, 100 million to 1,000 million cells, 150 million 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 to 500 million cells, or 200 million to 500 million cells. This population of cells is 4-1BBL + It may include cells. In some embodiments of such a cell population, at least 50% of the B cells in the cell population express 4-1BBL (4-1BBL + In some embodiments of such cell populations, 50% to 60%, 50% to 70%, 50% to 80%, or 50% to 90% of the B cells in the cell population express 4-1BBL.

[0166] As described herein, this population of cells is 4-1BBL + It can be applied to the selection of cells. In some embodiments, 4-1BBL +The population of such cells applied to the selection of cells may be present in the composition. Such a composition may comprise, for example, at least 1 million cells, at least 2 million cells, at least 3 million cells, at least 4 million cells, at least 5 million cells, at least 10 million cells, at least 15 million cells, at least 20 million cells, or at least 25 million cells. In some embodiments, the population comprises 1 million to 500 million cells, 2 million to 500 million cells, 3 million to 500 million cells, 4 million to 500 million cells, 5 million to 500 million cells, 10 million to 500 million cells, 15 million to 500 million cells, 20 million to 500 million cells, or 25 million to 500 million cells.

[0167] In some respects, 4-1BBL + Cells additionally selected for the cells, e.g., an expanded population of B cells (e.g., 4-1BBL) + A composition comprising an extended population of B cells, wherein at least 95% of the cells in the population are CD19 + It may be a composition in which 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 the B cells in the cell population express 4-1BBL.

[0168] In some aspects, at least 85% of the B cells in a cell population express 4-1BBL. In some aspects, at least 90% of the B cells in a cell population express 4-1BBL. In some aspects, at least 95% of the B cells in a cell population express 4-1BBL.

[0169] As described herein, cells (e.g., 4-1BBL +A population of expanded B cells selected for the cells may be exposed to tumor cells. In some aspects, in the resulting population of cells, at least 98% or at least 99% of the B cells in the population express MHC-class I and / or at least 98% or at least 99% of the B cells in the population express MHC-class II. In some aspects, a composition comprising the resulting population of tumor cells comprises one or more antigens from one or more tumor cells. In some aspects, the resulting population of cells is CD8 + Expand T cells and / or CD8 + It can promote GzmB expression in T cells and / or kill tumor cells.

[0170] As a result of the fact that the method for expanding B cells provided herein can be performed in the absence of feeder cells, in some aspects, the composition comprising the group of cells provided herein does not contain feeder cells.

[0171] Examples

[0172] Example 1.

[0173] Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density centrifugation and cultured at 37°C under 5% CO2 in RPMI medium containing 15% v / v heat-inactivated human AB serum (Sigma), HEPES buffer (Invitrogen), and penicillin / streptomycin / glutamine (Invitrogen), with the addition of 20% soluble 4-trimeric CD40L (SPD-CD40L fusion protein, UltraCD40L; Multimeric Biotherapeutics), and 400 ng / ml cyclosporine-A (Novartis). Various concentrations of IL-4 (R&D Systems) were used. Cells were cultured for a period of 14 days, and total cell count and B cell ratio were evaluated.

[0174] As shown in Figure 1, 40 ng / mL of IL-4 resulted in maximum fold expansion (>10-fold) of B cells in 14-day culture, indicating that this concentration of IL-4 may be beneficial for future B cell expansion.

[0175] Example 2.

[0176] Peripheral blood mononuclear cells (PBMCs) were isolated using picol-hypark density centrifugation and cultured at 37°C under 5% CO2 in RPMI medium containing 15% v / v heat-inactivated human AB serum (Sigma), HEPES buffer (Invitrogen), and penicillin / streptomycin / glutamine (Invitrogen), supplemented with 40 ng / mL IL-4 (R&D Systems), 20% soluble 4-trimeric CD40L (SPD-CD40L fusion protein, UltraCD40L; Multimeric Biotherapeutics), and 400 ng / mL cyclosporine-A (Novartis). Cells were cultured in T75 flasks (Corning), counted every 3–4 days, and 0.5–1 x 10⁶ 6The cell density was maintained at 1 cell / mL. After 14 days of culture, the expanded B cells were phenotypically characterized by flow cytometry.

[0177] As shown in Fig. 2, when PBMC was the starting population with an increasing proportion of CD19+ cells (i.e., B cells), there was a ~10-fold increase in the absolute number of cells after 14 days of culture (Figs. 2a-2b). The B cell culture also showed an increased percentage of cells expressing CD86 and CD80, and the majority expressed HLA-DR throughout the culture (Figs. 2c-2d).

[0178] These data demonstrate that B cells can be successfully expanded from PBMCs, and that these expanded "donor" B cells clearly exhibited the phenotype of mature APCs.

[0179] Example 3.

[0180] B cell expansion cultures were performed as described above. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated using Picoll-Hypark density centrifugation. All cell culture combinations were expanded at 37°C under 5% CO2 in RPMI medium containing 15% v / v heat-inactivated human AB serum (Sigma), HEPES buffer (Invitrogen), and penicillin / streptomycin / glutamine (Invitrogen), with the addition of 40 ng / mL IL-4 (R&D Systems). Cells were cultured in T75 flasks (Corning), counted every 3–4 days, and 0.5–1 x 10⁶ 6 The cell density was maintained at 1 cell / mL. After 14 days of culture, the expanded B cells were phenotypically characterized by flow cytometry.

[0181] Three culture conditions were tested:

[0182] (1) Ultra PBMC:PBMC was cultured in the presence of 20% soluble 4-trimeric CD40L and 400 ng / ml cyclosporine-A (Novartis).

[0183] (2) Ultra CD19+: B cells were isolated using a CD19 micro beads kit and cultured in the presence of 20% soluble 4-trimeric CD40L.

[0184] (3) MB CD19+ B cells were isolated using the CD19 microbead kit and cultured in the presence of CD40L multimer.

[0185] culture results Fig. 3 Presented in ). Culture in the presence of CD40L multimers following the B cell isolation step showed the highest duplicative expansion (3.2-fold). Ultra CD40L (multimeric) in both isolated B cells and PBMC cultures showed <2-fold expansion. These data suggest that optimal B cell expansion occurs with subsequent culture in the presence of CD40L multimers following prior B cell isolation.

[0186] The phenotypic characteristics of the expanded cells were also monitored by 5-color flow cytometry analysis performed on a Beckman-Coulter FC500 flow cytometer. The percentage of CD19+ cells was >80% in both Ultra CD19+ and MB CD19+ cultures on day 0, but was approximately 10% in the Ultra PBMC culture (Fig. 4, upper left panel). All culture conditions showed increased expression of co-activation markers (CD80 and CD86) over 14 days of culture (Fig. 4, lower panel). Contaminating CD3+ T cells were highest in the Ultra PBMC culture and lowest in the MB CD19+ culture (Fig. 4, upper right panel).

[0187] As shown in Figure 4, MB CD19+ culture conditions provided substantial activation of B cells. By using a B cell population isolated for culture rather than a PBMC starting population (containing cyclosporine supplemented in the medium to restrict T cell growth), a purer B cell culture with minimal contamination of CD3+ T cells was provided on day 14. Phenotypologically, these B cells are activated APCs.

[0188] Example 4.

