EBV-specific immune cells

Abstract

To provide methods for generating / expanding populations of immune cells comprising immune cells specific for an Epstein Barr Virus (EBV) lytic antigen.SOLUTION: The present invention provides methods comprising stimulating immune cells specific for an EBV lytic antigen 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. Also disclosed are populations of immune cells comprising immune cells specific for an EBV lytic antigen expanded according to such methods, and uses thereof.SELECTED DRAWING: Figure 1A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 16 / 388,776 (filed Apr. 18, 2019; The contents and elements thereof are incorporated herein by reference in their entirety for all purposes. The present application claims priority to the prior art (which may be incorporated herein by reference). [Technical field]

[0002] The present disclosure relates to at least the fields of molecular and cell biology, immunology, and medical treatment. Also relates to methods of prevention and treatment of cancer.

[0003] This invention was made in part under grant number CA126752 awarded by the National Institutes of Health. Made with Government support. The Government has certain rights in this invention. [Background technology]

[0004] Approximately 40% of lymphomas, all undifferentiated nasopharyngeal carcinomas (NPCs), and approximately 10% of gastric cancers The EBV genome was carried by the adoptive transfer of These cells express viral proteins that can be targeted by EBV-specific T cells (EBVST). do.

[0005] EBV+ malignancies arising outside of immunosuppressive settings express only 1 of approximately 90 EBV proteins. Although only four of these antigens are expressed, and these antigens are poorly immunogenic, they are still of value for EBVST. These provide target antigens for

[0006] Ongoing clinical studies on the use of EBV-specific T cells to treat EBV-positive malignancies. In the clinical trial, EBV-transformed B cells were used in the expansion of EBV-specific T cells (NC T02578641), or PBMCs were incubated with a peptide corresponding to an EBV type II latency antigen. This involves stimulation with (NCT01555892). Summary of the Invention

[0007] The present disclosure relates to peptides corresponding to EBV lytic antigens that are useful for the treatment of EBV-associated diseases. can be used to generate / expand a population of EBV-specific immune cells that are It is based on an unexpected discovery.

[0008] EBV-infected cells in EBV-associated diseases show substantial expression of EBV latent antigens. Despite the fact that only EBV lytic antigens are A population of immune cells that is generated / expanded by stimulating immune cells with EBs It is demonstrated herein that the cytolytic activity against V-infected cells. It is produced by stimulating immune cells with peptides of EBV lytic antigens. The population of immune cells that is generated / expanded stimulates immune cells with peptides from EBV latent antigens. EBV positive These results demonstrate similar or improved ability to treat human cancers in vivo. can be.

[0009] Another unexpected finding was the use of both EBV lytic and latent antigens in stimulation. The method involves quantifying the frequency of immune cells specific for EBV latent antigens in an expanded population. It did not result in a substantial decline.

[0010] The present inventors further identified immune cells specific for EBV lytic antigens and those specific for EBV latent antigens. and a population of immune cells specific for EBV for treating EBV-associated disease. More effective than a panel of immune cells that contains immune cells specific only for EBV latent antigens This makes it clear that:

[0011] In a first aspect, the present disclosure relates to an Epstein-Barr virus (EBV) lytic antigen. For generating or expanding a population of immune cells that includes immune cells specific for The method comprises: (i) subjecting peripheral blood mononuclear cells (PBMCs) to one or more EBV lytic antigens; or (ii) one or more peptides corresponding to all or a portion of Presents one or more peptides corresponding to all or part of multiple EBV lytic antigens By contacting the cells with antigen-presenting cells (APCs), specific immunity against EBV lytic antigens is generated. The method includes stimulating immune cells.

[0012] In some embodiments, the method further comprises detecting an EBV lytic antigen specific for the EBV lytic antigen. The immune cells are then subjected to one or more immunoreactive antigens corresponding to all or part of one or more EBV lytic antigens. By contacting the cells with APCs presenting a number of peptides, the cells were specifically targeted to EBV lytic antigens. The method includes selectively restimulating said immune cells.

[0013] In addition, immune cells specific for Epstein-Barr virus (EBV) lytic antigens and and an immune cell specific for EBV latent antigen. or expanding peripheral blood mononuclear cells (PBMCs), comprising: (i) one or one or more peptides corresponding to all or part of a plurality of EBV lytic antigens, and one or more peptides corresponding to all or part of one or more EBV latent antigens; or (ii) one or more antibodies corresponding to all or part of one or more EBV lytic antigens. or peptides, and antibodies corresponding to all or part of one or more EBV latent antigens. by contacting the antigen-presenting cell (APC) with one or more peptides that are The immune cells specific for EBV lytic antigens and the immune cells specific for EBV latent antigens were and stimulating the cells.

[0014] In some embodiments, the method further comprises detecting an EBV lytic antigen specific for the EBV lytic antigen. The immune cells specific for EBV latent antigens are then incubated with one or more EBV antibodies. One or more peptides corresponding to all or part of the EBV lytic antigen, and The antigen is contacted with APCs presenting one or more peptides corresponding to all or a portion of the antigen. By this, the immune cells specific for EBV lytic antigens and the immune cells specific for EBV latent antigens were isolated. The method includes re-stimulating specific immune cells with the antigen.

