ebv-specific immune cells

Abstract

Disclosed herein are methods for generating / expanding a population of immune cells comprising immune cells specific for Epstein-Barr virus (EBV) lytic antigens, the methods comprising stimulating immune cells specific for EBV lytic antigens by contacting peripheral blood mononuclear cells (PBMCs) with (i) one or more peptides corresponding to all or a portion of one or more EBV lytic antigens; or (ii) antigen presenting cells (APCs) presenting one or more peptides corresponding to all or a portion of one or more EBV lytic antigens. Also disclosed are populations of immune cells comprising immune cells specific for EBV lytic antigens expanded according to these methods, and uses thereof.

Description

[0001] This application claims priority to US16 / 388,776, filed April 18, 2019, the contents and elements of which are incorporated herein by reference in their entirety and for all purposes. Technical Field

[0002] This disclosure relates at least to the fields of molecular and cell biology and immunology, and also to methods of medical treatment and prevention.

[0003] Statement regarding federally funded research or development

[0004] This invention was completed with government support, funded by Grant CA126752 from the National Institutes of Health. The government holds certain rights to this invention.

[0005] background

[0006] Approximately 40% of lymphomas, all undifferentiated nasopharyngeal carcinomas (NPCs), and about 10% of gastric cancers carry the Epstein-Barr virus (EBV) genome and express viral proteins that can be targeted by EBV-specific T cells that can be adopted.

[0007] EBV+ malignancies occurring outside of immunosuppression express only 1 to 4 of the approximately 90 EBV proteins. Although these antigens have poor immunogenicity, they provide target antigens for EBVSTs.

[0008] Ongoing clinical trials on the use of EBV-specific T cells to treat EBV-positive malignancies use EBV-transformed B cells to expand EBV-specific T cells (NCT02578641), or involve stimulating PBMCs with peptides corresponding to the EBV type II latent antigen (NCT01555892). Invention Overview

[0010] This disclosure is based on the unexpected discovery that peptides corresponding to EBV cleavage antigens can be used to generate / amplify EBV-specific immune cell populations, which can be used to treat EBV-related diseases.

[0011] Although in reality, EBV-infected cells in EBV-related diseases only exhibit high expression of EBV latent antigens, the inventors have demonstrated that immune cell populations generated / expanded by stimulating immune cells with peptides containing EBV cleavage antigens exhibit cytolytic activity against EBV-infected cells. Furthermore, immune cell populations generated / expanded by stimulating immune cells with peptides containing EBV cleavage antigens have been shown to exhibit similar or improved therapeutic efficacy against EBV-positive cancers in vivo, compared to those generated / expanded by stimulating immune cells with peptides containing EBV latent antigens.

[0012] Another unexpected finding was that using both EBV lysis antigen and EBV latent antigen in the stimulation did not lead to a significant reduction in the frequency of immune cells specific to EBV latent antigen in the amplified population.

[0013] The inventors further demonstrated that an immune cell population containing immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens is more effective in treating EBV-related diseases than an immune cell population containing only immune cells specific to EBV latent antigens.

[0014] In a first aspect, this disclosure provides a method for generating or expanding a population of immune cells comprising immune cells specific to Epstein-Barr virus (EBV) lytic antigens, the method comprising stimulating immune cells specific to EBV lytic antigens by contacting peripheral blood mononuclear cells (PBMCs) with: (i) one or more peptides corresponding to all or part of one or more EBV lytic antigens; or (ii) antigen-presenting cells (APCs) presenting one or more peptides corresponding to all or part of one or more EBV lytic antigens.

[0015] In some embodiments, the method further includes restimulating the immune cells by contacting immune cells specific to EBV cleavage antigens with APCs that present all or part of one or more peptides corresponding to one or more EBV cleavage antigens.

[0016] A method is also provided for generating or expanding an immune cell population comprising immune cells specific to Epstein-Barr virus (EBV) lytic antigens and immune cells specific to EBV latent antigens, the method comprising stimulating the immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by contacting peripheral blood mononuclear cells (PBMCs) with: (i) one or more peptides corresponding to all or part of one or more EBV lytic antigens and one or more peptides corresponding to all or part of one or more EBV latent antigens; or (ii) presenting antigen-presenting cells (APCs) that present one or more peptides corresponding to all or part of one or more EBV lytic antigens and one or more peptides corresponding to all or part of EBV latent antigens.

[0017] In some embodiments, the method further includes restimulating the immune cells by contacting immune cells specific to EBV lysis antigens and immune cells specific to EBV latent antigens with antigen-presenting cells that present all or part of one or more peptides corresponding to one or more EBV lysis antigens and one or more peptides corresponding to one or more EBV latent antigens.

[0018] In some embodiments according to various aspects of this disclosure, the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, and BDLF3. In some embodiments according to various aspects of this disclosure, the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, and combinations thereof.

[0019] In some embodiments, the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2, and BDLF3. In some embodiments, the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2, BDLF3, and combinations thereof.

[0020] In some embodiments, the one or more EBV latent antigens are selected from EBNA1, EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1, LMP2A, and LMP2B. In some embodiments, the one or more EBV latent antigens are selected from EBNA1, EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1, LMP2A, LMP2B, and combinations thereof.

[0021] In some embodiments, the one or more EBV latent antigens are selected from EBNA1, LMP1, LMP2A, and LMP2B. In some embodiments, the one or more EBV latent antigens are selected from EBNA1, LMP1, LMP2A, LMP2B, and combinations thereof.

[0022] In some implementations, the PBMC is a PBMC that has depleted CD45RA-positive cells.

[0023] It also provides isolated populations of immune cells that can be obtained or obtained by means of the methods disclosed herein.

[0024] It also provides isolated populations of immune cells containing immune cells specific to Epstein-Barr virus (EBV) lytic antigens.

[0025] It also provides isolated populations of immune cells containing immune cells specific to Epstein-Barr virus (EBV) lytic antigens and immune cells specific to EBV latent antigens.

[0026] Pharmaceutical compositions comprising isolated immune cell populations according to the present disclosure are also provided.

[0027] The present disclosure also provides methods for treating or preventing diseases or conditions using isolated immune cell populations or pharmaceutical compositions.

[0028] The use of isolated immune cell populations or pharmaceutical compositions according to this disclosure in the preparation of medicaments for the treatment or prevention of diseases or conditions is also provided.

[0029] A method for treating or preventing a disease or condition is also provided, comprising administering to a subject an isolated population of immune cells or a pharmaceutical composition according to the present disclosure.

[0030] The present disclosure also provides methods for treating or preventing diseases or disorders associated with EBV infection using isolated immune cell populations or pharmaceutical compositions.

[0031] The use of isolated immune cell populations or pharmaceutical compositions according to this disclosure in the preparation of medicaments for the treatment or prevention of diseases or disorders associated with EBV infection is also provided.

[0032] A method for treating or preventing diseases or disorders associated with EBV infection is also provided, comprising administering to a subject an isolated population of immune cells or a pharmaceutical composition according to the present disclosure.

[0033] The present disclosure also provides methods for treating or preventing cancer using isolated immune cell populations or pharmaceutical compositions according to the present disclosure.

[0034] The use of isolated immune cell populations or pharmaceutical compositions according to this disclosure in the preparation of medicaments for the treatment or prevention of cancer is also provided.

[0035] A method for treating or preventing cancer is also provided, comprising administering to a subject an isolated population of immune cells or a pharmaceutical composition according to the present disclosure.

[0036] In some embodiments according to various aspects of this disclosure, the disease or condition associated with EBV infection is EBV-related cancer.

[0037] In some embodiments according to various aspects of this disclosure, the cancer is an EBV-related cancer.

[0038] In some implementations, the EBV-related cancer is selected from EBV-positive lymphoma, EBV-positive nasopharyngeal carcinoma, and EBV-positive gastric cancer.

[0039] A method for killing EBV-infected cells is also provided, comprising contacting the EBV-infected cells with an isolated population of immune cells or a pharmaceutical composition according to the present disclosure. This method may be in vitro or in vivo.

[0040] The use of isolated immune cell populations or pharmaceutical compositions according to this disclosure for killing cells infected with EBV is also provided.

[0041] A method for killing cancer cells is also provided, comprising contacting the cancer cells with an isolated population of immune cells or a pharmaceutical composition according to the present disclosure. This method may be in vitro or in vivo.

[0042] The use of isolated immune cell populations or pharmaceutical compositions according to this disclosure for killing cancer cells is also provided.

[0043] In some implementations, the cancer cells are infected with EBV. Invention Details

[0045] This disclosure is based on the unexpected discovery that immune cells specific to EBV lysis antigens can kill EBV-infected cells, and that a cell population containing immune cells specific to both EBV lysis antigens and EBV latency antigens shows an improved ability to control EBV-positive malignancies compared to a cell population containing only immune cells specific to EBV latency antigens.

[0046] Epstein-Barr virus replication

[0047] Virological descriptions of Epstein-Barr virus (EBV) can be found in, for example, Stanfield and Luftiq, F1000Res. (2017) 6:386 and Odumade et al., Clin Microbiol Rev (2011) 24(1):193-209, both of which are incorporated herein by reference.

[0048] EBV infects epithelial cells through the binding of the viral protein BMRF2 to β1 integrin, and the binding of the viral protein gH / gL to integrins avβ6 and avβ8. EBV infects B cells through the interaction of the viral glycoprotein gp350 with CD21 and / or CD35, followed by the interaction of viral gp42 with MHC class II. These interactions trigger the fusion of the viral envelope with the cell membrane, allowing the virus to enter the cell. Once inside the cell, the viral capsid dissolves, and the viral genome is transported to the nucleus.

[0049] EBV has two replication modes: latency and cleavage.

[0050] The incubation period does not lead to the production of virions and can occur in B cells and epithelial cells. The EBV genome circular DNA exists as an episome in the cell nucleus and is replicated by the host cell's DNA polymerase. During the incubation period, only a small fraction of the EBV genes are expressed in one of three different modes known as the incubation program, producing different groups of viral proteins and ribonucleic acid. The incubation period is described, for example, in Amon and Farrell, Reviews in Medical Virology (2004) 15(3):149–56, the full text of which is incorporated herein by reference.

[0051] The EBNA1 protein and the non-coding RNA EBER were expressed in latency programs I-III. Latency programs II and III further involved the expression of EBNALP, LMP1, LMP2A, and LMP2B proteins, while latency program III further involved the expression of EBNA2, EBNA3A, EBNA3B, and EBNA3C.

[0052] EBNA1 is multifunctional and plays a role in gene regulation, extrachromosomal replication, and maintenance of the EBV episome genome through positive and negative regulation by the viral promoter (Duellman et al., J Gen Virol. (2009); 90(Pt9):2251–2259). EBNA2 is involved in the regulation of latent viral transcription and contributes to the immortalization of EBV-infected cells (Kempkes and Ling, Curr Top Microbiol Immunol. (2015) 391:35-59). EBNA-LP is essential for native B cell transformation and recruits transcription factors for viral replication (Szymula et al., PLoS Pathog. (2018); 14(2):e1006890). EBNA3A, 3B, and 3C interact with RBPJ to influence gene expression, contributing to the survival and growth of infected cells (Wang et al., J Virol. (2016) 90(6):2906–2919). LMP1 regulates gene expression involved in B cell activation (Chang et al., J. Biomed. Sci. (2003) 10(5): 490–504). LMP2A and LMP2B inhibit normal B cell signaling by mimicking activated B cell receptors (Portis and Longnecker, Oncogene (2004) 23(53): 8619–8628). EBERs form ribonucleoprotein complexes with host cell proteins and are thought to play a role in cell transformation.

[0053] The latency cycle can proceed according to any of latency programs I through III in B cells, and typically progresses from III to II through I. When quiescent naive B cells are infected, EBV enters latency program III. Expression of the latent III gene activates the B cell, which becomes a proliferating progenitor. EBV then typically progresses to latency II by restricting expression to a subset of genes, which leads to the progenitor differentiating into a memory B cell. Further restriction of gene expression leads EBV into latency I. When the memory B cell divides, EBNA1 expression allows EBV to replicate. In epithelial cells, only latency II occurs.

[0054] In primary infection, EBV replicates in oropharyngeal epithelial cells and establishes latent phase III, II, and I infection in B lymphocytes. Latent EBV infection in B lymphocytes is necessary for viral persistence, subsequent replication in epithelial cells, and release of infectious virus into saliva. Latent phase III and II EBV infection in B lymphocytes, latent phase II infection in oral epithelial cells, and latent phase II infection in NK- or T-cells can lead to malignancies characterized by consistent EBV genome presence and gene expression.

[26]

[0055] Latent EBV in B cells can be reactivated and converted into lytic replication. The lysis cycle leads to the production of infectious virions, which can occur in B cells and epithelial cells, as reviewed by Kenney et al. in Human Herpesviruses: Biology (Therapy and Immunoprophylaxis; Cambridge University Press (2007)), which is incorporated herein by reference in its entirety.

[0056] Dissociative replication requires the EBV genome to be linear. Latent EBV genomes are free-living and therefore must be linearized for lytic reactivation. In B cells, lytic replication typically only occurs after reactivation from the latent phase.

[0057] Immediate-early cleavage gene products such as BZLF1 and BRLF1 act as trans-activators, enhancing their own expression and the expression of later cleavage cycle genes.