[0189] PMBCs were isolated from whole blood as described above, and B cells were isolated using a CD19 microbead kit (Miltenyi Biotec). Isolated B cells were cultured at 37°C under 5% CO2 in StemMACS HSC expansion medium (Miltenyi Biotec) supplemented with 10% v / v heat-inactivated human AB serum (Valley Biomedical), and stimulated with IL-4 (240 IU / mL medium; Miltenyi Biotec) and CD40L multimer (24 U / mL medium; Miltenyi) (N=3). These concentrations were determined based on the results of previous expansions performed and the biological activity of the specific lots of IL-4 and CD40L used in these experiments. The seeding concentration was 500,000 cells / mL medium. CD40L stimulation was performed on culture days 0, 7, and 9, and fresh IL-4 and medium were supplemented every 2-4 days of culture.

[0190] In one culture replica, Stemmax HSC expansion medium was replaced with HSC-Brew GMP medium (Milteny) to ensure GMP compliance, and a G-Rex® 10M bioreactor (Wilson Wolf) was used instead of a T25 or T75 flask. In these GMP-compliant replicas, CD19 selection was performed with GMP-grade CD19 micro beads (Milteny Biotech) on a Clinimax Plus closed system (Milteny Biotech).

[0191] As shown in Fig. 5, B cells expanded from a starting population of 14 million cells (Fig. 5a) to 111 million cells over 14 days, which was equivalent to an 8-fold expansion over 14 days (Fig. 5b). In GMP-compliant culture, 30 million B cells expanded to 213 million cells after 14 days (~7-fold expansion). In particular, the use of a bioreactor and GMP-compliant reagents provided a significant expansion of B cells, suggesting that the protocol can be applied to a clinical setting.

[0192] Flow cytometry analysis was performed on cell culture samples before 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 difluor-405 live / dead immobilizable growth dye (Invitrogen). Briefly, 5x10 5 Canine cells were stained with the above antibody for 30 minutes, washed with MACS buffer supplemented with 0.5% BSA, and fixed with 1% paraformaldehyde. Data were acquired on a Beckman Coulter Cytoplex. At least 50,000 cells per sample were analyzed. After gating doublets and apoptotic cells, data analysis was performed using FlowJo software. Descriptive statistics were performed on PRISM.

[0193] Flow cytometry data are presented in Fig. 6. Descriptive statistics from day 0 (Fig. 6a) and day 14 (Fig. 6b) of culture are provided. The cell population used for culture after B cell isolation showed high expression of CD20 (89.2% + / - 4.48) and HLA-DR (99.4% + / - 0.418), but low expression of CD80 (1.22% + / - 0.836) and CD86 (2.66% + / - 1.86) (Fig. 6c). On day 14, the samples showed high expression of CD19 (98.5% + / - 0.985), HLA-DR (99.9% + / - 0.117), CD80 (94.5% + / - 4.05), and CD86 (99.7% + / - 0.382).

[0194] Figure 7 provides representative flow cytometry data from a single B cell expansion experiment (this single dataset is included within the statistics provided in Figure 6). (Figure 7a) Low expression of CD80 and CD86 on day 0 (top panel). High expression of CD80, CD86, and HLA-DR is observed after 14 days of culture (bottom panel). (Figure 7b) Additionally, the expression of 4-1BBL was evaluated in this experiment because it is an activation marker and is present on activated APCs. (Top panel): Low expression of 4-1BBL on day 0 of culture. (Bottom panel): After 14 days, more than 50% of B cells express 4-1BBL.

[0195] These data show that expanded B cells uniformly express high levels of CD80, CD86, and HLA-DR, suggesting that these cells are mature APCs and can be used for downstream applications.

[0196] Example 5.

[0197] An exemplary protocol is as described in FIG. 8. The exemplary protocol of FIG. 8 is a representative embodiment of the method described herein. This exemplary protocol utilizes a G-Rex® 10M bioreactor and provides a culture period of 21 days (final volume of 50 mL). In particular, the bioreactor does not allow the removal of the medium but allows the addition of a medium component (e.g., IL-4).

[0198] Cells obtained from PMBCs (and purified using anti-CD19 GMP micro beads) are cultured in HSC-Brew GMP medium ("growth medium") supplemented with 10% v / v heat-inactivated human AB serum at 37°C under 5% CO2. CD40L multimer stimulation is performed on culture days 0, 7, 9, and 14, and IL-4 is supplemented throughout the culture duration at prescribed 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 throughout the experiment, taking into account the variability of biological activity from lot to lot. The seeding concentration of cells is 500,000 cells / mL, with a starting cell count of 10 million cells per G-Rex® 10M.

[0199] Example 6.

[0200] An exemplary protocol is also 9 As described in [the document]. The exemplary protocol of FIG. 9 is a representative embodiment of the method described herein. This exemplary protocol utilizes a G-Rex® 10M bioreactor and provides a culture period of 12 days (final volume of 100 mL).

[0201] Day 0: CD8 using anti-CD8 GMP micro beads (e.g., using the Clinimax® system) +T cells are isolated. Subsequently, the negative fraction is collected (e.g., using the Clinimax® system) CD19 + Isolate B cells. CD19 + Count the B cells for viability, then plate them in a bioreactor at 500,000 cells / mL with a starting cell count of 10 million cells. CD19 + B cells are cultured in HSC-Brew GMP medium (20 mL) supplemented with 10% v / v heat-inactivated human AB serum, CD40L multimer (480 IU), and IL-4 (4800 IU) (37°C, 5% CO2).

[0202] Day 5: Additional growth medium (total volume ≤ 30 mL) and IL-4 (7200 IU) in the culture CD19 + Add to B cells (37℃, 5% CO2).

[0203] Day 7: Additional growth medium (total volume ≤ 50 mL), IL-4 (12000 IU), and CD40L multimer (1200 UI) in the culture CD19 + Add to B cells (37℃, 5% CO2). Count the cells for growth.

[0204] Day 9: Additional growth medium (total volume ≤ 50 mL), IL-4 (18000 IU), and CD40L multimer (1800 UI) in the culture CD19 + Add to B cells (37℃, 5% CO2).

[0205] Day 12: CD19 + Collect B cells and check their viability. Isolate additional cells and 4-1BBL + Select B cells. After isolation, 4-1BBL + B cells were checked for growth potential and quality, and preserved via cryopreservation to obtain 4-1BBL +Produces an expanded population of B cells. In some embodiments, 4-1BBL + An expanded population of B cells may be useful as a vaccine. In some embodiments, 4-1BBL + An expanded population of B cells can be incubated with one or more tumor antigens (e.g., to produce a cancer vaccine).

[0206] Example 7.

[0207] Peripheral blood samples from glioblastoma patients are collected in EDTA-treated tubes, and PBMCs are isolated by a Ficoll gradient. B cells are isolated from PMBCs and cultured using the exemplary method described in Example 5 or 6.

[0208] 4-1BBL + 2 x 10 B cells in complete RPMI medium 6 Resuspend in dog cells / ml and stimulate with 5 μg / ml human anti-CD40. In some embodiments, after 24 hours, 1000 UI / ml recombinant human IFNγ is added and incubated for an additional 24-48 hours. In some embodiments, IFNγ is not added. B cells may be supplemented with 100 nM recombinant human BAFF throughout the entire in vitro activation process.

[0209] The final product B cell population is "B Vax It may be referred to as ". In some embodiments, B cells are co-cultured with cancer tumor lysate (or tumor antigen) (e.g., in a 1:1 ratio) for 5 to 72 hours.

[0210] Example 8.

[0211] The exemplary protocol in Table 1 is a representative embodiment of the method described herein. This exemplary protocol utilizes a G-Rex® 10M, 50M, or 100M bioreactor based on the seeded cell concentration and provides an 11-day culture period.

[0212] Table 1 .

[0213]

[0214] Table 2.

[0215]

[0216] Table 3.

