AU2018316253A1

AU2018316253A1 - LMP1-expressing cells and methods of use thereof

Info

Publication number: AU2018316253A1
Application number: AU2018316253A
Authority: AU
Inventors: Il-Kyu Choi; Zhe Wang; Baochun Zhang
Original assignee: Dana Farber Cancer Institute Inc
Current assignee: Dana Farber Cancer Institute Inc
Priority date: 2017-08-10
Filing date: 2018-08-09
Publication date: 2020-01-16
Also published as: WO2019032862A1

Abstract

The instant disclosure provides LMP1-expressing immunogenic B cells presenting exogenous antigens. Also provided are methods of activating T cells and treating cancer and infectious diseases by vaccination or adoptive cell transfer using the immunogenic B cells. The instant disclosure further provides methods and kits for producing the immunogenic B cells.

Description

LMP1-EXPRESSING CELLS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No.

62/543,479, filed on August 10, 2017, the entire disclosure of which is hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on January 5, 2018, is named 601517DFC-016PCSequence_Listing_ST25.txt and is 3,629 bytes in size.

FIELD OF INVENTION

The present invention relates generally to LMP1-expressing immunogenic B cells presenting exogenous antigens and methods of immunotherapy using the same.

BACKGROUND

Preclinical and clinical developments have shown that immunotherapy represents powerful means to treat or even cure cancer. The main approaches of immunotherapy include adoptive cell transfer (ACT), vaccination, and reinvigorating anti-tumor immunity, for example, through immune checkpoint blockade. The ACT and vaccination approaches largely rely on the activation of T lymphocytes, which can be elicited when an antigen is presented by major histocompatibility complex (MHC) molecules on the surface of an antigen-presenting cell (APC) along with co-stimulation signals simultaneously provided by the APC. Classical APCs include dendritic cells, macrophages, and B cells.

Epstein-Barr virus (EBV), also known as human herpes virus 4 (HHV-4), is a potent tumor virus. EBV specifically infects and transforms human B cells, but also some epithelial cells. EBV-infected B cells are rapidly eliminated by T cells, but EBV acquires a dormant state in a minute fraction of B cells, establishing a life-long latent infection in more than 90% of human beings. Under the conditions of immunosuppression, EBV can spread from these few cells, resulting in explosive expansion of infected B cells and their malignant transformation. Expression of EBV-encoded latent membrane protein 1 (LMP1) is essential for the transformation of human B cells by EBV and can by itself induce oncogenic transformation of rodent fibroblasts.

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There is a need in the art to develop more effective systems for presenting desired antigens and thereby producing therapeutic T cells for the treatment of cancer and other diseases.

SUMMARY

The present disclosure provides immunotherapies using LMP1-expressing immunogenic B cells presenting exogenous antigens for the treatment of cancer and other diseases. Also provided are methods and kits for producing the immunogenic B cells.

Accordingly, in one aspect, the present disclosure provides a method for producing an immunogenic cell, the method comprising:

(a) obtaining a B cell comprising a first nucleic acid encoding a first peptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1; and (b) contacting the B cell with a composition comprising an exogenous antigen or a precursor thereof, thereby producing an immunogenic B cell.

In certain embodiments, the B cell is a naive B cell. In certain embodiments, the B cell is immortalized. In certain embodiments, the B cell is a primary B cell.

In certain embodiments, the first peptide comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the first nucleic acid is operably linked to a transcriptional regulatory element.

In certain embodiments, the exogenous antigen is a second peptide. In certain embodiments, the precursor of the exogenous antigen is a protein comprising the amino acid sequence of the second peptide. In certain embodiments, the precursor of the exogenous antigen is a second nucleic acid encoding the second peptide.

In certain embodiments, the exogenous antigen is a tumor-associated antigen (TAA). In certain embodiments, the composition comprises a tumor cell comprising the TAA. In certain embodiments, the exogenous antigen is a neoantigen. In certain embodiments, the composition comprises a tumor cell comprising the neoantigen. In certain embodiments, the composition further comprises an agent capable of facilitating cell fusion.

In certain embodiments, the exogenous antigen is a bacterial antigen. In certain embodiments, the composition comprises a bacterium comprising the bacterial antigen. In certain embodiments, the bacterium is inactivated. In certain embodiments, the composition comprises a cell that has been infected with a bacterium. In certain embodiments, the composition further comprises an agent capable of facilitating cell fusion.

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In certain embodiments, the exogenous antigen is a viral antigen. In certain embodiments, the composition comprises a viral particle comprising the viral antigen. In certain embodiments, the viral particle is replication defective. In certain embodiments, the composition comprises a cell that has been infected with a viral particle. In certain embodiments, the composition further comprises an agent capable of facilitating cell fusion.

In certain embodiments, the immunogenic B cell comprises the exogenous antigen on the surface. In certain embodiments, the exogenous antigen is conjugated to a class II major histocompatibility complex (MHC-II) on the surface of the immunogenic B cell. In certain embodiments, the exogenous antigen is conjugated to a class I major histocompatibility complex (MHC-I) on the surface of the immunogenic B cell.

In certain embodiments, the method further comprises reducing the proliferative capacity of the immunogenic B cell. In certain embodiments, reducing the proliferative capacity of the immunogenic B cell comprises irradiating the immunogenic B cell.

In another aspect, the instant disclosure provides an immunogenic B cell produced by the method of any one of the preceding claims.

In another aspect, the instant disclosure provides an isolated B cell comprising:

(a) a first nucleic acid encoding a first peptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1; and (b) an exogenous antigen or a precursor thereof.

In certain embodiments, the exogenous antigen is a TAA. In certain embodiments, the exogenous antigen is a neoantigen. In certain embodiments, the exogenous antigen is a bacterial antigen. In certain embodiments, the exogenous antigen is a viral antigen.

In certain embodiments, the isolated B cell comprises the exogenous antigen on the surface. In certain embodiments, the exogenous antigen is conjugated to an MHC-II on the

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In another aspect, the instant disclosure provides a vaccine comprising the immunogenic B cell or the isolated B cell as disclosed herein. In certain embodiments, the vaccine further comprises an adjuvant.

In another aspect, the instant disclosure provides a method of activating a T cell, the method comprising contacting the T cell with the immunogenic B cell, the isolated B cell, or the vaccine as disclosed herein.

In certain embodiments, the T cell is a CD4⁺ T cell. In certain embodiments, the T cell is a CD8⁺ T cell. In certain embodiments, the activated T cell is cytotoxic.

In certain embodiments, the T cell is contacted with the isolated B cell ex vivo. In certain embodiments, the method further comprises culturing the activated T cell under suitable conditions to allow proliferation of the activated T cell. In certain embodiments, the method further comprises administering the activated T cell to a subject in need thereof. In certain embodiments, the subject has a tumor comprising the exogenous antigen. In certain embodiments, the subject is infected with a bacterium comprising the exogenous antigen. In certain embodiments, the subject is infected with a virus comprising the exogenous antigen. In certain embodiments, the immunogenic B cell or the isolated B cell is autologous to the subject. In certain embodiments, the immunogenic B cell or the isolated B cell is from a donor having an MHC matched with the subject. In certain embodiments, the T cell is autologous to the subject. In certain embodiments, the T cell is from a donor having an MHC matched with the subject.

In certain embodiments, the T cell is in a subject, and the immunogenic B cell, the isolated B cell, or the vaccine is administered to the subject in an amount effective to activate the T cell in the subject.

In another aspect, the instant disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic B cell, the isolated B cell, or the vaccine as disclosed herein.

In certain embodiments, the subject has a tumor comprising the exogenous antigen. In certain embodiments, the subject is infected with a bacterium comprising the exogenous antigen. In certain embodiments, the subject is infected with a virus comprising the exogenous antigen. In certain embodiments, the isolated cell is autologous to the subject. In certain embodiments, the isolated cell is from a donor having an MHC matched with the subject.

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In certain embodiments, the method of administering a T cell, a B cells, or a vaccine further comprises administering to the subject an immune co-stimulation therapy. In certain embodiments, the immune co-stimulation therapy is selected from the group consisting of an agonist of CD27, an agonist of 0X40, and an agonist of 4-IBB. In certain embodiments, the method of administering a T cell, a B cells, or a vaccine further comprises administering to the subject an immune checkpoint targeting therapy. In certain embodiments, the method of administering a T cell, a B cells, or a vaccine further comprises administering to the subject a Treg modulating therapy.

In another aspect, the instant disclosure provides a kit comprising:

(a) a first nucleic acid encoding a first peptide comprising the amino acid sequence at least 90% identical to SEQ ID NO: 1; and (b) a composition comprising an exogenous antigen or a precursor thereof.

In certain embodiments, the first peptide comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the nucleic acid is operably linked to a transcriptional regulatory element.

In certain embodiments, the exogenous antigen is a second peptide. In certain embodiments, the precursor of the exogenous antigen is a protein comprising the amino acid sequence of the second peptide. In certain embodiments, the precursor of the exogenous antigen is a second nucleic acid encoding the peptide.

In certain embodiments, the exogenous antigen is a TAA. In certain embodiments, the composition comprises a tumor cell comprising the TAA. In certain embodiments, the exogenous antigen is a neoantigen. In certain embodiments, the composition comprises a tumor cell comprising the neoantigen. In certain embodiments, the composition further comprises an agent capable of facilitating cell fusion.

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In certain embodiments, the kit further comprises an agent capable of reducing the proliferative capacity of a B cell. In certain embodiments, the kit further comprises an instruction to irradiate a B cell.

BRIEF DESCRIPTION OF DRAWINGS

Figure IA is a schematic diagram showing that LMP1 signaling in B cells (e.g., primary B cells) induces expression and presentation of cellular antigens (including many TAAs), and enhances co-stimulation function, thereby eliciting potent polyclonal cytotoxic T cell responses. In B cells, constitutive LMP1 signaling induces massive cellular gene expression. This leads to (1) upregulation of cellular machinery involved in antigen processing and presentation (e.g., MHCs), (2) induction of strong co-stimulation signals (B71, B7-2, ICAM-1, and particularly CD70, OX40L and 4-1BBL), and (3) induced and/or enhanced expression of certain cellular antigens (including a wide range of TAAs). Presentation of the LMP1 signaling-induced cellular antigens (TAAs) and simultaneous costimulations drive activation and cytotoxic differentiation of CD4⁺ and CD8⁺ T cells specific to these antigens. Thus, LMP1 signaling makes B cells hyperimmunogenic antigenpresenting cells (APCs).

Figure IB is a schematic diagram showing that LMP1 signaling in lymphoma B cells enhances presentation of lymphoma inherent TAAs and neoantigens. Some of these lymphoma inherent TAAs are LMP1 signaling-induced TAAs, whose expression is enhanced by LMP1 signaling, whereas other lymphoma inherent TAAs are not. The increased antigen presentation (1) along with enhanced co-stimulation signals (2) leads to cytotoxic T cell responses against these tumor antigens. Thus, LMP1 signaling turns lymphoma B cells into hyperimmunogenic antigen-presenting cells (APCs).

Figure 2A is a schematic diagram showing an expression cassette of LMP1 used in generating CD19-cre;LMPl^^STOP (CL) transgenic mice.

Figure 2B is a schematic diagram demonstrating the role of LMP1 in the surveillance and transformation of LMPl-expressing (EBV-infected) B cells.

Figure 3A is a graph showing dynamics of LMPl-expressing B cells (CD19⁺Fas⁺; Fas is induced by LMP1 signaling and consequently used as a reporter for LMP1 expression in B cells) and activated (CD69⁺) CD4 and CD8 T cells, analyzed by FACS, in the spleen of CL mice compared to those in CD19-cre/+ control (‘C’) mice. The respective mean values of at least three mice of each genotype, at each time point are plotted.

Figure 3B is a graph showing dynamics of LMPl-expressing B cells and activated (CD69⁺) CD4 and CD8 T cells, analyzed by FACS, in the bone marrow (BM) of CL mice

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PCT/US2018/046060 compared to those in CD19-cre/+ control (‘C’) mice. The respective mean values of at least three mice of each genotype, at each time point are plotted.

Figure 4 is a graph showing cytolytic activity of CD4⁺ and CD8⁺ T cells to LMP1expressing B cells. CD4 and CD8 T cells from day 6-8 CL mice kill LMP1-expressing lymphoma cells, upon co-culture for 4 hours. E:T ratio, effector to target cell ratios.

Figure 5A shows FACS analysis of the indicated effector molecules in primary CD4 T cells isolated from day 6-8 CL mice spleen, compared to primary CD4 T cells from adult CL spleen, demonstrating tumor-killing T cells express key cytotoxic molecules.

Figure 5B shows mean fluorescence intensities (MFI) of the indicated effector molecules detected as in the Figure 5A FACS analysis.

Figure 5C shows FACS analysis of the indicated effector molecules in primary CD8 T cells isolated from day 6-8 CL mice spleen, compared to primary CD8 T cells from adult CL spleen, demonstrating tumor-killing T cells express key cytotoxic molecules.

Figure 5D shows mean fluorescence intensities (MFI) of the indicated effector molecules detected as in the Figure 5C FACS analysis.

Figure 6A is a graph showing cytotoxicity of the indicated T cells assayed on LMP1expressing lymphoma cells as targets. CD4 and CD8 T cells were from adult (day 42-84) CL mice BM; the adoptive CD4 T cells were those initially isolated from adult CL mice BM, adoptively transferred (along with LMPl-expressing lymphoma cells) into Rag2^yc^ recipients, and then recovered from the latter. Representative data from three independent experiments are shown. All mice used here are on a (C57BL/6xBALB/c) Fl (CB6F1) background, while the lymphoma cells are on a C57BL/6xBALB/c mixed background.

Figure 6B is a representative series of graphs showing the flow cytometry analysis of the indicated effector molecules in the adoptive CD4 cells compared to primary CD4 cells from adult CL mice BM (chronic state) and spleens (negative control).

Figure 6C is a set of survival curves showing the therapeutic efficacies of adoptive CD4 and CD8 cells in combination with radiation therapy (RT) in mice bearing aggressive LMPl-driven primary lymphomas. TCRfi~~S~~ CL mice on a C57BL/6xBALB/c mixed background at 8-week old were treated with 500 Rad of irradiation. One day later, some mice were further treated (by intravenous injection) with the indicated T cells isolated from CL mice on a CB6F1 background at the dose of 1 x 10⁶ cells/recipient. Survival curves were compared using the log-rank test.

Figure 7A is a bar graph showing TCR Υβ chains in CD8 T cells from the indicated mice that were stained with a panel of monoclonal antibodies for the indicated TCR Υβ

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PCT/US2018/046060 chains. These νβ specific antibodies collectively detected 85-95% of TCRs in all the samples. Control d8, day 8 old CD19-cre/+ mice. Data are shown as mean ± SEM.

Figure 7B is a bar graph showing TCR Υβ chains in CD4 T cells (excluding CD25⁺Foxp3⁺ Tregs) from the indicated mice that were stained with a panel of monoclonal antibodies for the indicated TCR Υβ chains. These Υβ specific antibodies collectively detected 85-95% of TCRs in all the samples. Control d8, day 8 old CD19-cre/+ mice; the adoptive CD4 T cells were those initially isolated from adult CL mice BM, adoptively transferred (along with LMP1-expressing lymphoma cells) into Rag2^Yc^ recipients, and then recovered from the latter. Data are shown as mean ± SEM.

Figure 7C is a graph showing\n vitro killing activity of the indicated CD4 T cells from day 6-8 CL mice, assayed on LMP1-expressing lymphoma cells. Data are shown as mean ± SEM of duplicates. Representative data from two independent experiments are shown. CL and control mice used here are on a CB6F1 background.

Figure 8 shows FACS analysis of naive B cells, CD40-activated B cells from wildtype (WT) mice, LMP1-expressing lymphoma B cells and B cells from LMPlfl^srop mice treated with TAT-Cre to turn on LMP1 expression in vitro (LMP 1-expressing B cells).

Figure 9A shows fluorescent microscopy imaging of B cells expressing LMP1-GFP fusion, LMPl™^lm-GFP fusion or GFP, respectively. Note that wild-type LMP1 aggregates into large complexes on cell membrane, while the mutant LMPl™^lm loses its ability to aggregate.

Figure 9B is a pair of graphs showing CD4 T cells (left panel) and CD8 T cells (right panel) from day 6-8 CL mice assayed for killing activity on B cells (from WT B6 mice) transduced with retroviral vectors expressing wild-type LMP1 or a signaling-dead mutant LMP I™¹'. B cells untransduced or transduced with the empty vector as controls.

Figure 10A is a pair of graphs showing that CD4 and CD8 T cells from day 6-8 CL mice lyse LMP 1-expressing B cells/lymphoma cells as well as anti-CD40 pretreated WT B cells, but not naive B cells.

Figure 10B is a graph showing the results of an in vitro killing assay performed with CD4 T cells from day 6-8 CL mice on CD40-activated WT B cells (from B6 mice), in the presence of Fas-Fc (to block FasL-mediated killing) and/or MHCII blocking antibody.