[0058] Early cleavage gene products play roles in viral replication (e.g., BALF5, a catalytic component of EBV DNA polymerase; BMRF1, a DNA-binding protein; BALF2, a helicase; BBLF4, a primase; BSLF1, and primase-associated proteins BBLF2 / 3) and deoxynucleotide metabolism (e.g., thymidine kinase BXLF1, dUTP enzyme BORF2). Other early cleavage gene products act as transcription factors (e.g., BMRF1, BRRF1), play roles in RNA stability and processing (e.g., BMLF1), or are involved in immune evasion (e.g., BHRF1, which inhibits apoptosis).

[0059] Late lysis gene products are traditionally classified as those expressed after the onset of viral replication. They typically encode structural components of the virion, such as nucleocapsid proteins, and glycoproteins that mediate EBV binding and fusion (e.g., gp350 / 220, gp85, gp42, gp25). Other late lysis gene products play a role in immune evasion; BCLF1 encodes a viral homolog of IL-10, and BALF1 encodes a protein homologous to the anti-apoptotic protein Bcl2.

[0060] EBV antigen expression in EBV-related cancers

[0061] EBV antigens expressed in EBV-positive cancers are described in, for example, Craddock and Heslop Update Cancer Ther. (2008) Mar; 3(1):33–41, Gottschalk and Rooney Curr Top Microbiol Immunol. (2015) 391:427–454, and Shinozaki-Ushiku et al., Int J Oncol. (2015) 46(4):1421-34.

[0062] EBV-associated lymphomas in immunocompromised subjects, such as those following HSCT or solid organ transplantation, in subjects with congenital immunodeficiency or HIV infection, exhibit type III latency and express EBNA1, EBNA2, EBNA-LP, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1, and LMP2. EBV-associated EBV-positive Hodgkin lymphoma, non-Hodgkin lymphoma, certain types of T-cell lymphoma, NK-cell lymphoma, some cases of B-cell lymphoma, and nasopharyngeal carcinoma exhibit type II latency and express EBNA1, BARF1, LMP1, and LMP2. EBV-positive Burkitt lymphoma exhibits type I latency and expresses EBNA1 and BARF1. EBV-positive gastric cancer exhibits type I or type II latency.

[0063] Previously, it was thought that in immunocompetent subjects, EBV-positive cancer cells expressed only EBV latent antigens (and not cleavage cycle antigens). Transcripts encoding cleavage cycle gene products have recently been detected in EBV-positive malignancies, including gastric cancer, nasopharyngeal carcinoma, and B-cell lymphoma.

[0064] It has not been determined whether it is possible to generate a population of immune cells specific to EBV cleavage antigens from patients with EBV-positive cancer or healthy donor subjects, whether such immune cells would exhibit effector activity against EBV-infected cells, or whether a population of immune cells specific to EBV cleavage antigens could be used to treat EBV-related cancers.

[0065] EBV antigen

[0066] The aspects disclosed herein employ peptides corresponding to EBV antigens.

[0067] In some embodiments according to various aspects of this disclosure, the EBV cleavage antigen is selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU, EBNA1-FUK, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, and gp350. The EBV cleavage antigen may be selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU, EBNA1-FUK, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, gp350, and combinations thereof. In some embodiments, the EBV cleavage antigen is selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, and BDLF3. The EBV cleavage antigen may be selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, and combinations thereof. In some embodiments, the EBV cleavage antigen is selected from BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2, and BDLF3. The EBV cleavage antigen may be selected from BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2, BDLF3, and combinations thereof.

[0068] In some embodiments, the EBV cleavage antigen is selected from BZLF1, BRLF1, BMRF1, BMLF1, BXLF1, BALF1, BLLF2, BALF2, BNLF2A, BNLF2B, and BMRF2. The EBV cleavage antigen may be selected from BZLF1, BRLF1, BMRF1, BMLF1, BXLF1, BALF1, BLLF2, BALF2, BNLF2A, BNLF2B, BMRF2, and combinations thereof. In some embodiments, the EBV cleavage antigen is selected from BZLF1, BRLF1, BMRF1, BMLF1, BXLF1, BALF1, BLLF2, BALF2, and BNLF2A. The EBV cleavage antigen may be selected from BZLF1, BRLF1, BMRF1, BMLF1, BXLF1, BALF1, BLLF2, BALF2, BNLF2A, and combinations thereof. In some embodiments, the EBV cleavage antigen is selected from BZLF1, BRLF1, BMRF1, BMLF1, BALF2, BNLF2A, BNLF2B, and BMRF2. The EBV cleavage antigen may be selected from BZLF1, BRLF1, BMRF1, BMLF1, BALF2, BNLF2A, BNLF2B, BMRF2, and combinations thereof.

[0069] In some implementations, the EBV cleavage antigen is selected from immediate-early cleavage antigens, early cleavage antigens, or late cleavage antigens.

[0070] In some embodiments, the immediate early lysis antigen is selected from BZLF1, BRLF1, and BMRF1. The immediate early lysis antigen may be selected from BZLF1, BRLF1, BMRF1, and combinations thereof.

[0071] In some embodiments, the early lysis antigen is selected from BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU, and EBNA1-FUK. The early lysis antigen may be selected from BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU, EBNA1-FUK, and combinations thereof. In some embodiments, the early lysis antigen is selected from BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, and BMRF2. The early lysis antigen may be selected from BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, and combinations thereof. In some embodiments, the early lysis antigen is selected from BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, and BMRF2. The early lysis antigen may be selected from BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2, and combinations thereof.

[0072] In some embodiments, the late lysis antigen is selected from BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, and gp350. The late lysis antigen may be selected from BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, gp350, and combinations thereof. In some embodiments, the late lysis antigen is selected from BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, and BDLF3. The late lysis antigen may be selected from BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, and combinations thereof. In some embodiments, the late lysis antigen is BDLF3.

[0073] In some embodiments according to various aspects of this disclosure, the EBV latent antigen is selected from EBNA1, EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1, LMP2A, and LMP2B. The EBV latent antigen may be selected from EBNA1, EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1, LMP2A, LMP2B, and combinations thereof. In some embodiments, the EBV latent antigen is selected from EBNA1, LMP1, LMP2A, and LMP2B. The EBV latent antigen may be selected from EBNA1, LMP1, LMP2A, LMP2B, and combinations thereof. In some embodiments, the EBV latent antigen is selected from EBNA1, LMP1, and LMP2A. The EBV latent antigen may be selected from EBNA1, LMP1, LMP2A, and combinations thereof.

[0074] In some implementations, the EBV latent antigen is selected from type III latent antigen, type II latent antigen, or type I latent antigen.

[0075] In some embodiments, the type III latent antigen is selected from EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA3B, and EBNA3C. The type III latent antigen may be selected from EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and combinations thereof. In some embodiments, the type II latent antigen is selected from EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, and BARF1. The type II latent antigen may be selected from EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, and combinations thereof. In some embodiments, the type I latent antigen is selected from EBNA1 and BARF1. The type I latent antigen may be selected from EBNA1, BARF1, and combinations thereof.

[0076] When referring to EBV antigens herein, this disclosure also considers isotypes, fragments, and variants (including mutants) of a given EBV antigen. The amino acid sequence of a given EBV antigen, such as the EBV antigen referred to herein, can be obtained, for example, from The UniProt Knowledgebase (UniProtKB), see The UniProt Consortium, Nucleic Acids Research (2019), 47(D1):D506-D515.

[0077] The “isotype,” “fragment,” or “variant” of the reference EBV antigen may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the reference EBV antigen (e.g., the reference isotype of the antigen).

[0078] A “fragment” typically refers to a portion of a reference protein and can be of any length (in terms of amino acid number), although it may optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment is derived), and can have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the length of the reference protein. A “variant” typically refers to a protein having an amino acid sequence containing one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of the reference protein, but maintaining a comparable degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. An “isotype” typically refers to a variant of the reference protein expressed by EBV.

[0079] In some implementations according to the various aspects described herein, references are made to “one or more” antigens from a given list. A reference to “one or more” antigens means anywhere between one or all of the antigens listed. Depending on the number of antigens listed, “one or more” can refer to, for example, antigens 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., listed.

[0080] Peptides and peptide mixtures (pepmix)

[0081] The present disclosure discloses a method for generating / amplifying a population of immune cells specific to EBV antigens using peptides corresponding to EBV antigens.

[0082] As used herein, a "peptide" is a chain of two or more amino acid monomers linked by peptide bonds. Peptides are typically between 2 and 50 amino acids in length. A "polypeptide" is a polymer chain composed of two or more peptides. Polypeptides are typically longer than about 50 amino acids.

[0083] As used herein, a peptide “corresponding to” a reference antigen contains or is composed of the amino acid sequence of the reference antigen. For example, a peptide “corresponding to” EBNA1 contains or is composed of an amino acid sequence found in the amino acid sequence of EBNA1 (i.e., a subsequence of the amino acid sequence of EBNA1).

[0084] The peptides used herein typically have a length of 5-30 amino acids, such as 5-25 amino acids, 10-20 amino acids, or 12-18 amino acids. In some embodiments, the peptides have a length of one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the peptides have a length of approximately 15 amino acids.

[0085] The term "peptide" as used in this article can refer to a group of different peptides.

[0086] In some embodiments according to various aspects of this disclosure, the method uses peptides corresponding to more than one antigen. In such embodiments, at least one peptide corresponds to each antigen. For example, when the method uses peptides corresponding to EBNA1 and LMP1, the peptides comprise at least one peptide corresponding to EBNA1 and at least one peptide corresponding to LMP1.

[0087] In some embodiments, the method uses peptides corresponding to all or part of a reference antigen. Peptides corresponding to the entirety of a given antigen cover the full length of the antigen's amino acid sequence. That is, these peptides together contain all the amino acids of the given antigen's amino acid sequence.

[0088] A peptide corresponding to a portion of a given antigen covers a portion of the amino acid sequence of the antigen. In some embodiments where the peptide covers a portion of the amino acid sequence of the antigen, the peptides together may cover, for example, more than 10%, such as more than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the amino acid sequence of the antigen.

[0089] In some embodiments, the method uses overlapping peptides. "Overlapping" refers to shared amino acids, more typically amino acid sequences. For example, the first peptide consists of amino acids corresponding to positions 1-15 of the EBNA1 amino acid sequence, and the second peptide consists of amino acids corresponding to positions 5-20 of the EBNA1 amino acid sequence. The first and second peptides are overlapping peptides corresponding to EBNA1, overlapping by 11 amino acids.

[0090] In some embodiments, the overlapping peptide overlaps one of 1-20, 5-20, 8-15, or 10-12 amino acids. In some embodiments, the overlapping peptide overlaps one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, the overlapping peptide overlaps 11 amino acids.

[0091] In some implementations, the method uses peptides of 5-30 amino acids in length with 1-20 overlapping amino acids, corresponding to all or part of a given reference antigen.

[0092] In some embodiments, the method uses peptides of 15 amino acids in length with 11 overlapping amino acids, corresponding to all given reference antigens. For a given antigen, mixtures of these peptides may be referred to herein as a “peptide mixture (peptide mixture) peptide library” or “peptide mixture”.

[0093] For example, “EBNA1 pempmix” used in Example 1 of this document refers to a library of 158 15-peptides, which overlap by 11 amino acids and span the full length of the amino acid sequence of EBNA1, as shown in UniProt:P03211-1, v1.

[0094] In some embodiments according to various aspects of this disclosure, the peptide “corresponding to” a given EBV antigen may be a mixture of peptides of that antigen.

[0095] Methods for generating / expanding EBV-specific immune cell populations

[0096] This disclosure relates to the generation / amplification of EBV-specific immune cell populations.

[0097] "EBV-specific immune cells" express / contain receptors (preferably T-cell receptors) that recognize peptides that can recognize EBV antigens (e.g., when presented by MHC molecules). EBV-specific immune cells can express / contain such receptors due to the expression of endogenous nucleic acids encoding such antigen receptors, or because they have been engineered to express such receptors.

[0098] Immune cells can be hematopoietic cells, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. Lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells, or innate lymphocytes (ILCs), or their precursors. Immune cells can express, for example, CD3 peptides (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), TCR peptides (TCRα or TCRβ), CD27, CD28, CD4, or CD8.

[0099] Technicians are familiar with methods for generating / amplifying virus-specific immune cell populations in vitro / in vitro. Typical culture conditions (i.e., cell culture medium, additives, temperature, gas environment), cell numbers, culture cycles, etc., can be determined by referring to, for example, Ngo et al., J Immunother. (2014) 37(4):193–203, the entire contents of which are incorporated herein by reference.

[0100] Conveniently, the cell cultures according to this disclosure can be maintained at 37°C in a humid atmosphere containing 5% CO2. Cells of the cell cultures according to this disclosure can be established and / or maintained at any suitable density, which can be readily determined by those skilled in the art. For example, at a density of about 0.5 × 10⁻⁶ cells / year... 6 Approximately 5×10 6 Initial density of cells / mL culture (e.g., approximately 1 × 10⁻⁶ cells / mL culture) 6 (Cells / mL) to establish cultures.

[0101] Culture can be carried out in any container suitable for the volume of the culture, such as in the wells of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, cells are cultured in a bioreactor, such as the bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, the entire contents of which are incorporated herein by reference. In some embodiments, cells are cultured in GRex cell culture containers, such as GRex flasks or GRex 100 bioreactors.