[0217]

[0218] Day 0:

[0219] 1. CD19 using anti-CD19 GMP micro beads (e.g., using the Clinimax® system) + Isolate B cells. Count CD19+ B cells for viability, then plate them in a G-Rex® 100M bioreactor at 500,000 cells / mL according to Table 2. CD19 + B cells are cultured in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, CD40L multimer (8 IU / mL), and BAFF (100 ng / mL) (37℃, 5% CO2).

[0220] a. Lower doses of CD40L multimer and BAFF are used during this step to promote healthy B cell culture while preventing cell overactivation and proliferation. The inventors' study found that 8 IU / mL of CD40L multimer, with the addition of BAFF, CD19 + It was shown to be sufficient to promote the survival of B cells. The inventors chose to use a set dose of BAFF of 100 ng / mL instead of IL-4 to avoid the unnecessary proliferation and expansion of cells observed with IL-4 + CD40L multimer therapy. The goal of the 4-day culture is to promote healthy cultures but to limit cell fragility when harvested on the 4th day to avoid cell membrane rupture during self-selection, as observed when isolating cells from 4-day cultures with IL-4 and CD40L multimers.

[0221] 2. Next, now CD19 + Collect the negative fraction of the depleted original Leucopec product (e.g., using the Clinimax® system) CD8 + Isolate T cells. CD8 + T cells are counted for viability and cryopreserved in a GMP cryopreservation agent containing 10% DMSO for future use.

[0222] Day 4:

[0223] 1. CD19 + Collect B-cell populations and 4-1BBL using Milteny Biotech CD137L (4-1BBL) biotinylated antibody and Clinimax® anti-biotin cGMP micro beads (e.g., using the Clinimax® system). + Magnetically label cells.

[0224] 2. Freshly isolated 4-1BBL + Count the B cells for viability, then plate them in a G-Rex® bioreactor at 250,000 cells / mL according to the seeding conditions in Table 2. 4-1BBL + B cells are cultured in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, CD40L multimer (12 IU / mL), and IL-4 (50 IU / mL) (37℃, 5% CO2).

[0225] a. The goal of this process stage is 4-1BBL achieved with co-therapy of IL-4 and CD40L multimers. + The goal is to promote the robust proliferation and activation stage of B cells. Based on data collected during the optimization stage, CD40L multimer capacity is increased at this stage to influence greater cell expansion and proliferation.

[0226] Day 7:

[0227] 1. Carefully remove the depleted GM from the bottom of the bioreactor until the flask minimum is reached, without disturbing the cells, in accordance with the guidelines in Table 3. Resuspend the cells in the remaining medium, count the viability, and remove the cells for flow analysis. Based on the counted viability, resuspend 4-1BBL+ cells in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, CD40L multimer (12 IU / mL), and IL-4 (50 IU / mL) to obtain 0.25 x 10⁶ 6 Re-establish cells / mL (37℃, 5% CO2). Seeding conditions are described in detail in Table 2. If expansion exceeds the maximum seeding capacity, divide the cells into multiple flasks according to the cell range specifications in Table 2.

[0228] a. During culture, clustered 4-1BBL + B cells will settle at the bottom of the G-Rex® bioreactor within the gas-permeable membrane. This enables the removal of excess or depleted medium during culture. To promote the proliferation and expansion of 4-1 BBL+ B cells, remove the depleted culture medium and resuspend the cells in fresh GM to achieve a consistent cell concentration (0.25 x 10⁶). 6 The count (cells / mL) will be re-established. This fresh GM will contain fresh supplements of CD40L multimer (12 IU / mL) and IL-4 (50 IU / mL) to promote further activation and proliferation. Removal of excess medium also enables easier resuspension in the G-Rex® bioreactor, as less medium allows for greater displacement from the pipette to break up cell clumps. This technique is used to avoid cell clumps that can interfere with obtaining accurate and reliable cell counts.

[0229] Day 11:

[0230] 1. 4-1BBL + Collect B cells, count viability, and remove cells for flow analysis.

[0231] a. In some embodiments, 4-1BBL + An expanded population of B cells may be useful as a vaccine. In some embodiments, 4-1BBL + An expanded population of B cells can be incubated with one or more tumor antigens (e.g., to produce a cancer vaccine).

[0232] Example 9. CD40L Multimer Optimization (Fig. 12b-c)

[0233] purpose: This study aimed to understand how different concentrations of CD40L multimers affect the proliferation, activation, and expansion of CD19+ B cells. The only variable tested in this study was the different concentrations of CD40L multimers in the culture.

[0234] Method and material: Using cryopreserved CD19+ B cells (Fig. 12a) and freshly isolated CD19+ B cells (Fig. 12b), different concentrations of CD40L multimers in culture with IL4 to promote cell expansion and proliferation were tested. CD19+ B cells were cultured at 0.5 x 10⁶ in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum. 6Cells were plated at 10 cells / mL. The concentration of IL-4 was maintained constant at 360 IU / mL prior to the time point of this experiment, as previously described. In Figure 12a, CD40L multimers in the 8–12 IU / mL range were tested in comparison to a previously developed dose of 24 IU / mL. In Figure 12b, CD40L multimers in the 6–12 IU / mL range were tested to further verify the previous findings. On day 7, cells were resuspended, counted, and HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum was added along with the new IL-4 and new CD40L multimers according to specific well conditions. Cells were collected on day 12, and population viability was measured by counting the cells.

[0235] Results and Conclusions: Original results from cryopreserved CD19+ B cells revealed that a high concentration of 24 IU / mL was not necessary and could be reduced. Results from freshly isolated CD19+ B cells, similar to those in the clinical protocol, demonstrated that the CD19+ B cells were healthy and proliferative in all groups regardless of a decrease in protein concentration. However, the cells expanded at a greater rate when 12 IU / mL of CD40L multimer was added to the culture. These newly developed lower concentrations of CD40L multimer reduce the expansion costs and the amount of reagent required.

[0236] Example 10. IL-4 optimization (Fig. 12d)

[0237] purpose: This study aimed to understand how different concentrations of IL-4 affect the proliferation, activation, and expansion of CD19+ B cells. The only variable tested in this study was the different concentrations of IL-4 in the culture.

[0238] Method and material:Different concentrations of IL-4 in the culture were tested along with a set concentration of CD40L multimer to promote cell expansion and proliferation using cryopreserved CD19+ B cells. CD19+ B cells were placed in 0.5 x 10⁶ HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum. 6 Cells were plated at 12 IU / mL. The concentration of CD40L multimer was maintained constant at 12 IU / mL based on previous optimization. In Figure 12d, IL-4 in the range of 50–275 IU / mL was used and compared to wells utilizing a previously developed dose of 360 IU / mL. CD19+ B cells were expanded in the culture for 7 days. On day 5, cells were resuspended, counted, and fresh IL-4 was added to HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum according to specific well conditions. No additional CD40L multimer was added on day 5, as the current 12-day protocol does not account for this.

[0239] Results and Conclusions: The results from these experiments were consistent across all groups, and expansion was slightly increased at lower concentrations of IL-4. This experiment revealed that the original concentration of IL-4 in the culture was too high and could be reduced to 50 IU / mL over the course of the expansion period. This newly developed lower concentration of IL-4 reduces the cost of expansion and decreases the amount of reagent required.

[0240] Example 11. Cell density optimization (Fig. 12e)

[0241] purpose: This study aimed to understand how different cell densities used during culture affect the proliferation, activation, and expansion of CD19+ B cells.