Figure 11 shows FACS analysis of CD4⁺ effector/memory T cells (excluding Tregs) from Foxp3^GFP;CL male mice that recognize and proliferate on CD40-activated WT B cells in an MHC-II restricted manner.

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Figure 12A shows FACS analysis of CD40 expression on LMP1-expressing B cells from a 6-day old CL mouse, compared to that on B cells from a littermate control (CD19cre/+). Note that LMP1 signaling in B cells upregulates CD40.

Figure 12B shows FACS analysis of CD40 expression on B cells from the indicated mice at 6 weeks old. Note that the B cells in CL and CD40~^/~;CL mice represent residual B cells (which do not express LMP1) after clearance of LMP1-expressing B cells.

Figure 12C shows FACS analysis of B cells and T cells in spleens of the indicated mice at 6 weeks old.

Figure 12D shows FACS analysis of activation marker CD69 on CD4 and CD8 T cells from the BM of the indicated mice at 6 weeks old. Data in FIGs. 12A-12D represent 2-3 mice analyzed for each genotype.

Figure 13A is a heat map showing expression of co-stimulatory and co-inhibitory molecules in LMP1-expressing B cells compared to control B cells. Splenic B cells from LMP f^!STO‘’/YFpfl^STOP _and Yppfl^STOP/+ mice (both on a CB6F1 background) were treated with TAT-Cre to generate LMP 1-expressing B cells and YFP control B cells. All treated B cells were collected at day 2 post-treatment for array analysis.

Figure 13B shows FACS plots (upper panel) and mean fluorescence intensities (MFI; lower panel) of the indicated co-stimulatory ligands in LMP 1-expressing B cells from day 68 CL mice, compared to splenic B cells from WT control (ctr) mice. Data are representative of 2-6 mice analyzed for each group. The mice (CZ and control) are on a CB6F1 background. Each symbol represents an individual mouse; bars show the respective mean values; ****, p<0.0001; ***, p<0.001 (unpaired two-tailed student’s t-test).

Figure 13C is a heat map showing cytokine genes expressed in LMP 1-expressing B cells compared to control B cells. Splenic B cells from LMP 1^^STOF/YFP^^STOP and Yppf^ISTOP/+mice (both on a CB6F1 background) were treated with TAT-Cre to generate LMP1expressing B cells and YFP control B cells. All treated B cells were collected at day 2 posttreatment for array analysis. Mean-centered log2 gene expression ratios are depicted by color scale.

Figure 14A shows FACS analysis of Eomes and GzmB expression in CD4 T cells from day 6-8 CL mice and WT control (ctr) mice. GzmB levels in Eomes⁺ CD4 cells from CL mice were compared to that in total CD4 cells from control mice and shown on the right.

Figure 14B shows FACS analysis of Eomes vs. T-bet (upper panel) and GzmB vs. IFN-γ (lower panel) in CD4 T cells from day 6-8 CL mice and WT control (ctr) mice. The frequencies (mean ± SEM) of indicated populations are shown within the gates.

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Figure 14C shows FACS analysis of Eomes vs. T-bet (upper panel) and GzmB vs.

IFN-γ (lower panel) in CD8 cells from day 6-8 CL mice and WT control (ctr) mice. Data in (A-C) are representative of 3-4 mice of each group (all on a CB6F1 background), analyzed in two independent experiments.

Figure 15A shows FACS analysis of Eomes vs. GATA-3 in CD4 cells from day 6-8 CL mice and WT control (ctr) mice. Data are representative of 3-4 mice of each group (all on a CB6F1 background), analyzed in two independent experiments.

Figure 15B shows FACS analysis of Eomes vs. RORyt in CD4 cells from day 6-8 CL mice and WT control (ctr) mice. Data are representative of 3-4 mice of each group (all on a CB6F1 background), analyzed in two independent experiments.

Figure 16A is a graph showing numbers (mean ± SEM) of recovered T cells after coculturing for 7 days with B cells expressing LMP1 or LMPl™¹¹¹¹. The cell culture was begun with 1.5 x 10⁶ purified CD4 T cells together with the indicated B cells (irradiated at 500 RAD before co-culturing) at 1:1 ratio in triplicate wells of 12-well plates. No exogenous cytokines were added. ***, p<0.001 (unpaired two-tailed student’s t-test). B cells and T cells are from 2-3 months old naive WT B6 mice spleens.

Figure 16B shows FACS analysis of Eomes and T-bet expression in CD4 cells cocultured with the indicated B cells (as in (A)).

Figure 16C is a graph showing cytotoxicity of CD4 cells expanded on LMP1-B cells (as in (A)) against B cells transduced with the MSCV-LMP1-IRES-GFP retrovirus, which contained GFP⁺ (LMP1-B cells) and GFP cells (not successfully transduced cells and thus representing LPS-activated B cells, see Materials and Methods; these cells served as control).

Figure 16D shows proliferation of CD4 T cells expanded on LMP1-B cells (as in (A)) assayed on CD40-activated B cells from WT or CIITA ^ mice. Data in (A-D) are representative of 2-4 independent experiments using splenic B cells and T cells from 2-3 months old naive WT B6 mice.

Figure 16E shows FACS analysis of Eomes expression in CD4 cells either freshly isolated from naive B6 mice (Ex vivo), or after co-culturing for 7 days with LMP1-B cells in the presence of the indicated blocking antibodies or corresponding isotype controls. Representative data from one of triplicate wells are shown, with the frequency of Eomes⁺cells in the gate.

Figure 16F shows numbers (mean ± SEM) of Eomes⁺ CD4 cells recovered from culture wells treated with the indicated blocking antibodies relative to those from corresponding isotype control treated wells.

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Figure 16G shows numbers (mean ± SEM) of recovered CD4 cells after co-culturing for 7 days with LMP1⁺ B cells in the presence of the indicated blocking antibodies or corresponding isotype controls. The cell culture was begun with 1 x 10⁶ purified CD4 T cells in triplicate wells of 24-well plates.

Figure 16H shows FACS analysis of Eomes expression in CD8 cells either freshly isolated from naive B6 mice, or after co-culturing for 3 days with LMP1-B cells in the presence of the indicated blocking antibodies or corresponding isotype controls. Representative data from one of triplicate wells are shown, with the frequency of Eomes⁺cells in the gate.

Figure 161 shows numbers (mean ± SEM) of Eomes⁺ CD8 cells recovered from culture wells treated with the indicated blocking antibodies relative to those from corresponding isotype control treated wells.

Figure 16J shows numbers (mean ± SEM) of recovered CD8 cells after co-culturing for 3 days with LMP1⁺ B cells in the presence of the indicated blocking antibodies or corresponding isotype controls. The cell culture was begun with 0.5 x 10⁶ purified CD 8 T cells in triplicate wells of 24-well plates.

Figure 17 is a representative flow cytometry analysis that shows detection of specific T cell response to TAAs expressed by LMP1-expressing B cells. CD8 T cells reactive to a Survivin-derived epitope (ATFKNWPFL) and an EphA2-derived epitope (VVSKYKPM) were detected by MHC-I tetramers bearing the corresponding epitope peptides in CD19cre^E^²;LMPl^flSTOP (C^ERr2L) and CD19-cre'^J<r2 (C^PT2) control mice at day 5 following Tamoxifen treatment (to turn on LMP1 expression initially in a small fraction of B cells). The frequencies of tetramer⁺ CD8 T cells are shown within the gates. All mice are on a B6 background.

Figure 18A shows analysis of the frequency of CD4 Tregs (CD25⁺Foxp3⁺) in the CD4 T cell compartment in day-8 old CL and control (CDl 9-cre ) mice. The percentage (average ± SEM) of CD4 Tregs in CD4⁺ T cells is shown above the gate.

Figure 18B shows analysis of the frequency of CD4 Tregs in the CD4⁺ T cells in adult (day 42-84) CL mice BM (left panel) or in recipient mice transplanted with adult CL mice BM CD4⁺ T cells and LMP1⁺ lymphoma cells (right panel). CD4⁺ T cells were recovered from recipients at day 10 post-transfer for FACS analysis.

Figure 18C shows direct killing activity of the indicated T cells isolated from adult Foxp3^DTR/GFP;CL male mice (on a CB6F1 background), assayed using LMP1⁺ lymphoma cells as targets. CD4 dep Tregs, CD4 T cells depleted of Tregs.

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Figure 18D shows direct killing activity of the CD8 T cells isolated from adult

Foxp3^DTR/GFP;CL male mice (on a CB6F1 background), with or without addition (at 1:1 ratio) of CD4 Tregs from the same mice, assayed using CD40-activated WT B cells (on a B6 background) as targets.

Figure 19A shows a scheme depicting the use of LMP1-expressing cells to activate/expand T cells for adoptive cell transfer (ACT) therapy for cancers.

Figure 19B shows a scheme of ACT in which CD8 and/or CD4 T cells primed by LMP1-expressing B cells are used to treat tumor-bearing mice. Before tumor implantation, mice receive 600 Rad of total body irradiation (TBI) to create a lymphopenic condition favorable for adoptive T cell expansion.

Figure 19C is a graph showing that ACT of CD8 T cells primed by LMP1-expressing B cells delays tumor (A20) growth. Control mice received no ACT. Error bars represent means ± SEM.

Figure 19D is a graph showing that ACT of CD4 T cells primed by LMP1-expressing B cells delays tumor (A20) growth. Control mice received no ACT. Error bars represent means ± SEM.

Figure 19E is a graph showing that CD4 T cells primed by LMP1-expressing A20 lymphoma cells kill the unmodified (parental) A20 cells, but not control naive B cells. Unprimed (naive) CD4 T cells have no killing activity.

Figure 19F is a graph showing that CD4 T cells primed by LMP1-expressing A20 lymphoma cells kill the A20 cells in an MHC-II-dependent manner. This indicates that the CD4 T cells recognize (and kill) the A20 cells through endogenous antigens presented on MHC-II. Note: OITA ^z. lacking MHC-II expression.

Figure 19G shows a scheme of ACT in which CD4 T cells primed by LMP1expressing A20 lymphoma cells are used to treat tumor (A20)-bearing mice.

Figure 19H is a graph showing that ACT of CD4 T cells primed by LMP1-expressing A20 lymphoma cells (these primed CD4 cells are referred to as CD4 CTLs) markedly delays tumor (A20) growth. Control mice received either no ACT or naive CD4 T cells. Error bars represent means ± SEM.

Figure 191 shows a scheme of ACT in which CD8 T cells primed by LMP1expressing A20 lymphoma cells are used to treat tumor (A20)-bearing mice.

Figure 19J is a graph showing that ACT of CD8 T cells primed by LMP1-expressing A20 lymphoma cells delays tumor (A20) growth. Control mice received no ACT.

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Figure 20A shows a scheme depicting vaccination strategy with LMPl-expressing B cells or tumor cells for treatment of cancers.

Figure 20B shows a vaccination scheme in which lymphoma cells are transduced to express LMP1 and used as vaccine to treat the unmodified (parental) B cell lymphoma.

Figure 20C is a graph showing that vaccination with LMPl-expressing A20 lymphoma cells markedly delays tumor (A20) growth. A20 lymphoma cells expressing the signaling-dead mutant LMPl™¹¹¹¹ serve as control vaccine.

Figure 20D shows a vaccination scheme in which tumor cells (B16-F10) are transduced to express LMP1 and used as vaccine to treat the unmodified (parental) tumor (melanoma).

Figure 20E is a graph showing that vaccination with LMPl-expressing B16-F10 melanoma cells markedly delays tumor (melanoma) growth. B16-F10 cells expressing the signaling-dead mutant LMPl™^lmor transduced with the empty vector serve as control vaccine.

Figure 21A shows a scheme depicting the use of LMPl-expressing B cells as an APC system to produce desired antigen (such as OVA)-specific cytotoxic T cells.

Figure 21B is a graph showing that CD8⁺ OT-I T cells primed by OVA peptideloaded LMPl-expressing B cells lyse OVA-expressing EL4 cells, but not control EL4 cells.

Figure 21C is a graph showing that CD8⁺ OT-I T cells primed by OVA peptideloaded LMPl-expressing B cells lyse OVA peptide-loaded naive B cells, but not unloaded naive B cells.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to be understood that this disclosure is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only in the appended claims. It is readily apparent to one skilled in the art that various embodiments and modifications can be made to the disclosure of the present application without departing from the scope and spirit of the instant application.

I. Definitions

In describing and claiming the instant application, the following terminology will be used in accordance with the definitions set forth below.

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As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

Still further, the terms “having,” “including,” “containing,” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.

As used herein, the term “antigen” is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates. Any macromolecules, including virtually all proteins or peptides, can serve as antigens. Furthermore, antigens can be derived from recombinant or genomic DNA. In certain embodiments, an antigen includes a fragment of a protein that elicits an immune response.

As used herein, the term “exogenous antigen” refers to an antigen that is expressed from a polynucleotide in a cell, wherein the polynucleotide is not present in the genome of the cell (e.g. it is present in a vector in the cell that does not integrate into the genome of the cell), is not present in the genome of the cell (e.g. is not a native gene of the cell) or is not present at the native genomic location of the gene in the genome of the cell (e.g. a gene from the cell that is provided in a vector that integrates into the genome of the cell, but not at the location where the native gene is located in the genome of the cell). For example, an exogenous antigen expressed in a human cell could be identical in amino acid sequence to an antigen naturally expressed by the human cell, however, the exogenous antigen would be expressed from a vector that either did not integrate into the genome of that cell or integrated at a distinct location from the endogenous gene. The term “exogenous antigen” also refers to an antigen that is contacted to a cell wherein the antigen is a protein that is expressed outside the contacted cell.

Exogenous antigens include without limitation unmodified peptides, peptides with one or more post-translational modifications (e.g., phosphorylation, glycosylation, or Snitrosylation), glycoproteins, and proteoglycans. A skilled person in the art would appreciate that the exogenous antigen need not be absent in a wild-type B cell of the same subset (e.g., a primary naive B cell), and may be present in a B cell of the same subset (e.g., a primary naive B cell) at a substantially lower amount, wherein the B cell comprises a polynucleotide encoding LMP1 but is otherwise wild-type. In certain embodiments, the exogenous antigen is absent in a wild-type cell of the species (e.g., human). In certain embodiments, the

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PCT/US2018/046060 exogenous antigen is substantially absent (e.g., not significantly expressed from the native genomic locus) in a wild-type B cell of the same subset (e.g., a primary naive B cell).

As used herein, the term “precursor” of an exogenous antigen refers to one or more molecules from which the exogenous antigen can be derived. In certain embodiments, the exogenous antigen is a modified (e.g., phosphorylated, S-nitrosylated, methylated, acetylated, glycosylated, ubiquitinated, or SUMOylated) peptide or protein, and the precursor is the peptide or protein without the modification. A skilled person in the art would appreciate that the precursor need not be physically converted to the exogenous antigen. Instead, the information carried on the precursor can be converted into the exogenous antigen. For example, in certain embodiments, the exogenous antigen is a peptide or protein, and the precursor is a polynucleotide encoding the peptide or protein, or the reverse complement thereof. A skilled person in the art would also appreciate that a precursor of a precursor of an exogenous antigen is a precursor of the exogenous antigen.

As used herein, the term “LMP1” refers to Epstein-Barr virus (EBV) latent membrane protein 1. In certain embodiments, LMP1 comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, LMP1 consists of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, LMP1 is encoded by the gene with NCBI Gene ID No. 3783750. In further embodiment, LMP1 is a polypeptide with a sequence identity ranging from 70% to 80%, from 81% to 85%, from 86% to 90%, from 91% to 95%, from 96% to 100%, or 100% to SEQ ID NO. 1. In other embodiments, LMP1 is a polypeptide with a sequence identity of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO. 1.

SEQ ID NO: 1 (LMP1 polypeptide sequence from GenBank Accession No. YP_401722.1): MEHDLERGPPGPRRPPRGPPLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYS FALMLIIIILIIFIFRRDLLCPLGALCILLLMITLLLIALWNLHGQALFLGIVLFIFGCLLVL GIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFL AILIWMYYHGQRHSDEHHHDDSLPHPQQATDDSGHESDSNSNEGRHHLLVSGAGDG PPLCSQNLGAPGGGPDNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNG PQDPDNTDDNGPHDPLPHSPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGH SHDSGHGGGDPHLPTLLLGSSGSGGDDDDPHGPVQLSYYD.