[0102] This method typically involves culturing a population of immune cells (e.g., a heterogeneous population of immune cells, such as peripheral blood mononuclear cells, PBMCs) in the presence of antigen-presenting cells (APCs) that present viral antigen peptide:MHC complexes, under conditions of appropriate co-stimulation and signal amplification to induce activation and expansion; the population contains cells with antigen-specific receptors. The process of T cell activation is well known to those skilled in the art and described, for example, in Immunobiology, 5th Edition, Janeway CA Jr, Travers P, Walport M et al., New York: Garland Science (2001), Chapter 8, the entire contents of which are incorporated herein by reference.

[0103] Specifically, the method includes the step of presenting T cells containing an EBV antigen peptide:MHC complex-specific T cell receptor (TCR) to an APC that stimulates the TCR-specific EBV antigen peptide:MHC complex. The APC is infected with a virus that encodes or contains / expresses the EBV antigen / peptide and presents the EBV antigen peptide in the context of an MHC molecule. Stimulation induces T cell activation and promotes cell division (proliferation), leading to the generation and / or expansion of a population of T cells specific to the EBV antigen.

[0104] Compared to the pre-stimulation population, the post-stimulation cell population is enriched with EBV antigen-specific T cells (i.e., EBV antigen-specific T cells are present in the population at an increased frequency after stimulation). In this way, a population of EBV antigen-specific T cells is expanded / generated from heterogeneous T cell populations with different specificities. An EBV antigen-specific T cell population can be generated from a single T cell through stimulation and subsequent cell division. Existing EBV antigen-specific T cell populations can be expanded through stimulation of the EBV antigen-specific T cell population and subsequent cell division.

[0105] It should be understood that, in embodiments of various aspects of this disclosure, the EBV antigen-specific immune cells are preferably T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are helper T cells (T cells). H (Cells). In some embodiments, the T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)). The immune cells preferably express / contain TCRs specific to peptides against EBV antigens.

[0106] EBV-specific T cells can exhibit certain functional properties of T cells in response to a specific viral antigen or in response to a cell containing / expressing the virus / antigen. In some embodiments, these properties are functional characteristics associated with effector T cells, such as cytotoxic T cells.

[0107] In some implementations, EBV-specific T cells may exhibit one or more of the following properties: cytotoxicity against cells containing / expressing EBV or EBV antigens specific to the T cell; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granzyme expression, and / or FAS ligand (FASL) expression in response to cells containing / expressing EBV or EBV antigens specific to the T cell.

[0108] EBV-specific T cells express / contain TCRs that, when presented by appropriate MHC molecules, can recognize the T cell-specific EBV antigen peptide. EBV-specific T cells can be CD4+ T cells and / or CD8+ T cells.

[0109] The method disclosed herein includes stimulating EBV antigen-specific immune cells by contacting a population of immune cells with a peptide corresponding to an EBV antigen or with an APC presenting a peptide corresponding to an EBV antigen. This method step may be referred to herein as “stimulation” or “stimulation step.” This method step typically involves maintaining cells in in vitro / ex vivo culture and may be referred to as a “stimulation culture.”

[0110] In some embodiments, the method includes one or more additional stimulation steps. That is, in some embodiments, the method includes one or more further steps to restimulate the cells obtained through the stimulation steps. Such further stimulation steps may be referred to herein as "restimulation" or "restimulation step". This method step typically involves maintaining the cells in in vitro / ex vivo culture and may be referred to as "restimulation culture".

[0111] It should be understood that “contacting” PBMCs or cell populations obtained through the stimulation steps described herein (for restimulation) with a peptide corresponding to the EBV antigen typically involves culturing the PBMCs / cell populations in an in vitro / ex vivo cell culture medium containing said peptide. Similarly, it should be understood that “contacting” PBMCs / cell populations with a presenting peptide corresponding to the EBV antigen typically involves co-culturing PBMCs and PBMCs / cell populations in an in vitro / ex vivo cell culture medium.

[0112] In some embodiments, the method includes contacting PBMCs with peptides corresponding to EBV antigens. In such embodiments, APCs (e.g., dendritic cells, macrophages, and B cells) within the PBMC population internalize (e.g., through phagocytosis, pinocytosis), process the antigen, and present it on MHC class I molecules (cross-presentation) and / or MHC class II molecules for subsequent activation of CD8+ and / or CD4+ T cells within the PBMC population.

[0113] In some embodiments, the method includes contacting a cell population obtained through the stimulation steps described herein with a peptide corresponding to an EBV antigen. In such embodiments, APCs in the cell population (e.g., dendritic cells, macrophages, and B cells) internalize (e.g., through phagocytosis, pinocytosis), process the antigen, and present it on MHC class I molecules (cross-presentation) and / or MHC class II molecules for subsequent restimulation of CD8+ and / or CD4+ T cells in the cell population.

[0114] In some embodiments, the method includes contacting PBMCs with APCs that present peptides corresponding to EBV antigens. In some embodiments, the method includes contacting a cell population obtained through the stimulation steps described herein with APCs that present peptides corresponding to EBV antigens.

[0115] The co-culture of T cells and APCs in stimulation and restimulation according to the present invention is carried out in a cell culture medium. The cell culture medium can be any cell culture medium in which T cells and APCs can be maintained in vitro / in vitro culture. Culture media suitable for lymphocyte culture are well known to those skilled in the art, including, for example, RPMI-1640 medium, AIM-V medium, Iscoves medium, etc.

[0116] In some embodiments, the cell culture medium may comprise RPMI-1640 medium and / or Click's medium (also known as Eagle's Ham's amino acid (EHAA) medium). The composition of these media is well known to those skilled in the art. The formulation of RPMI-1640 medium is described, for example, in Moore et al., JAMA (1967) 199:519-524, and the formulation of Click's medium is described, for example, in Click et al., Cell Immunol (1972) 3:264-276. RPMI-1640 medium may be obtained from, for example, Thermo Fisher Scientific, while Click's medium may be obtained from, for example, Sigma-Aldrich (catalog number C5572).

[0117] In some embodiments, the method of this disclosure includes culturing PBMCs that have been contacted with a peptide corresponding to an EBV antigen in a cell culture medium comprising RPMI-1640 medium and Click's medium, or culturing PBMCs in the presence of APCs that present a peptide corresponding to an EBV antigen. In some embodiments, the method of this disclosure includes culturing a cell population obtained through the stimulation steps described herein that has been contacted with a peptide corresponding to an EBV antigen in a cell culture medium comprising RPMI-1640 medium and Click's medium, or culturing a cell population obtained through the stimulation steps described herein in the presence of APCs that present a peptide corresponding to an EBV antigen.

[0118] In some embodiments, the cell culture medium comprises (by volume) 25-65% RPMI-1640 medium and 25-65% Click's medium. In some embodiments, the cell culture medium comprises 30-60% RPMI-1640 medium and 30-60% Click's medium. In some embodiments, the cell culture medium comprises 35-55% RPMI-1640 medium and 35-55% Click's medium. In some embodiments, the cell culture medium comprises 40-50% RPMI-1640 medium and 40-50% Click's medium. In some embodiments, the cell culture medium comprises 45% RPMI-1640 medium and 45% Click's medium.

[0119] In some embodiments, the cell culture medium may contain one or more cell culture medium additives. Cell culture medium additives are well known to those skilled in the art and include antibiotics (such as penicillin, streptomycin), serum (such as fetal bovine serum (FBS), bovine serum albumin (BSA)), L-glutamine, cytokines / growth factors, etc.

[0120] In some embodiments, the cell culture medium contains 5-20% FBS (by volume), such as 7.5-15% FBS, or 10% FBS. In some embodiments, the cell culture medium contains 0.5-5% GlutaMax, such as 1% GlutaMax. In some embodiments, the cell culture medium contains 0.5-5% Pen / Strep, such as 1% Pen / Strep.

[0121] The present disclosure discloses the use of antigen-presenting cells (APCs) in a method for generating / expanding a population of immune cells specific to EBV.

[0122] According to this disclosure, APCs can be specialized APCs. Specialized APCs are dedicated to presenting antigens to T cells; they efficiently process and present MHC peptide complexes on the cell surface and express high levels of co-stimulatory molecules. Specialized APCs include dendritic cells (DCs), macrophages, and B cells. Non-specialized APCs are other cells capable of presenting MHC peptide complexes to T cells, particularly MHC class I peptide complexes to CD8+ T cells.

[0123] In some implementations, an APC is an APC capable of cross-presenting antigens internalized by the APC (e.g., taken up via endocytosis / phagocytosis) at MHC class I. Cross-presentation of MHC class I antigens to CD8+ T cells is described, for example, in Alloatti et al., Immunological Reviews (2016), 272(1):97-108, which is incorporated herein by reference in its entirety. Antigen-presenting cells capable of cross-presentation include, for example, dendritic cells (DCs), macrophages, B cells, and sinus endothelial cells.

[0124] As explained herein, in some embodiments, the APC used to stimulate EBV antigen-specific immune cells is contained within a cell population (e.g., PBMCs) comprising EBV antigen-specific immune cells, and the EBV antigen-specific cell population is amplified according to the methods of this disclosure. In such embodiments, the APC can be, for example, dendritic cells, macrophages, B cells, or any other cell type within the cell population capable of presenting antigens to EBV antigen-specific immune cells.

[0125] In some embodiments, the method uses an APC that has been modified to express / contain the EBV antigen / its peptide. In some embodiments, the APC can present a peptide corresponding to the EBV antigen because it has already been contacted with and internalized the peptide. In some embodiments, the APC may have been "pulsated" by the peptide, which typically involves culturing the APC in vitro in the presence of the peptide for a period of time sufficient for the APC to internalize the peptide.

[0126] In some implementations, the APC can present a peptide corresponding to the EBV antigen due to the expression of a nucleic acid encoding the antigen within the cell. Since the APC has already been infected with EBV (e.g., in the case of B cells, such as LCLs), it can contain a nucleic acid encoding the EBV antigen. Since the nucleic acid encoding the antigen has already been introduced into the cell, for example through transfection, transduction, electroporation, etc., the APC can contain a nucleic acid encoding the EBV antigen. The nucleic acid encoding the EBV antigen can be provided in a plasmid / vector.

[0127] In some embodiments, the APCs used in the methods of this disclosure are selected from activated T cells (ATCs), dendritic cells, B cells (including, for example, LCLs), and artificial antigen-presenting cells (aAPCs), such as those described in Neal et al., J Immunol ResTher (2017) 2(1):68-79 and Turtle and Riddell, Cancer J. (2010) 16(4):374-381.

[0128] In some implementations, APCs are autologous in relation to the cell populations they will be co-cultured with to generate / expand an immune cell population containing immune cells specific to the EBV antigen. That is, in some implementations, the APCs are derived from the same subject (or derived from cells obtained from) the subject with whom they are to be co-cultured.

[0129] The use of polyclonal activated T cells (ATCs) as APCs and methods for their preparation are described, for example, in Ngo et al., J Immunother. (2014) 37(4):193–203, which is incorporated herein by reference. In short, ATCs can be generated in vitro by nonspecific activation of T cells in the presence of IL-2, through stimulation of PBMCs with agonist antiCD3 and agonist antiCD28 antibodies.

[0130] Dendritic cells can be generated according to methods known in the art, such as those described in Ngo et al., J Immunother. (2014) 37(4): 193–203. Dendritic cells can be prepared from monocytes, which can be obtained by CD14 selection from PBMCs. Monocytes can be cultured in a cell culture medium, resulting in their differentiation into immature dendritic cells, which may contain, for example, IL-4 and GM-CSF. Immature dendritic cells can be matured by culturing in the presence of IL-6, IL-1β, TNFα, PGE2, GM-CSF, and IL-4.

[0131] LCL can be produced according to methods well known in the art, such as those described in Hui-Yuen et al., J Vis Exp (2011) 57:3321, and Hussain and Mulherkar, Int J Mol Cell Med (2012) 1(2):75-87, both of which are incorporated herein by reference in their entirety. In short, LCL can be produced in the presence of cyclosporine A by incubating PBMCs with concentrated cell culture supernatant of EBV-producing cells (e.g., B95-8 cells).

[0132] Artificial antigen-presenting cells (aAPCs) include, for example, K562cs cells, which are engineered to express co-stimulatory molecules CD80, CD86, CD83 and 4-1BBL (described, for example, Suhoski et al., Mol Ther. (2007) 15(5):981-8).

[0133] In some embodiments, the APC is not an EBV-infected cell. In some embodiments, the APC is not an EBV-infected B cell. In some embodiments, the APC is not an EBV-LCL.

[0134] In some embodiments, the stimulation step includes contacting PBMCs with peptides corresponding to EBV antigens. In some embodiments, the restimulation step includes contacting EBV antigen-specific immune cells with APCs that present peptides corresponding to EBV antigens. In some embodiments, the restimulation step includes contacting EBV antigen-specific immune cells with ATCs that present peptides corresponding to EBV antigens.

[0135] In some embodiments, the method further uses agents for enhancing co-stimulation during stimulation and / or restimulation. Such agents include, for example, cells expressing co-stimulatory molecules (e.g., CD80, CD86, CD83, and / or 4-1BBL), such as LCLs or K562cs cells. In some embodiments, the cells expressing the co-stimulatory molecules are HLA-negative, EBV-incapable LCLs, also known as “uLCLs” or “uLCLs.” uLCLs are described, for example, in US2018 / 0250379 A1.