[0015] In some embodiments according to various aspects of the present disclosure, one or more EBV lysis The antigens are BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BL LF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5 and BDLF3. In some embodiments according to the aspect, the one or more EBV lytic antigens are BZLF 1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BG LF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF 4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BA LF3, BALF5, BDLF3 and combinations thereof.

[0016] In some embodiments, the one or more EBV lytic antigens are BZLF1, BR LF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF 2 and BDLF3. In some embodiments, one or more E BV lytic antigens include BZLF1, BRLF1, BMLF1, BMRF1, BALF2, and BN The group consisting of LF2A, BNLF2B, BMRF2, BDLF3, and combinations thereof is selected from.

[0017] In some embodiments, the one or more EBV latent antigens are EBNA1, EB NA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1, L In some embodiments, the 1 One or more of the EBV latent antigens are EBNA1, EBNA-LP, EBNA2, EBNA 3A, EBNA3B, EBNA3C, BARF1, LMP1, LMP2A, LMP2B and combinations thereof.

[0018] In some embodiments, the one or more EBV latent antigens are EBNA1, LM P1, LMP2A and LMP2B. or multiple EBV latent antigens, EBNA1, LMP1, LMP2A, LMP2B and combinations thereof.

[0019] In some embodiments, the PBMCs are PBMCs that have been depleted of CD45RA positive cells. He is the MC.

[0020] Isolated immune cell populations obtained or obtainable by the methods according to the present disclosure is also provided.

[0021] Isolation of immune cells specific for Epstein-Barr Virus (EBV) lytic antigens Populations of isolated immune cells are also provided.

[0022] Epstein-Barr virus (EBV) lytic antigen-specific immune cells and EBV Also provided is an isolated immune cell population comprising an immune cell specific for a latent antigen.

[0023] Pharmaceutical compositions comprising isolated immune cell populations according to the present disclosure are also provided.

[0024] The present disclosure provides a method for treating or preventing a disease or disorder. Also provided is an isolated immune cell population or pharmaceutical composition comprising the

[0025] In the manufacture of a pharmaceutical for use in a method for treating or preventing a disease or disorder Uses of the isolated immune cell populations or pharmaceutical compositions according to the present disclosure are also provided.

[0026] A method for treating or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of ... compound according to the present disclosure. Methods are also provided that include administering the isolated immune cell population or pharmaceutical composition to a subject. can be.

[0027] The present invention relates to a method for treating or preventing a disease or disorder associated with EBV infection. An isolated immune cell population or pharmaceutical composition according to the disclosure is also provided.

[0028] Medicament for use in a method for treating or preventing a disease or disorder associated with EBV infection Also provided is the use of an isolated immune cell population or a pharmaceutical composition according to the present disclosure in the manufacture of a product. It will be offered.

[0029] A method for treating or preventing a disease or disorder associated with EBV infection. and administering to the subject an isolated immune cell population or a pharmaceutical composition according to the present disclosure. A method is also provided.

[0030] Isolated immune cells according to the present disclosure for use in a method of treating or preventing cancer Populations or pharmaceutical compositions are also provided.

[0031] The present disclosure provides a method for the manufacture of a medicament for use in a method for the treatment or prevention of cancer. Use of the isolated immune cell population or pharmaceutical composition in a therapeutic agent is also provided.

[0032] A method for treating or preventing cancer, comprising administering to a subject an isolated Methods are also provided that include administering the immune cell population or pharmaceutical composition to a subject.

[0033] In some embodiments according to various aspects of the present disclosure, a disease associated with EBV infection or The disorder is EBV-associated cancer.

[0034] In some embodiments according to various aspects of the present disclosure, the cancer is an EBV-associated cancer. .

[0035] In some embodiments, the EBV-associated cancer is EBV-positive lymphoma, EBV-positive nasal Selected from pharyngeal cancer and EBV-positive gastric cancer.

[0036] A method for killing cells infected with EBV, comprising: Also provided are methods comprising contacting an isolated immune cell population or pharmaceutical composition according to The method may be an in vitro method or an in vivo method.

[0037] An isolated immune cell population according to the present disclosure for killing cells infected with EBV or Use of the pharmaceutical composition is also provided.

[0038] A method for killing cancer cells, comprising infecting the cancer cells with isolated immune cells according to the present disclosure. Also provided are methods comprising contacting an in vitro population or pharmaceutical composition with the It may be an in vivo method or an in vivo method.

[0039] The use of an isolated immune cell population or pharmaceutical composition according to the present disclosure for killing cancer cells. Uses are also provided.

[0040] In some embodiments, the cancer cells are infected with EBV.

[0041] The present disclosure provides evidence that immune cells specific for EBV lytic antigens can kill EBV-infected cells. In addition, they contain immune cells specific for EBV latent and lytic antigens. A population of cells that only contains immune cells specific for EBV latent antigens. The unexpected finding was that it showed an improved ability to inhibit EBV-positive malignancies when compared Based on.

[0042] Epstein-Barr virus replication The virology of the Epstein-Barr virus (EBV) is described, for example, by Stanfield and Luftiq, F1000Res.(2017)6:386, and Odumad e et al., Clin Microbiol Rev (2011) 24(1):193-209 (both of which are incorporated herein by reference in their entireties).