[0242] Method and material:Cells plated at different densities during different extension periods were tested using cryopreserved CD19+ B cells. CD19+ B cells were plated at 0.15 x 10⁶ in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, 50 IU / mL IL-4, and 12 IU / mL CD40L multimer. 6 - 0.5 x 10 6 Cells were plated at densities ranging from 1 cell / mL. The concentrations of CD40L multimer and IL-4 were newly developed from previous optimization experiments. CD19+ B cells were expanded for 7 days of culture. On day 5, cells were resuspended, counted, and fresh IL-4 was added to HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum according to specific well conditions. No additional CD40L multimer was added on day 5, as the current 12-day protocol does not account for this.

[0243] Results and Conclusions: The expansion of each group was consistent across all conditions; however, as cell density decreased across all groups, there was a tendency toward higher growth potential. This experiment was 0.15 x 10⁶ 6 - 0.25 x 10 6 It was found that plating cells at a lower density of 0.15 x 10⁶ cells / mL induced healthier expansion. 6 To reduce incidental costs incurred when plating at a lower density of cells / mL, 0.25 x 10 6 A density of cells / mL was recommended.

[0244] Example 12. Isolated background (Fig. 13c-d)

[0245] purpose: This study aimed to understand how optimized doses of IL-4 and CD40L multimers can help alleviate previous problems associated with the isolation of 4-1BBL+ from an expanded CD19+ B-cell population.

[0246] Method and material: Freshly isolated CD19+ B cells were expanded with updated doses of IL-4 and CD40L, and attempts were made to isolate 4-1 BBL+ B cells on different days during expansion. Both self-isolation and flow-based sorting were tested as shown in Fig. 13c. All results were unsuccessful, confirming that less than 20% of cells were viable after isolation.

[0247] Results and Conclusions: The data collected from this experiment helps illustrate the need to reconstruct the inventors' extension and isolation processes. Even after optimizing the concentrations of the supplemented IL-4 and CD40L multimers, the cells remained in a very fragile state, and the cell membranes could easily rupture as they passed through a magnetic field.

[0248] Example 13. Optimization of microbead isolation (Figs. 14 and 15)

[0249] purpose: This experiment aimed to understand how long the original CD19 micro beads remain on the surface of CD19+ B cells after self-selection (Clinimax® system). This information helped the inventors design an optimized method for the isolation of 4-1BBL+.

[0250] Method and material:CD19+ B cells were isolated from Leucopecs freshly mobilized via CD19 microbeads. Cells were collected every 24 hours from Day 0 to Day 4 and stained with fluorescent dye-conjugated anti-CD19 antibody. Sorting was performed when the CD19 marker became available and recognizable by flow cytometry. The reappearance of the flow-labeled CD19 population was possible only when the CD19 microbeads detached from the cell surface prior to staining. Cells were placed in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, 50 IU / mL IL-4, and 12 IU / mL CD40L multimer at a rate of 0.25 x 10⁶ throughout the entire experiment. 6 Plated at a density of cells / mL.

[0251] Results and Conclusions: The CD19 population fully reappeared around day 4 (96 hours of culture). Based on these data, the inventors concluded that the original CD19 microbeads had detached from the entire CD19+ B-cell population by this time. These data revealed that 4 days after the isolation of the original CD19+ B-cells was the earliest point in time at which 4-1BBL+ B-cell isolation could be performed. This was because the Clinimax® system could not distinguish between different microbeads (4-1BBL or CD19); therefore, if populations of both remained as microbeads, the isolated population would not be purely 4-1BBL+ B cells.

[0252] Example 14. Optimization of 4-day CD19+ culture (Figs. 17-20)

[0253] purpose: This study was designed to evaluate how the use of IL-4 or BAFF in combination with CD40L multimers can promote the growth of CD19+ B cells over a 4-day long culture period while limiting their activation and expansion. The goal was to preserve cells capable of undergoing additional rounds of self-selection.

[0254] Method and material: CD19+ B cells were isolated from freshly mobilized Leucopec via CD19 microbead-positive selection, and cryopreserved CD19+ B cells were used. In the figure above, CD19+ B cells were 0.50 x 10⁶ in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum and 8-10 IU / mL of CD40L multimer. 6 The cells were cultured at a density of 1 cell / mL. One group of plated cells received an additional supplement of 50 IU / mL IL-4, while the other group received an additional supplement of 100 ng / mL BAFF instead. After 4 days of culture, CD19+ B cells were collected and counted for cell viability. Subsequently, 4-1BBL+ B cells were magnetically labeled and isolated from the collected CD19+ B-cell population. The isolated 4-1BBL+ B-cell population was collected and counted for cell viability.

[0255] Results and Conclusions: The viability of CD19+ B cells was high across both IL-4 and CD40L multimer conditions at harvest on Day 4. However, CD19+ B cells from the BAFF + CD40L condition maintained significantly greater viability throughout the 4-1BBL+ cell self-labeling process. Additionally, isolated 4-1BBL+ cells maintained significantly greater viability only in the group originating from CD19+ B cells previously cultured with BAFF and CD40L. These data demonstrated that the isolation of 4-1BBL+ B cells was now possible via self-separation technology (Clinimax® system) from CD19+ B cells cultured with BAFF + CD40L over a 4-day period. During these 4 days, the cells remained healthy, and the original CD19 microbeads detached from the cell surface.

[0256] Example 15. 4-1BBL+ extended culture (Fig. 21)

[0257] purpose: This experiment was designed to evaluate how 4-1BBL+ cells isolated from a newly developed isolation protocol can expand, proliferate, become activated, and retain their surface 4-1BB ligands.

[0258] Method and material: CD19+ B cells were isolated from freshly mobilized Leucopec via CD19 microbead-positive selection. CD19+ B cells were cultured at 0.50 x 10⁶ in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, 8 IU / mL CD40L multimer, and 100 ng / mL BAFF. 6 They were cultured at a density of 1 cell / mL. On day 4, CD19+ B cells were collected, and 4-1BBL+ B cells were subsequently isolated. Subsequently, 4-1BBL+ B cells were cultured at 0.25 x 10⁶ 6 Dog cells / mL or 0.50 x 10⁶ 6 The extent of their expansion was determined by culturing at different time ranges at cell / mL. On the third day of the group expanded for up to 6 days, cells were resuspended, counted, and re-plated to their original plating density in HSC-Brew GMP medium supplemented with 10% v / v heat-inactivated human AB serum, IL-4, and CD40L. The expansion status over the 5-day course did not include a second restimulation. CD40L multimer administration was consistently maintained at 12 IU / mL, and IL-4 was consistently maintained at 50 IU / mL based on previous optimization. Subsequently, 4-1BBL+ B cells were harvested after expansion, and purity was measured by staining 4-1BBL with a fluorescent dye-conjugated anti-4-1BBL antibody.

[0259] Results and Conclusions: All 4-1BBL+ B cells expanded; however, the greatest expansion was observed under lower density conditions, which was 0.25 x 10⁶ 6This further supports the claim that a cell / mL density is the most effective and cost-effective for maintaining the most proliferative environment. The best results were obtained from a group extended over a 6-day course and restimulated to the middle. Flow results from this group demonstrated 58% recognition of the 4-1BBL+ B cell population. The inventors' most updated protocol utilizes the above methodology; however, the inventors have made slight adjustments to add an additional extension day to alleviate concerns regarding the 4-1BBL+ B cell culture needing to be manipulated over the weekend.

[0260] Other embodiments

[0261] Embodiment 1. A method for producing an expanded population of B cells,

[0262] A method comprising culturing a population of B cells for a certain period in the presence of 20-60 ng / mL interleukin-4 (IL-4), 5-15% v / v serum and CD40 ligand (CD40L), wherein the population of B cells is cultured in a bioreactor.