[0136] Other examples of reagents for enhancing co-stimulation include, for instance, agonist antibodies specific to co-stimulatory receptors expressed by T cells (e.g., 4-1BB, CD28, OX40, ICOS, etc.), and co-stimulatory molecules capable of activating co-stimulatory receptors expressed by T cells (e.g., CD80, CD86, CD83, 4-1BBL, OX40L, ICOSL, etc.). Such reagents can be provided, for example, in the form of beads.

[0137] In some implementations, the restimulation step includes, in the presence of uLCLs, contacting EBV antigen-specific immune cells with ATCs that present peptides corresponding to EBV antigens.

[0138] Contact between immune cell populations and peptides corresponding to EBV antigens or APCs presenting peptides corresponding to EBV antigens can occur in the presence of one or more cytokines to promote T cell activation and proliferation. In some embodiments, stimulation occurs in the presence of one or more of IL-7, IL-15, IL-6, IL-12, IL-4, IL-2, and / or IL-21. It should be understood that the cytokines are exogenously added to the culture and are not cytokines produced by the cells in the culture. In some embodiments, the added cytokines are recombinant cytokines.

[0139] Therefore, in some embodiments, the method of this disclosure includes culturing PBMCs that have been contacted with a peptide corresponding to an EBV antigen in the presence of one or more of IL-7, IL-15, IL-6, IL-12, IL-4, IL-2 and / or IL-21, or culturing PBMCs in the presence of APCs that present a peptide corresponding to an EBV antigen.

[0140] In some embodiments, the culture is performed in the presence of IL-7, IL-15, IL-6, IL-12, IL-4, IL-2, and / or IL-21. In some embodiments, the culture is performed in the presence of IL-7, IL-15, IL-6, and / or IL-12. In some embodiments, the culture is performed in the presence of IL-7 and / or IL-15.

[0141] In some embodiments, the final concentration of IL-7 in the culture is 1-100 ng / ml, such as 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml. In some embodiments, the final concentration of IL-7 in the culture is approximately 10 ng / ml.

[0142] In some embodiments, the final concentration of IL-15 in the culture is 1-100 ng / ml, such as 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml. In some embodiments, the final concentration of IL-15 in the culture is approximately 10 ng / ml.

[0143] In some embodiments, the final concentration of IL-15 in the culture is 10-1000 ng / ml, such as 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml. In some embodiments, the final concentration of IL-15 in the culture is approximately 100 ng / ml.

[0144] In some embodiments, the final concentration of IL-6 in the culture is 10-1000 ng / ml, such as 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml. In some embodiments, the final concentration of IL-6 in the culture is approximately 100 ng / ml.

[0145] In some embodiments, the final concentration of IL-12 in the culture is 1-100 ng / ml, such as 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml. In some embodiments, the final concentration of IL-12 in the culture is 10 ng / ml.

[0146] In some implementations, the final concentration of IL-7 is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), and the final concentration of IL-15 is 10-1000 ng / ml (e.g., one of 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml, such as about 100 ng / ml).

[0147] In some implementations, the final concentration of IL-7 is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), and the final concentration of IL-15 is 10-1000 ng / ml (e.g., one of 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml, such as about 100 ng / ml).

[0148] In some implementations, the final concentration of IL-7 is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), the final concentration of IL-6 is 10-1000 ng / ml (e.g., one of 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml, such as about 100 ng / ml), the final concentration of IL-12 is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), and the final concentration of IL-15 is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml).

[0149] In some implementations, the final concentration of IL-7 in the stimulation culture is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), and the final concentration of IL-15 in the stimulation culture is 10-1000 ng / ml (e.g., one of 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml, such as about 100 ng / ml).

[0150] In some embodiments, the final concentration of IL-7 in the stimulation culture is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), the final concentration of IL-6 in the stimulation culture is 10-1000 ng / ml (e.g., one of 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml, such as about 100 ng / ml), the final concentration of IL-12 in the stimulation culture is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), and the final concentration of IL-15 in the stimulation culture is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml).

[0151] In some implementations, the final concentration of IL-7 in the restimulated culture is 1-100 ng / ml (e.g., one of 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml, such as 10 ng / ml), and the final concentration of IL-15 in the restimulated culture is 10-1000 ng / ml (e.g., one of 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml, such as about 100 ng / ml).

[0152] The stimulation and restimulation according to this method typically involves co-culturing T cells and APCs for a period of time sufficient for the APCs to stimulate the T cells and for the T cells to undergo cell division.

[0153] In some embodiments, the method of this disclosure includes culturing PBMCs that have been contacted with a peptide corresponding to an EBV antigen, or culturing PBMCs in the presence of an APC that presents a peptide corresponding to an EBV antigen, for a period of at least 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, or at least 7 days. In some embodiments, the culture lasts from 24 hours to 20 days, for example, one day from 48 hours to 14 days, 3 days to 12 days, 4 days to 11 days, 6 days to 10 days, or 7 days to 9 days.

[0154] In some embodiments, the method of this disclosure includes culturing a cell population obtained by the stimulation steps described herein, for a period of at least 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, or at least 7 days, wherein the cell population has been contacted with a peptide corresponding to the EBV antigen, or cultured in the presence of APCs that present a peptide corresponding to the EBV antigen. In some embodiments, the culture lasts from 24 hours to 20 days, for example, from 48 hours to 14 days, 3 days to 12 days, 4 days to 11 days, 6 days to 10 days, or 7 days to 9 days.

[0155] Stimulation and restimulation can be terminated by separating the cells in the culture from the culture medium in which they were cultured, or by diluting the culture, for example, by adding cell culture medium. In some embodiments, the method includes a step of collecting the cells at the end of the stimulation or restimulation culture.

[0156] In some embodiments, the restimulation step according to this disclosure can be established by adding cell culture medium (and any other additives described herein) in an amount suitable for the percentage / concentration of cell culture medium, conditioned medium (and any additives) required to achieve the restimulation step.

[0157] At the end of the culture period for a given stimulation or restimulation step, cells can be collected and separated from the cell culture supernatant. Cells can be collected by centrifugation, separating the cell culture supernatant from the cell pellet. The cell pellet can then be resuspended in cell culture medium, for example, for the restimulation step. In some embodiments, cells may undergo a washing step after collection. The washing step may include resuspending the cell pellet in an isotonic buffer such as phosphate-buffered saline, collecting the cells by centrifugation, and discarding the supernatant.

[0158] Methods for generating and / or amplifying populations of immune cells specific to EBV antigens according to this disclosure typically include more than one stimulation step. There is no upper limit to the number of stimulation steps that can be performed in a method according to this disclosure. In some embodiments, the method includes more than 2, 3, 4, or 5 stimulation steps. In some embodiments, the method includes one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 stimulation steps. The stimulation steps in a method according to this disclosure can be different from each other.

[0159] In some embodiments, the PBMCs used in the method of the present invention are depleted of CD45RA-positive cells. That is, in some embodiments, the PBMCs are "PBMCs depleted of CD45RA-positive cells" or "CD45RA-negative PBMCs". The depletion of CD45RA-positive cells is intended to reduce the number of natural killer cells and / or regulatory T cells in the cell population generated / expanded according to the method of the present disclosure.

[0160] In some embodiments, the method includes, for example, a step of depleting CD45RA-positive cells in PBMCs prior to the stimulation step according to this disclosure. In some embodiments, the method includes, for example, a step of depleting CD45RA-positive cells in cells obtained through the stimulation step according to this disclosure prior to the restimulation step according to this disclosure. Depletion of CD45RA-positive cells can be achieved by any suitable method, such as by MACS, for example using... Biotec columns and magnetic anti-CD45RA antibody-coated beads.

[0161] In some embodiments, the cell population used to derive the APCs used in the methods of this disclosure is depleted of CD45RA-positive cells. That is, in some embodiments, the cell population used to derive the APCs is a "CD45RA-depleted" or "CD45RA-negative" population. For example, in embodiments using ATCs as APCs, the ATCs may be derived from a population of PBMCs that are depleted of CD45RA-positive cells, or from a population of CD45RA-negative PBMCs.

[0162] In some embodiments, the method further includes modifying EBV antigen-specific immune cells to increase IL-7-mediated signaling in the cells. IL-7-mediated signaling has been shown to improve the survival and antitumor activity of tumor-specific T cells—see, for example, Shum et al., Cancer Discov. (2017) 7(11):1238–1247, and WO 2018 / 038945 A1.

[0163] Specific exemplary implementations of the method steps

[0164] In conjunction with this disclosure, the following specific exemplary method steps are explicitly considered:

[0165] (A) By contacting PBMCs with mixtures of BZLF1 peptides, BRLF1 peptides, BMRF1 peptides, BMLF1 peptides, BALF2 ​​peptides, BNLF2A peptides, BNLF2B peptides and / or BMRF2 peptides, immune cells specific to EBV cleavage antigens are stimulated.

[0166] (B) By contacting PBMCs with mixtures of BZLF1 peptides, BRLF1 peptides, BMRF1 peptides, BMLF1 peptides, BXLF1 peptides, BALF1 peptides, BLLF2 peptides, BALF2 ​​peptides and / or BNLF2A peptides, immune cells specific to EBV cleavage antigens are stimulated.

[0167] (C) By contacting PBMCs with a mixture of BZLF1 peptides, BRLF1 and / or BMRF1 peptides, immune cells specific to EBV cleavage antigens are stimulated.

[0168] (D) By contacting PBMCs with a mixture of BMRF1 peptides, a mixture of BMLF1 peptides, a mixture of BALF2 ​​peptides, a mixture of BNLF2A peptides, a mixture of BNLF2B peptides and / or a mixture of BMRF2 peptides, immune cells specific to EBV cleavage antigens are stimulated.

[0169] (E) Stimulating immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by exposing PBMCs to the following substances:

[0170] BZLF1 peptide mixture, BRLF1 peptide mixture, BMRF1 peptide mixture, BMLF1 peptide mixture, BALF2 ​​peptide mixture, BNLF2A peptide mixture, BNLF2B peptide mixture and / or BMRF2 peptide mixture; and

[0171] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0172] (F) Stimulate immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by exposing PBMCs to the following substances:

[0173] BZLF1 peptide mixture, BRLF1 peptide mixture, BMRF1 peptide mixture, BMLF1 peptide mixture, BXLF1 peptide mixture, BALF1 peptide mixture, BLLF2 peptide mixture, BALF2 ​​peptide mixture and / or BNLF2A peptide mixture; and

[0174] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0175] (G) Stimulating immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by exposing PBMCs to the following substances:

[0176] BZLF1 peptide mixture, BRLF1 peptide mixture and / or BMRF1 peptide mixture; and

[0177] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0178] (H) By exposing PBMCs to the following substances, immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens are stimulated:

[0179] Mixtures of BMRF1 peptides, BMLF1 peptides, BALF2 ​​peptides, BNLF2A peptides, BNLF2B peptides, and / or BMRF2 peptides; and

[0180] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0181] (I) In the presence of IL-7, IL-15, IL-6 and / or IL-12, stimulate immune cells according to any one of (A) to (H).

[0182] (J) In the presence of IL-7 and / or IL-15, stimulate immune cells according to any one of (A) to (I).

[0183] (K) Stimulate immune cells according to any one of (A) to (J), wherein the PBMC depletes CD45RA-positive cells.

[0184] (1) Stimulate immune cells specific to EBV cleavage antigens by co-culturing with APCs that have been pulsed with BZLF1 peptide mixture, BRLF1 peptide mixture, BMRF1 peptide mixture, BMLF1 peptide mixture, BALF2 ​​peptide mixture, BNLF2A peptide mixture, BNLF2B peptide mixture and / or BMRF2 peptide mixture.

[0185] (M) Stimulate immune cells specific to EBV cleavage antigens by co-culturing with APCs that have been pulsed with BZLF1 peptide mixture, BRLF1 peptide mixture, BMRF1 peptide mixture, BMLF1 peptide mixture, BXLF1 peptide mixture, BALF1 peptide mixture, BLLF2 peptide mixture, BALF2 ​​peptide mixture and / or BNLF2A peptide mixture.

[0186] (N) Immune cells specific to EBV cleavage antigens are stimulated by co-culturing with APCs pulsed with a mixture of BZLF1 peptides, BRLF1 and / or BMRF1 peptides.

[0187] (O) Stimulate immune cells specific to EBV cleavage antigens by co-culturing with APCs that have been pulsed with a mixture of BMRF1 peptides, BMLF1 peptides, BALF2 ​​peptides, BNLF2A peptides, BNLF2B peptides and / or BMRF2 peptides.

[0188] (P) Stimulation of immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by co-culturing with APCs that have been pulsed with the following substances:

[0189] BZLF1 peptide mixture, BRLF1 peptide mixture, BMRF1 peptide mixture, BMLF1 peptide mixture, BALF2 ​​peptide mixture, BNLF2A peptide mixture, BNLF2B peptide mixture and / or BMRF2 peptide mixture; and

[0190] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0191] (Q) Stimulate immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by co-culturing with APCs that have been pulsed with the following substances:

[0192] BZLF1 peptide mixture, BRLF1 peptide mixture, BMRF1 peptide mixture, BMLF1 peptide mixture, BXLF1 peptide mixture, BALF1 peptide mixture, BLLF2 peptide mixture, BALF2 ​​peptide mixture and / or BNLF2A peptide mixture; and

[0193] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0194] (R) Stimulation of immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by co-culturing with APCs that have been pulsed with the following substances:

[0195] BZLF1 peptide mixture, BRLF1 peptide mixture and / or BMRF1; and

[0196] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0197] (S) Stimulation of immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by co-culturing with APCs that have been pulsed with the following substances:

[0198] Mixtures of BMRF1 peptides, BMLF1 peptides, BALF2 ​​peptides, BNLF2A peptides, BNLF2B peptides, and / or BMRF2 peptides; and

[0199] Mixtures of EBNA1 peptides, LMP1 peptides, and / or LMP2 peptides.