[0043] EBV binds to the β1 integrin via the viral protein BMRF2. In addition, the viral proteins gH / gL bind to the integrins avβ6 and avβ8. EBV infects epithelial cells via the viral glycoprotein gp350, which binds to C Viral gp4 then interacts with D21 and / or CD35. 2 infects B cells by interacting with MHC class II. These interactions causes the viral envelope to fuse with the cell membrane, allowing the virus to enter the cell. Once inside the cell, the viral capsid dissolves and the viral genome is released. is transported to the nucleus.

[0044] EBV has two modes of replication: latent and lytic.

[0045] The latent cycle does not result in the production of virions and can occur in B cells and epithelial cells The circular DNA of the EBV genome exists in the cell nucleus as an episome and binds to the DNA polymerase of the host cell. During latency, only some of the EBV genes are copied by different A latency program that produces a diverse set of viral proteins and RNA This latent cycle can occur in one of three distinct patterns, known as For example, Amon and Farrell, Reviews in Medical Vir and Physiology (2004) 15(3):149-56, which is incorporated by reference in its entirety. is incorporated herein).

[0046] EBNA1 protein and non-coding RNA EBER mediate latency programs I and II In latent programs II and III, EBNALP is also expressed. , accompanied by expression of LMP1, LMP2A and LMP2B proteins, and initiates latency program I In addition, expression of EBNA2, EBNA3A, EBNA3B, and EBNA3C was increased in II. Accompany.

[0047] EBNA1 is multifunctional and plays a role in gene regulation, extrachromosomal replication, and viral promoters. plays a role in maintaining the EBV episomal genome through positive and negative regulation of (Duellman et al., J Gen Virol. (2009); 90 (Pt 9):2251-2259). EBNA2 is involved in the regulation of latent viral transcription and is involved in the regulation of EBV contributes to the immortalization of infected cells (Kempkes and Ling, Curr T op Microbiol Immunol. (2015)391:35-59). E.B. NA-LP is required for innate B cell transformation and mediates transcription factors for viral replication. (Szymula et al., PLoS Pathog. (2018);14(2): e1006890). EBNA3A, EBNA3B, and EBNA3C interact with RBPJ. They act to affect gene expression, thereby contributing to the survival and growth of infected cells. (Wang et al., J Virol. (2016) 90(6):2906-2919). LMP1 regulates the expression of genes involved in B cell activation (Chang et al., J. Biomed.Sci.(2003)10(5):490-504). LMP2A and LMP2B regulates normal B cell signaling by mimicking the activated B cell receptor. (Portis and Longnecker, Oncogene (2 004)23(53):8619-8628). Various EBERs bind to host cell proteins. It has been proposed that the ribonucleoprotein complex plays a role in cell transformation. is.

[0048] The latency cycle in B cells can occur in any of three stages: latency program I, latency program II, and latency program III. Thus, progression is possible, and in the normal case, progression is III → II → I. Upon infecting cells, EBV enters latency program III. Expression of latency III genes EBV then typically induces the activation of B cells, which then become proliferating blasts. It regulates expression of a subset of genes that induce blast differentiation into memory B cells. The EBV progresses to latency stage II by restricting its expression. EBNA1 expression induces the virus to enter latency phase I. EBNA1 expression inhibits EBV replication. However, this is only possible when memory B cells divide. In epithelial cells, only latency II occurs. do.

[0049] During primary infection, EBV replicates in oropharyngeal epithelial cells and progresses to latency stages III, II, and EBV infection is established in B lymphocytes. Latent EBV infection of B lymphocytes leads to viral persistence. required for viral replication, subsequent replication in epithelial cells, and release of infectious virus into saliva. EBV latent stage III and latent stage II infection of B lymphocytes and oral epithelial cells Latency II infection, as well as latency II infection of NK cells or T cells, is associated with a uniform EBV genotype. can result in a variety of malignancies characterized by the presence and gene expression of

[26]

[0050] Latent EBV in B cells can be reactivated to switch to lytic replication The lytic cycle results in the production of infectious virions and occurs in B cells and epithelial cells. For example, Arvin et al., Human Herpesviruses: Biology ology,Therapy and Immunoprophylaxis(Camb In Chapter 25 of the book "The Great Passage of the American Indian," published by Keith University Press (2007), Nney, which is incorporated herein by reference in its entirety.

[0051] Lytic replication requires that the EBV genome be linear. The lysosomes are thus linearized for reactivation to induce lysis. In B cells, lytic replication normally only occurs after reactivation from latency. do.

[0052] Immediate-early lytic gene products, such as BZLF1 and BRLF1, regulate their own expression. Acts as a transactivator enhancing expression of early and late lytic cycle genes .

[0053] Different early lytic gene products play different roles in viral replication (e.g., D BALF5, a DNA polymerase catalytic component; BMRF1, a DNA polymerase processivity factor , DNA-binding protein BALF2, helicase BBLF4, primase BSLF1, and primase-related proteins BBLF2 / 3), and deoxynucleotide metabolism (e.g., thymidine kinase BXLF1, dUTPase BORF 2) Other early lytic gene products behave like transcription factors (e.g., BMRF1, BR RF1), which has a role in RNA stability and processing (e.g., BMLF1 ), or involved in immune evasion (e.g., BHRF1, which inhibits apoptosis).