[0263] Embodiment 2. A method for producing an expanded population of B cells, comprising culturing a population of B cells for a certain period in the presence of 200-400 IU / mL interleukin-4 (IL-4), 5-15% v / v serum, and CD40 ligand (CD40L), wherein the population of B cells is cultured in a bioreactor.

[0264] Embodiment 3. A method in which, in Embodiment 1 or 2, the fixed period is 12-25 days, optionally 12-21 days or 12-16 days.

[0265] Embodiment 4. A method in any one of embodiments 1 to 3, wherein the specified period is at least 12 days, at least 13 days, at least 14 days, or at least 21 days.

[0266] Embodiment 5. A method in any one of Embodiments 1 to 4, wherein the fixed period is 14 days.

[0267] Embodiment 6. In any one of Embodiments 1 to 5, the expanded population of B cells is at least 5x10 6 Dogs or at least 10x10 6 A method involving dog B cells.

[0268] Embodiment 7. A method in any one of Embodiments 1 to 6 in which at least 90%, at least 95%, or at least 98% of the cells in the expanded population are B cells.

[0269] Embodiment 8. A method in any one of Embodiments 1 to 7, wherein the expanded population of B cells does not contain a detectable amount of non-B cells, optionally wherein the expanded population of B cells does not contain a detectable amount of T cells.

[0270] Embodiment 9. A method in any one of Embodiments 1 to 8, wherein the concentration of IL-4 is maintained at 20-60 ng / mL, 40 ng / mL, or 200-400 IU / mL for a certain period.

[0271] Embodiment 10. A method in any one of Embodiments 1 to 9, wherein a population of B cells is cultured in the presence of 30-60 ng / mL IL-4 or 40-60 ng / mL IL-4.

[0272] Embodiment 11. A method in any one of Embodiments 1 to 10, wherein a population of B cells is cultured in the presence of 40 ng / mL IL-4 or 240 IU / mL IL-4.

[0273] Embodiment 12. A method in any one of Embodiments 1 to 11, wherein the serum concentration is maintained at 5-15% v / v for a certain period.

[0274] Embodiment 13. A method in any one of Embodiments 1 to 12, wherein a population of B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum.

[0275] Embodiment 14. A method in any one of Embodiments 1 to 13, wherein the serum is AB serum, optionally human AB serum.

[0276] Embodiment 15. A method in any one of Embodiments 1 to 14, wherein the serum is heat-inactivated serum.

[0277] Embodiment 16. A method in any one of Embodiments 1 to 15 in which the concentration of CD40L is maintained at 0.1-1 mg / mL.

[0278] Embodiment 17. A method in any one of Embodiments 1 to 16, wherein the CD40L is a macromeric CD40L.

[0279] Embodiment 18. The method of Embodiment 17, wherein the polymeric CD40L is a soluble 4-trimeric CD40L, optionally wherein the trimer is linked by surfactant protein D.

[0280] Embodiment 19. A method in any one of Embodiments 1 to 18, wherein a population of B cells is stimulated with CD40L on the 0th, 7th, and 9th days of a certain period.

[0281] Embodiment 20. A method in any one of Embodiments 1 to 19, wherein the method comprises seeding a population of B cells at a density of 400,000 to 600,000 cells / mL before the culture step.

[0282] Embodiment 21. Any one of Embodiments 1 to 20, wherein the method comprises seeding a population of B cells at a density of about 500,000 cells / mL before the culture step.

[0283] Embodiment 22. A method in any one of Embodiments 1 to 21, wherein the population of B cells is obtained from a human subject.

[0284] Embodiment 23. A method in any one of Embodiments 1 to 22 in which a population of B cells is cultured in the presence of a cell medium, optionally a GMP-grade cell medium.

[0285] Embodiment 24. The method of Embodiment 23 in which the cell medium is not removed from the population of B cells for the entire duration of a certain period.

[0286] Embodiment 25. The method of Embodiment 23, wherein 20%, 10%, 5%, or 3% or less of the cell medium is removed from the population of B cells over the entire duration of the specified period.

[0287] Embodiment 26. A method in any one of embodiments 23 to 25, wherein a population of B cells is supplemented with a growth medium for a certain period, optionally on the 5th, 7th, 9th and / or 12th day of the certain period.

[0288] Embodiment 27. A method in any one of Embodiments 1 to 26, wherein a population of cells is further cultured in the presence of cyclosporine.

[0289] Embodiment 28. A method in any one of Embodiments 1 to 27, wherein a population of B cells is supplemented with IL-4 at any 2-4 days and / or on the 5th, 7th, 9th and / or 12th days of the period.

[0290] Embodiment 29. A method in any one of Embodiments 1 to 28, wherein a population of B cells is supplemented with IL-4 on the 5th, 7th, 9th and / or 12th days of a certain period.

[0291] Embodiment 30. A method in any one of Embodiments 1 to 29, wherein the bioreactor comprises a gas-permeable rapid-expanding cell culture membrane.

[0292] Embodiment 31. A method in any one of Embodiments 1 to 30, wherein the population of B cells is a population of human B cells.

[0293] Embodiment 32. The method of Embodiment 31, wherein the population of human B cells is isolated from whole blood.

[0294] Embodiment 33. The method of Embodiment 32 in which a population of human B cells is isolated using micro beads.

[0295] Embodiment 34. The method of Embodiment 33, wherein the micro beads comprise an antibody, optionally an anti-CD19 antibody.

[0296] Embodiment 35. In any one of Embodiments 1 to 34, 4-1BBL from an expanded population of B cells + A method further comprising isolating B cells.

[0297] Embodiment 36. A method in any one of Embodiments 1 to 35 in which the population of B cells is not treated with IFN-γ.

[0298] Embodiment 37. A composition comprising a population of expanded B cells produced according to any one of the methods of Embodiments 1 to 36.

[0299] Embodiment 38. A composition comprising a population of at least 50 million cells, wherein

[0300] At least 95% of the cells in the population are CD19 + It is a B cell, and

[0301] At least 90% of the cells in the population express HLA-DR, and

[0302] At least 75% of the cells in the population express CD80;

[0303] At least 75% of the cells in the population express CD86

[0304] Composition.

[0305] Embodiment 39. A composition according to Embodiment 37 or 38, wherein at least 20% of the B cells in the cell population express 4-1BBL.

[0306] Embodiment 40. A composition according to Embodiment 37 or 38, wherein 20% to 95% of the B cells in the cell population express 4-1BBL.

[0307] Embodiment 41. A composition in any one of embodiments 37 to 40, wherein the cell population comprises at least 75 million cells.

[0308] Embodiment 42. The composition of Embodiment 41, wherein the cell population comprises at least 100 million cells.

[0309] Embodiment 43. The composition of Embodiment 42, wherein the cell population comprises at least 150 million cells.

[0310] Embodiment 44. The composition of Embodiment 43, wherein the cell population comprises at least 200 million cells.

[0311] Embodiment 45. A composition in any one of embodiments 37 to 44, wherein the cell population comprises 1,000 million or fewer cells.

[0312] Embodiment 46. The composition of Embodiment 45, wherein the cell population comprises 750 million or fewer cells.

[0313] Embodiment 47. The composition of Embodiment 46, wherein the cell population comprises 500 million or fewer cells.

[0314] Embodiment 48. A composition comprising a population of at least 25 million cells, wherein

[0315] At least 95% of the cells in the population are CD19+ It is a B cell, and

[0316] At least 90% of the cells in the population express HLA-DR, and

[0317] At least 75% of the cells in the population express CD80;

[0318] At least 75% of the cells in the population express CD86;

[0319] At least 80% of B cells in a cell population express 4-1BBL

[0320] Composition.