[0200] (T) Stimulate immune cells according to any one of (L) to (S), wherein the APC is an ATC.

[0201] (U) Stimulate immune cells according to (T), wherein the ATCs are derived from PBMCs that have depleted CD45RA positive cells.

[0202] (V) In the presence of uLCLs, stimulate immune cells according to any one of (L) to (U).

[0203] (W) In the presence of IL-7 and / or IL-15, stimulate immune cells according to any one of (L) to (V).

[0204] (X) Stimulate immune cells according to any one of (A) to (K), and then stimulate immune cells according to any one of (L) to (W).

[0205] Method / Characteristics of the immune cell population generated / expanded by this method

[0206] The methods disclosed herein may optionally be characterized by reference to the characteristics of the method and / or the characteristics of the immune cell population generated / expanded by the method.

[0207] In some embodiments, the immune cell population generated / expanded according to the method of this disclosure has one or more of the following characteristics:

[0208] a) Cells that produce IFNγ in response to stimulation by peptides corresponding to one or more EBV cleavage antigens;

[0209] b) Includes cells that produce IFNγ in response to stimulation by peptides corresponding to one or more EBV cleavage antigens, and includes cells that produce IFNγ in response to stimulation by peptides corresponding to one or more EBV latent antigens.

[0210] c) Includes cells that produce IFNγ in response to stimulation by EBV-infected cells;

[0211] d) Cell lysis activity against EBV-infected autologous cells;

[0212] e) In vivo anticancer activity against EBV-positive cancers;

[0213] f) Inhibit the growth of EBV-positive tumors in vivo; and

[0214] g) Reduce the metastasis of EBV-positive cancers in the body.

[0215] Immune cell populations can be assessed, for example, by ELISPOT analysis to determine whether they contain cells that produce interferon-γ in response to stimulation by peptides corresponding to EBV antigens and / or EBV-infected cells (e.g., LCLs), as described in Example 2.

[0216] Immune cell populations can be analyzed using the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, such as... 51 The Cr release assay, which assesses cytolytic activity against autologous EBV-infected cells, is incorporated herein by reference in its entirety. Autologous EBV-LCLs can be used for analysis, such as those described in Example 3.

[0217] By analyzing in a suitable model, the anticancer activity against EBV-positive cancers and / or inhibition of tumor growth in EBV-positive tumors can be assessed in vivo against immune cell populations. Suitable models and analyses include the EBV-LCL xenograft model approach used in Example 4. Metastasis can be assessed, for example, by monitoring the location of cancer cells in the animal within such a model, as performed in Example 4.

[0218] In some embodiments, the immune cell population generated / expanded according to the method of this disclosure may have one or more of the following characteristics, compared to the immune cell population generated / expanded according to a reference method for generating / expanding EBV-specific immune cells:

[0219] h) Contains a larger number / proportion of cells that produce IFNγ in response to stimulation by peptides corresponding to one or more EBV antigens;

[0220] i) Containing a larger number / proportion of cells that produce IFNγ in response to stimulation by peptides corresponding to one or more EBV cleavage antigens;

[0221] j) Contains cells that are specific to a larger number of different EBV antigens (i.e., a wider range of EBV antigens);

[0222] k) Contains a larger number / proportion of cells that produce IFNγ in response to stimulation by EBV-infected cells;

[0223] l) Greater anti-cancer activity against EBV-positive cancers in vivo;

[0224] m) has a greater inhibitory effect on the growth of EBV-positive tumors in vivo;

[0225] n) Greater reduction in EBV-positive cancer metastasis in the body;

[0226] o) Lasts longer in the body; and

[0227] p) Induces increased production of one or more pro-inflammatory cytokines (e.g., GM-CSF and / or IFNγ) in subjects who are administered the drug with immune cell populations.

[0228] p) In subjects who received administration of immune cell populations, it caused a reduction in the production of one or more anti-inflammatory cytokines (e.g., IL-10).

[0229] The survival / persistence of a given population of immune cells in vivo can be assessed, for example, by labeling cells with detectable markers or reporter substances and monitoring their survival over time. These methods include, for example, labeling cells with firefly luciferase and measuring activity at different time points.

[0230] Cytokine production in subjects administered a given population of immune cells can be assessed, for example, by analyzing blood-derived samples (e.g., whole blood, plasma, serum) obtained from the subject. Cytokine levels can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), as described in Example 4.

[0231] A reference method for generating / amplifying EBV-specific immune cells may be the method described in Example 1 of this document, which uses a latent peptide mixture (only) in the stimulation.

[0232] Methods using immune cell populations generated / expanded by this method

[0233] Immune cell populations containing EBV-specific immune cells that are generated / expanded as described herein can be used for therapeutic and / or preventative approaches.

[0234] A method for treating / preventing a disease / condition in a subject is provided, comprising administering to the subject an EBV-specific immune cell population generated / amplified according to the method of this disclosure. The use of the EBV-specific immune cell population generated / amplified according to the method of this disclosure in medical treatment / prevention methods is also provided. The use of the EBV-specific immune cell population generated / amplified according to the method of this disclosure in methods for treating / preventing diseases / conditions is also provided. The use of the EBV-specific immune cell population generated / amplified according to the method of this disclosure in the preparation of a medicament for methods of treating / preventing diseases / conditions is also provided.

[0235] Specifically, a method is envisioned for using EBV-specific immune cell populations generated / amplified according to this disclosure to treat / prevent diseases / conditions via adoptive cell transfer (ACT).

[0236] Adoptive cell transfer typically refers to the process of obtaining cells (e.g., immune cells) from a subject, usually by drawing a blood sample for cell isolation. The cells are then typically modified and / or expanded before being administered to the same subject (in the case of autologous / allogeneic cell adoptive transfer) or to different subjects (in the case of allogeneic cell adoptive transfer). This treatment generally aims to provide the subject with a population of cells possessing certain desired characteristics, or to increase the frequency of cells with those characteristics in the subject. The purpose of adoptive transfer can be to introduce cells or cell populations into the subject and / or to increase the frequency of cells or cell populations in the subject.

[0237] Adoptive transfer of immune cells is described, for example, in Kalos and June, 2013, Immunity 39(1):49-60 and Davis et al., 2015, Cancer J. 21(6):486–491, both of which are incorporated herein by reference in their entirety. Those skilled in the art can determine, based on this disclosure, appropriate reagents and procedures for adoptive transfer of cells, for example by reference to Dai et al., 2016 J Nat Cancer Inst 108(7):djv439, the entire contents of which are incorporated herein by reference.

[0238] In some implementations, the method includes:

[0239] (a) Generating / amplifying EBV-specific immune cell populations according to the methods of this disclosure, and

[0240] (b) Administration of the generated / expanded population of EBV-specific immune cells to the subject.

[0241] In some implementations, the method includes:

[0242] (a) Isolating / obtaining immune cell populations (e.g., PBMCs) from subjects;

[0243] (b) To generate / amplify an EBV-specific immune cell population according to the methods of this disclosure, and

[0244] (c) Administration of the generated / expanded population of EBV-specific immune cells to the subject.

[0245] In some embodiments, the subject from whom the immune cells (e.g., PBMCs) are isolated is the same subject who was administered the generated / expanded population of EBV-specific immune cells (i.e., the adoptive transfer can be autologous / autologous cells). In some embodiments, the subject from whom the immune cells (e.g., PBMCs) are isolated is a different subject from the subject who was administered the generated / expanded population of EBV-specific immune cells (i.e., the adoptive transfer can be allogeneic cells).

[0246] In some implementations, the method may include one or more of the following:

[0247] Blood samples were obtained from the subjects;

[0248] Isolate immune cells (such as PBMCs) from blood samples;

[0249] Generate / amplify EBV-specific immune cell populations according to the methods of this disclosure;

[0250] Collect / isolate EBV-specific immune cell populations;

[0251] Mix EBV-specific immune cell populations with adjuvants, diluents, or carriers;

[0252] Subjects were given a population of EBV-specific immune cells.

[0253] This method can effectively reduce the development / progression of a disease / condition, alleviate its symptoms, or reduce its pathology. It can effectively prevent the progression of a disease / condition, such as preventing its worsening or slowing its rate of development. In some embodiments, the method can lead to improvement in the disease / condition, such as a reduction in symptoms or some other associated reduction in severity / activity. In some embodiments, the method can prevent the disease / condition from progressing to later stages (e.g., a chronic stage or metastasis).

[0254] It should be understood that the therapeutic and preventive efficacy of the cell populations generated / expanded according to this disclosure can be extended to the treatment / prevention of any disease / condition from which therapeutic or preventive benefits will be derived from a reduction in the number / activity of EBV-loaded and / or EBV-infected cells.

[0255] For example, the disease / symptom can be a disease / symptom in which EBV or EBV-infected cells are pathologically involved, such as a disease / symptom in which EBV infection is positively correlated with the onset, development or progression of the disease / symptom and / or the severity of one or more symptoms of the disease / symptom, or a disease / symptom in which EBV infection is a risk factor for the onset, development or progression of the disease / symptom.

[0256] The treatment can target one or more of the following: reducing EBV burden, reducing the number / proportion of EBV-positive cells, reducing the activity of EBV-positive cells, delaying / preventing the onset / progression of disease / symptoms, reducing the severity of disease / symptoms, reducing the survival / growth of EBV-positive cells, and increasing the survival rate of the subject.

[0257] In some implementations, the disease / condition that is treated / prevented according to this disclosure is a disease / condition characterized by EBV infection.

[0258] In some embodiments, subjects may be selected for the treatment described herein based, for example, on the detection of EBV / EBV-infected cells in the periphery or in organs / tissues affected by the disease / condition (e.g., organs / tissues in which symptoms of the disease / condition are manifested), or by the detection of EBV-positive cancer cells (e.g., EBV-positive cells in a tumor). The disease / condition may affect any tissue or organ or organ system. In some embodiments, the disease / condition may affect several tissues / organs / organ systems.

[0259] In some implementations, subjects may be selected for treatment / prevention according to this disclosure based on a determination of EBV infection or cells containing EBV infection.

[0260] EBV is associated with a variety of cancers, as reviewed in Jha et al., Front Microbiol. (2016) 7:1602, the entire contents of which are incorporated herein by reference.

[0261] Therefore, in some implementations, the disease to be treated / prevented according to this disclosure is cancer.

[0262] Cancer can refer to any unwanted cell proliferation (or any disease characterized by unwanted cell proliferation), growth, or tumor. Cancer can be benign or malignant, and can be primary or secondary (metastatic). A tumor can be any abnormal cell growth or proliferation and can be located in any tissue. Cancer can originate from, for example, the adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, greater omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, and / or leukocytes.

[0263] Tumors can be nervous system or non-nervous system tumors. Nervous system tumors can originate from the central or peripheral nervous system, such as gliomas, medulloblastomas, meningiomas, neurofibromas, ependymomas, schwannomas, neurofibrosarcomas, astrocytomas, and oligodendrogliomas. Non-nervous system cancers / tumors can originate from any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, non-small cell lung cancer, hematologic malignancies, and sarcomas.

[0264] In some implementation schemes, the cancer is selected from: colon cancer, colon cancer tumor, colorectal cancer, nasopharyngeal cancer, cervical cancer, oropharyngeal cancer, gastric cancer, hepatocellular carcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urothelial carcinoma, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer, or mesothelioma.

[0265] In some implementation schemes, the cancer to be treated / prevented is EBV-related cancer. “EBV-related” cancer can be cancer caused or aggravated by EBV infection, cancer in which infection is a risk factor, and / or cancer in which infection is positively correlated with onset, development, progression, severity, or metastasis.

[0266] EBV-related cancers that can be treated / prevented according to the present invention include B-cell-related cancers such as Burkitt lymphoma, post-transplant lymphoproliferative disorder (PTLD), central nervous system lymphoma (CNS lymphoma), Hodgkin lymphoma, non-Hodgkin lymphoma, and EBV-related lymphomas associated with immunodeficiency (including, for example, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection / AIDS, and oral hairy leukoplakia), as well as epithelial-related cancers such as nasopharyngeal carcinoma (NPC) and gastric cancer (GC).

[0267] In some implementations, the cancer is selected from lymphoma (e.g., EBV-positive lymphoma), head and neck squamous cell carcinoma (HNSCC; e.g., EBV-positive HNSCC), nasopharyngeal carcinoma (NPC; e.g., EBV-positive NPC), and gastric cancer (GC; e.g., EBV-positive GC).

[0268] EBV infection is also associated with the development / progression of a variety of autoimmune diseases, such as multiple sclerosis and systemic lupus erythematosus (SLE, see, for example, Ascherio and Munger Curr Top Microbiol Immunol. (2015); 390(Pt 1):365-85), and the EBV antigen EBNA2 has recently been shown to be associated with genetic regions that are suggested to be risk factors for the development of SLE, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, juvenile idiopathic arthritis and celiac disease (Harley et al., Nat Genet. (2018) 50(5):699–707).

[0269] Therefore, in some implementations, the diseases / conditions to be treated / prevented according to this disclosure are selected from systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, juvenile idiopathic arthritis, and celiac disease.