[0054] A variety of late lytic gene products have been previously identified as being expressed after the initiation of viral replication. They are generally classified as glycoproteins that mediate EBV attachment and fusion. (e.g., gp350 / 220, gp85, gp42, gp25) as well as virions Other late lytic proteins encode structural components of the Gene product plays a role in immune evasion; BCLF1 is a viral homolog of IL-10 BALF1 encodes a β-amyloid β-blocker protein (BALF1), which shares homology with the anti-apoptotic protein Bcl2. Encodes proteins.

[0055] Expression of EBV antigens in EBV-associated cancers EBV antigens expressed in EBV-positive cancers are described, for example, by Craddock and He slop, Update Cancer Ther.(2008)Mar;3(1):3 3-41, Gottschalk and Rooney, Curr Top Microb iol Immunol. (2015) 391: 427-454, and Shinoza Ki-Ushiku et al., Int J Oncol.(2015)46(4):1421- It is described in 34.

[0056] Immunocompromised subjects, e.g., subjects following HSCT or solid organ transplantation, congenital immune deficiencies EBV-associated lymphomas occurring in subjects with HIV infection or EBV-associated lymphomas have a type III latency period. EBNA1, EBNA2, EBNA-LP, EBNA3A, EBNA3B, EBN It expresses A3C, BARF1, LMP1 and LMP2. Hodgkin's lymphoma, non-Hodgkin's lymphoma, some types and T-cell lymphoma, NK cell Lymphoma, some cases of B-cell lymphoma and nasopharyngeal carcinoma, exhibit type II latency; Expresses EBNA1, BARF1, LMP1 and LMP2. EBV-positive Burkitt Lymphoma EBV-positive gastric cancer shows type I latency, expressing EBNA1 and BARF1. The disease may present with type I or type II latency.

[0057] EBV positivity in subjects who are immunocompetent only for EBV latency antigens (but not lytic cycle antigens) Lytic cycle genes have long been thought to be expressed by cells in cancer cells. Transcripts encoding these products have recently been identified in a variety of cancers, including gastric cancer, nasopharyngeal carcinoma, and B-cell lymphoma. It has been detected in many EBV-positive malignant tumors.

[0058] A population of immune cells specific for EBV lytic antigens was isolated from patients with EBV-positive cancer or Can EBV lytic antigens be generated from healthy donor subjects? Whether specific immune cells will exhibit effector activity against EBV-infected cells, Alternatively, a population of immune cells specific for EBV lytic antigens could treat EBV-associated cancers. Whether this will be useful for

[0059] EBV antigen Various aspects of the present disclosure employ peptides corresponding to different EBV antigens.

[0060] In some embodiments according to various aspects of the disclosure, the EBV lytic antigen is BZLF. 1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BA RF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF 2, BKRF4, BMRF2, FU, EBNA1-FUK, BALF4, BILF1, B ILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3 and gp3 50. EBV lytic antigens are selected from BZLF1, BRLF1, BMLF1, BMR F1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, B NLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU, EBNA1-FUK, BALF4, BILF1, BILF2, BNFR1, BVRF2, The group consisting of BALF3, BALF5, BDLF3, gp350, and combinations thereof In some embodiments, the EBV lytic antigen is selected from BZLF. 1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BG LF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF 4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BA LF3, BALF5 and BDLF3. EBV lytic antigens are selected from BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF 5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF 3, BALF5, BDLF3, and combinations thereof. In some embodiments, the EBV lytic antigen is BZLF1, BRLF1, BM LF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2 and BD EBV lytic antigens are selected from BZLF1, BRLF1, BMLF1, BM RF1, BALF2, BNLF2A, BNLF2B, BMRF2, BDLF3 and The compound may be selected from the group consisting of:

[0061] In some embodiments, the EBV lytic antigen is BZLF1, BRLF1, BMRF 1, BMLF1, BXLF1, BALF1, BLLF2, BALF2, BNLF2A, B NLF2B and BMRF2. EBV lytic antigens are selected from BZLF1, BRLF 1, BMRF1, BMLF1, BXLF1, BALF1, BLLF2, BALF2, BN LF2A, BNLF2B, BMRF2, and combinations thereof. In some embodiments, the EBV lytic antigen is BZLF1, BRLF 1, BMRF1, BMLF1, BXLF1, BALF1, BLLF2, BALF2 ​​and BNLF2A. EBV lytic antigens are selected from BZLF1, BRLF1, BMRF1 , BMLF1, BXLF1, BALF1, BLLF2, BALF2, BNLF2A and In some embodiments, the compound may be selected from the group consisting of: , EBV lytic antigens are BZLF1, BRLF1, BMRF1, BMLF1, BALF2, The EBV lytic antigen is selected from BNLF2A, BNLF2B and BMRF2. LF1, BRLF1, BMRF1, BMLF1, BALF2, BNLF2A, BNLF2 B, BMRF2, and combinations thereof.

[0062] In some embodiments, the EBV lytic antigen is an immediate early lytic antigen, an early lytic antigen or is selected from late lytic antigens.