The term “MHC” refers to “major histocompatibility antigen.” In humans, the MHC genes are known as HLA (“human leukocyte antigen”) genes. The most intensely studied HLA genes are the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLADPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRBL In humans,

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PCT/US2018/046060 the MHC is divided into three regions: Class I, II, and III. The A, B, and C genes belong to MHC class I, whereas the six D genes belong to class II. MHC class I molecules are made of a single polymorphic chain containing 3 domains (alpha 1, 2 and 3), which associates with beta 2 microglobulin at cell surface. Class II molecules are made of 2 polymorphic chains, each containing 2 domains (alpha 1 and 2, and beta 1 and 2). Class I MHC molecules are expressed on virtually all nucleated cells. Peptide fragments presented in the context of class I MHC molecules are recognized by CD8⁺ T lymphocytes (traditionally called cytotoxic T lymphocytes or CTLs). CD8⁺ T lymphocytes frequently mature into cytotoxic effectors which can lyse cells bearing the stimulating antigen. Class II MHC molecules are expressed primarily on activated lymphocytes and professional APCs. CD4⁺ T lymphocytes (traditionally called helper T lymphocytes or HTLs) are activated with recognition of a unique peptide fragment presented by a class II MHC molecule, usually found on an APC, like a macrophage, dendritic cell, or B cell. CD4⁺ T lymphocytes proliferate and secrete cytokines that either support an antibody-mediated response through the production of IL-4 or support a cell-mediated response through the production of IL-2 and IFN-gamma, or acquire direct killing activity (cytotoxicity).

The term “co-stimulatory molecule” refers to molecules on APCs that engage particular co-stimulatory receptors on T cells, providing the so-called second signal necessary for T cell proliferation and functional differentiation. Representative co-stimulatory molecules include, but are not limited to, CD80/B7-1, CD86/B7-2, 0X40 ligand, 4-1BB ligand and CD70.

As used herein, the term “cytokine” is defined as growth, differentiation or chemotropic factors secreted by immune or other cells, whose action is on cells of the immune system, such as, but not limited to, T cells, B cells, NK cells and macrophages or other cell types, such as endothelial cells, hematopoietic cells, etc. Representative cytokines include, but are not limited to, the group consisting of IFN-γ, TNF-α, IL-2 and IL-17.

The term “sequence identity” or “sequence homology” of two sequences when used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, when the two sequences are aligned. In particular embodiments, the sequence identity is from 70% to 80%, from 81% to 85%, from 86% to 90%, from 91% to 95%, from 96% to 100%, or 100%. In certain embodiments, the sequence identity is 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

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The terms “cell,” “cell line,” and “cell culture” as used herein include progeny, which are any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.

The term “nucleic acid” and “polynucleotide” are used interchangeably and refer to deoxyribonucleic acids or ribonucleic acids and variants thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixedbase and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994))

The term “transcriptional regulatory element” refers to a nucleic acid sequence, usually found upstream (5') to a coding sequence, which directs transcription of a nucleic acid sequence into mRNA. Exemplary transcriptional regulatory elements include promoters and enhancers. A promoter typically provides a recognition site for RNA polymerase and the other factors necessary for proper initiation of transcription. As contemplated herein, a promoter or promoter region includes promoters derived from a sequence in a wild-type genome by inserting or deleting regulatory regions, subjecting the promoter to random or site-directed mutagenesis, etc. The activity or strength of a promoter may be measured in terms of the amounts of RNA it produces, or the amount of protein generated in a cell or tissue, relative to a promoter whose transcriptional activity has been previously assessed. An enhancer typically provides a recognition site for one or more transcription factors that increase or decrease the transcription of a gene operably linked thereto. The enhancer can be located either upstream or downstream from the gene. A transcriptional regulatory element can be a constitutive (e.g. MSCV promoter) tissue-specific (e.g., active only in certain cells and/or at certain developmental stages), or regulatable (e.g., can be switched on and/or off by one or more exogenous molecules).

The term “operably linked” refers to the functional spatial arrangement of two or more nucleic acid sequences. For example, where a promoter is operably linked to a nucleic

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PCT/US2018/046060 acid sequence encoding a polypeptide, the promoter is positioned relative to the nucleic acid sequence such that transcription of the nucleic acid sequence is directed by the promoter region.

As used herein, the term “autologous” in the context of ACT refers to any material (e.g., cells) administered to a subject from whom the material was derived.

As used herein, the term “polypeptide” is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is interchangeable with the term “peptide.” A protein may comprise one or more polypeptides.

As used herein, the term “treating” includes prophylaxis of the specific disorder or condition, or alleviation of one or more symptoms associated with a specific disorder or condition and/or preventing or eliminating the symptoms.

As used herein, an “effective” amount or a “therapeutically effective amount” of a pharmaceutical refers to a nontoxic but sufficient amount of the pharmaceutical to provide the desired effect. For example, one desired effect would be the prevention or treatment of a tumor (e.g., breast cancer). The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term “in vivo” refers to a process taking place inside a living subject. The term “in vitro” refers to a process taking place outside a living subject.

The term “proliferative capacity” refers to the ability of cells to undergo cell division. The proliferative capacity of cells may be measured by any method known in the art including, but not limited to, the enumeration of cells before and after stimulation with a suitable growth factor, fluorescent dye assays, incorporation of BrdU in the DNA of proliferating cells, incorporation of radio-labeled analogues such as 3H-thymidine into the DNA of proliferating cells and/or the detection of cellular markers of proliferation.

“A subject” encompasses, but is not limited to, a mammal, e.g. a human, a domestic animal or a livestock including a cat, a dog, a cow, and a horse. As used herein the term “patient” without further designation is intended to encompass any warm-blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs, and other pets) and humans.

II. Isolated B cells comprising exogenous antigens

The instant disclosure provides isolated B cells comprising a nucleic acid encoding LMP1 and capable of presenting exogenous antigens. Such B cells can be used as antigen18

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PCT/US2018/046060 presenting cells. As used herein, the term “antigen-presenting cell” encompasses without limitation a variety of cells capable of displaying, acquiring, and/or presenting at least one antigen or antigenic fragment on its cell surface. In general, the term “antigen-presenting cell” can be any cell that enhances an immune response (i.e., from the T cell or B cell arms of the immune system) against an antigen or antigenic composition. An APC can display or present an antigen with a class I major histocompatibility complex (MHC-I) or a class II major histocompatibility complex (MHC-II). In certain embodiments, the MHC is selected from the group consisting of MHC-I, MHC-II, HLA-A, HLA-B, HLA-C, HLA-DP (e.g., HLA-DPA1 or HLA-DPB1), HLA-DQ (e.g., HLA-DQA1 or HLA-DQB1), and HLA-DR (e.g., HLA-DRa or HLA-DRP). In certain embodiments, a B cell as disclosed herein displays or presents an antigen with an MHC-II (e.g., HLA-DP, HLA-DQ, or HLA-DR).

APCs generally activate T cells that bind to the antigens presented. In certain embodiments, a B cell as disclosed herein is capable of activating a CD4⁺ T cell or a CD8⁺ T cell. In certain embodiments, the CD4⁺ T cell or CD8⁺ T cell is a cytotoxic T cell. In certain embodiments, the B cell further comprises a co-stimulatory signal on the surface capable of engaging one or more co-stimulatory receptors on the T cell. Accordingly, in certain embodiments, the B cell is capable of eliciting an effective adaptive immunity against the exogenous antigen. Exemplary adaptive immunity includes T cell proliferation, differentiation and survival. In certain embodiments, the B cell further comprises one or more additional molecules capable of aiding or enhancing an immune response. Exemplary additional molecules include secreted soluble molecules (e.g., cytokines, chemokines, and cytotoxic molecules).

In certain embodiments, LMPlcomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In certain embodiments, the nucleic acid encoding LMP1 is less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to an Epstein-Barr virus (EBV) genome. In certain embodiments, at least 50% of an Epstein-Barr virus (EBV) genome is absent from the nucleic acid. In certain embodiments, the nucleic acid is operably linked to a transcription regulatory element (e.g., a promoter and/or an enhancer). In certain embodiments, the transcription regulatory element comprises a promoter and/or an enhancer.

The nucleic acid encoding LMP1 can be present in any form and in any subcellular compartment of the B cell. In certain embodiments, the nucleic acid is a DNA. In certain embodiments, the nucleic acid is an RNA. In certain embodiments, the nucleic acid is modified from a DNA or RNA, optionally wherein the modification increases the stability

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PCT/US2018/046060 and/or half-life of the nucleic acid in the B cell. In certain embodiments, the nucleic acid is integrated into the genome of the B cell. In certain embodiments, the nucleic acid is present as an extrachromosomal nucleic acid.

In certain embodiments, the B cell is a naive B cell (e.g., a B cell that has not been exposed to a foreign antigen so that it has not committed differentiation into a clone of memory or plasma cells). In certain embodiments, the B cell is a plasma cell. In certain embodiments, the B cell is a memory B cell. In certain embodiments, the B cell is a primary B cell. In certain embodiments, the B cell is immortalized. In certain embodiments, the B cell further comprises a polynucleotide encoding a polypeptide capable of inducing cell death. In certain embodiments, the polypeptide is a chimeric polypeptide comprising a multimerization (e.g., dimerization or oligomerization) region and a cell death-inducing region, wherein the cell death-inducing region is activated by multimerization. In certain embodiments, the cell death-inducing region comprises a sequence of a caspase (e.g., caspase-9) that has protease activity. In certain embodiments, the cell death-inducing region comprises the full-length human caspase-9 polypeptide. In certain embodiments, the cell death-inducing region comprises a truncated human caspase-9 polypeptide (e.g., wherein the CARD domain of caspase-9 is deleted).

In certain embodiments, the B cell further comprises an exogenous antigen or a precursor thereof. In certain embodiments, the exogenous antigen is absent in a wild-type cell of the species (e.g., human), and is from a different species (e.g., a bacterium or a virus). In certain embodiments, the exogenous antigen is substantially absent (e.g., not significantly expressed from the native genomic locus) in a wild-type B cell of the same subset (e.g., a primary naive B cell), but can be substantially expressed in a different cell (e.g., a tumor cell with a B cell or non-B cell origin) of the subject. In one embodiment, the exogenous antigen is expressed at a substantially lower amount in a B cell of the same subset (e.g., a primary naive B cell), wherein the B cell comprises a polynucleotide encoding LMP1 but is otherwise wild-type. In another embodiment, the exogenous antigen is expressed in a wild-type B cell of the same subset (e.g., a primary naive B cell) primarily in a different isoform or with a different modification. In yet another embodiment, the exogenous antigen is expressed in a wild-type B cell of the same subset (e.g., a primary naive B cell) primarily in a different subcellular compartment.

In certain embodiments, the exogenous antigen is a peptide or a protein. In certain embodiments, the precursor comprises at least a portion of the sequence of the peptide or protein. In certain embodiments, the precursor further comprises a membrane penetrating

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PCT/US2018/046060 sequence (e.g., a Tat domain from HIV, an angiopep peptide, a receptor-binding domain of apolipoprotein B, and a receptor-binding domain of apolipoprotein E). In certain embodiments, the precursor is cleaved or partially degraded to generate the peptide or protein. In certain embodiments, the precursor can be converted to the exogenous antigen by one or more enzymes present in the B cell.

In certain embodiments, the exogenous antigen is a peptide or protein. Either a fulllength polypeptide or a truncated peptide of the antigen can be employed. Where the exogenous antigen is a truncated peptide, the truncation that leads to desirable expression, antigen presentation, and/or immunogenicity can be identified by methods known in the art (e.g., the methods described in the Example section). In certain embodiments, the peptide or protein is unmodified. In certain embodiments, the peptide or protein comprises one or more post-translational modifications (e.g., phosphorylation, glycosylation, or S-nitrosylation, methylated, acetylated, ubiquitinated, and/or SUMOylated). In certain embodiments, the precursor is a peptide or protein without the modifications or comprising different modifications.

In certain embodiments, the precursor is a polynucleotide encoding the peptide or protein, or a reverse complement thereof, from which the exogenous antigen is expressed. In certain embodiments, the polynucleotide is not present in the genome of the B cell. In certain embodiment, the polynucleotide is present in the genome, but is not present at the genomic location of the genome in a wild-type cell of the species (e.g., human).

In certain embodiments, the exogenous antigen is a glycoprotein or a proteoglycan. In certain embodiments, the precursor comprises at least a portion of the glycoprotein or proteoglycan. For example, in certain embodiments, the precursor comprises at least a portion of the peptide or protein part of the glycoprotein or proteoglycan. In certain embodiments, the precursor comprises at least a portion of a carbohydrate group of the glycoprotein or proteoglycan. The precursor can further comprise a different peptide or protein, from which the carbohydrate group can be transferred to the exogenous antigen.

In certain embodiments, the exogenous antigen is a tumor-associated antigen (TAA). In certain embodiments, the TAA is specifically expressed in a tumor cell. In certain embodiments, the TAA is specifically expressed in a cell type that has undergone neoplasm (e.g., prostate cell). Exemplary TAAs of these two types include without limitation Survivin, NY-ESO-1, CEA, PSA, PSCA, SCP-1, PSMA, PRAME, tyrosinase, melan-A/MART-1, an SSX protein, such as SSX-2 or SSX-4, a MAGE protein, such as MAGE-1 or MAGE-3, LAGE, mesothelin, Her-2/Neu, PLK1, VEGF-A, VEGFR2, and Tie-2. In certain

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PCT/US2018/046060 embodiments, the TAA is associated with non-cancerous cells of the tumor such as tumor neovasculature or other stromal cells within the tumor microenvironment. Exemplary TAAs of this type include without limitation NY-ESO-1, SSX-2, LAGE, and PRAME.

In certain embodiments, the exogenous antigen is a neoantigen. As used herein, the term “neoantigen” refers to tumor-specific mutation-derived antigens. In certain embodiments, neoantigens can be purified from a tumor sample. In other embodiments, a neoantigen can be prepared by sequencing the nucleic acids in a tumor sample, identifying the tumor-specific mutations, and synthesizing peptides or polypeptides comprising the mutant sequences. In certain embodiments, the composition is useful in treating the patient from whom the tumor sample is obtained.

In certain embodiments, the isolated B cell comprises one or more TAAs and/or one or more neoantigens. In certain embodiments, the isolated B cell comprises one or more nucleic acids encoding TAAs and/or neoantigens. In certain embodiments, at least one of the nucleic acids encodes two or more (e.g., three or more, four or more, or five or more) TAAs and/or neoantigens. In certain embodiments, the TAAs and/or the neoantigens are present in a same tumor sample. In certain embodiments, the isolated B cell is useful for treating the patient from whom the tumor sample is obtained.

In certain embodiments, the exogenous antigen is a bacterial antigen. In certain embodiments, the bacterial antigen is derived from a bacterium selected from the group consisting of Salmonella, Escherichia, Pseudomonas, Bacillus, Vibrio, Campylobacter, Heliobacter, Erwinia, Borrelia, Pelobacter, Clostridium, Serratia, Xanothomonas, Yersinia, Burkholdia, Listeria, Shigella, Pasteurella, Enterobacter, Corynebacterium, Mycobacterium tuberculosis, and Streptococcus.

In certain embodiments, the exogenous antigen is a viral antigen. In certain embodiments, the viral antigen is expressed from a virus selected from the group consisting of human immunodeficiency virus (HIV), human T-lymphotropic virus, herpes virus, papillomavirus, Ebola virus, picornavirus, enterovirus, measles virus, mumps virus, influenza virus, parainfluenza virus, rabies virus, vesicular stomatitis virus (VSV), dengue virus, hepatitis virus, rhinovirus, yellow fever virus, bunga virus, polyoma virus, coronavirus, rubella virus, echovirus, pox virus, varicella zoster virus, and African swine fever virus.

In certain embodiments, the exogenous antigen is a fungal antigen. In certain embodiments, the fungal antigen is derived from a fungus selected from the group consisting of Aspergillus, Coccidoides, Cryptococcus, Candida, Nocardia, Pneumocystis, and Chlamydia.

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In certain embodiments, the exogenous antigen is a parasite antigen. In certain embodiments, the parasite antigen is derived from a parasite selected from the group consisting of Babesia, Entamoeba, Leishmania, Plasmodium, Trypanosoma, Toxoplasma, Giarda, flat worms, and round worms.

In certain embodiments, the B cell comprises an exogenous antigen on the cell surface. In certain embodiments, the exogenous antigen is conjugated to a major histocompatibility complex (MHC). In certain embodiments, the MHC is selected from the group consisting of MHC-I, MHC-II, HLA-A, HLA-B, HLA-C, HLA-DP (e.g., HLA-DPA1 or HLA-DPB1), HLA-DQ (e.g., HLA-DQA1 or HLA-DQB1), and HLA-DR (e.g., HLADRa or HLA-DRP). In certain embodiments, a B cell as disclosed herein displays or presents an antigen with an MHC-II (e.g., HLA-DP, HLA-DQ, or HLA-DR).

III. Methods of producing immunogenic B cells

In another aspect, the present disclosure provides a method of producing an immunogenic cell, the method comprising obtaining a B cell comprising a nucleic acid encoding LMP1 as disclosed herein, and contacting the B cell with a composition comprising an exogenous antigen or a precursor thereof, thereby producing an immunogenic B cell.

The B cell can be contacted with a composition comprising an exogenous antigen as disclosed herein using any methods known in the art, including the methods disclosed herein.