[0321] Embodiment 49. The composition of Embodiment 48, wherein at least 98% of the B cells in the population express MHC-class I.

[0322] Embodiment 50. A composition according to Embodiment 49, wherein at least 99% of the B cells in the population express MHC-class I.

[0323] Embodiment 51. A composition in any one of embodiments 48 to 50, wherein at least 98% of the B cells in the population express MHC-class II.

[0324] Embodiment 52. The composition of Embodiment 51, wherein at least 99% of the B cells in the population express MHC-class II.

[0325] Embodiment 53. A composition comprising one or more tumor antigens in any one of embodiments 48 to 52.

[0326] Embodiment 54. A composition in any one of Embodiments 48 to 53, wherein the cell population is capable of expanding CD8+ T cells.

[0327] Embodiment 55. A composition in any one of Embodiments 48 to 54, wherein the cell population is capable of promoting GzmB expression in CD8+ T cells.

[0328] Embodiment 56. A composition in any one of embodiments 48 to 55, wherein a population of cells is capable of killing tumor cells.

[0329] Embodiment 57. A composition in any one of embodiments 37 to 56, wherein at least 98% of the cells in the population are CD19+ B cells.

[0330] Embodiment 58. The composition of Embodiment 57, wherein at least 99% of the cells in the population are CD19+ B cells.

[0331] Embodiment 59. A composition in which, in any one of embodiments 37 to 58, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population express HLA-DR.

[0332] Embodiment 60. A composition in any one of embodiments 37 to 59, wherein at least 80%, at least 85%, at least 90%, or at least 95% of the cells in the population express CD80.

[0333] Embodiment 61. A composition according to Embodiment 60, wherein at least 96% of the cells in the population express CD80.

[0334] Embodiment 62. The composition of Embodiment 61, wherein at least 97% of the cells in the population express CD80.

[0335] Embodiment 63. The composition of Embodiment 62, wherein at least 98% of the cells in the population express CD80.

[0336] Embodiment 64. The composition of Embodiment 63, wherein at least 99% of the cells in the population express CD80.

[0337] Embodiment 65. A composition in any one of embodiments 37 to 64, wherein at least 80%, at least 85%, or at least 90% of the cells in the population express CD86.

[0338] Embodiment 66. The composition of Embodiment 68, wherein at least 95% of the cells in the population express CD86.

[0339] Embodiment 67. The composition of Embodiment 66, wherein at least 96% of the cells in the population express CD86.

[0340] Embodiment 68. The composition of Embodiment 67, wherein at least 97% of the cells in the population express CD86.

[0341] Embodiment 69. The composition of Embodiment 68, wherein at least 98% of the cells in the population express CD86.

[0342] Embodiment 70. The composition of Embodiment 69, wherein at least 99% of the cells in the population express CD86.

[0343] Embodiment 71. A composition in any one of embodiments 37 to 70, wherein 2% or less of the cells in the population are T cells.

[0344] Embodiment 72. The composition of Embodiment 71, wherein 1% or less of the cells in the population are T cells.

[0345] Embodiment 73. The composition of Embodiment 72, wherein 0.5% or less of the cells in the population are T cells.

[0346] Embodiment 74. A composition in any one of embodiments 37 to 73, wherein the cells within the group are human cells.

[0347] Embodiment 75. A composition in any one of embodiments 37 to 74, wherein the cell population does not contain feeder cells.

[0348] Embodiment 76. A composition in any one of embodiments 37 to 75, wherein the cell population is expanded from a cell population obtained from a patient.

[0349] Embodiment 77. The composition of Embodiment 76, wherein the patient has cancer.

[0350] Embodiment 78. The composition of Embodiment 77, wherein the cancer is a glioma.

[0351] Embodiment 79. A B cell vaccine comprising at least a subset of B cells from an extended population of B cells of any one of Embodiments 1 to 36.

[0352] Embodiment 80. In Embodiment 79, at least 50%, 60%, 70%, 80%, 90%, or 95% of the total cells of the subset of B cells are 4-1BBL + B cell vaccine, a B cell.

[0353] Embodiment 81. A B cell vaccine according to Embodiment 79 or 80, wherein the antigen, optionally additionally comprising a tumor antigen.

[0354] Embodiment 82. A B cell vaccine according to Embodiment 81, wherein a subset of B cells is incubated with a tumor lysate containing a tumor antigen.

[0355] Embodiment 83. A B cell vaccine comprising a CD40 agonist in any one of embodiments 79 to 82.

[0356] Embodiment 84. A method for treating a disease or disorder in a human subject, comprising administering a B cell vaccine of any one of embodiments 79 to 83.

[0357] Embodiment 85. The method of Embodiment 84 in which the disease or disorder is cancer.

[0358] Embodiment 86. The method of Embodiment 85, wherein the cancer is brain cancer.

[0359] Embodiment 87. The method of Embodiment 86, wherein the cancer is a glioblastoma.

[0360] Embodiment 88. A method comprising administering any one of the B cell vaccines of Embodiments 79 to 83 to a human subject.

[0361] Embodiment 89. A method in which, in any one of embodiments 84 to 88, the human subject has cancer.

[0362] Embodiment 90. The method of Embodiment 89, wherein the human subject has brain cancer, optionally glioblastoma.

[0363] Embodiment 91. Any one of embodiments 84 to 90, wherein the method further comprises administering a therapeutic agent, optionally wherein the therapeutic agent is an immune checkpoint inhibitor.

[0364] Equivalents and Categories

[0365] Although various embodiments of the present invention have been described and illustrated herein, a person skilled in the art will readily conceive of various other means and / or structures to perform the functions described herein and / or obtain results and / or one or more advantages, and each such modification and / or variation is considered to be within the scope of the embodiments of the present invention described herein. More generally, a person skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will vary depending on the specific applications or uses in which the teachings of the present invention are applied. A person skilled in the art will be able to recognize or identify many equivalents to the specific embodiments of the present invention described herein by using only commercial experimentation. Accordingly, the embodiments are presented merely as examples, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced differently from those specifically described and claimed. The embodiments of the present invention in this disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more of these features, systems, articles, materials, kits and / or methods is included within the scope of the invention of this disclosure, provided that these features, systems, articles, materials, kits and / or methods are not mutually contradictory.

[0366] All definitions as defined and used herein shall be understood to take precedence over dictionary definitions, definitions in the literature incorporated by reference, and / or the ordinary meaning of the defined terms.

[0367] All references, patents, and patent applications disclosed herein are incorporated by reference in relation to the subject matter for which they are cited, and in some cases, may encompass the entirety of the literature.

[0368] As used herein in the specification and claims, the singular form should be understood to mean "at least one" unless otherwise clearly indicated.

[0369] The phrase “and / or” as used herein in the specification and claims should be understood to mean “either one or both” of the elements thus combined, that is, elements that exist jointly in some cases and separately in others. Multiple elements enumerated with “and / or” should be interpreted in the same way, that is, “one or more” of the elements thus combined. Whether or not they relate to the elements specifically identified, other elements other than those specifically identified by the “and / or” clause may arbitrarily exist. Accordingly, as a non-limiting example, a reference to “A and / or B,” when used with open language such as “comprising,” may, in one embodiment, refer to A alone (arbitrarily including elements other than B); in another embodiment, refer to B alone (arbitrarily including elements other than A); or in yet another embodiment, refer to both A and B (arbitrarily including other elements).