[0063] In some embodiments, the immediate early lytic antigen is BZLF1, BRLF1, and BM The immediate early lytic antigen is selected from BZLF1, BRLF1, BMRF1 and It may be selected from the group consisting of combinations thereof.

[0064] In some embodiments, the early lytic antigen is BMLF1, BMRF1, BXLF1 , BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BN LF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU and EBNA1- The early lytic antigen is selected from BMLF1, BMRF1, BXLF1, BAL F1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B , BHLF1, BLLF2, BKRF4, BMRF2, FU, EBNA1-FUK and In some embodiments, the compound may be selected from the group consisting of: , early lytic antigens are BMLF1, BMRF1, BXLF1, BALF1, BALF2, B GLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKR The early lytic antigen is selected from BMLF1, BMRF1, BX LF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2 B, BHLF1, BLLF2, BKRF4, BMRF2, and combinations thereof In some embodiments, the early lysis antigen is selected from the group consisting of BML Select from F1, BMRF1, BALF2, BNLF2A, BNLF2B and BMRF2 The early lytic antigens are BMLF1, BMRF1, BALF2, BNLF2A, and BNL F2B, BMRF2, and combinations thereof.

[0065] In some embodiments, the late lytic antigen is BALF4, BILF1, BILF2 , BNFR1, BVRF2, BALF3, BALF5, BDLF3 and gp350 Late lytic antigens are selected: BALF4, BILF1, BILF2, BNFR1, BV RF2, BALF3, BALF5, BDLF3, gp350, and combinations thereof In some embodiments, the late lytic antigen is selected from the group consisting of B ALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5 and BDLF3. The late lytic antigen is selected from BALF4, BILF1, BILF 2. BNFR1, BVRF2, BALF3, BALF5, BDLF3, and their combinations In some embodiments, the late lytic antibody may be selected from the group consisting of a combination of The original is BDLF3.

[0066] In some embodiments according to various aspects of the present disclosure, the EBV latent antigen is EBNA. 1, EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BAR The EBV latent antigen is selected from EBV F1, LMP1, LMP2A and LMP2B. NA1, EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, B ARF1, LMP1, LMP2A, LMP2B, and combinations thereof In some embodiments, the EBV latent antigen is selected from EBNA1, The EBV latent antigen is selected from LMP1, LMP2A and LMP2B. , LMP1, LMP2A, LMP2B, and combinations thereof. In some embodiments, the EBV latent antigen is EBNA1, LMP1 and LMP2A. The EBV latent antigen is selected from EBNA1, LMP1, LMP2 A and combinations thereof.

[0067] In some embodiments, the EBV latent antigen is a type III latent antigen, a type II latent antigen, antigen or type I latency antigen.

[0068] In some embodiments, the type III latency antigen is EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA Type III latency antigens are selected from EBNA1, EBNA 3B, and EBNA3C. -LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and combinations thereof. In some embodiments, the type II latency antigen is EBNA1, EBNA-LP , LMP1, LMP2A, LMP2B and BARF1. Hara, EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1 and and combinations thereof. In the present invention, the type I latency antigen is selected from EBNA1 and BARF1. is selected from the group consisting of EBNA1, BARF1, and combinations thereof. be.

[0069] When an EBV antigen is referred to herein, the present disclosure refers to the isotype of a given EBV antigen, Fragments, variants (including mutants) are also contemplated. Any of the amino acid sequences, e.g., the amino acid sequences for the EBV antigens referred to herein. For example, The UniProt Knowledgebase (UniProtK B) can be searched from the UniProt Consortium, Nu Cleic Acids Research (2019), 47(D1):D506-D See 515.

[0070] "Isotype", "fragment" or "variant" of a reference EBV antigen, as the case may be, At least 60 amino acid sequences to the amino acid sequence of a reference EBV antigen (e.g., a reference isoform of the antigen). % amino acid sequence identity, preferably 70%, 75%, 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. be.

[0071] A "fragment" generally refers to a portion of a reference protein, such as a However, optionally, a reference protein (i.e., may be at least 20% of the length of the protein from which the fragment is derived, and may have a maximum length of The length is 50%, 75%, 80%, 85%, 90%, 95% or 99% of the total copies of a reference protein. Compared with the amino acid sequence, one or more amino acid substitutions, amino acid insertions, amino acid deletions or has an amino acid sequence that contains other modifications, but is The term "proteins" refers to proteins that share a significant degree of sequence identity (eg, at least 60%) with the target protein. "Isoform" generally refers to a variant of a reference protein expressed by EBV.

[0072] In some embodiments according to various aspects described herein, a given list References to "one or more" antigens from means anything between one and all of the antigens shown in the list. Depending on the number of antigens, the reference to "one or more" may refer to, for example, one or more of the antigens listed. It can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0073] Peptides and pepmixes In various aspects of the disclosure, a peptide corresponding to an EBV antigen is provided that is specific for the EBV antigen. The present invention is used in methods for generating / expanding populations of specific immune cells.

[0074] As used herein, a "peptide" refers to two peptides linked by a peptide bond. A peptide typically has a length of about 2 to 50 amino acids. A "polypeptide" is a polymeric chain of two or more peptides. Polypeptides are typically greater than about 50 amino acids in length.