For example, in certain embodiments, the composition comprises an exogenous antigen or precursor thereof (e.g., a polynucleotide, peptide, protein, or proteoglycan), and

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PCT/US2018/046060 further comprises a carrier (e.g., a liposomes, a lipoplexes, or a nanoparticle) that can facilitate introduction of the exogenous precursor into the B cell. In certain embodiments, the composition comprises a peptide or protein comprising a membrane penetrating sequence (e.g., a Tat domain from HIV, an angiopep peptide, a receptor-binding domain of apolipoprotein B, and a receptor-binding domain of apolipoprotein E), wherein the peptide or protein can enter the B cell directly.

In certain embodiments, the composition comprises a cell comprising an exogenous antigen or a precursor thereof, and the cell is contacted with the LMP1-expressing B cell to induce cell fusion. In certain embodiments, the composition further comprises an agent capable of facilitating cell fusion (e.g., polyethylene glycol). In certain embodiments, the exogenous antigen or precursor thereof is expressed in the cell. In certain embodiments, the exogenous antigen is a TAA, and the composition comprises a tumor cell comprising the TAA. In one embodiment, the TAA is expressed from the genome of the tumor cell. In another embodiment, the TAA is a peptide or protein with one or more modifications, wherein the modifications are specific to the tumor cell or are present at substantially higher levels in the tumor cell relative to in a non-tumorous cell or a B cell. In other embodiments, the exogenous antigen or precursor thereof is from a microbe or pathogen (e.g., bacterium, virus, fungus, or parasite), and the cell comprised in the composition is infected with the microbe or pathogen.

In certain embodiments, the TAA is specifically expressed in a tumor cell. In certain embodiments, the TAA is specifically expressed in a cell type that has undergone neoplasm (e.g., prostate cell). Exemplary TAAs of these two types include without limitation Survivin, NY-ESO-1, CEA, PSA, PSCA, SCP-1, PSMA, PRAME, tyrosinase, melan-A/MART-1, an SSX protein, such as SSX-2 or SSX-4, a MAGE protein, such as MAGE-1 or MAGE-3, LAGE, mesothelin, Her-2/Neu, PLK1, VEGF-A, VEGFR2, and Tie-2. In certain embodiments, the TAA is associated with non-cancerous cells of the tumor such as tumor neovasculature or other stromal cells within the tumor microenvironment. Exemplary TAAs of this type include without limitation NY-ESO-1, SSX-2, LAGE, and PRAME.

In certain embodiments, the exogenous antigen is a neoantigen. As used herein, the term “neoantigen” refers to tumor-specific mutation-derived antigens. In certain embodiments, neoantigens can be purified from a tumor sample. In other embodiments, neoantigen can be prepared by sequencing the nucleic acids in a tumor sample, identifying the tumor-specific mutations, and synthesizing peptides or polypeptides comprising the

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PCT/US2018/046060 mutant sequences. In certain embodiments, the composition is useful in treating the patient from whom the tumor sample is obtained.

In certain embodiments, the composition comprises one or more TAAs and/or one or more neoantigens. In certain embodiments, the composition comprises one or more nucleic acids encoding TAAs and/or neoantigens. In certain embodiments, at least one of the nucleic acids encodes two or more (e.g., three or more, four or more, or five or more) TAAs and/or neoantigens. In certain embodiments, the TAAs and/or the neoantigens are present in a same tumor sample. In certain embodiments, the composition is useful for treating the patient from whom the tumor sample is obtained.

In certain embodiments, the exogenous antigen is a bacterial antigen, and the composition comprises a bacterium. In certain embodiments, the bacterium is selected from the group consisting of Salmonella, Escherichia, Pseudomonas, Bacillus, Vibrio, Campylobacter, Heliobacter, Erwinia, Borrelia, Pelobacter, Clostridium, Serratia, Xanothomonas, Yersinia, Burkholdia, Listeria, Shigella, Pasteurella, Enterobacter, Corynebacterium, Mycobacterium tuberculosis, and Streptococcus. In certain embodiments, the B cell is infected by the bacterium. In certain embodiments, the bacterium is inactivated (e.g., by heat, chemicals, or radiation).

In certain embodiments, the exogenous antigen is a viral antigen, and the composition comprises a vims. In certain embodiments, the vims is selected from the group consisting of human immunodeficiency vims (HIV), human T-lymphotropic vims, herpes vims, papillomavirus, Ebola vims, picornavims, enterovims, measles vims, mumps vims, influenza vims, parainfluenza vims, rabies vims, vesicular stomatitis vims (VSV), dengue vims, hepatitis vims, rhinovims, yellow fever vims, bunga vims, polyoma vims, coronavims, rubella vims, echovims, pox vims, varicella zoster vims, and African swine fever vims. In certain embodiments, the B cell is infected by the vims. In certain embodiments, the vims is replication defective (e.g., lacking at least one viral gene necessary for replication).

In certain embodiments, the exogenous antigen is a fungal antigen, and the composition comprises a fungus. In certain embodiments, the fungus is selected from the group consisting of Aspergillus, Coccidoides, Cryptococcus, Candida, Nocardia, Pneumocystis, and Chlamydia. In certain embodiments, the fungus is inactivated (e.g., by heat, chemicals, or radiation).

In certain embodiments, the exogenous antigen is a parasite antigen, and the composition comprises a parasite. In certain embodiments, the parasite is selected from the group consisting of Babesia, Entomoeba, Leishmania, Plasmodium, Trypanosoma,

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Toxoplasma, Giarda, flat worms, and round worms. In certain embodiments, the parasite is inactivated (e.g., by heat, chemicals, or radiation).

Some microbes or pathogens have means of immunosuppression. For example, some bacteria and viruses encode genes that can suppress antigen presentation by MHC molecules, or genes that can inhibit T cell activation. Accordingly, in certain embodiments, the microbe or pathogen is modified (e.g., by heat, chemicals, or genetic modification) to inhibit or reduce the immunosuppressive function.

In certain embodiments, the method disclosed herein produces an immunogenic B cell comprising an exogenous antigen on the cell surface. In certain embodiments, the exogenous antigen is conjugated to a major histocompatibility complex (MHC). In certain embodiments, the MHC is selected from the group consisting of MHC-I, MHC-II, HLA-A, HLA-B, HLA-C, HLA-DP (e.g., HLA-DPA1 or HLA-DPB1), HLA-DQ (e.g., HLA-DQA1 or HLA-DQB1), and HLA-DR (e.g., HLA-DRa or HLA-DRP). In certain embodiments, a B cell as disclosed herein displays or presents an antigen with an MHC-II (e.g., HLA-DP, HLADQ, or HLA-DR).

In another aspect, the present disclosure provides a method of producing an immunogenic cell, the method comprising obtaining a B cell comprising an exogenous antigen or a precursor thereof, and contacting the B cell with a vector (e.g., expression vector) encoding LMP1, thereby producing an immunogenic B cell. The B cell comprising an exogenous antigen or a precursor thereof is described in section II, except that the B cell does not comprise a polynucleotide encoding LMP1.

As used herein, “vector” refers to a composition of matter which comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including without limitation linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. The term “vector” includes an autonomously replicating plasmid or a vims. This term should also be constmed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated vims vectors, retroviral vectors, lentiviral vectors, and the like.

As used herein, the term “expression vector” refers to a vector comprising a polynucleotide operatively linked to a nucleotide sequence that can drive the expression of the polynucleotide. An expression vector comprises sufficient cis-acting elements (e.g., transcriptional regulatory elements) for expression. Trans-acting elements for expression can

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PCT/US2018/046060 be supplied by the host cell or in an in vitro expression system. Expression vectors are known in the art, including without limitation cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. The expression vector, as used herein, lacks at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of an EBV genome.

A viral vector can be contacted with a B cell disclosed herein, optionally in the presence of one or more agents facilitating viral infection, to be introduced into the B cell. As used herein, the term “viral vector” refers to a viral genome (e.g., DNA or RNA) or a viral particle comprising the viral genome. Viral vectors can be replication-competent, or can be genetically disabled so as to be replication-defective or replication-impaired. The term “viral particle” refers to the viral genome as well as one or more capsid proteins encapsidating the viral genome. In certain embodiments, the viral particle also includes an envelope of lipids that surrounds the capsid. Exemplary viral vectors include adenoviral vectors, adenoassociated viral vectors, and retroviral vectors (e.g., lentiviral vectors).

Non-viral vectors can be contacted with a B cell disclosed herein to be transfected into the B cell. As used herein, “non-viral vectors” include, but are not limited to, nucleic acids encapsulated within or associated with liposomes, lipoplexes, nanoparticles, and other carriers. In certain embodiments, a liposome or lipoplex has a neutral, negative or positive charge and can comprise cardolipin, anisamide-conjugated polyethylene glycol, dioleoyl phosphatidylcholine, or other neutral, anionic, or cationic lipids or lipid conjugates. A vector can be complexed with cationic polymers (e.g., polyethylenimine (PEI)), biodegradable cationic polysaccharide (e.g., chitosan), or cationic polypeptides (e.g., atelocollagen, poly lysine, and protamine).

In certain embodiments of any one of the aspects disclosed in this section, the method further comprises reducing the proliferative capacity of the B cell comprising a nucleic acid encoding LMP1, the B cell comprising an exogenous antigen or a precursor thereof, or an immunogenic B cell produced by the method. The proliferative capacity of the B cell can be reduced by any methods known in the art, including treatment with a cytostatic agent (e.g., a chemotherapeutic agent), a fixative (e.g., formaldehyde, or an alcohol), or irradiation (e.g., with 30 Gy).

IV. Methods of using immunogenic B cells

The isolated B cell as disclosed herein or an immunogenic B cell produced using a method as disclosed herein can be used in any composition (e.g., a vaccine). Accordingly,

TI

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PCT/US2018/046060 the present disclosure provides a vaccine comprising the B cell, optionally further comprising an adjuvant.

A variety of adjuvants may be employed, including, for example, systemic adjuvants and mucosal adjuvants. A systemic adjuvant is an adjuvant that can be delivered parenterally. Systemic adjuvants include adjuvants that create a depot effect, adjuvants that stimulate the immune system and adjuvants that do both. An adjuvant that creates a depot effect is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. In some embodiments, the adjuvant stimulates the immune system, for instance, cause an immune cell to produce and secrete cytokines or IgG. This class of adjuvants includes immunostimulatory nucleic acids, such as CpG oligonucleotides; saponins purified from the bark of the Q. saponaria tree, such as QS-21; poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); RNA mimetics such as polyinosinic:polycytidylic acid (poly I:C) or poly I:C stabilized with poly-lysine (poly-ICLC [HILTONOL; Oncovir, Inc.]; derivatives of lipopolysaccharides (LPS) such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

In another aspect, the instant disclosure provides a method of activating a T cell, the method comprising contacting the T cell with the B cell or vaccine as disclosed herein. In certain embodiments, the T cell is activated by contact with the B cell.

In certain embodiments, the T cell is a cytotoxic T cell. As used herein, the term “cytotoxic T cell” refers to a T lymphocyte that can kill cells expressing a MHC-presented antigen, such as cancer cells or cells infected by a microbe or pathogen. Cytotoxic T cells include CD8⁺ T cells (traditionally referred to as CTLs or CD8⁺ CTLs) and a subtype of CD4⁺ T cells (CD4⁺ CTLs) that have direct killing activity as described in the instant disclosure. CTLs have specificity for peptide antigens that are presented by MHC proteins expressed on the cell surface. The traditional method of activating T cells using EBVtransformed B cells, also known as lymphoblastoid cell lines (LCLs), predominantly expands and activates CD8⁺ CTLs. By contrast, the method disclosed herein promotes the proliferation and activation of a substantial amount of both CD4⁺ CTLs and CD8⁺ CTLs. Since the frequency of CD4⁺ CTLs strongly correlates with responses to T cell infusion in a clinical study of post-transplant lymphoproliferative disorder, CD4⁺ CTLs hold great

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PCT/US2018/046060 potential for treating this malignant disease and non-EBV-related cancers. Accordingly, in certain embodiments, the T cell is a CD4⁺ T cell, thereby producing a CD4⁺ cytotoxic T cell. In certain embodiments, the T cell is a CD8⁺ T cell, thereby producing a CD8⁺ cytotoxic T cell. In certain embodiments, the CD4⁺ or CD8⁺ cytotoxic T cell can lyse a cancer cell. In certain embodiments, the CD4⁺ or CD8⁺ cytotoxic T cell can lyse a cell infected with a microbe or pathogen (e.g., bacterium, virus, fungus, or parasite), thereby inducing and promoting the destruction of the intracellular microbe or pathogen.

Accordingly, the method as disclosed herein can be used for treating cancer. As used herein, the terms “cancer” and “tumor” refer to a hyperproliferation of cells whose unique trait—loss of normal controls— results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. A tumor can be benign or malignant. Examples include, but are not limited to, melanoma, hepatocarcinoma, leukemia, lymphoma, retinoblastoma, astrocytoma, glioblastoma, neuroblastoma, sarcoma, lung, breast, uterine, pancreatic, prostate, renal, bone, testicular, uterine, ovarian, cervical, gastrointestinal, brain, colon, or bladder cancer. In certain embodiments, the cancer is a solid tumor (e.g., sarcoma, carcinoma, melanoma, gastric cancer, or nasopharyngeal carcinoma). In certain embodiments, the method is employed after the tumor is surgically resected.

The method as disclosed herein can also be used to treat an infectious disease. In certain embodiments, the infectious disease is caused by or associated with infection with a bacterium, virus, fungus, or parasite. The infection can be either acute or chronic, and can be either local or systemic. In certain embodiments, the bacterium is selected from the group consisting of Salmonella, Escherichia, Pseudomonas, Bacillus, Vibrio, Campylobacter, Heliobacter, Erwinia, Borrelia, Pelobacter, Clostridium, Serratia, Xanothomonas, Yersinia, Burkholdia, Listeria, Shigella, Pasteurella, Enterobacter, Corynebacterium, Mycobacterium tuberculosis, and Streptococcus. In certain embodiments, the virus is selected from the group consisting of human immunodeficiency virus (HIV), human T-lymphotropic virus, herpes virus, papillomavirus, Ebola virus, picomavirus, enterovirus, measles virus, mumps virus, influenza virus, parainfluenza virus, rabies virus, vesicular stomatitis virus (VSV), dengue virus, hepatitis virus, rhinovirus, yellow fever virus, bunga virus, polyoma virus, coronavirus, rubella virus, echovirus, pox virus, varicella zoster virus, and African swine fever virus. In certain embodiments, the fungus is selected from the group consisting of Aspergillus, Coccidoides, Cryptococcus, Candida, Nocardia, Pneumocystis, and Chlamydia. In certain embodiments, the parasite is selected from the group consisting of Babesia, Entomoeba, Leishmania, Plasmodium, Trypanosoma, Toxoplasma, Giarda, flat worms, and round worms.

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The method disclosed herein can be employed either ex vivo or in vivo. The ex vivo approach is generally known as adoptive cell transfer (ACT). The in vivo approach is generally known as vaccination.

Where the B cell is contacted with the T cell ex vivo, the method can further comprise culturing the T cell with the B cell or vaccine under suitable conditions to allow proliferation of the T cell. The suitable conditions can include certain factors that promote or enhance the survival, proliferation, or differentiation of T cells. Exemplary factors include cytokines (e.g., IL-2, IL-1, IL-6, IL-12, or IL-18), anti-CD3 antibodies, anti-CD28 antibodies, phytohemagglutinin, calcium ionophores, inhibitors to cell death (e.g., FasL/Fas neutralizing antibodies), and cells that can facilitate T cell activation (e.g., macrophages or dendritic cells). In contrast to the traditional method of activating T cells using LCL, which generally takes 2-3 months, the method disclosed herein can take about 11 days for preparation of LMP1-B cells and subsequent generation of antigen-specific T cells. Accordingly, in certain embodiments, the T cell is cultured for a suitable length of time (e.g., about 3-5 days, 5-7 days, or 7-14 days; equal to or less than 3, 4, 5, 6, 7, 8, 9, or 10 days; or, equal to or less than 1, 2, 3, or 4 weeks). The T cell can be co-cultured with the B cell during the entire length of time or a portion thereof. In certain embodiments, the B cell that is contacted with the T cell is replenished (e.g., every2-3 days, 3-4 days, or 4-5 days). The factors can be added and withdrawn anytime in the course of the culture. For example, IL-2 may be added from day 3 onward.

The ACT strategy disclosed herein can be employed by administering the T cell that has been contacted with the B cell to a subject in need thereof. In certain embodiments, the B cell is autologous to the subject. In certain embodiments, the B cell is from a donor having an MHC matched with the subject. In certain embodiments, the T cell is autologous to the subject. In certain embodiments, the T cell is from a donor having an MHC matched with the subject. In certain embodiments, a population of cells that are substantially pure (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the population are T cells) are administered to the subject. In certain embodiments, the method further comprises isolating the T cell (e.g., by affinity-based or fluorescence-based cell sorting) from the mixed culture with the B cell.