[0270] The cell populations generated / expanded according to this disclosure can also be used independently of EBV infection for the treatment / prevention of diseases / conditions.

[0271] As described above, the cell population generated / expanded according to this disclosure can be used in methods for treating cancer.

[0272] In some embodiments, the cancer may express cancer antigens. A “cancer antigen” is an antigen expressed or overexpressed by cancer cells. Cancer antigens can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of cancer antigens may be cancer-related. Cancer antigens may be aberrantly expressed by cancer cells (e.g., expressed at an aberrant location) or may be expressed by cancer cells at an aberrant structure. Cancer antigens may be able to elicit an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer antigen may be a cancer cell surface antigen). In some embodiments, the antigen portion bound to the antigen-binding molecule described herein is displayed on the outer surface of the cancer cell (i.e., extracellularly). In some embodiments, the cancer antigen is an antigen whose expression is associated with the development, progression, or severity of cancer symptoms. Cancer-related antigens may be associated with the etiology or pathology of cancer or may be aberrantly expressed due to cancer. In some embodiments, the cancer antigen is an antigen whose expression is upregulated by cancer cells (e.g., at RNA and / or protein levels), for example, compared to the expression levels of comparable non-cancer cells (e.g., non-cancer cells derived from the same tissue / cell type). In some embodiments, cancer antigens are preferentially expressed by cancer cells and not by equivalent non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, cancer antigens are products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer antigens are products of overexpressed cellular proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, or cell surface molecules (e.g., cell surface proteins or glycoproteins). In some embodiments, cancer antigens are cell signaling molecules, such as cytokines, chemokines, interferons, interleukins, or lymphokines. In some embodiments, cancer antigens are growth factors or hormones.

[0273] In some implementations, a cancer being "positive" for the cancer antigen means that the cancer contains cells expressing the cancer antigen (e.g., cells expressing the cancer antigen on their cell surface). Cancer antigen-positive cancers may overexpress the cancer antigen. Overexpression of the cancer antigen can be determined by detecting the gene or protein expression level of the cancer antigen, which is higher than the expression level in equivalent non-cancer / non-tumor tissue. Gene / protein expression of the cancer antigen may be a risk factor and / or positively correlated with the occurrence, development, progression, or severity of symptoms of cancer and / or metastasis.

[0274] Subjects

[0275] Subjects described in the aspects described in this disclosure may be any animal or human. Subjects are preferably mammals, more preferably humans. Subjects may be non-human mammals, but are more preferably humans. Subjects may be of any sex. Subjects may be patients. Subjects may have been diagnosed with a disease / condition requiring treatment, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0276] In embodiments according to this disclosure, the subject is preferably a human subject. In some embodiments, the subject to be treated according to the treatment or prevention method disclosed herein is a subject suffering from a disease / condition or at risk of developing a disease / condition. In embodiments according to this disclosure, subjects may be selected for treatment according to the method based on characteristics of certain markers of such disease / condition.

[0277] Sequence identity

[0278] Pairwise and multiple sequence alignments performed to determine the percentage of identity between two or more amino acid or nucleic acid sequences can be performed in various ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega. J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al., 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772–780). When using such software, it is preferable to use the default parameters, such as gap penalty and extension penalty.

[0279] Numbered paragraphs

[0280] The following numbered paragraphs provide further statements regarding the features and combinations of features covered by this disclosure:

[0281] 1. A method for generating or expanding a population of immune cells comprising immune cells specific to Epstein-Barr virus (EBV) lytic antigens, the method comprising stimulating EBV lytic antigen-specific immune cells by contacting PBMCs (PBMCs) with: (i) one or more peptides corresponding to all or part of one or more EBV lytic antigens; or (ii) antigen-presenting cells (APCs) presenting one or more peptides corresponding to all or part of one or more EBV lytic antigens.

[0282] 2. The method of claim 1, wherein the method further comprises restimulating immune cells specific to EBV cleavage antigens by contacting the immune cells with an APC that presents all or part of one or more peptides corresponding to one or more EBV cleavage antigens.

[0283] 3. The method according to claim 1, wherein the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, and BDLF3.

[0284] 4. The method according to claim 1, wherein the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2 and BDLF3.

[0285] 5. The method according to claim 1, wherein the PBMC is a PBMC that has depleted CD45RA-positive cells.

[0286] 6. A method for generating or expanding a population of immune cells comprising immune cells specific to Epstein-Barr virus (EBV) lytic antigens and immune cells specific to EBV latent antigens, the method comprising stimulating the immune cells specific to EBV lytic antigens and immune cells specific to EBV latent antigens by contacting PBMCs (PBMCs) with: (i) one or more peptides corresponding to all or part of one or more EBV lytic antigens and one or more peptides corresponding to all or part of one or more EBV latent antigens; or (ii) presenting antigen-presenting cells (APCs) corresponding to all or part of one or more peptides corresponding to one or more EBV lytic antigens and one or more peptides corresponding to all or part of one or more EBV latent antigens.

[0287] 7. The method of claim 6, wherein the method further comprises restimulating the immune cells specific to EBV cleavage antigens and the immune cells specific to EBV latent antigens by contacting the immune cells with APCs that present all or part of one or more peptides corresponding to one or more EBV cleavage antigens and one or more peptides corresponding to one or more EBV latent antigens.

[0288] 8. The method according to claim 6, wherein the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, and BDLF3.

[0289] 9. The method according to claim 6, wherein the one or more EBV cleavage antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2 and BDLF3.

[0290] 10. The method according to claim 6, wherein the one or more EBV latent antigens are selected from EBNA1, EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1, LMP2A, and LMP2B.

[0291] 11. The method of claim 6, wherein one or more EBV latent antigens are selected from EBNA1, LMP1, LMP2A and LMP2B.

[0292] 12. The method of claim 6, wherein the PBMC is a PBMC that has depleted CD45RA-positive cells.

[0293] 13. An isolated population of immune cells obtained or obtainable by the method of claim 1.

[0294] 14. A method for treating or preventing diseases or disorders associated with EBV infection, comprising administering to a subject the isolated immune cell population as described in claim 13.

[0295] 15. The method of claim 14, wherein the disease or disorder is cancer.

[0296] 16. The method of claim 14, wherein the disease or disorder is an EBV-related cancer selected from EBV-positive lymphoma, EBV-positive nasopharyngeal carcinoma, and EBV-positive gastric cancer.

[0297] ***

[0298] The present invention includes combinations of the described aspects and preferred features, unless such combinations are obviously not permitted or explicitly avoided.

[0299] The chapter headings used here are for organizational purposes only and should not be construed as limiting the topics described.

[0300] Aspects and embodiments of the invention will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0301] Throughout this specification, including the following claims, unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” shall be understood to imply inclusion of the said integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0302] It should be noted that, as used in the specification and appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly specifies otherwise. A range herein may be expressed as “about” a particular value, and / or to “about” another particular value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another embodiment.

[0303] When this article discloses the nucleic acid sequence, it also explicitly includes its reverse complementary sequence.

[0304] The methods described herein can be performed in vitro or in vivo. In some embodiments, the methods described herein are performed in vitro. The term "in vitro" is intended to include experiments performed with cultured cells, while the term "in vivo" is intended to include experiments performed with whole multicellular organisms. Brief description of the attached diagram

[0306] Examples and experiments illustrating the principles of the methods and compositions of this disclosure will now be discussed with reference to the accompanying drawings.

[0307] Figure 1A and 1BThe data. The bar chart shows the number of spot-forming cells (SFCs) per 100,000 cells specific to the indicated EBV antigen in the expanded cell populations of PBMCs obtained from four different EBV-positive lymphoma patients. Figure 1A The number of SFCs per 100,000 cells in response to stimulation with a specified antigen is shown, including EBVSTs generated by stimulation with a mixture of latent peptides (T2 EBVSTs) or EBVSTs generated by stimulation with a mixture of latent + cleaved peptides (full EBVSTs). Figure 1B The number of SFCs per 100,000 cells in response to stimulation with a specified antigen is shown, including EBVSTs generated by stimulation with a mixture of cleaved peptides (cleaved EBVSTs) or a mixture of latent + cleaved peptides (full EBVSTs).

[0308] Figure 2 The bar chart shows the number of spot-forming cells (SFCs) per 100,000 cells specific to a given EBV antigen in EBVSTs expanded from PBMCs of two EBV-positive lymphoma patients after stimulation with latent peptide mixtures (T2-EBVSTs), cleaved peptide mixtures (cleaved-EBVSTs), or latent + cleaved peptide mixtures (BR-EBVSTs). The total number of cells is shown in bars.

[0309] Figure 3 The bar chart shows the number of spot-forming cells (SFCs) per 100,000 cells specific to the indicated EBV antigen within EBVSTs expanded from PBMCs of three healthy donor subjects by stimulation with latent peptide mixtures (T2-EBVSTs), immediate-early cleavage peptide mixtures (IE-EBVSTs), early cleavage peptide mixtures (E-EBVSTs), or latent + cleavage peptide mixtures (BR-EBVSTs).

[0310] Figure 4 The bar graph shows the percentage of autologous EBV-LCLs dissolved by EBVSTs expanded from PBMCs of three healthy donor subjects in an in vitro cell lysis activity assay, using latent peptide mixtures (T2-EBVSTs), immediate-early cleavage peptide mixtures (IE-EBVSTs), early cleavage peptide mixtures (E-EBVSTs), or latent + cleavage peptide mixtures (BR-EBVSTs).

[0311] Figure 5 The bar chart shows the number of spot-forming cells (APCs) per 100,000 cells specific to the indicated EBV antigen or LCLs in EBVSTs amplified from PBMCs by stimulation with a mixture of latent peptides (T2-EBVSTs) or a mixture of latent + cleaved peptides (BR-EBVSTs).

[0312] Figures 6A to 6C The figures and images show the in vivo anticancer activity of EBVSTs expanded from PBMCs by stimulation of autologous EBV-LCLs with a mixture of latent peptides (T2-EBVSTs) or a mixture of latent and cleaved peptides (BR-EBVSTs) in a mouse xenograft model of EBV-positive cancer. The control group received PBS instead of EBVSTs. Figure 6A Show tumor volume (mm) 3 As time changes, Figure 6B and 6C The tumor burden was shown to change over time by measuring the luciferase activity of EBV-LCLs expressing firefly luciferase.

[0313] Figure 7 The images show the load and location of EBV-LCLs expressing firefly luciferase in mice on a specified day of an experiment investigating the anticancer activity of EBV-LCLs amplified from PBMCs by stimulation with autologous EBV-LCLs using a mixture of latent peptides (T2-EBVSTs) or a mixture of latent and cleaved peptides (BR-EBVSTs). Control groups received PBS instead of EBVSTs.

[0314] Figures 8A to 8D Images and graphs show the in vivo anticancer activity of EBVSTs expanded from PBMCs against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer, stimulated with a mixture of latent peptides (T2-EBVSTs), a mixture of cleaved (2) peptides (cleaved (2)-EBVSTs), or a mixture of cleaved (2)+latent peptides (BR(2)-EBVSTs). The control group received PBS instead of EBVSTs. Figures 8A to 8C The changes in tumor burden over time are shown by measuring the luciferase activity of EBV-LCLs expressing firefly luciferase. Figure 8A A rear-side view is shown. Figure 8B A ventral view is shown relative to a specified date of EBVST injection. Figure 8C The changes in total tumor burden over time are shown. Figure 8D The tumor volume (mm) was displayed. 3 (Changes over time)

[0315] Figures 9A to 9CThe block diagram shows the cytokine levels detected in the serum of mice treated with EBVSTs amplified from PBMCs at specified days after EBVST injection in a mouse xenograft model of EBV-positive cancer, by stimulation with a latent peptide mixture (T2-EBVSTs), a cleaved peptide mixture (cleaved(2)-EBVSTs), or a cleaved peptide mixture (2) + a latent peptide mixture (BR(2)-EBVSTs). Figure 9A The level of GM-CSF was displayed. Figure 9B It showed the level of interferon-γ. Figure 9C The IL-10 level is displayed. ***P = 0.0001.

[0316] Figure 10 The bar chart shows the number of spot-forming cells (SFCs) per 100,000 EBV antigen-specific cells in a cell population expanded from PBMCs obtained from four different healthy donor subjects (D#1 to D#4) using a mixture of latent peptides. WW = EBVST expanded from whole PBMCs and restimulated using ATCs from whole PBMCs; WD = EBVST expanded from whole PBMCs and restimulated using ATCs from PBMCs depleted of CD45RA-positive cells; DW = EBVST expanded from PBMCs depleted of CD45RA-positive cells and restimulated using ATCs from whole PBMCs; and DD = EBVST expanded from PBMCs depleted of CD45RA-positive cells and restimulated using ATCs from PBMCs depleted of CD45RA-positive cells.

[0317] Figure 11 The histogram shows the expression of CD80 and HLA-DR in ATCs from four different healthy donor subjects (D#1 to D#4), from intact PBMCs, or from PBMCs depleted of CD45RA-positive cells, as determined by flow cytometry. Example

[0318] In the following embodiments, the inventors describe the generation of cell populations comprising EBV-specific T cells from a PBMC population by stimulation with peptides containing different EBV antigens. The inventors identified the EBV reactivity of the expanded cell populations, their ability to exhibit effector activity against EBV-infected cells, and their in vivo anticancer activity against EBV-positive cancers. The inventors also investigated methods for increasing the proportion of EBV-reactive cells in the expanded populations, as well as their anticancer activity and in vivo persistence.