[0370] The term “or” as used herein in the specification and claims should be understood to have the same meaning as “and / or” as defined above. For example, when separating items from a list, “or” or “and / or” should be interpreted as inclusive, that is, including not only at least one of the number of elements or the list, but also more than one, and optionally additional unlisted items. Only terms otherwise explicitly indicated, such as “only one of” or “exactly one of”, or “consisting of” when used in the claims, will refer to including exactly one of the number of elements or the list. In general, the term “or” as used herein will be interpreted only to indicate an exclusive alternative (i.e., “one or the other, but not both”) when preceded by terms of exclusivity such as “either one,” “one of,” “only one of,” or “exactly one of”. “Consisting of essentially” will have its ordinary meaning as used in the field of patent law when used in the claims.

[0371] With respect to a list of one or more elements, the phrase “at least one” as used herein in the specification and claims means at least one element selected from any one or more of the elements in the list of elements, but it should be understood that it does not necessarily include at least one of each and all of the elements specifically enumerated within the list of elements, nor exclude any combination of elements in the list of elements. This definition also allows for the possibility that elements other than the specifically identified elements may exist within the list of elements referred to by the phrase “at least one,” whether or not they are related to the specifically identified elements. Accordingly, 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”) may refer, in one embodiment, to at least one A (and optionally including elements other than B) including optionally more than one where B is absent; or in another embodiment, to at least one B (and optionally including elements other than A) including optionally more than one where A is absent; In another embodiment, at least one A including optionally more than one, and at least one B including optionally more than one (and optionally including other elements) may be referred to.

[0372] Additionally, unless otherwise clearly indicated, in any method claimed herein comprising more than one step or action, it should be understood that the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are mentioned.