The cytotoxicity of the T cell can be examined using any methods known in the art, including the methods described in the instant disclosure. For example, in an in vitro killing assay, the T cell can be contacted with a test cell presenting the exogenous antigen on the cell surface. Cell death of the test cell can be examined using any methods known in the art (e.g.,

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PCT/US2018/046060 by measuring the metabolic parameters, activation of caspases, or release of intracellular contents). In certain embodiments, the T cell is administered to the subject after its cytotoxicity reaches a certain standard (e.g., each T cell is capable of killing at least 1, 2, 3, 4,

5, 10, 15, 20, 30, 40, 50, 60, 70 80, 90, or 100 test cells in 24 hours).

Where the B cell is contacted with the T cell by vaccination in vivo, the method can be employed by administering an effective amount of the B cell or the vaccine to a subject in need thereof. In certain embodiments, the B cell is autologous to the subject. In certain embodiments, the B cell is from a donor having an MHC matched with the subject. In certain embodiments, the subject has a tumor comprising (e.g., on the cell surface) the exogenous antigen. In certain embodiments, the subject is infected with a microbe or pathogen comprising the exogenous antigen, optionally wherein a cell of the subject comprises the exogenous antigen on the cell surface. In certain embodiments, the microbe or pathogen is a bacterium, virus, fungus, or parasite.

In certain embodiments, the ability of the B cell to activate T cells (e.g., CTLs) is examined by any methods known in the art, including the methods described in the instant disclosure. For example, the B cell can be contacted with a population of T cells under suitable conditions to allow proliferation of the T cells. In certain embodiments, the T cells are further examined for their cytotoxicity. In certain embodiments, the B cell is administered to the subject after its ability to activate T cells reaches a certain standard (e.g., each B cell is capable of activating at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70 80, 90, or 100 T cells after 3, 4, 5, 6, or 7 days, and/or each B cell is capable of activating at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70 80, 90, or 100 CD4⁺ or CD8⁺ CTLs after 3, 4, 5, 6, or 7 days).

An LMP1-expressing B cell can take up an exogenous antigen from a neighboring cell (e.g., tumor cell or pathogen-infected cell) in vivo. In certain embodiments, the exogenous antigen is released from the neighboring cell, and is taken up by the LMP1expressing B cell by direct internalization (e.g., through B cell receptor). In certain embodiments, the exogenous antigen is on the surface of or encapsulated in a membrane structure (e.g., the neighboring cell or a vesicle released therefrom, such as an apoptotic body), and is taken up by fusion of the B cell with the membrane structure. Accordingly, in one aspect, the instant disclosure provides a method of treating a subject having a lesion (e.g., tumor or pathogen infection), the method comprising administering an LMP1-expressing B cell to the subject, wherein the administration site is at or close to (e.g., no greater than 1, 2, 5, 10, 20, 50, 100, or 200 mm away from) the lesion or a draining lymph node thereof. In

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PCT/US2018/046060 certain embodiments, the B cell is a naive B cell. In certain embodiments, the B cell is immortalized. In certain embodiments, the B cell is a primary B cell. In certain embodiments, the B cell has not been contacted with an exogenous antigen (e.g., an exogenous antigen as disclosed herein) in vitro.

In certain embodiments, the ACT or vaccination method further comprises administering to the subject an adjuvant. In certain embodiments, the adjuvant is administered prior to, at about the same time as, or subsequent to the administration of the isolated cells or T cells. In certain embodiments, the adjuvant is administered within the same patient visit as the administration of the isolated cells or T cells. In certain embodiments, the adjuvant is administered in the same composition (e.g., vaccine) as the B cell or T cell. In certain embodiments, the adjuvant is administered in a different composition from the B cell or T cell.

In certain embodiments, the ACT or vaccination method further comprises administering to the subject one or more immune checkpoint targeting therapies. As used herein, the term “immune checkpoints” refers to a group of molecules on the cell surface of CD4⁺ and CD8⁺ T cells or other cells, such as tumor cells or other immunoregulatory cells, that effectively serve as “brakes” to down-modulate or inhibit an anti-tumor immune response. In certain embodiments, the immune checkpoint targeting therapy is selected from the group consisting of an antagonist anti-PD-1 antibody, an antagonist anti-PD-Ll antibody, an antagonist anti-PD-L2 antibody, an antagonist anti-CTLA-4 antibody, an antagonist antiTIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-CEACAMl antibody, and an IDO inhibitor, i.e., an agent that inhibits the enzymatic activity of IDO (indoleamine(2,3)-dioxygenase) and/or TDO (tryptophan 2,3-dioxygenase).

In certain embodiments, the immune checkpoint targeting therapy is an anti-PD-1 antibody, optionally wherein the anti-PD-1 antibody is pembrolizumab, nivolumab, Pidilizumab, MEDI0680, PDR001, REGN2810, PF-06801591, BGB-A317, TSR-042, or SHR-1210. In certain embodiments, the immune checkpoint targeting therapy is an anti-PDLl antibody, optionally wherein the anti-PD-Ll antibody is atezolizumab, durvalumab, avelumab (MSB0010718C), MDX-1105, or AMP-224. In certain embodiments, the immune checkpoint targeting therapy is an anti-CTLA-4 antibody, optionally wherein the anti-CTLA4 antibody is ipilimumab. In some embodiments, the immune checkpoint targeting therapy is an IDO inhibitor, optionally wherein the IDO inhibitor is epacadostat, FOO 1287, indoximod, orNLG919.

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In certain embodiments, the ACT or vaccination method further comprises administering to the subject an immune co-stimulation therapy. As used herein, the term “immune co-stimulation therapy” refers to a therapeutic agent that induces or promotes the expression and/or activity of an immune co-stimulation molecule, wherein immune costimulation molecule provides a signal for T cell proliferation, functional differentiation, and/or activation. Exemplary immune co-stimulation molecules include without limitation CD80, CD86, 0X40, 0X40 ligand, 4-IBB, 4-IBB ligand, CD70, CD27, and GITR. In certain embodiments, the immune co-stimulation therapy is selected from the group consisting of an agonist of CD27 (e.g., an agonistic anti-CD27 antibody), an agonist of 0X40 (e.g., an agonistic anti-OX40 antibody), and an agonist of 4-IBB (e.g., an agonistic anti-41BB antibody), and an agonist of GITR (e.g., an agonistic anti-GITR antibody). In certain embodiments, the immune co-stimulation therapy is a multi-specific antibody that specifically binds two or more immune co-stimulation molecules.

In certain embodiments, the ACT or vaccination method further comprises administering to the subject a Treg modulating therapy. As used herein, the term “Treg modulating therapy” refers to a therapeutic agent that inhibits or decreases the viability and/or activity of a regulatory T cell (Treg). Treg modulating therapies are known in the art, and include without limitation antibodies (e.g., full antibodies, and antigen-binding fragments thereof) that specifically bind to CTLA-4, GITR, CCR4, PD-1, LAG3, CD25, or CD15s.

The immune checkpoint targeting therapy, immune co-stimulation therapy, and/or Treg modulating therapy, as an additional therapy, can be administered prior to, contemporaneously with (e.g., during the same doctor visit), or subsequent to the administration of the vaccination or ACT therapy. If the additional therapy is administered subsequent to the administration of the vaccination or ACT therapy, the patient’s response to the vaccination or ACT therapy can be examined to determine the necessity and dose of the additional therapy.

Both the ACT and the vaccination strategies disclosed herein can be validated and optimized in preclinical models (e.g., cancer models or infection models) either alone or in combination with one or more additional therapies (e.g., an immune checkpoint targeting therapy, an immune co-stimulation therapy, and/or a Treg modulating therapy).

V. Kits

In another aspect, the instant disclosure provides kits for producing immunogenic B cells or vaccines, comprising (a) a nucleic acid encoding LMP1 as disclosed herein; and (b) a composition comprising an exogenous antigen or a precursor thereof as disclosed herein. In

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PCT/US2018/046060 certain embodiments, the kit comprises a first container comprising a nucleic acid encoding LMP1 as disclosed herein, and a second container comprising an exogenous antigen or a precursor thereof as disclosed herein. The two components of the kit can be introduced to a B cell contemporaneously or sequentially. In certain embodiments, where the composition comprises a cell (e.g., a tumor cell, or a cell infected with a microbe or pathogen), the kit further comprises an agent capable of facilitating cell fusion (e.g., polyethylene glycol). In certain embodiments, the kit further comprises an agent capable of reducing the proliferative capacity of a B cell (e.g., a cytostatic agent or a fixative) or an instruction to irradiate a B cell (e.g., with 30 Gy).

Furthermore, in accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J.Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

The following examples are provided to further elucidate the advantages and features of the present application, but are not intended to limit the scope of the application. The examples are for illustrative purposes only.

EXAMPLES

Materials and Methods

Mice

C57BL/6J (B6), CD19-cre, CIITA '-, CD4(T^g, Foxp3^DTR/GFP, YFP^flSTOP, CD19-cre'^PT2, OT-I (all on a B6 background), and BALB/c were obtained from the Jackson Laboratory. Rag2 common γchairT^G (Rag2 yc ) mice were bred in our mouse colony or purchased from Taconic. LMPl^flSTOP allele on a BALB/c background has been described previously (B. Zhang et al., Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model. Cell 148, 739 (Feb 17, 2012)). Foxp3^DTR/GFP ;CD19cre;IMPl^flSTOP (Foxp3^DTR/GFP;CL) mice on a (C57BL/6xBALB/c) Fl (CB6F1) background

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PCT/US2018/046060 were generated by crossing CD19-cre;Foxp3^DTR/GFP to LMP1^^STOP mice. Only male Foxp3^DTR/GFP;CL mice were used in experiments. (1)40 ;CD19-cre mice were crossed with CD40^+/-;LMPl^flSTOP mice to generate CD40 ^/ ;CL mice and their corresponding controls. LMPl^flSTOP allele backcrossed to a B6 background was used in some experiments. All mice were bred and maintained in animal facilities under specific pathogen-free conditions. All animal experiments were conducted according to protocols approved by the DFCI Institutional Animal Care and Use Committee.

Flow cytometry

Lymphoid single-cell suspensions were stained with the following monoclonal antibodies specific for CD3e (145-2C11), CD4 (L3T4), CD8 (53-6.7), CD19 (1D3), CD25 (PC61.5), CD40 (3/23), CD43 (S7), CD69 (H1.2F3), CD70 (FR70), CD80 (16-10A1), CD86 (GL1), 4-1BBL (TKS-1), OX40L (RM134L), Fas (Jo2), H-2Kb (AF6-88.5), I-Ab (AF6120.1), ICAM-1 (3E2), TCRb (H57-597), TCR Vb5 (MR9-4), TCR Vbll (RR3-15), TCR Vbl2 (MRU-1), IFN-g (XMG1.2), Granzyme B (GzmB, NGZB), Perforin (eBioOMAK-D), CD107a (1D4B), FasL (MFL3), TRAIL (N2B2), Foxp3 (FJK-16s), Eomes (Danllmag), Tbet (4B10), GATA-3 (TWAJ) and RORgt (Q31-378) from BD Biosciences, Biolegend or eBioscience. Topro3 (Invitrogen) or eFluor 506 (eBioscience) was used to exclude dead cells. Intracellular staining for GzmB, perforin, Foxp3, Eomes, T-bet, GATA-3 and RORgt was done with the Foxp3 staining buffer set (eBioscience). Intracellular staining for GzmB and IFN-g was conducted using the IC Fixation/Permeabilization buffer (eBioscience). TCR Vft repertoire was analyzed with the mouse νβ TCR screening panel (BD Biosciences) according to the manufacturer’s instructions. All samples were acquired on a FACSCanto II (BD Biosciences), and analyzed by FlowJo software (Tree Star). Fluorescence-activated cell sorting (FACS sorting) was performed using a FACSAria II (BD Biosciences). In all T cell sorting experiments, CD Id tetramer (NIH tetramer facility) was employed to exclude natural killer T cells.

Retroviral constructs and transduction

LMP1 cDNA was cloned into the MSCV-IRES-GFP or MSCV-Puro retroviral vector to generate MSCV-LMP1-IRES-GFP or MSCV-LMPl-Puro. To generate a retrovirus expressing the signaling-defective LMP1 mutant LMP I™¹'. amino acids FWLY(38-41) of the transmembrane domain 1 (TM1) of LMP 1 were altered to AALA by QuikChange sitedirected mutagenesis (Stratagene), and the resultant mutant was cloned into the MSCV-IRESGFP or MSCV-Puro retroviral vector. CD43-depleted (by using anti-CD43 microbeads from Miltenyi Biotec) splenic B cells were activated in vitro by 20 pg/ml lipopolysaccharide (LPS,

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Sigma) for 24 hrs, infected with retroviruses, and continually cultured in the presence of LPS. For B cells transduced with GFP-carrying retroviruses, at 48 or 72 hrs post-infection the cells were extensively washed and then used in downstream experiments (GFP⁺ indicates successfully transduced cells). For B cells transduced with Puro-carrying retroviruses, at 24 hrs post-infection the cells were selected with Puromycin (6 pg/ml; Sigma) for 18 hrs, followed by extensive wash and recovery in fresh medium for 1 day before using in downstream experiments.

In vitro killing assay

Various target cells were labeled with CellTrace Violet (Invitrogen) before use. CD4 or CD8 T cells were purified from the bone marrow (BM) or spleen of mice by FACS sorting. The T cells were then co-cultured with 2 x 10³ target cells at different effectorlarget ratios for 4 hrs (on LMP1-expressing B cells/lymphoma cells and corresponding control cells) or 6 hrs (on CD40-activated B cells and resting B cells) in 96-well round-bottomed plates, followed by active Caspase-3 staining (BD Biosciences) (B. Zhang et al., Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model. Cell 148, 739 (Feb 17, 2012), L. He et al., A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells. Journal of Immunological Methods 304, 43 (Sep, 2005)). For blocking assay, the target cells were pre-incubated with anti-IA/IE (M5/114.15.2) blocking antibody or isotype control rat IgG2b (both at 10 gg/ml; Biolegend) for 20 min at 37 °C, whereas the CD4 T cells were preincubated with Fas-ligand neutralizing fusion protein rmFas-Fc or isotype control human IgGl (both at 10 gg/ml; R&D Systems) under the same conditions. In all killing assays, effector-target mixtures in 96-well plates were spun down at 200 rpm for 2 min prior to the incubation at 37°C, and cultures were stained for CD4 or CD8 to exclude effector cells and analyzed for active Caspase-3 levels in CellTrace-labeled target cells. Active Caspase3⁺CellTrace⁺ cells represent apoptotic target cells. % specific killing = % apoptotic target cells of cultures with both effectors and targets - % apoptotic target cells of cultures with targets alone.

T cell proliferation assay for MHC restriction

CD43-depleted splenic B cells were isolated from wild-type (WT) or CIITA^G mice (both on a C57BL6 background) and activated by anti-CD40 antibody (HM40-3, eBioscience) at 1 gg/ml for 48 hrs. CD4 effector T cells (excluding GFP⁺ regulatory T cells (Tregs)) from the BM of adult Foxp3^DTR/GFP;CL mice or CD4 T cells primed in vitro by LMP 1-expressing B cells were sorted and stained with CellTrace (Invitrogen), followed by a

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PCT/US2018/046060 hrs incubation in fresh RPMI media to ensure the T cells were at rest before co-culture with target cells. The CD4 T cells (1 x 10⁵ cells) were subsequently co-cultured with target cells,

CD40-activated WT or CIITA ^ B cells (1 x 10⁵ cells), in 96-well U-bottom plate for 4 days, followed by staining with Topro3, anti-TCRp, -CD4 and -CD19 and FACS analysis of

CellTrace dilution in CD4 cells.

LMP1 localization analysis

LMP1 or LMPl™^lm cDNA was each subcloned into the pCAG-GFP vector (Addgene, #11150) to obtain C-terminally GFP-tagged constructs. The plasmids (pCAGLMP1-GFP, pCAG-LMPl™^lm-GFP or vector control pCAG-GFP) were then electroporated into mouse lymphoma B cells (line 775) (B. Zhang et al., An oncogenic role for alternative NF-kappaB signaling in DLBCL revealed upon deregulated BCL6 expression. Cell reports 11, 715 (May 5, 2015)). 24 hrs after electroporation, the cells were counterstained with the DNA-specific fluorescent dye Hoechst 33342 (blue, Sigma) and imaged with fluorescence microscopy.