[0319] Example 1: Production of EBV-specific T cells

[0320] PBMCs were isolated from blood samples obtained from healthy donors or lymphoma patients using the standard Ficoll-Paque density gradient centrifugation method.

[0321] The generation of ATCs

[0322] Anti-CD3 (clone OKT3) and anti-CD28 agonist antibodies were coated onto the wells of tissue culture plates by adding 0.5 mL of 1 mg / mL antibody diluted 1:1000 and incubating at 37°C for 2–4 hours or overnight at 4°C. Anti-CD3 / CD28 agonist antibody-coated plates were then cultured in CTL cell culture medium (containing RPMI-1640 medium, 50% Click medium, 10% FBS, 1% GlutaMax, and 1% Pen / Strep) supplemented with 10 ng / mL IL-7 and 5 ng / mL IL-15, and stimulated at 1 × 10⁶ cells per cell line. 6 One PBMC (in 2 ml of cell culture medium). Maintain the cells at 37°C under a 5% CO2 atmosphere. The next day, replace 1 ml of cell culture medium with fresh CTL medium containing 20 ng / ml IL-7 and 200 ng / ml IL-15. Maintain the ATCs in culture, then harvest and use for experiments or freeze for storage on days 5–7.

[0323] General LCLs

[0324] LCLs lacking HLA class I and HLA class II surface expression (i.e., HLA-negative LCLs) were obtained by targeting and knocking out genes encoding HLA class I and HLA class II molecules in lymphoblastoid cell lines prepared from EBV-transformed B cells. The HLA-negative cells were further modified to knock out genes essential for EBV replication. The resulting cells obtained through this method are referred to herein as universal cells (uLCLs).

[0325] Expansion of EBV-specific T cells

[0326] In a cell culture medium containing 50% Advanced RPMI, 50% Click medium, 10% FBS, 1% GlutaMax, 1% Pen / Strep, supplemented with IL-7 (10 ng / ml) and IL-15 (100 ng / ml), 2 × 10⁶ cells were stimulated with one of the following combinations of peptide mixtures obtained from JPT Technologies (overlapping 15-amino acid peptide libraries, overlapping 11 amino acids, spanning the entire amino acid sequence of the relevant antigen). 6 PBMCs for 9 days to expand EBV-specific T cells:

[0327] (i) EBNA1 peptide mixture (JPT catalog number PM-EBV-EBNA1) + LMP1 peptide mixture (JPT catalog number PM-EBV-LMP1) + LMP2 peptide mixture (JPT catalog number PM-EBV-LMP2) – “Latent peptide mixture”

[0328] (ii) BZLF1 peptide mixture (JPT catalog number PM-EBV-BZLF1) + BRLF1 peptide mixture (JPT catalog number PM-EBV-BRLF1) + BMRF1 peptide mixture (JPT catalog number PM-EBV-BMRF1) + BMLF1 peptide mixture* + BALF2 ​​peptide mixture* + BNLF2A peptide mixture* + BNLF2B peptide mixture* + BMRF2 peptide mixture* – “Cleavage peptide mixture”

[0329] (iii) EBNA1 peptide mixture + LMP1 peptide mixture + LMP2 peptide mixture + BZLF1 peptide mixture + BRLF1 peptide mixture + BMRF1 peptide mixture + BMLF1 peptide mixture + BALF2 ​​peptide mixture + BNLF2A peptide mixture + BNLF2B peptide mixture + BMRF2 peptide mixture – “Latency + Cleavage Peptide Mixture”

[0330] A mixture of peptides (i.e., a mixture of peptides) is used for stimulation, with a final dosage of 10 ng of the mixture of peptides / 1 × 10⁻⁶. 6 One PBMC.

[0331] *The peptide mixtures used for BMLF1, BALF2, BNLF2A, BNLF2B, and BMRF2 are prepared by combining individual constituent peptides obtained from Genemed.

[0332] (iv) BZLF1 peptide mixture + BRLF1 peptide mixture + BMRF1 peptide mixture – “Immediately Early Cleavage Peptide Mixture”

[0333] (v) BMRF1 peptide mixture + BMLF1 peptide mixture + BALF2 ​​peptide mixture + BNLF2A peptide mixture + BNLF2B peptide mixture + BMRF2 peptide mixture (JPT Cat.No.PM-EBV-BMRF1) – “Early Cleavage Peptide Mixture”

[0334] During the 9-day process, additional cell culture medium is added as needed, and cytokines are supplemented on days 5, 6, or 7.

[0335] At the end of the 9-day culture period, the cells were restimulated in the presence of uLCLs by co-culturing with irradiated, peptide-pulsed autologous activated T cells (ATCs). In short, 2 × 10⁶ cells were cultured together. 6ATCs and peptide mixture (10 ng peptide mixture / 1 × 10) 6 ATCs were incubated in CTL medium at 37°C for 30 minutes, followed by irradiation with 30 Gy and harvested. Then, in CTL medium containing IL-7 (10 ng / ml) and IL-15 (100 ng / ml), ATCs responsive to the peptide pulse were mixed with cells and uLCL (irradiated with 100 Gy) in the culture at a ratio of 1:1:5 (responsive cells: peptide pulsed ATCs: irradiated uLCL). Specifically, 1 × 10⁶ ATCs were incubated in CTL medium at 37°C for 30 minutes, followed by irradiation with 30 Gy and harvested. 5 One responder cell, 1×10 5 One peptide pulse ATCs and 0.5 × 10 6 One irradiated uLCL was cultured in 2 mL of CTL medium in the wells of a 24-well tissue culture plate.

[0336] Cells were maintained at 37°C in a 5% carbon dioxide atmosphere. After 3-4 days, cell culture medium containing IL-7 (10 ng / ml) and IL-15 (100 ng / ml) was added as needed. On day 5 or 6, additional cell culture medium containing IL-7 (10 ng / ml) and IL-15 (100 ng / ml) was added as needed. After 6-7 days, the expanded EBVST was harvested for analysis or experiments.

[0337] In all embodiments and figures:

[0338] EBVSTs generated by using a mixture of latent peptides (i.e., above (i)) are called “type 2 latent antigen (T2)-EBVSTs”;

[0339] EBVSTs produced by using a mixture of cleavable peptides (i.e., (ii) above) are referred to as “cleavable EBVSTs”;

[0340] EBVSTs produced using the method of latent period + cleavable peptide mixture (i.e., (iii) above) are called "broad-spectrum (BR)-EBVSTs";

[0341] EBVSTs produced using the immediate-early cleavage peptide mixture method (i.e., above (iv)) are referred to as "immediate early (IE)-EBVSTs"; and

[0342] EBVSTs produced by using an early cleavage peptide mixture (i.e., the above (v)) are called "early (E)EBVSTs".

[0343] Example 2: Analysis of the specificity of EBVSTs for EBV antigen

[0344] EBVSTs prepared from PBMCs of EBV-positive lymphoma patients using different peptide mixtures were analyzed by ELISPOT to determine their ability to recognize different EBV antigens.

[0345] In short, in 96-well plates pre-coated with anti-IFNγ capture antibody at 1×10 5 EBVSTs were seeded at a density of 10 cells / well and stimulated with a peptide mixture corresponding to the indicated EBV peptide. After 18–20 hours of culture, IFNγ+ spots were developed on plates, dried overnight in the dark at room temperature, and quantified. The frequency of T cells specific to each antigen was expressed as the number of specific spot-forming cells (SFCs) per input cell.

[0346] Figure 1A and 1B This indicates that stimulation with a peptide mixture of individual lysis cycle antigens can generate T cells specific to the lysis cycle antigens. Combining peptide mixtures of latent and lysis antigens does not impair specificity for either antigen; in fact, the overall frequency of antigen-specific T cells is higher when the peptide mixtures are combined.

[0347] When used alone, either the latent antigen peptide mixture or the cleavage antigen peptide mixture exhibits poor specificity in some patients' T cells, but good specificity can be obtained when the latent antigen peptide mixture and the cleavage antigen peptide mixture are used in combination (see Donor 2). This is thought to be a result of activated T cells producing cytokines, which work synergistically.

[0348] Figure 2 The results showed that, compared to EBVST generated using either the latent antigen peptide mixture alone or the cleavage antigen peptide mixture alone, the number of cells secreting IFNγ in response to stimulation by the peptide mixture corresponding to the EBV latent and cleavage antigens was significantly greater for EBVST generated using the combination of latent antigen and cleavage antigen peptide mixtures. There was a super-additive increase in the number of interferon-γ-producing cells when using the combination of latent antigen and cleavage antigen peptide mixtures.

[0349] In further experiments, EBVSTs prepared from PBMCs of healthy donors using different peptide mixtures were analyzed by ELISPOT to determine their ability to recognize different EBV antigens, as described above.

[0350] The results are as follows Figure 3 As shown, it demonstrates the possibility of expanding T cells specific to latent cycle antigens, immediate-early lysis antigens, and early lysis antigens from PBMCs of healthy donors.

[0351] Example 3: Cytotoxic components of EBVSTs obtained by stimulating PBMCs with a mixture of peptides corresponding to different EBV antigens Analysis

[0352] The ability of EBVST obtained by stimulating PBMCs with different peptide mixtures to kill EBV-transformed autologous B cell lines was analyzed.

[0353] 10 μl of Cr 51 Add to 1×10 6 Autologous LCLs were pulsed with 10 ng of an EBV latency + cleavage peptide mixture (see Example 1, (iii)) and incubated at 37°C for 1 h. The LCLs were then washed three times with CTL medium and resuspended in CTL medium to 50,000 cells / mL. EBVSTs were seeded with the pulsed autologous LCLs in 200 μl of CTL medium in the wells of a 96-well V-shaped plate at an effector cell:target cell ratio of 20:1 (100,000 EBVSTs + 5,000 LCLs). The co-cultures were incubated at 37°C and 5% CO2 for 4 h. The supernatant was then harvested, and the Cr released by the killed target cells was measured using a gamma-ray counter. 51 To determine the percentage of specific lysis of target cells.

[0354] The results are as follows Figure 4 As shown, EBV antigen-specific T cells obtained by stimulating PBMCs with different peptide mixtures were able to kill EBV-transformed autologous B cell lines.

[0355] The potent ability of EBVSTs obtained using a mixture of lysing antigenic peptides to kill EBV-LCLs is surprising, as only a small fraction of LCLs are expected to be in the lysis cycle (and thus expressing the target antigen). This result can be explained by endocytosis and antigen presentation from lysed dead cells.

[0356] Example 4: In vivo anticancer activity of EBVSTs obtained by stimulating PBMCs with a mixture of peptides corresponding to different EBV antigens. Sexual analysis

[0357] The inventors used a mouse xenograft model to study the comparative efficacy of BR-EBVSTs and T2-EBVSTs in treating EBV-positive cancers in vivo.

[0358] In short, by using 3.5 × 10 in matrigel 6 Autologous LCLs expressing firefly luciferase were subcutaneously implanted into the flank of NSG mice to establish EBV-positive tumors. Eight days later, when the tumors were visible, the mice were administered PBS (control group) and 5×10⁻⁶ oz.kJ / mL via intravenous injection. 6 One BR-EBVSTs or 5×10 6 T2-EBVSTs.

[0359] Throughout the experiment, tumors were monitored using bioluminescence imaging; luciferase activity was monitored by intraperitoneal injection of D-luciferin (1.5 mg per mouse), and the mice were imaged using an IVIS imaging system (Xenogen) 10 minutes later. Tumor volume was also monitored using calipers.

[0360] Prior to infusion, the ability of BR-EBVSTs and T2-EBVSTs to produce interferon-γ in response to stimulation by different EBV antigens or EBV-LCLs was analyzed using ELISPOT. Results showed... Figure 5 In this study, it was demonstrated that, compared to immune cell populations amplified with peptides corresponding to both EBV latency and cleavage antigens, immune cell populations amplified with peptides corresponding to EBV latency antigens alone contained cells that responded to both EBV latency and cleavage antigens, and contained a larger proportion of cells that produced IFNγ in response to EBV-LCL stimulation.

[0361] Figure 6 and Figure 7 The results showed that BR-EBVSTs controlled EBV-positive tumors faster than T2-EBVSTs.

[0362] Figure 7 It was also shown that mice treated with BR-EBVST had fewer metastases than mice treated with T2-EBVST.

[0363] In further experiments, as described above, 3.5 × 10⁻⁶ stypomas were implanted subcutaneously. 6 EBV-positive tumors were established using autologous LCLs expressing firefly luciferase. Eight days later, mice were administered PBS (control group) and 1×10⁻⁶ PBS via intravenous injection. 6 BR(2)-EBVSTs, 1×10 6 One cleavage (2)-EBVSTs or 1×10 6 T2-EBVSTs.