[0075] As used herein, a peptide "corresponding to" a reference antigen is one that contains the amino acid sequence of the reference antigen. The antigen may comprise or consist of the amino acid sequence of a reference antigen. For example, The peptide that is "binding to EBNA1" is found within the amino acid sequence of EBNA1 (i.e., or a sequence of amino acids that is a subsequence of the amino acid sequence of It consists of an array of.

[0076] As used herein, peptides are typically 5-30 amino acids in length, e.g. , 5-25 amino acids, 10-20 amino acids, or 12-18 amino acids. In some embodiments, the peptides are 5, 6, 7, 8, 9, 10, 1 one of 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids In some embodiments, the peptide is about 15 amino acids in length.

[0077] "Peptide" as used herein refers to a population that includes non-identical peptides. There are cases.

[0078] In some embodiments according to various aspects of the disclosure, the method comprises administering more than one antigen to a subject. In such an embodiment, a peptide corresponding to each of the antigens is used. At least one corresponding peptide is present. For example, in the method, EBNA1 and When various peptides corresponding to LMP1 and EBNA1 are used, the peptides At least one peptide corresponding to LMP1 and at least one peptide corresponding to LMP2. include.

[0079] In some embodiments, the method comprises the step of: A peptide corresponding to the entirety of a given antigen is a peptide that is a sequence of the amino acid sequence of this antigen. That is, together these peptides span the entire amino acid sequence of a given antigen. Contains all of the amino acids.

[0080] A peptide corresponding to a portion of a given antigen spans a portion of the amino acid sequence of this antigen. In some embodiments, the peptides span a portion of the amino acid sequence of the antigen. For example, it may cover more than 10% of the amino acid sequence of the antigen, e.g. 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 95% This may cover one of the following:

[0081] In some embodiments, the methods use overlapping peptides. " share amino acids in common, and more typically share different sequences of amino acids. As an example, the first peptide may be an amino acid sequence corresponding to positions 1 to 15 of the amino acid sequence of EBNA1. The second peptide corresponds to the 5th to 20th amino acid sequence of EBNA1. The first peptide and the second peptide are composed of an amino acid sequence of 11 amino acids. These are overlapping peptides corresponding to EBNA1, which overlap with each other.

[0082] In some embodiments, the overlapping peptides are 1-20, 5-20, 8-15, or overlap by one of 10 to 12 amino acids. , overlapping peptides are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 In some embodiments, the nucleotide sequence overlaps with the nucleotide sequence of ... , the overlapping peptides overlap by 11 amino acids.

[0083] In some embodiments, the method comprises: Peptides having a length of 5 to 30 amino acids overlapping by 1 to 20 amino acids are used. can be.

[0084] In some embodiments, the method includes the step of generating 11 antigens corresponding to all given reference antigens. Peptides having a length of 15 amino acids overlapping each other by amino acids are used. A mixture of peptides is called a "pepmix peptide pool" or "pepmix peptide pool" for a given antigen. These may be referred to herein as "mixes."

[0085] For example, the "EBNA1 pepmix" used in Example 1 herein contains Amino acid sequence for EBNA1 as shown in niProt:P03211-1(v1) The sequence of 158 15-mer peptides overlapping by 11 amino acids spans the entire length of the sequence. Shows the pool.

[0086] In some embodiments according to various aspects of the present disclosure, a given EBV antigen "corresponding The "peptides of interest" may be a pepmix for this antigen.

[0087] Methods for generating / expanding populations of immune cells specific for EBV Various aspects of the present disclosure are directed to generating / expanding populations of immune cells specific for EBV. It relates to.

[0088] "EBV-specific immune cells" are cells that express EBV antigen peptides (e.g., MHC a receptor (preferably a T cell receptor) that can recognize the EBV-specific immune cells express / contain such receptors and such antigens. as a result of expression of an endogenous nucleic acid encoding a receptor, or expressing such a receptor may be manifested / included as a result of being manipulated in such a way.

[0089] Immune cells include cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, and lymphocytes. Lymphocytes may be, for example, T cells, B cells, NK cells, N They may be KT cells or innate lymphoid cells (ILCs), or their precursors. Immune cells can express, for example, CD3 polypeptides (e.g., CD3γ, CD3ε, CD3ζ or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD2 8, and may express CD4 or CD8.

[0090] To generate / expand virus-specific immune cell populations in vitro / ex vivo Various methods for this purpose are well known to those skilled in the art. Typical culture conditions (i.e., cell culture Culture medium, additives, temperature, gas atmosphere), cell number, culture period, etc. are determined as described, for example, in Ngo et al., J. Immunother. (2014) 37(4):193-203 (which is incorporated in its entirety) This can be determined by reference to the US Pat. No. 6,393,431, which is incorporated herein by reference.

[0091] Advantageously, the various cultures of cells according to the present disclosure are maintained in a humidified atmosphere containing 5% CO2. The cells of the cell culture according to the present disclosure may be cultured at 37° C. Any suitable density can be established, as can be readily determined, and For example, the culture may be cultured at about 0.5×1 per ml of culture. 0 6 ~Approx. 5×10 6 cells / ml (e.g., approximately 1 x 10 6 The initial density of the cells was established at 100%. This may be the case.