Gene expression profiling

B cells were isolated from spleens of Yppfl^STOP/+ _and LMP f^p’^TO‘’/γρρΑ^8ΊΌΡ mice by CD43 depletion using magnetic-activated cell sorting (Miltenyi Biotec) and treated with TAT-Cre as previously described (S. B. Koralov et al., Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 132, 860 (Mar 7, 2008)). At day 2 post-treatment, total RNA was extracted from the cells with TRIzol reagent (Invitrogen) according to manufacturer's specifications, followed by microarray analysis at the Molecular Biology Core Facility atDFCI, using GeneChip Mouse Gene 2.0 ST arrays (Affymetrix). In vitro generation of cytotoxic CD4 T cells on LMP 1-expressing B cells

Sorted CD4 T cells from the spleens of naive B6 mice were plated in 12-well plates at 1.5 x 10⁶ per well with irradiated (500 Rad) LMP1⁺ or LMPl™^lm+ β cells at a 1:1 ratio. Five days later, the CD4 T cells were re-stimulated with 0.75 x 10⁶ of the same target B cells for an additional 2 days. All cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Sigma), 100 lU/ml penicillin (Gibco), 10 mM HEPES (Coming), lx nonessential amino acids (Coming), 1 mM sodium pymvate (Gibco) and 50 μΜ β-mercaptoethanol (Sigma), and without addition of any growth factors or cytokines. Blockade of co-stimulatory ligands during LMP 1⁺ B cell-driven cytotoxic T cell production

Irradiated LMP 1-expressing B cells were pre-incubated with blocking antibodies against CD70 (FR70, rat IgG2b), OX40L (RM134L, rat IgG2b) and/or 4-1BBL (TKS-1, rat IgG2a), or the corresponding isotype controls (all at 10 gg/ml; Biolegend), for 50 min at

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37°C. Splenic CD4 (1 x 10⁶) or CD8 cells (0.5 x 10⁶) sorted from naive B6 mice were subsequently co-cultured with the target B cells at 1:1 ratio in 24-well plates. The CD8 T cells were harvested for FACS analysis after 3 days of co-culture, whereas the CD4 T cells were re-stimulated at day 5 with 0.5 x 10⁶ of the same target B cells for an additional 2 days, followed by FACS analysis.

Statistical analysis

Statistical significance was determined by unpaired two-tailed Student’s t test, except where indicated; a p value < 0.05 was considered significant (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Example 1. Generation and characterization of a B cell specific LMP1 transgenic mouse model

LMP1 coding sequence derived from the EBV B95-8 strain, preceded by a loxPflanked Neo^r-STOP cassette, was placed into Rosa26 locus to generate a conditional LMP1 knockin allele, LMP1^:>1STOP, which allows expression of LMP1 through excision of a transcriptional/translational STOP cassette via Cre/loxP-mediated recombination (Figure 2A). The LMPl^flSTOP strain was generated from BALB/c-derived embryonic stem (ES) cells. Splenic B cells isolated from LMPl^flSTOP mice expressed LMP1 following treatment with TAT-Cre and proliferated in cell culture, whereas TAT-Cre treated wild-type B cells died over time. The induction of LMP1 was accompanied by the upregulation of CD95/Fas. Subsequently, Fas was used as a reporter for LMP1 expression in B cells.

To generate B cell specific LMP1 transgenic mouse model, the LMPF^stof (BALB/c) strain was bred with CD19-cre (C57BL/6) strain. Homozygous CD19-cre mice were crossed with homozygous or heterozygous IMP I¹¹''¹⁰'¹ or BALB/c mice to produce CD19cre-,IMPl^flSTOP mice (hereafter referred as “CZ”) or CD19-cre/+ control mice (hereafter referred to as “C”), all on a CB6F1 background (Fl offspring of a cross between C57BL/6 χ BALB/c). CL mice expressed LMP1 transgene specifically in B cells. Analysis of CL mice revealed that LMP1-expressing B cells were eliminated by T cells, similar to EBV-infected B cells in humans; T cell depletion resulted in rapid, fatal B cell proliferation and lymphomagenesis in the mice, resembling EBV-driven malignancies in immunosuppressed patients (Figure 2B). These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo.

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Example 2. Both CD4 and CD8 T cells develop cytotoxic response to LMPl-expressing

B cells

The detailed time course and nature of immune surveillance in CL mice were investigated. Analysis of the dynamics of LMPl-expressing B cell and T cell responses revealed a peak T cell response against LMPl-expressing B cells on days 6-8 after birth, followed by rapid elimination of LMPl-expressing B cells (Figures 3A and 3B). T cells contracted afterwards, but long-term memory formed and persisted, and continued to eliminate newly arising LMPl-expressing B cells in the bone marrow (BM, the primary organ for B cell development). Accordingly, a small population of LMPl-expressing B cells was detected in the BM, but not in the spleen, of adult mice (Figures 3A and 3B).

Particularly striking was the high level of cytotoxic activity by CD4 cells which had similar cytotoxic function as CD8 cells. CD4 and CD8 cells from the BM and spleen of day 6-8 CL mice displayed potent killing activity on LMPl-expressing lymphoma cells (derived from T cell-deficient CL mice) ex vivo (Figure 4). Remarkably, CD4 cells isolated from day 6-8 CL mice expressed perforin, granzyme B (GzmB), and CD107a, at levels similar to those of the CD8 cells (Figures 5A-D). In addition, these cells expressed high levels of Fas ligand (FasL) but not TRAIL (Figures 5A-D and data not shown), suggesting that they kill LMP 1expressing B cells through perforin-granzyme as well as FasL mediated pathways. Yet, given that LMPl-expressing B cells remain controlled in mice deficient for Fas but not in mice deficient for perforin, the perforin-granzyme pathway appears to be the predominant killing mechanism of these cytotoxic T cells. Overall, our data demonstrate that LMP 1 expression by B cells induces potent cytotoxic CD4 and CD8 T cell-mediated immunity.

Although CD4 and CD8 cells in the BM of adult CL mice remain an activated state (CD69⁺), these CD4 cells exhibited little cytotoxicity, in contrast to CD8 cells from the same mice (Figure 6A). Nevertheless, when the CD4 cells were co-transferred with LMPlexpressing lymphoma cells into lymphopenic hosts, they exhibited superior anti-tumor activity relative to that of the CD8 cells, and their antitumor activity remained intact in the presence of antibodies blocking IFNy and TNFa. Remarkably, CD4 cells that were recovered from the adoptive hosts displayed potent killing activity ex vivo (Figure 6A), associated with expression of cytotoxic molecules - perforin, granzyme B, CD107a and FasL, in sharp contrast to the donor cells prior to transfer (Figure 6B).

The finding that, upon co-transfer with LMPl-expressing lymphoma cells, chronic state CD4 cells regain cytotoxicity and mediate superior antitumor activity relative to that of their CD8 counterparts, prompted us to test and compare these CD4 and CD8 cells for their

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PCT/US2018/046060 therapeutic efficacy in LMP1-driven primary lymphomas. Considering that the heavy tumor burden in these mice may establish an immunosuppressive environment and thereby impede the expansion and function of adoptive T cells, we pre-treated the mice with radiation therapy (RT) to reduce the tumor burden and create a lymphopenic condition favorable for adoptive T cell expansion and function, followed by transfer of a single dose (lx 10⁶/recipient) of CD4 or CD8 cells. We found that RT alone moderately improved survival of tumor-bearing mice. The combination with adoptive CD8 cells further prolonged mice survival, and CD4 cells displayed even stronger antitumor activity than the CD8 cells (Figure 6C). Thus, CD4 cells, upon developing into cytotoxic effectors, can be superior to CD8 cells in tumor control, as demonstrated in this primary lymphoma model.

Example 3. CD4 and CD8 T cells mount a polyclonal response to LMPl-expressing B cells

To assess the diversity of T cells involved in the immune response, we assessed the TCR νβ repertoire on CD4 (excluding CD25⁺Foxp3⁺ Tregs) and CD8 cells from day 6-8 CL mice (these cells have high killing activity and express the effector memory marker CD44), in comparison with those from control mice (CD19-cre/+\ We also examined T cells from the BM of adult CL mice, in which CD4 cells exhibit minimum killing activity, while CD8 cells retain good killing activity (the majority of these CD4 and CD8 cells are antigenspecific). CD8 cells from day 6-8 and adult CL mice displayed polyclonal VPs (day 6-8 CL mice showed a modest increase in νβ13, while in adult CL mice νβ13 levels were similar to those in control mice; Figure 7A). CD4 cells from day 6-8 CL mice also displayed a grossly polyclonal response, though a few νβ TCRs (νβ5, -11 and -12) showed variable degrees of enrichment compared to those in control mice (Figure 7B). By in vitro killing assay, CD4 cells bearing νβ5, -11 and -12 TCRs displayed similar killing activity as cells carrying the other TCRs (Figure 7C), indicating that the killing activity of CD4 cells in CL mice is not associated with restricted TCR νβ chains, and making it unlikely that the response is mediated by a superantigen. In the BM of adult CL mice, the frequencies of the νβ5, -11 and -12 TCRs had diminished to levels comparable to those seen in control mice, while νβ8.1/8.2 TCRs were skewed at this chronic stage (Figure 7B). Upon adoptive transfer, CD4 cells from the BM of adult CL mice carried over their broad TCR repertoire (Figure 7B), but they had regained killing activity (Figure 6). The further skewing of νβ8.1/8.2 TCRs might be due to their dominance in the donor cells (Figure 7B). These observations reiterate that the killing activity of the T cells is not associated with restricted TCR νβ chains. Overall, these data

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PCT/US2018/046060 indicate that both CD4 and CD8 T cells mount a polyclonal response to LMP 1-expressing B cells.

Example 4. T cells recognize CD40-activated B cells that lack LMP1 expression

LMP 1 has been characterized as a functional analog of constitutively active CD40, which is a major co-stimulatory receptor for the functional maturation of antigen-presenting cells (APCs). We found that, similar as activation of CD40, LMP1 expression in B cells resulted in upregulation of key proteins critical for the induction of a productive T cell response, including MHC-I, MHC-II, CD80/B7-1, CD86/B7-2 and ICAM-1 (many of these molecules were even higher than those in CD40-activated B cells (Figure 8). These would presumably lead to enhanced antigen presentation and co-stimulation, including presentation of endogenous antigens (Rowe et al., 1995; Schultze etal., 1995; Schultze et al., 1997; Smith et al., 2009).

To determine if LMP 1 signaling-induced B cell hyper-immunogenicity is essential for the T cell response, we constructed an LMP1 mutant in which amino acids FWLY(38-41) of transmembrane domain 1 (TM1) were changed to AALA (referred to as LMP I™¹'): this abolishes LMP1 clustering and signaling (Yasui et al., 2004) (Figure 9A) and presumably its immune-stimulatory function (Smith et al., 2009). In an in vitro killing assay, cytotoxic CD4 and CD8 T cells from day 6-8 CL mice efficiently recognized and killed B cells expressing wild-type LMP1 but not B cells expressing the signaling-dead mutant LMP I ™¹', or the vector-transduced or untransduced control B cells (the latter cells are in fact LPS-activated B cells) (Figure 9B). Thus, T cell recognition of LMPl-expressing B cells requires LMP1 signaling, which renders the B cells highly immunogenic.

Because LMP1 is a functional analog of constitutively active CD40, and because LMP 1 and CD40 both activate the immunogenicity of B cells and possibly enhance endogenous antigen presentation (see above), we tested whether primed T cells from CL mice recognize CD40-activated wild-type (WT) B cells via the cellular antigens that they share with LMPl-expressing B cells. We found that cytotoxic CD4 and CD8 T cells from day 6-8 CL mice lysed WT B cells that were pre-activated with anti-CD40, but not resting (naive) B cells (Figure 10A). These data suggest that B cells with LMP1 signaling provide endogenous antigens to be targeted by cytolytic T cells. The CD4 T cell killing activity of CD40-activated WT B cells was suppressed by blocking recognition of MHC class II (Figure 10B). Killing could also be decreased by blocking the FasL-Fas apoptotic pathway (CD40-activated B cells express Fas, as do LMPl-expressing B cells (Figure 8)), and blocking both MHC-II and FasL led to a more substantial reduction in the killing activity (Figure 10B). These data suggest

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PCT/US2018/046060 that cytotoxic T cells target LMP1-expressing B cells by recognizing self-peptide/MHC complex and exert their cytolytic activity by perforin-granzyme and FasL-Fas dependent pathways.

Unambiguous evidence that the T cells in CL mice recognize self-peptide/MHC complexes was obtained by analyzing the proliferative responses of CD4 effector/memory T cells (excluding Foxp3⁺ Tregs which are known to be self-reactive) on CD40-activated B cells, derived from WT versus OITA ' (lacking MHC-II expression) mice. A significant fraction of the effector/memory CD4 cells proliferated vigorously on CD40-activated WT B cells in an MHC-II restricted manner (Figure 11).

Together, our data indicate that T cells recognize and lyse LMP1-expressing B cells via cellular antigens, some of which are also presented on WT B cells that are activated through the analogous CD40 pathway (Figures 10-11). Because the cytotoxic T cells from CL mice do not lyse resting B cells (Figure 10A) nor WT B cells activated by LPS (through a pathway unrelated to LMP1 signaling; Figure 9B), it appears that cellular antigens induced by LMP1 signaling, rather than common B cell antigens, are the main targets of T cells. Given that the TCR repertoire during the acute phase of the immune response is very diverse (similar to that in naive mice) and that there is no clonal deletion of any νβ TCR afterwards (Figure 7A-C), it can be inferred that the T cells target a large number of LMP1 signalinginduced cellular antigens, but not a superantigen. At present, we cannot exclude the involvement of LMP 1-derived peptides in the T cell response in CL mice. However, such response might be too small to be detectable with our previous peptide screening assay.

Example 5. LMP1 induces immune surveillance independent of CD40 signaling

Although LMP 1 signaling and constitutive CD40 activation enhanced cellular antigen presentation as well as co-stimulation to a certain degree, immune surveillance was only seen in mice whose B cells expressed LMP1, but not in mice whose B cells expressed an LMP1CD40 fusion protein (LMP 1 transmembrane region fused to the intracellular signaling domain of CD40, thereby making CD40 pathway constitutively active; both mouse models used the same gene expression strategy, namely knocking-in to the Rosa26 locus) (HomigHolzel et al., 2008; Zhang et al., 2012). These results suggest that the LMP1 signaling domain is distinct from that of CD40, in its ability to induce immune surveillance. However, considering that LMP1 signaling in B cells upregulates CD40 expression (Figure 12A), we addressed the possibility that LMP1 induces immune surveillance by potently amplifying CD40 signaling by breeding CL mice to a CD40 ' background. Comparing CL mice on a CD40-null versus -WT background indicated that LMP 1-expressing B cells were efficiently

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PCT/US2018/046060 eliminated by activated CD4 and CD8 T cells irrespective of CD40 status (Figure 12B-D). In other words, LMP1 induces immune surveillance independent of CD40 signaling.

Example 6. LMP1-B cells drive cytotoxic T cells via co-stimulation by CD70, OX40L and 4-1BBL

We next sought to uncover the molecular mechanisms via which LMP1 signaling induces potent cytotoxic T cell responses. While CD8 T cells inherently develop cytotoxic capacity upon priming with antigens and various co-stimulatory signals, CD4 T cells are multipotential yet uniquely polarized towards the cytotoxic phenotype in our system, we thus focused on identifying co-stimulatory molecules that were expressed on LMP1-expressing B cells and able to induce the cytotoxic differentiation of CD4 cells. Recently, similar granzyme/perforin-featured cytotoxic CD4 T cells have been described, whose differentiation is fully dependent on the T-box transcription factor Eomesodermin (Eomes), but not on the Thl polarizing T-bet (Curran et al., 2013; Qui et al., 2011; Swain et al., 2012). Furthermore, systemic activation of 4-IBB and/or 0X40 co-stimulatory pathways (by agonist antibodies) induces high levels of Eomes in antigen-primed CD4 cells, which then drives their cytotoxic differentiation (Curran et al., 2013; Qui et al., 2011). Systemic CD27 activation also induces Eomes expression in CD4 cells (Curran et al., 2013). Our data show that LMP1-expressing B cells express greatly enhanced levels of 4-IBB ligand (4-1BBL), 0X40 ligand (OX40L) and CD70 (CD27 ligand), compared to control B cells (Figure 13A-B). Proinflammatory cytokines, including IL27 and IL 15, may also play a supportive role in cytotoxic CD4 cell generation (Curran et al., 2013). However, with the exception of the gene for the IL27 subunit β, the other cytokine genes were only marginally, if at all, induced in LMP1-B cells (Figure 13C).