[0364] As described in Example 1, cleaved (2)-EBVSTs and BR(2)-EBVSTs used in this experiment were generated, except that the EBVSTs were amplified using a combination of the following peptide mixtures during stimulation:

[0365] (vi) BZLF1 peptide mixture + BRLF1 peptide mixture + BMRF1 peptide mixture + BMLF1 peptide mixture + BXLF1 peptide mixture + BALF1 peptide mixture + BLLF2 peptide mixture + BALF2 ​​peptide mixture + BNLF2A peptide mixture – “Cleavage (2) peptide mixture”

[0366] (vii) EBNA1 peptide mixture + LMP1 peptide mixture + LMP2 peptide mixture + BZLF1 peptide mixture + BRLF1 peptide mixture + BMRF1 peptide mixture + BMLF1 peptide mixture + BXLF1 peptide mixture + BALF1 peptide mixture + BLLF2 peptide mixture + BALF2 ​​peptide mixture + BNLF2A peptide mixture – “Cleavage (2) + Latent peptide mixture”

[0367] The peptide mixtures of BMLF1, BXLF1, BALF1, BLLF2, BALF2, and BNLF2A were prepared by combining individual constituent peptides obtained from Genemed. The peptide mixtures used for EBNA1, LMP1, LMP2, BZLF1, BRLF1, and BMRF1 were obtained from JPT Technologies, as shown in Example 1.

[0368] EBVSTs produced using the method of cleavage peptide mixture (i.e., above (vi)) are called “cleavage (2)-EBVSTs”, and EBVSTs produced using the method of cleavage (2) + latency peptide mixture (i.e., above (vii)) are called “BR(2)-EBVSTs”.

[0369] Throughout the experiment, tumors were monitored using bioluminescence imaging as described above, and tumor volume was also monitored using caliper measurements.

[0370] Plasma samples were also collected from mice on days 3 and 8 after EBVST administration and analyzed by enzyme-linked immunosorbent assay to determine the levels of GM-CSF, interferon-γ, and IL-10.

[0371] The results are shown in Figures 8 and 9.

[0372] EBVSTs amplified by peptide stimulation with EBV latency and cleavage antigen strongly inhibited tumor growth. Figures 8A to 8D EBVSTs expanded by stimulation with EBV cleavage antigens alone can also inhibit tumor growth, and the degree of inhibition is similar to or greater than that of EBVSTs expanded by stimulation with EBV latency antigens alone.

[0373] Compared to mice treated with EBVSTs stimulated by EBV latency antigen alone, mice treated with EBVSTs stimulated by peptides of EBV latency antigen and cleavage antigen also showed increased serum pro-inflammatory cytokine levels. Furthermore, mice treated with EBVSTs stimulated by peptides of EBV cleavage antigen showed similar or increased serum pro-inflammatory cytokine levels compared to mice treated with EBVSTs stimulated by EBV latency antigen alone. Figure 9A and 9B ).

[0374] In contrast, mice treated with EBVSTs stimulated by peptides of EBV latency antigen and cleavage antigen had lower serum IL-10 levels compared to mice treated with EBVSTs stimulated by EBV latency antigen alone, while mice treated with EBVSTs stimulated by peptides of EBV cleavage antigen had similar or lower serum IL-10 levels compared to mice treated with EBVSTs stimulated by EBV latency antigen alone. Figure 9C ).

[0375] Therefore, BR-EBVSTs have been found to kill more tumor cells and produce more pro-inflammatory cytokines. Without being bound by any particular theory, this could lead to changes in the tumor microenvironment, increased epitope proliferation in vivo, and additional tumor cell killing by non-viral tumor antigen-specific T cells.

[0376] Example 5: Conclusion

[0377] In short, the inventor has shown:

[0378] A population of T cells containing T cells specific to cleavage and latent EBV antigens can be obtained by stimulating PBMCs from healthy donors and lymphoma patients with a mixture of peptides corresponding to cleavage and latent EBV antigens (see, for example, Figures 1 and 3).

[0379] Stimulation of PBMCs with a peptide mixture corresponding to both lytic and latent EBV antigens produced more EBV antigen-responsive T cells compared to stimulation with a peptide mixture corresponding to either lytic or latent EBV antigens alone (see e.g.). Figure 2 );

[0380] T cell populations obtained by stimulating PBMCs with peptide mixtures corresponding to lytic + latent EBV antigens, or immediate early lytic antigens, or early lytic antigens, were able to kill EBV-LCLs, with an ability similar to that of T cell populations obtained by stimulating PBMCs with peptide mixtures corresponding to latent EBV antigens (see, for example). Figure 4 );

[0381] Compared to the T cell population obtained by stimulating PBMCs with a peptide mixture corresponding to both lytic and latent EBV antigens, the T cell population obtained by stimulating PBMCs with a peptide mixture corresponding to both lytic and latent EBV antigens contained a larger proportion of cells that responded to EBV-infected cells (see, for example). Figure 5 );

[0382] Compared with the T cell population obtained by stimulating PBMCs with a peptide mixture corresponding to latent EBV antigens, the T cell population obtained by stimulating PBMCs with a peptide mixture corresponding to lytic + latent EBV antigens showed improved control of tumor growth and reduced metastasis in EBV-positive cancers (see, for example, Figures 6 and 7).

[0383] Compared with the T cell population obtained by stimulating PBMCs with a peptide mixture corresponding to the latent EBV antigen, the T cell population obtained by stimulating PBMCs with a peptide mixture corresponding to the lysed EBV antigen showed similar or improved control over tumor growth in EBV-positive cancers (see Figure 8 for example).

[0384] Compared with subjects treated with T cells obtained by stimulating PBMCs with a peptide mixture corresponding to latent EBV antigens, subjects with EBV-positive cancer treated with T cells obtained by stimulating PBMCs with a peptide mixture corresponding to cleaved + latent EBV antigens had elevated levels of pro-inflammatory cytokines (GM-CSF, IFNγ) and decreased levels of anti-inflammatory cytokines (IL-10) in their peripheral blood (see Figure 9, e.g.).

[0385] Compared with subjects treated with T cells obtained by stimulating PBMCs with a peptide mixture corresponding to latent EBV antigens, subjects with EBV-positive cancer treated with T cells obtained by stimulating PBMCs with a peptide mixture corresponding to cleaved EBV antigens had similar or elevated levels of pro-inflammatory cytokines (GM-CSF, IFNγ) and similar or decreased levels of anti-inflammatory cytokines (IL-10) in their peripheral blood (see Figure 9, for example).

[0386] Example 6: Generation of EBV-specific T cells from PBMCs depleted of CD45RA-positive cells

[0387] Next, the inventors investigated the effect of depleting CD45RA-positive cells from the PBMC population on the expansion of the EBV-specific T cell population.

[0388] Because of IL-15-mediated stimulation of natural killer cell proliferation, the growth of natural killer cells from the PBMC population may be problematic in methods for expanding EBVST from a natural killer cell population. CD45RA is an immature T cell marker, also expressed on native T regulatory cells and natural killer cells; therefore, it can be inferred that depletion of CD45RA+ cells will remove natural killer cells from the initial PBMC population. Depletion of CD45RA+ cells also removes regulatory T cells that can suppress the growth of antigen-specific T cells, especially in cancer patients, and also removes immature cells that can grow as bystander cells and dilute antigen-specific T cells.

[0389] use The columns and CD45RA-conjugated beads were used to deplete CD45RA-expressing cells in PBMCs, and the PBMCs depleted of CD45RA-positive cells were then used to expand EBV-specific T cells by stimulation with a mixture of latent peptides, essentially as described in Example 1.

[0390] PBMCs depleted of CD45RA-positive cells were also used as a starting population to generate ATCs for use in restimulation, which were generated essentially as described in Example 1.

[0391] The following experimental conditions were compared:

[0392] (i) EBVST+ expanded from whole PBMCs (i.e., PBMCs that have not depleted CD45RA-positive cells) was restimulated with ATCs generated from whole PBMCs - in Figure 10 In Chinese, it is referred to as "WW" (meaning all + all);

[0393] (ii) EBVST+ expanded from whole PBMCs was restimulated with ATCs generated from PBMCs depleted of CD45RA-positive cells. Figure 10 In Chinese, this is referred to as "WD" (meaning total exhaustion).

[0394] (iii) EBVST+ expanded from PBMCs depleted of CD45RA-positive cells was restimulated using ATCs generated from whole PBMCs. Figure 10 In Chinese, this is referred to as "DW" (meaning exhausted + full).

[0395] (iv) EBVST+ expanded from PBMCs depleted of CD45RA-positive cells was restimulated with ATCs generated from PBMCs depleted of CD45RA-positive cells. Figure 10 In Chinese, this is referred to as "DD" (i.e., exhaustion + exhaustion).

[0396] The ability of EBVSTs prepared from PBMCs obtained from four different healthy donors (D#1 to D#4) to recognize different EBV antigens was determined by ELISPOT analysis according to (i) to (iv) above. The ELISPOT analysis was performed essentially as described in Example 2.

[0397] The results are as follows Figure 10As shown. For each donor, compared to using the entire PBMC population, using PBMCs depleted of CD45RA-positive cells as the starting population for EBVST expansion resulted in an increased proportion of cells in the expanded population that secreted interferon-γ in response to stimulation by the EBV peptide mixture. Similarly, using PBMCs depleted of CD45RA-positive cells as the starting population for generating ATCs for restimulation resulted in a greater proportion of cells in the expanded population that secreted interferon-γ in response to stimulation by the EBV peptide mixture.

[0398] The inventors analyzed the expression of co-stimulatory molecules CD80 and HLA-DR (MHC class II) in ATCs produced by whole PBMCs or by PBMCs depleted of CD45RA-positive cells using flow cytometry.

[0399] The results are as follows Figure 11 As shown, ATCs generated from PBMCs depleted of CD45RA-positive cells exhibited higher expression of CD80 and HLA-DR compared to ATCs generated from whole PBMCs, thus improving antigen presentation and co-stimulatory properties.

Claims

1. A method for generating or expanding a population of immune cells comprising immune cells specific for Epstein Barr Virus (EBV) lytic antigens and immune cells specific for EBV latent antigens, the method comprising stimulating immune cells specific for EBV lytic antigens and immune cells specific for EBV latent antigens by contacting peripheral blood mononuclear cells (PBMCs) with (i) peptides corresponding to all of EBNA1, LMP1, LMP2A, LMP2B, BZLF1, BRLF1, BMRF1, BMLF1, BALF2, BNLF2A, BNLF2B, and BMRF2, or (ii) antigen presenting cells (APCs) that present peptides corresponding to all of EBNA1, LMP1, LMP2A, LMP2B, BZLF1, BRLF1, BMRF1, BMLF1, BALF2, BNLF2A, BNLF2B, and BMRF2.

2. The method of claim 1, wherein the method comprises contacting PBMCs with peptides corresponding to all of EBNA1, LMP1, LMP2A, LMP2B, BZLF1, BRLF1, BMRF1, BMLF1, BALF2, BNLF2A, BNLF2B, and BMRF2.

3. The method of claim 1 or 2, wherein the method comprises contacting PBMCs with an EBNA1 peptide mixture, an LMP1 peptide mixture, an LMP2 peptide mixture, a BZLF1 peptide mixture, a BRLF1 peptide mixture, a BMRF1 peptide mixture, a BMLF1 peptide mixture, a BALF2 peptide mixture, a BNLF2A peptide mixture, a BNLF2B peptide mixture, and a BMRF2 peptide mixture.

4. The method of claim 1 or 2, wherein the PBMCs are CD45RA positive cell depleted PBMCs.

5. The method of claim 1 or 2, wherein the stimulation of immune cells specific for EBV lytic antigens and immune cells specific for EBV latent antigens is in the presence of IL-7 and / or IL-15.

6. The method of claim 1, wherein the method further comprises re-stimulating immune cells specific for EBV lytic antigens and immune cells specific for EBV latent antigens by contacting these immune cells with antigen presenting cells (APCs) that present peptides corresponding to all or a portion of EBNA1, LMP1, LMP2A, LMP2B, BZLF1, BRLF1, BMRF1, BMLF1, BALF2, BNLF2A, BNLF2B, and BMRF2.

7. The method of claim 6, wherein the re-stimulation of immune cells specific for EBV lytic antigens and immune cells specific for EBV latent antigens is in the presence of IL-7 and / or IL-15.

8. The method of claim 6 or claim 7, wherein restimulating the immune cells comprises contacting specific immune cells against EBV antigens with activated T cells (ATC) presenting peptides corresponding to EBV antigens in the presence of uLCLs.

9. A population of immune cells obtained by the method of any one of claims 1-8.

10. A pharmaceutical composition comprising a population of immune cells according to claim 9.

11. Use of a population of immune cells according to claim 9 or a pharmaceutical composition according to claim 10 for the manufacture of a medicament for use in a method of treating an EBV-positive cancer.

12. Use according to claim 11, wherein the EBV-positive cancer is selected from the group consisting of EBV-positive lymphoma, EBV-positive nasopharyngeal carcinoma and EBV-positive gastric cancer.

13. An in vitro method of killing cells infected with EBV, comprising contacting cells infected with EBV with a population of immune cells according to claim 9 or a pharmaceutical composition according to claim 10.

14. Use of a population of immune cells according to claim 9 or a pharmaceutical composition according to claim 10 for the in vitro killing of cells infected with EBV.

15. Use of a population of immune cells according to claim 9 or a pharmaceutical composition according to claim 10 for the manufacture of a preparation for killing cells infected with EBV.

16. An in vitro method for killing cancer cells infected with EBV, comprising contacting the cancer cells with a population of immune cells according to claim 9 or a pharmaceutical composition according to claim 10.

17. Use of a population of immune cells according to claim 9 or a pharmaceutical composition according to claim 10 for the in vitro killing of cancer cells infected with EBV.

18. Use of a population of immune cells according to claim 9 or a pharmaceutical composition according to claim 10 for the manufacture of a preparation for killing cancer cells infected with EBV.

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