[0092] The culture is grown in any vessel suitable for the volume of the culture, e.g. For example, the wells of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, the cells are cultured in a bioreactor. See, e.g., Somerville and Dudley, Oncoimmunology ( 2012) 1(8):1435-1437, which is incorporated herein by reference in its entirety. In some embodiments, the cells are cultured in a bioreactor as described in The cells are then cultured in a GRex cell culture vessel, e.g., a GRex flask or a GRex1 The cells are cultured in a 00 bioreactor.

[0093] The methods of the disclosure generally involve screening a population of immune cells (e.g., cells that have an antigen-specific receptor). A heterogeneous population of immune cells (e.g., peripheral blood mononuclear cells; PBMCs) containing appropriate costimulatory and conditions that result in signal amplification, resulting in activation and expansion. Under these conditions, the presence of antigen-presenting cells (APCs) that present viral antigen peptide:MHC complexes was investigated. The process of T cell activation is well known to those skilled in the art and includes, for example, For example, Immunobiology (5th ed., Janeway CA Jr., Travel rs P, Walport M et al., New York: Garland Science. (2001), Chapter 8, which is incorporated by reference in its entirety. (I).

[0094] Specifically, the method comprises detecting T cell receptors specific for EBV antigenic peptide:MHC complexes. T cells containing the TCR bind to the EBV antigen peptide:MHC complex for which the TCR is specific. The process involves stimulating the APCs with EBV antigens / peptides (1 or by infection with a virus encoding EBV antigens / peptides (one or more) and presenting EBV antigenic peptides in the context of MHC molecules. Stimulation activates T cells, promoting cell division (proliferation), resulting in EBV formation. This results in the generation and / or expansion of a population of T cells specific for the V antigen.

[0095] The population of cells obtained after stimulation is specific for EBV antigens when compared to the pre-stimulation population. Allogeneic T cells are enriched (i.e., EBV antigen-specific T cells are increased in the population after stimulation). In this way, a population of T cells specific for EBV antigens is generated. They are expanded / generated from a heterogeneous population of various T cells with different specificities. A population of T cells specific for EBV antigens is generated that responds to stimulation and subsequent cell division. Thus, they can be generated from only one T cell. The pre-existing population of cells stimulates and, as a result, stimulates the expression of a population of EBV antigen-specific T cells. The cells may expand by cell division.

[0096] In embodiments of various aspects of the disclosure, specific for the EBV antigens herein It will be appreciated that the target immune cell is preferably a T cell. In some embodiments, the T cells are CD3+ and CD4+ T cells. In some embodiments, the T cells are CD3+ and CD8+ T cells. are T helper cells (T H In some embodiments, the T cells are cytotoxic The immune cell is preferably a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)). Expresses / contains a TCR specific for an EBV antigen peptide.

[0097] EBV-specific T cells are produced in response to viral antigens for which they are specific, or in response to the virus. may display certain functional properties of T cells in response to cells containing / expressing a specific antigen In some embodiments, the property is that of an effector T cell (e.g., a cytotoxic T It is a functional property related to a cell.

[0098] In some embodiments, the EBV-specific T cells exhibit one or more of the following characteristics: May be directed against EBV or cells that contain / express EBV antigens for which T cells are specific. cytotoxicity against EBV, or cells that contain / express EBV antigens for which T cells are specific Proliferation in response to IFNγ expression, CD107a expression, IL-2 expression, and TNFα expression , perforin expression, granzyme expression, granulysin expression and / or FAS expression Gand (FASL) expression.

[0099] EBV-specific T cells bind peptides of EBV antigens for which they are specific, in the appropriate MHC express / contain a TCR that can recognize when presented by a molecule. EBV The specific T cells may be CD4+ T cells and / or CD8+ T cells.

[0100] The disclosed method involves subjecting a population of immune cells to a peptide corresponding to an EBV antigen(s). or a peptide corresponding to EBV antigen(s). EBV antigen-specific APCs are contacted with APCs presenting EBV antigens or APCs presenting EBV antigens. Such method steps are referred to as "stimulation" or "stimulation steps." Such method steps may be referred to herein as These typically require maintenance in ex vivo culture and are referred to as "stimulator cultures." There are cases.

[0101] In some embodiments, the method includes one or more additional stimulation steps. That is, in some embodiments, the method includes restimulating the cells obtained by the stimulation step. Such a further stimulation step may be referred to as "restimulation". Such method steps may be referred to herein as "restimulation steps." Cells typically require maintenance in in vitro / ex vivo culture and are referred to as "restimulators." This is sometimes referred to as "intense culture."

[0102] PBMCs (for stimulation) or obtained by the stimulation process described herein A population of cells is then transfected (for restimulation) with a peptide (or peptides) corresponding to the EBV antigen(s). "Contacting" the PBMC / cell population with one or more of the peptides generally refers to Culturing in vitro / ex vivo in cell culture medium containing Similarly, it will be understood that the present invention involves the administration of EBV antigens (one or more) to the PBMC / cell population. The peptide or peptides are "contacted" with an APC presenting a peptide or peptides corresponding to the peptide or peptides. Generally, this means in vitro / in vitro incubation of the APCs and the PBMCs / cell population in cell culture medium. It will be appreciated that this involves ex vivo co-culturing.