Consistent with the plausible roles of 4-IBB and 0X40 (and also CD27) pathways in inducing Eomes-Granzyme program in T cells, high levels of Eomes and GzmB were expressed in a major population of CD4 cells in day 6-8 CL mice (Figure 14A). Systemic 41BB activation is known to result in selective expression of Eomes, without T-bet expression (Curran et al., 2013), while simultaneous activation of 4-1BB and 0X40 induces both Eomes and T-bet in CD4 cells (Qui et al., 2011). Because LMP1-B cells express ligands for both pathways, we also examined T-bet expression in the CD4 cells: analysis of Eomes and T-bet expression by CD4 cells from CL mice revealed three populations of effector cells— Eomes⁴ T-bct . Eomes⁺T-bet⁺, and Eomes T-bct⁴—in sharp contrast to CD4 cells from control naive mice (Figure 14B). Furthermore, CD4 cells from CL mice expressed GzmB and/or IFN-γ, in contrast to those from control naive mice (Figure 14B). GzmB expression

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PCT/US2018/046060 depends on Eomes (but not T-bet) (Curran et al., 2013; Qui et al., 2011), while IFN-γ is mainly driven by T-bet (Swain et al., 2012); thus, our FACS analyses revealed three subtypes of effector CD4 cells in CL mice: (i) Eomes/GzmB-featured cytotoxic cells (similar to those described in (Curran et al., 2013)); (ii) T-bet/IFN-γ featured Thl cells (Swain et al., 2012); (iii) a population that displayed features of both the cells described in (i) and (ii) (these cells were similar to the ‘cytotoxic CD4 Thl cells’ described in (Qui et al., 2011)). CD4 cells from CL mice exhibited no expression of GATA3 or RORyt (Figure 15A-B), indicating no commitment towards the Th2 or Th 17 subsets. The co-stimulation pathways may similarly affect CD8 cells (Curran et al., 2013; Qui et al., 2011), but in contrast to their CD4 counterparts, the CD8 cells in day 6-8 CL mice developed into a single, nearly uniform population, that was Eomes⁺T-bet⁺GzmB⁺IFN-y⁺ (Figure 14C).

The finding that LMP1⁺ B cells efficiently present cellular antigens, and simultaneously provide high levels of co-stimulatory ligands (4-1BBL, OX40L and CD70) that are implicated in cytotoxic T cell programming, suggests that these B cells may suffice, as an APC system, to induce CTL responses to cellular antigens. Indeed, we found that upon a short period (7 days) of co-culture with LMP1⁺ B cells in vitro (without addition of any exogenous cytokine), a sizable fraction of CD4 T cells from naive WT mice was activated/expanded; this effect depended on LMP1 signaling in B cells, as CD4 cells failed to expand on LMP I™¹ '-expressing B cells (Figure 16A). A sizable fraction of CD4 cells activated/expanded by LMP1-B cells turned on the Eomes and/or T-bet programs (Figure 16B), developed cytotoxicity (Figure 16C), and recognized CD40-activated WT B cells in an MHC-II dependent manner (Figure 16D).

This in vitro system provided unique opportunities for assessing the roles of 4-1BBL, OX40L and CD70 in the LMP1⁺ B cell-driven cytotoxic T cell generation. In this system, we observed that, when co-cultured with LMP1⁺ B cells, CD4 cells gave rise to an optimal Eomes⁺ population on day 7, while CD8 cells readily differentiated into Eomes⁺ by day 3. With use of antibody-mediated blocking in culture, we found that 4-1BBL blockade did not alter the fraction of CD4 cells with the Eomes⁺ phenotype (Figure 16E), or the absolute number of Eomes⁺ CD4 cells (Figure 16F); OX40L blockade led to a slight reduction in the fraction of Eomes⁺ cells, but a significant decrease in the number; and CD70 blockade caused an even more severe reduction of the fraction and total number of Eomes⁺ CD4 cells (Figures 16E, 16F, and 16G). With regard to their CD8 counterparts, blocking OX40L and CD70 each reduced the frequency and number of the Eomes⁺ population, to an extent similar to that seen with CD4 cells; however, 4-1BBL blockade also reduced the frequency and significantly

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PCT/US2018/046060 decreased the number of Eomes⁺ CD8 cells (Figures 16H, 161, and 16J), in sharp contrast to the lack of effect seen with the CD4 cells. Furthermore, blocking all three co-stimulatory ligands altogether almost completely abrogated the generation of Eomes⁺ CD8 cells (Figures 16H, 161, and 16J). Together, these results demonstrate that LMP1-expressing B cells drive the differentiation and expansion of CD4 CTLs via CD70 and OX40L mediated costimulation, and of CD8 CTLs via CD70, OX40L, as well as 4-1BBL. CD70 has a more pronounced role in the generation of both types of CTLs.

Overall, our findings indicate that LMP1 signaling turns B cells into highly immunogenic APCs, by enhancing endogenous antigen presentation and potent costimulation (via CD70, OX40L and 4-1BBL), and drives cytotoxic CD4 and CD8 T cell responses. The target antigens appear to comprise a large array of LMP1 signaling-induced cellular antigens (see schematic in Figure 1A).

Example 7. A novel concept: LMP1 signaling induces potent tumor immunity mediated by CD4⁺ and CD8⁺ cytotoxic T cells against wide range of TAAs

Our findings presented herein show that LMP1 signaling activates B cells to present cellular antigens and simultaneously provide co-stimulatory signals through CD70, 0X40 ligand and 4-IBB ligand, resulting in the induction of cytotoxic CD4 and CD8 T cells that kill LMP1-expressing B cells. This work provides a mechanism whereby T cells can recognize and eliminate EBV-infected or transformed cells via cellular as well as viral antigens.

The polyclonal TCRs on reactive T cells in CL mice indicate that diverse cellular antigens are being targeted. This raises the question of why the virus would evolve a strategy to induce host immune surveillance that target broad cellular antigens. Perhaps, this is favorable for long-term virus-host coexistence. EBV rapidly drives B cell proliferation and transformation, during which LMP1 turns on multiple cellular oncogenic pathways. Meanwhile, LMP1 signaling renders infected cells highly immunogenic, by efficient presentation of viral antigens and LMP1 signaling-induced cellular antigens, and strong costimulation for the differentiation of cytotoxic CD8 and CD4 cells (and also Thl type CD4 cells). Consequently, a much larger TCR repertoire and multiple arms of effector cells are recruited in the immune response, which enables rapid elimination of EBV/LMP1-expressing B cells, and prevents deadly lymphoproliferation and lymphomagenesis. B cells harboring dormant virus are spared, allowing the virus to persist in the host, and efficiently spread in the human population.

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Cytotoxic T cells recognize LMP1⁺ B cells (and LMP1-driven lymphoma cells) through diverse cellular antigens, which appear mainly induced by LMP1 signaling. Because LMP1 is the key oncoprotein for EBV-driven tumorigenesis (Kaye, et al. (1993) Proc Natl Acad Sei USA. 90(19):9150-54), the cellular antigens induced by LMP1 and recognized by T cells would be TAAs belonging to the subgroup of “overexpression antigens” (Coulie et al. (2014) Nat Rev Cancer 14(2): 135-46). Our studies presented herein lead us to raise a novel concept: signaling by the Epstein-Barr virus LMP1 protein induces potent tumor immunity mediated by CD4⁺ and CD8⁺ cytotoxic T cells against wide range of TAAs. The underlying molecular processes are illustrated in a schematic model in Figure 1A: In B cells, constitutive LMP1 signaling induces massive cellular gene expression. This leads to upregulation of antigen processing, presenting function (MHCs), strong co-stimulation signals (B7-1, B7-2, ICAM-1, and particularly CD70, OX40L and 4-1 BBL), and induced and/or enhanced expression of certain cellular antigens (including a wide range of TAAs). Presentation of these antigens and simultaneous co-stimulations drive activation and cytotoxic differentiation of CD4⁺ and CD8⁺ T cells specific to these antigens.

Example 8. T cell responses to exemplary TAAs

Some of the T cell targets presented by LMP 1-expressing B cells were also induced in normal B cells upon constitutive CD40 signaling. By microarray, -2,120 genes were upregulated >2 folds in LMP 1-expressing B cells, and -50% of those genes were also upregulated in CD40-activated B cells. These aberrantly expressed LMP1 signaling-induced cellular antigens included many known TAAs. A few of such TAAs were chosen to demonstrate that LMP 1 signaling-induced cellular antigens, particularly TAAs, were indeed T cell target antigens (Table 1). Their potential epitopes bound to MHC-I H-2D^b or H-2K^bwere either known from literature (for Survivin and EphA2) or predicted through IEDB (www.immuneepitope.org). Tetramers loaded with a Survivin epitope peptide (ATFKNWPFL/H-2D^b) or an EphA2 epitope peptide (VVSKYKPM/H-2K^b) were obtained from the NIH Tetramer Facility.

Table 1. Examples of LMP 1 signaling-induced cellular genes known as immunogenic TAAs

Gene	mRNA fold changes relative to naive B cells
LMP1-B	CD40-B
p21	16.3	2.7
Survivin	7.8	3.4

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Epha2	4.9	0.9
Kil'20a	3.9	6.9

For detection of TAA-specific T cell response, we used the CD19-cre^ERT2-fMPE^STOF(C Z) model system. The inducible 6 L system allows for LMP 1 expression to be turned on initially in a small fraction of B cells upon Tamoxifen treatment, thus mimicking primary EBV infection (Yasuda et al., 2013). Flow cytometry analysis with the Survivinand EphA2-Tetramers clearly identified epitope-specific CD8 T cell expansion in mice which peaked at day 5 after Tamoxifen treatment, but not in treated control mice (Figure 17 and data not shown). Of note, these T cells have low/medium affinity to the TAA peptide/MHC complexes, as expected for T cells specific to TAAs (Blankenstein et al., 2012); the detection of small populations of T cells recognizing individual epitopes is consistent with the finding that LMPl-expressing B cells elicit polyclonal T cell responses and further strengthens our prediction that wide range of LMP 1 signaling-induced cellular antigens/TAAs are targeted by T cells.

Example 9. Control of cellular antigen-specific T cells by CD4 Tregs leads to immune homeostasis

The broadly autoantigen (TAA)-reactive cytotoxic T cells ensure rapid elimination of LMPl-expressing B cells, but may also damage other host tissues. Importantly, after clearing the first wave of LMPl-expressing B cells, the immune system returns to a homeostatic state, as observed in adult CL mice in which the newly developing LMPl-expressing B cells are under constant surveillance. To understand how the homeostatic state is reached/maintained, we interrogated the role of CD4 Tregs, which are critical players in peripheral tolerance. We found that the frequency of CD4 Tregs was inversely correlated with the killing activity of bulk CD4 cells from CL mice: during the acute phase (day 6-8) of the immune response, CD4 cells displayed a high killing activity (Figure 4) and a low frequency (~7%) of Tregs (Figure 18A), whereas during the chronic phase (in adult CL mice BM), CD4 cells exhibited minimum killing activity (Figure 6) and a strikingly high frequency (-50%) of Tregs (Figure 18B, left panel); moreover, when co-transferred with LMPl-expressing lymphoma cells into lymphopenic hosts, chronic phase CD4 cells regained killing activity (Figure 6), and also displayed a sharp decrease of CD4 Tregs (Figure 18B, right panel). In vitro studies provided direct evidence that CD4 Tregs control the cytotoxicity of CD4 and CD8 effectors in the chronic phase: CD4 cells from the BM of adult CL mice exhibited pronounced cytotoxicity on LMPl-expressing B cells, but only after removing CD4 Tregs (Figure 18C), whereas

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PCT/US2018/046060 killing of CD40-activated WT B cells by CD8 cells was suppressed by adding CD4 Tregs to the cell culture (Figure 18D). Thus, during the chronic phase CD4 Tregs control the autoreactive effector T cells, allowing the effector cells to continuously eliminate newly arising LMP1-expressing B cells, but preventing the destruction of self tissues.

Example 10. Use of LMPl-expressing cells for Adoptive Cell Transfer (ACT) Therapy

Based on the concept that LMP1 expression in primary or lymphoma B cells induces cellular antigen expression and presentation, and elicits cytotoxic T cell responses against LMP1 signaling-induced cellular antigens (including many TAAs), lymphoma inherent TAAs, and neoantigens (Figures 1A and IB), patient-derived primary or lymphoma B cells, upon LMP 1 expression, could be used (after irradiation) to activate and expand autologous or donor-derived T cells for ACT to treat EBV-associated B cell lymphomas in immunocompetent hosts and immunosuppressed hosts (e.g., post-transplant and AIDS patients). The EBV-infected lymphoma cells express LMP1, and thus would present the same array of antigens on the surface as the antigens recognized by the infused T cells. The ACT strategy described herein could be similarly applied to EBV-unrelated B cell lymphomas by generating T cells targeting shared LMP1 signaling-induced TAAs, lymphoma inherent TAAs, and neoantigens, thereby eliciting anti-tumor cellular immunity. Other lineages (i.e., non-B lineage) of cells (e.g., tumor cells) expressing LMP1 could also be used in the ACT strategy described herein (Figure 19A).

To demonstrate use of LMPl-expressing B cells for ACT, syngeneic wild-type BALB/c mice were treated with a single dose of total body irradiation (TBI) at 600 Rad to create a lymphopenic condition favorable for adoptive T cell expansion, followed by transplantation of the A20 lymphoma cells (3xl0⁵ cells) on the same day. One day later, 3x10⁶ CD8 T cells primed by LMPl-expressing B cells for 3 days in culture, or 3x10⁶ CD4 T cells primed by LMPl-expressing B cells for 7 days in culture, were administered intravenously to the mice (Figure 19B). A single dose of CD8 T cells (containing ~50% of Eomes⁺ cytotoxic effectors) reduced the growth of the A20 lymphoma (Figure 19C). Similarly, a single dose of CD4 T cells (containing -10% of Eomes⁺ cytotoxic effectors) reduced the growth of the A20 lymphoma (Figure 19D). These results demonstrate that expressing LMP1 in B cells can produce therapeutic T cells against the A20 tumor (through shared TAAs).

Demonstrating use of LMPl-expressing lymphoma B cells for ACT, we showed that cultivating naive CD4 T cells (from WT mice) with LMPl-expressing A20 lymphoma cells for 7 days led to generation of CD4 CTLs, which lysed the A20 lymphoma cells in an MHC48

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Il-dependent manner, but not control naive B cells (Figures 19E and 19F). Adoptive transfer of a single low dose (0.4xl0⁶) of such CD4 CTLs into syngeneic mice pre-transplanted with A20 lymphoma cells (3xl0⁵ cells; SC, 7 days prior) led to markedly delayed tumor growth (naive CD4 T cells had no therapeutic effect; Figures 19G and 19H). Furthermore, we showed that adoptive transfer of CD8 CTLs (2xl0⁶), generated by cultivating naive CD8 T cells (from WT mice) on LMP 1-expressing A20 lymphoma cells for 3 days, into syngeneic mice pre-transplanted with A20 lymphoma cells (3xl0⁵ cells; SC, 7 days prior) resulted in delayed tumor growth (Figures 191 and 19J). Collectively, these data demonstrate that expressing LMP1 in A20 lymphoma cells can produce therapeutic T cells against the A20 tumor (through tumor endogenous antigens, including TAAs and neoantigens).

Example 11. “LMPl-cell vaccine” for cancer therapy

Based on the concept that LMP1 expression in primary or lymphoma B cells induces cellular antigens expression, presentation and elicits cytotoxic T cell responses against LMP1 signaling-induced cellular antigens (including many TAAs), lymphoma inherent TAAs, and neoantigens (Figures 1A and IB), LMP 1-expressing autologous primary or lymphoma B cells could be used as a “LMPl-cell vaccine” to prime T cells in vivo to treat EBV-associated B cell lymphomas in immunocompetent hosts. The EBV-infected lymphoma cells express LMP1, and thus would present the same array of antigens on the surface as the antigens presented by the LMPl-cell vaccine. Therefore, the T cells activated by the vaccine would exhibit cytotoxicity to the EBV-infected lymphoma cells. The vaccination strategy described herein could be similarly applied to EBV-unrelated B cell lymphomas by eliciting anti-tumor T cell immunity in vivo against shared LMP1 signaling-induced TAAs, lymphoma inherent TAAs, and neoantigens. Other lineages (i.e., non-B lineage) of cells (e.g., tumor cells) expressing LMP1 could also be used for generating LMPl-cell vaccines as described herein (Figure 20A).

To demonstrate use of a “LMPl-cell vaccine” for cancer therapy in vivo, poorly immunogenic A20 lymphoma and B16-F10 melanoma cell lines were chosen.

A20 lymphoma cells were transduced with wild-type LMP 1 or the signaling-dead mutant LMPl™^lm (as control). Syngeneic BALB/c mice were transplanted with 4* 10⁵ live A20 lymphoma cells subcutaneously (S.C.). Following the transplantation, the mice were vaccinated with A20 cells expressing LMP1 or LMPl™¹¹¹¹ at various time points (1 x 10⁶irradiated cells /S.C.) (Figure 20B). Vaccination with A20 lymphoma cells expressing widetype LMP1 markedly delayed A20 lymphoma growth (Figure 20C).

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B16-F10 melanoma cells were transduced with LMP1, LMPl™¹¹¹¹ or vector control. Syngeneic C57BL6 mice were transplanted with 1 χ 10⁵ live B16-F10 melanoma cells subcutaneously. Following the transplantation, the mice were vaccinated with B16-F10 cells expressing LMP1, LMPl™^lm or vector control at various time points (lx 10⁶ irradiated cells /S.C.) (Figure 20D). Vaccination with B16-F10 cells expressing wild-type LMP1 markedly delayed or abrogated Bl6-F10 melanoma tumor growth (Figure 20E).