Claims

Claim 1 4-1BBL including the following steps + Method for producing an expanded population of B cells: (i) 4-1 BBL in a bioreactor + A population of B cells was cultured for a certain period in the presence of interleukin-4 (IL-4), 5-15% v / v serum, and CD40 ligand (CD40L) to produce 4-1 BBL + The stage of producing an expanded population of B cells. Claim 2 In claim 1, the 4-1BBL cultured in step (i) + A population of B cells in a bioreactor CD19 + A method prepared by culturing a population of B cells in the presence of B-cell activating factor (BAFF) and CD40L. Claim 3 In paragraph 2, CD19 + A method in which a population of B cells is cultured in the presence of 50-200 nM, 50-150 nM, or 50-100 nM BAFF. Claim 4 In paragraph 1 or 2, 4-1BBL + A method in which a population of B cells is not cultured in the presence of BAFF. Claim 5 In paragraph 2, CD19 + A method in which a population of B cells is cultured in the presence of 100 nM BAFF. Claim 6 In paragraph 2, CD19 + A method in which a population of B cells is cultured in the presence of 100 nM recombinant human BAFF. Claim 7 In any one of paragraphs 2 through 6, CD19 + A method in which a population of B cells is not cultured in the presence of IL-4. Claim 8 In any one of paragraphs 2 through 5, CD19 + B cell populations and 4-1BBL + A method in which populations of B cells are cultured in the presence of different amounts of CD40L. Claim 9 In any one of paragraphs 2 through 5, CD19 + B cell populations and 4-1BBL + A method in which a population of B cells is cultured in the presence of equal amounts of CD40L. Claim 10 In any one of paragraphs 2 through 5, CD19 + B cell populations and 4-1BBL + A method in which a population of B cells is cultured in the presence of 5-20 U / mL CD40L or 5-15 U / mL CD40L. Claim 11 In any one of paragraphs 2 through 5, CD19 + B cell populations and 4-1BBL + A method in which a population of B cells is cultured in the presence of 8-12 U / mL CD40L. Claim 12 In any one of paragraphs 2 through 5, CD19 + A method in which a population of B cells is cultured in the presence of 8 U / mL CD40L or about 8 U / mL CD40L. Claim 13 In any one of paragraphs 1 to 5, 4-1BBL + A method in which a population of B cells is cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L. Claim 14 In any one of paragraphs 2 through 13, CD19 + A population of B cells is cultured in the presence of 8 U / mL CD40L or approximately 8 U / mL CD40L, and 4-1BBL + A method in which a population of B cells is cultured in the presence of 12 U / mL CD40L or about 12 U / mL CD40L. Claim 15 In any one of paragraphs 1 to 13, 4-1BBL + A method in which a population of B cells is cultured in the presence of 20 - 400 U / mL IL-4, 20 - 300 U / mL IL-4, 20 - 200 U / mL IL-4, 40 - 400 U / mL IL-4, 40 - 300 U / mL IL-4, 40 - 200 U / mL IL-4, 40 - 100 U / mL IL-4, 40 - 60 IU / mL IL-4, 20 - 100 U / mL IL-4, 20 - 60 U / mL IL-4, 50 U / mL IL-4, 30-60 ng / mL IL-4, or 40-60 ng / mL IL-4. Claim 16 In any one of paragraphs 2 through 15, CD19 + 4-1BBL from a population of B cells + A method further comprising isolating B cells. Claim 17 A method according to any one of paragraphs 1 to 16, wherein the specified period is at least 4 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 21 days. Claim 18 In paragraph 1, a method in which the fixed period is 12 to 14 days. Claim 19 In any one of paragraphs 1 through 18, 4-1BBL in (i) + An expanded population of B cells is at least 5x10 6 Dogs or at least 10x10 6 A method involving dog B cells. Claim 20 In any one of paragraphs 2 to 19, at least 90%, at least 95%, or at least 98% of the cells of the expanded population are 4-1BBL + B cell method. Claim 21 In any one of paragraphs 1 to 12, 4-1BBL + A population of B cells is at least 5x10 6 Dogs or at least 10x10 6 A method involving dog B cells. Claim 22 In any one of paragraphs 1 to 21, 4-1BBL + An extended population of B cells does not contain detectable amounts of non-B cells, and optionally here 4-1BBL + A method in which an expanded population of B cells does not contain a detectable amount of T cells. Claim 23 A method according to any one of claims 1 to 22, wherein the serum concentration is maintained at 5-15% v / v for a certain period. Claim 24 In any one of paragraphs 1 through 23, CD19 + A method in which a population of B cells is cultured in the presence of 8-15% v / v serum, optionally 10% v / v serum. Claim 25 A method in which, in any one of paragraphs 1 to 24, the serum is AB serum, optionally human AB serum. Claim 26 A method according to any one of claims 1 to 25, wherein the serum is heat-inactivated serum. Claim 27 A method in which CD40L is a multimeric CD40L in any one of claims 1 to 26. Claim 28 In paragraph 27, the method wherein the polymeric CD40L is a soluble 4-trimeric CD40L, optionally wherein the trimer is linked by surfactant protein D. Claim 29 In any one of paragraphs 1 through 28, CD19 + A population of B cells is stimulated with CD40L at least once on days 0–4 of a certain period and / or 4–1BBL + A method in which a population of B cells is stimulated with CD40L at least once on days 4–11 of a certain period. Claim 30 In any one of paragraphs 2 through 29, the method CD19 before the culture step + A method comprising seeding a population of B cells at a density of 400,000 to 800,000 cells / mL. Claim 31 In any one of paragraphs 2 through 29, the method CD19 before the culture step + A method comprising seeding a population of B cells at a density of approximately 750,000 cells / mL. Claim 32 In any one of claims 1 to 31, the method before the culture step 4-1BBL + A method comprising seeding a population of B cells at a density of 100,000 to 800,000 cells / mL. Claim 33 In any one of claims 1 to 31, the method before the culture step 4-1BBL + A method comprising seeding a population of B cells at a density of about 250,000 to 500,000 cells / mL. Claim 34 In any one of paragraphs 2 through 33, CD19 + A method in which a population of B cells is isolated and expanded from a population of B cells obtained from a human subject. Claim 35 In any one of paragraphs 2 through 34, CD19 + B cell populations and 4-1BBL + A method in which a population of B cells is cultured in the presence of cell medium, optionally Good Manufacturing Practice (GMP) grade cell medium. Claim 36 In paragraph 35, 20%, 10%, 5%, or 3% or less of the cell medium is CD19 + Culture of B cell populations and / or 4-1 BBL + A method of removing from a culture of B cells. Claim 37 In paragraph 35 or 36, CD19 + B cell population and / or 4-1BBL + A method in which a population of B cells is supplemented with growth medium on the 0th, 4th, and / or 7th days of a certain period, at random. Claim 38 In any one of paragraphs 2 through 37, CD19 + B cell population and / or 4-1BBL + A method in which a population of B cells is further cultured in the presence of cyclosporine. Claim 39 In any one of paragraphs 2 through 38, CD19 + B cells and / or 4-1BBL + A method in which a population of B cells is supplemented with IL-4 at random every 2-4 days and / or on the 5th, 7th, 9th and / or 12th days of a certain period. Claim 40 A method according to any one of claims 2 to 39, wherein a population of 4-1BBL cells is supplemented with IL-4 on the 5th, 7th, 9th and / or 12th days of a certain period. Claim 41 A method according to any one of claims 2 to 40, wherein the bioreactor comprises a gas-permeable rapid-expanding cell culture membrane. Claim 42 In any one of paragraphs 2 through 41, CD19 + A method in which a population of B cells is a population of human B cells. Claim 43 A method according to paragraph 42 in which a population of human B cells is isolated from whole blood, optionally in which a population of human B cells is isolated using micro beads, wherein the micro beads comprise an antibody, optionally an anti-CD19 antibody. Claim 44 In any one of paragraphs 1 through 43, CD19 + B cell population and / or 4-1BBL + A method in which a population of B cells is not treated with IFN-γ. Claim 45 4-1BBL produced according to the method of any one of paragraphs 1 to 44 + A composition comprising an extended population of B cells. Claim 46 A composition comprising a population of at least 50 million cells, wherein at least 95% of the cells in the population are CD19 + A composition in which 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. Claim 47 A composition according to claim 46, wherein 20% to 95% of the B cells in the cell population express 4-1BBL. Claim 48 A composition according to claim 46 or 47, wherein the cell population comprises at least 75 million cells, at least 100 million cells, at least 150 million cells, at least 200 million cells, at least 500 million cells, at least 750 million cells, or at least 1 billion cells. Claim 49 A composition comprising a population of at least 25 million cells, wherein at least 95% of the cells in the population are CD19 + A composition comprising B cells, wherein at least 90% of the cells in the population express HLA-DR, at least 75% of the cells in the population express CD80, at least 75% of the cells in the population express CD86, and at least 80% of the B cells in the cell population express 4-1BBL. Claim 50 A composition according to claim 49 in which at least 98% or 99% of the B cells in the population express MHC-class I. Claim 51 A composition according to claim 49 or 50, wherein at least 98% or 99% of the B cells in the population express MHC-class II. Claim 52 A composition comprising one or more tumor antigens in addition to any one of claims 49 to 51. Claim 53 In any one of paragraphs 46 to 52, at least 98% or 99% of the cells in the population are CD19 + Composition of B cells. Claim 54 A composition according to any one of claims 46 to 53, wherein at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population express HLA-DR, and optionally 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. Claim 55 A composition according to any one of claims 46 to 54, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the population express CD86. Claim 56 A composition according to any one of claims 46 to 55, wherein 2% or less, 1% or less, or 0.5% or less of the cells in the population are T cells. Claim 57 A composition according to any one of claims 46 to 56, wherein the composition does not contain feeder cells. Claim 58 A composition according to any one of claims 46 to 57, wherein the cell population is expanded from a cell population obtained from a patient, optionally wherein the patient has cancer and the cancer is a glioma. Claim 59 A method for producing a B cell vaccine comprising the following steps: (i) CD19 in a bioreactor + CD19 by culturing a population of B cells in the presence of B-cell activating factor (BAFF), serum, and CD40 ligand (CD40L). + Step of producing an expanded population of B cells; (ii) CD19 in (i) + 4-1BBL from an expanded population of B cells + Step of isolating B cells; (iii) 4-1BBL isolated from (ii) in a bioreactor + B cells were cultured in the presence of interleukin-4 (IL-4) and CD40L to produce 4-1BBL + A step of producing an expanded population of B cells, and (iv) 4-1BBL from (iii) + An expanded population of B cells is incubated with a tumor lysate containing at least one tumor antigen to produce 4-1BBL containing at least one tumor antigen. + A step of producing an expanded population of B cells and thereby producing a B cell vaccine. Claim 60 In paragraph 59, CD19 in (i) + A method in which a population of B cells is cultured for 0 to 4 days. Claim 61 In paragraph 59 or 60, the isolated 4-1BBL in (iii) + A method in which B cells are cultured for 4 to 11 days. Claim 62 In any one of paragraphs 59 through 61, CD19 in (i) + A method in which a population of B cells is cultured in the presence of 100 nM BAFF or 100 ng / mL BAFF and 8 U / mL CD40L or about 8 U / mL CD40L. Claim 63 In any one of paragraphs 59 through 62, the isolated 4-1BBL in (iii) + A method in which B cells are cultured in the presence of 50 U / mL or about 50 U / mL IL-4 and 12 U / mL or about 12 U / mL CD40L. Claim 64 In any one of paragraphs 59 through 63, CD19 in (i) + A method in which a population of B cells is isolated from a population of B cells using micro beads, wherein the micro beads contain antibodies, optionally anti-CD19 antibodies. Claim 65 In any one of paragraphs 59 through 63, the isolated 4-1BBL in (ii) + CD19 using B cell-labeled anti-4-1BBL antibody + A method of isolating from an extended population of B cells. Claim 66 At least 25% growth possible 4-1BBL + B cell vaccine containing an expanded population of B cells. Claim 67 In Clause 66, 25% - 75% growth possible 4-1BBL + B cell vaccine containing an expanded population of B cells. Claim 68 In Clause 66, 40% - 60% growth possible 4-1BBL + B cell vaccine containing an expanded population of B cells. Claim 69 A B cell vaccine in which the tumor antigen is associated with glioblastoma in any one of paragraphs 59 to 66. Claim 70 A B cell vaccine comprising a CD40 agonist in any one of paragraphs 59 to 66. Claim 71 A method for treating a disease or disorder in a human subject, comprising administering a B cell vaccine according to any one of claims 66 to 70. Claim 72 In paragraph 71, the method in which the disease or disorder is cancer. Claim 73 In paragraph 72, the cancer is brain cancer; optionally, the cancer is glioblastoma. Claim 74 A method comprising administering a B cell vaccine of any one of claims 66 to 70 to a human subject. Claim 75 A method in which, in any one of paragraphs 72 through 74, the human subject has cancer. Claim 76 In paragraph 75, the method in which a human subject has brain cancer, optionally glioblastoma. Claim 77 A method according to any one of paragraphs 71 to 76, wherein the method further comprises administering a therapeutic agent, optionally wherein the therapeutic agent is an immune checkpoint inhibitor. Claim 78 A method for producing a B cell vaccine comprising the following steps: (i) 4-1BBL according to the method of any one of claims 1 to 44 + A step of producing an expanded population of B cells; and (ii) 4-1BBL in (i) + A step of incubating an expanded population of B cells with a tumor lysate containing at least one tumor antigen. Claim 79 In paragraph 78, the method in which the tumor antigen in (ii) is associated with glioblastoma.

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