These results demonstrated that expressing LMP1 in otherwise poorly immunogenic A20 and B16 tumor cells could turn them into a powerful therapeutic vaccine against the respective unmodified (parental) tumors.

Example 12. Producing an antigen-specific APC by introducing an exogenous antigen to an LMPl-expressing B cell.

Examples 1-11 describe LMPl-expressing B cells useful in activating T cell immunity against a repertoire of endogenous antigens, such as p21, Survivin, Epha2, and Kif20a. This example describes four methods of modifying LMPl-expressing B cells to generate immunogenic cells useful in activating T cell immunity against specific exogenous antigens.

The first method relates to ectopically expressing an exogenous antigen in LMPlexpressing B cells, for example, using a vector encoding the exogenous antigen. The vector can be a non-viral vector introduced to the B cells by transfection or electroporation. Alternatively, the vector can be a viral vector (e.g., an adenoviral vector, an adeno-associated viral vector, or a retroviral vector such as a lentiviral vector) introduced to the B cells by transduction. The vector can also encode LMP1, obviating the need of introducing a second vector to the B cells.

The exogenous antigen expressed from the vector can be a TAA, a neoantigen, or an antigen specific to a microbe or pathogen (e.g., bacterium, virus, fungus, or parasite). Either a full-length polypeptide or a truncated peptide of the antigen can be expressed. Where a truncated peptide is expressed, the truncation that leads to desirable expression, antigen presentation, and/or immunogenicity can be identified, for example, by testing a series of truncated peptides, and examining the expression levels of the peptides, the levels of MHC complex presenting the peptides, and the activation of T cells in contact with the B cells (e.g., the ability of the T cells to kill cells expressing the full-length polypeptide).

The efficacy of this method can be demonstrated by the following experiments: an LMPl-expressing B lymphoma cell line is transduced with a viral vector encoding chicken ovalbumin (OVA). For in vitro activation, the B cells are co-cultured with a population of T

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PCT/US2018/046060 cells from wild-type mice, naive CD8⁺ OT-I T cells, or naive CD4⁺ OT-II T cells. For in vivo activation, the B cells are injected intravenously or subcutaneously to wild-type mice, or ΟΤΙ or OT-II mice. The generation of activated/cytotoxic CD8⁺ OT-I T cells and CD4⁺ OT-II T cells can be assessed by in vitro T cell proliferation assays and cytotoxicity assays.

The second method relates to uptake of an exogenous antigen by LMP 1-expressing B cells. The exogenous antigen can be any antigen obtained by chemical synthesis or purification. For example, the exogenous antigen can be a synthetic peptide either unmodified or modified with desired post-translational modifications. Amino acid analogs and modified amino acids can be introduced at specific positions. Alternatively, the exogenous antigen can be purified from a population of cells or a tissue that has undergone neoplasm or has been infected with a microbe or pathogen. For example, the exogenous antigen can be purified from the membrane fraction. The exogenous antigen can also be purified from a population of microbe or pathogen cells. This method requires a greater amount of materials to start with relative to the first method, but can introduce non-peptide molecules and polypeptides with modifications not readily present in B cells. LMP 1-expressing B cells can be injected into a tumor tissue or a microbe/pathogen-infected tissue, wherein the B cells can uptake an exogenous antigen released from the neighboring dead tumor cells or infected cells.

Methods to deliver the exogenous antigen to the B cells include direct uptake and transfection. For direct uptake, the exogenous antigen may have a property (e.g., positive charges at the N terminus of a peptide antigen) that allows it to penetrate the plasma membrane. For transfection, the exogenous antigen may be encapsulated in a transfection agent (e.g., liposome) or conjugated to a membrane-penetrating agent (e.g., a nanoparticle).

To demonstrate the efficacy of this method, LMPl-expressing B cells were incubated with chicken ovalbumin (OVA) peptide (SIINFEKLL; 1 pg/ml) for 2 hrs, and then cocultured with naive CD8⁺ OT-I T cells for 3 days; the generation of activated cytotoxic CD8⁺OT-I T cells was assessed by in vitro cytotoxicity assays (Figure 21A). The primed CD8⁺OT-I T cells exhibited potent killing activity against OVA-expressing EL4 cells but not the control EL4 cells (a T cell lymphoma line on a B6 background; Figure 2IB); the primed CD8⁺ OT-I T cells exhibited high killing activity against OVA peptide-loaded naive B cells but not the unloaded naive B cells (Figure 21C). Thus, an LMPl-expressing B cell, upon uptake of a desired exogenous antigen, can be used as an APC to produce antigen-specific cytotoxic T cells.

The third method relates to fusion of LMPl-expressing B cells with cells that comprise the exogenous antigen. The cell can be a tumor cell expressing a TAA or

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PCT/US2018/046060 neoantigen, or a bacterium- or virus-infected cell having one or more bacterial or viral antigens. The fusion can be induced by polyethylene glycol. The fused cells will present the exogenous antigen from the parent cell, and have improved immunogenicity conferred by

LMP1 expression.

The fourth method relates to infection of LMP1-expressing B cells with bacteria or virus. The infected B cells will express bacterial or viral proteins and present them on the cell surface, thereby activating the T cells in contact therewith. Some bacteria and viruses encode genes that can suppress antigen presentation by MHC molecules, or genes that can inhibit T cell activation. To increase the immunogenicity of the B cells, the bacterium or virus can be modified (e.g., by heat, chemicals, or genetic modification) to inhibit or reduce its immunosuppressive function.

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B. Zhang, S. Kracker, T. Yasuda, S. Casola, M. Vanneman, C. Homig-Holzel, Z. Wang, E. Derudder, S. Li, T. Chakraborty, S.E. Cotter, S. Koyama, T. Currie, G.J. Freeman, J.L. Kutok, S.J. Rodig, G. Dranoff, K. Rajewsky, Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model, Cell 148 (2012) 739-751.

M. Rowe, R. Khanna, C.A. Jacob, V. Argaet, A. Kelly, S. Powis, M. Belich, D. CroomCarter, S. Lee, S.R. Burrows, et al., Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein-1: coordinate up-regulation of peptide transporters and HLA-class I antigen expression, Eur J Immunol 25 (1995) 13741384.

C. M. Bollard, S. Gottschalk, V. Torrano, O. Diouf, S. Ku, Y. Hazrat, G. Carrum, C. Ramos,

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M. K. Brenner, H.E. Heslop, C.M. Rooney, Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins, J Clin Oncol 32 (2014) 798-808.

B. Zhang et al., An oncogenic role for alternative NF-kappaB signaling in DLBCL revealed upon deregulated BCL6 expression. Cell reports 11, 715 (May 5, 2015).

S. B. Koralov et al., Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 132, 860 (Mar 7, 2008).

C. M. Bollard, C. M. Rooney, Η. E. Heslop, T-cell therapy in the treatment of post-transplant lymphoproliferative disease. Nature reviews. Clinical oncology 9, 510-519 (2012).

T. Yasuda et al., Studying Epstein-Barr Virus Pathologies and Immune Surveillance by Reconstructing EBV Infection in Mice. Cold Spring Harbor symposia on quantitative biology, (2013).

C. Smith et al., Discerning regulation of cis- and trans-presentation of CD8+ T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation. Blood 113, 6148-6152 (2009).

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J. L. Schultze et al., Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity. Proceedings of the National

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J. L. Schultze, M. J. Seamon, S. Michalak, J. G. Gribben, L. M. Nadler, Autologous tumor infiltrating T cells cytotoxic for follicular lymphoma cells can be expanded in vitro. Blood 89,3806-3816 (1997).

T. Yasui, M. Luftig, V. Soni, E. Kieff, Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling. Proceedings of the National Academy of Sciences of the United States of America 101, 278-283 (2004).

C. Homig-Holzel et al., Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-kappaB pathway and promotes lymphomagenesis. The Journal of experimental medicine 205, 1317-1329 (2008).

J. Rastelli et al., LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgGl. Blood 111, 1448-1455 (2008).

Η. Z. Qui et al., CD134 plus CD137 dual costimulation induces Eomesodermin in CD4 T cells to program cytotoxic Thl differentiation. Journal of immunology 187, 3555-3564 (2011).

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Claims

1. A method for producing an immunogenic cell, the method comprising:

2. The method of claim 1, wherein the B cell is a naive B cell.

3. The method of claim 1 or 2, wherein the B cell is immortalized.

4. The method of claim 1 or 2, wherein the B cell is a primary B cell.

5. The method of any one of the preceding claims, wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 1.

6. The method of any one of the preceding claims, wherein the first nucleic acid is operably linked to a transcriptional regulatory element.

7. The method of any one of the preceding claims, wherein the exogenous antigen is a second peptide.

8. The method of claim 7, wherein the precursor of the exogenous antigen is a protein comprising the amino acid sequence of the second peptide.

9. The method of claim 7, wherein the precursor of the exogenous antigen is a second nucleic acid encoding the second peptide.

10. The method of any one of claims 1-9, wherein the exogenous antigen is a tumorassociated antigen (TAA).

11. The method of claim 10, wherein the composition comprises a tumor cell comprising the TAA.

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12. The method of any one of claims 1-9, wherein the exogenous antigen is a neoantigen.

13. The method of claim 12, wherein the composition comprises a tumor cell comprising the neoantigen.

14. The method of claim 11 or 13, wherein the composition further comprises an agent capable of facilitating cell fusion.

15. The method of any one of claims 1-9, wherein the exogenous antigen is a bacterial antigen.

16. The method of claim 15, wherein the composition comprises a bacterium comprising the bacterial antigen.

17. The method of claim 16, wherein the bacterium is inactivated.

18. The method of any one of claims 1-9, wherein the exogenous antigen is a viral antigen.

19. The method of claim 18, wherein the composition comprises a viral particle comprising the viral antigen.

20. The method of claim 19, wherein the viral particle is replication defective.

21. The method of any one of the preceding claims, wherein the immunogenic B cell comprises the exogenous antigen on the surface.

22. The method of claim 21, wherein the exogenous antigen is conjugated to a class II major histocompatibility complex (MHC-II) on the surface of the immunogenic B cell.

23. The method of claim 21, wherein the exogenous antigen is conjugated to a class I major histocompatibility complex (MHC-I) on the surface of the immunogenic B cell.

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24. The method of any one of the preceding claims, further comprising reducing the proliferative capacity of the immunogenic B cell.

25. The method of claim 24, wherein reducing the proliferative capacity of the immunogenic B cell comprises irradiating the immunogenic B cell.

26. An immunogenic B cell produced by the method of any one of the preceding claims.

27. An isolated B cell comprising:

28. The isolated B cell of claim TI. wherein the B cell is a naive B cell.

29. The isolated B cell of claim TI or 28, wherein the B cell is immortalized.

30. The isolated B cell of claim TI or 28, wherein the B cell is a primary B cell.

31. The isolated B cell of any one claims 27-30, wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 1.

32. The isolated B cell of any one of claims 27-31, wherein the first nucleic acid is operably linked to a transcriptional regulatory element.

33. The isolated B cell of any one of claims 27-32, wherein the exogenous antigen is a second peptide.

34. The isolated B cell of claim 33, wherein the precursor of the exogenous antigen is a protein comprising the amino acid sequence of the second peptide.

35. The isolated B cell of claim 33, wherein the precursor of the exogenous antigen is a second nucleic acid encoding the second peptide.

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36. The isolated B cell of any one of claims 27-35, wherein the exogenous antigen is a TAA.

37. The isolated B cell of any one of claims 27-35, wherein the exogenous antigen is a neoantigen.

38. The isolated B cell of any one of claims 27-35, wherein the exogenous antigen is a bacterial antigen.

39. The isolated B cell of any one of claims 27-35, wherein the exogenous antigen is a viral antigen.

40. The isolated B cell of any one of the claims 27-39, wherein the isolated B cell comprises the exogenous antigen on the surface.

41. The isolated B cell of claim 40, wherein the exogenous antigen is conjugated to an MHC-II on the surface.

42. The isolated B cell of claim 40, wherein the exogenous antigen is conjugated to an MHC-I on the surface.

43. A vaccine comprising the immunogenic B cell of claim 26 or the isolated B cell of any one of claims 27-42.

44. The vaccine of claim 43, further comprising an adjuvant.

45. A method of activating a T cell, the method comprising contacting the T cell with:

(a) the immunogenic B cell of claim 26;

(b) the isolated B cell of any one of claims 27-42; or (c) the vaccine of claim 43 or 44, thereby producing an activated T cell.

46. The method of claim 45, wherein the T cell is a CD4⁺ T cell.

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47. The method of claim 45, wherein the T cell is a CD8⁺ T cell.

48. The method of any one of claims 45-47, wherein the activated T cell is cytotoxic.

49. The method of any one of claims 45-48, wherein the T cell is contacted with the isolated B cell ex vivo.

50. The method of any one of claims 45-49, further comprising culturing the activated T cell under suitable conditions to allow proliferation of the activated T cell.

51. The method of claim 49 or 50, further comprising administering the activated T cell to a subject in need thereof.

52. The method of claim 51, wherein the subject has a tumor comprising the exogenous antigen.

53. The method of claim 51, wherein the subject is infected with a bacterium comprising the exogenous antigen.

54. The method of claim 51, wherein the subject is infected with a virus comprising the exogenous antigen.

55. The method of any one of claims 51-54, wherein the immunogenic B cell or the isolated B cell is autologous to the subject.

56. The method of any one of claims 51-54, wherein the immunogenic B cell or the isolated B cell is from a donor having an MHC matched with the subject.

57. The method of any one of claims 51-56, wherein the T cell is autologous to the subject.

58. The method of any one of claims 51-56, wherein the T cell is from a donor having an MHC matched with the subject.

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59. The method of any one of claims 45-48, wherein the T cell is in a subject, and the immunogenic B cell, the isolated B cell, or the vaccine is administered to the subject in an amount effective to activate the T cell in the subject.

60. A method of treating a subject in need thereof, the method comprising administering to the subject:

(a) a therapeutically effective amount of the immunogenic B cell of claim 26;

(b) a therapeutically effective amount of the isolated B cell of any one of claims ΊΊ42; or (c) a therapeutically effective amount of the vaccine of claim 43 or 44.

61. The method of claim 60, wherein the subject has a tumor comprising the exogenous antigen.

62. The method of claim 60, wherein the subject is infected with a bacterium comprising the exogenous antigen.

63. The method of claim 60, wherein the subject is infected with a virus comprising the exogenous antigen.

64. The method of any one of claims 60-63, wherein the isolated cell is autologous to the subject.

65. The method of any one of claims 60-63, wherein the isolated cell is from a donor having an MHC matched with the subject.

66. The method of any one of claims 51-65, further comprising administering to the subject an immune co-stimulation therapy.

67. The method of claim 66, wherein the immune co-stimulation therapy is selected from the group consisting of an agonist of CD27, an agonist of 0X40, and an agonist of 41BB.

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68. The method of any one of claims 51-67, further comprising administering to the subject an immune checkpoint targeting therapy.

69. The method of any one of claims 51-68, further comprising administering to the subject a Treg modulating therapy.

70. A kit comprising:

71. The kit of claim 70, wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 1.

72. The kit of claim 70 or 71, wherein the nucleic acid is operably linked to a transcriptional regulatory element.

73. The kit of any one of claims 70-72, wherein the exogenous antigen is a second peptide.

74. The kit of claim 73, wherein the precursor of the exogenous antigen is a protein comprising the amino acid sequence of the second peptide.

75. The kit of claim 73, wherein the precursor of the exogenous antigen is a second nucleic acid encoding the peptide.

76. The kit of any one of claims 70-75, wherein the exogenous antigen is a TAA.

77. The kit of claim 76, wherein the composition comprises a tumor cell comprising the TAA.

78. The kit of any one of claims 70-75, wherein the exogenous antigen is a neoantigen.

79. The kit of claim 78, wherein the composition comprises a tumor cell comprising the neoantigen.

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80. The kit of claim 77 or 79, wherein the composition further comprises an agent capable of facilitating cell fusion.

81. The kit of any one of claims 70-75, wherein the exogenous antigen is a bacterial antigen.

82. The kit of claim 81, wherein the composition comprises a bacterium comprising the bacterial antigen.

83. The kit of claim 82, wherein the bacterium is inactivated.

84. The kit of any one of claims 70-75, wherein the exogenous antigen is a viral antigen.

85. The kit of claim 84, wherein the composition comprises a viral particle comprising the viral antigen.

86. The kit of claim 85, wherein the viral particle is replication defective.

87. The kit of any one of claims 70-86, further comprising an agent capable of reducing the proliferative capacity of a B cell.

88. The kit of any one of claims 70-87, further comprising an instruction to irradiate a B cell.