[0103] In some embodiments, the method comprises: In such an embodiment, the method further comprises contacting the antibody with a peptide or peptides corresponding to the antibody. In addition, APCs (e.g., dendritic cells, macrophages, and B cells) contained in the PBMC population ) is the subsequent proliferation of CD8+ and / or CD4+ T cells in the PBMC population. For activation, antigens must be internalized (e.g., by phagocytosis or pinocytosis) and processed. and presented on the surface of MHC class I molecules (cross-presentation) and / or MHC class II molecules do.

[0104] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the ... The population of cells to be cultured is subjected to a peptide(s) corresponding to the EBV antigen(s). In such an embodiment, the APCs ( For example, dendritic cells, macrophages and B cells are included in the population of CD8+ T cells. For subsequent restimulation of cells and / or CD4+ T cells, antigens can be delivered (e.g., by phagocytosis) , by pinocytosis), processing, and expression of MHC class I molecules (cross-presentation) and and / or presented on the surface of MHC class II molecules.

[0105] In some embodiments, the method comprises: The method includes contacting the antibody with an APC that presents a peptide or peptides corresponding to the antibody. In an embodiment of the method, the method further comprises the step of: The population is then subjected to a ELISA to present a peptide(s) corresponding to an EBV antigen(s). The method includes contacting the APC with an APC that is

[0106] Co-culture of T cells with APCs in stimulation and restimulation according to the present disclosure in cell culture medium The cell culture medium is used to culture T cells and APCs in vitro / ex vivo depending on the conditions. The cell culture medium may be any medium that is maintained in the in vivo culture. Suitable culture media for use in culturing lymphocytes are well known to those skilled in the art. Examples of the medium include RPMI-1640 medium, AIM-V medium, and Iscove's medium. .

[0107] In some embodiments, the cell culture medium is RPMI-1640 medium and / or Crick medium (also known as Eagle-Ham amino acid (EHAA) medium) The composition of these media is well known to those skilled in the art. The formulation of -1640 medium is described, for example, in Moore et al., JAMA (1967) 199:519 The formulation of the Click medium is described in Click et al., Cell Immunology. Nol (1972) 3:264-276. RPMI-1640 medium was For example, ThermoFisher Scientific and Clippings are available. Culture medium can be obtained, for example, from Sigma-Aldrich (catalog no. C 5572).

[0108] In some embodiments, the methods of the present disclosure involve the use of a method for detecting EBV antigen(s) PBMCs contacted with a peptide or peptides that bind to EBV antigens or or more), in the presence of APCs presenting a peptide(s) corresponding to RPM The method involves culturing the cells in a cell culture medium containing I-1640 medium and Click medium. In some embodiments, the methods of the present disclosure involve the use of antibodies corresponding to EBV antigen(s). By the stimulation step described herein, the cells are contacted with a peptide(s). The resulting population of cells is then subjected to immunoprecipitation or immunoprecipitation with a peptide (s) corresponding to the EBV antigen (s). The cells were incubated in RPMI-1640 medium and Click medium in the presence of APCs expressing IgG1 or IgG2. The method involves culturing the cells in a cell culture medium containing the

[0109] In some embodiments, the cell culture medium is 25-65% (by volume) RPMI In some embodiments, the medium contains 25% to 65% of Crick medium. The cell culture medium was 30-60% RPMI-1640 medium and 30-60% Click In some embodiments, the cell culture medium comprises 35-55% RPMI. In some embodiments, the medium contains 35% to 55% Click medium. The cell culture medium was 40-50% RPMI-1640 medium and 40-50% Click In some embodiments, the cell culture medium comprises 45% RPMI-16 40 medium and 45% Click medium.

[0110] In some embodiments, the cell culture medium comprises one or more cell culture medium supplements. A variety of cell culture media additives are well known to those skilled in the art and include: , antibiotics (e.g., penicillin, streptomycin), serum (e.g., fetal bovine serum (FBS), bovine serum albumin (BSA), L-glutamine, cytokines / growth factors This includes children, etc.

[0111] In some embodiments, the cell culture medium contains 5-20% (by volume) FBS, e.g. For example, the medium contains 7.5 to 15% FBS, or 10% FBS. Therefore, cell culture medium should contain 0.5-5% GlutaMax, e.g., 1% Gluta In some embodiments, the cell culture medium contains 0.5-5% Pen / S Contains trep, e.g., 1% Pen / Strep.

[0112] In various aspects of the disclosure, antigen presenting cells (APCs) are immunoreactive cells specific for EBV. It is used in methods for generating / expanding populations of cells.

[0113] An APC according to the present disclosure may be a professional APC. L-APCs are specialized for presenting antigens to T cells and therefore have MHC-peptide It is efficient in processing and presenting tide complexes on the cell surface at high levels. Professional APCs include dendritic cells (DCs), macrophages, and inflammatory cells (ICCs). Non-professional APCs include MHC-peptide receptors, phages, and B cells. The complex can be presented to T cells, particularly MHC class I-peptide complexes, by CD8 + T cells.

[0114] In some embodiments, the APC is internalized (e.g., endothelial cells) by the APC. Cross-presentation of antigens (taken up by endocytosis / phagocytosis) on MHC class I CD8+ T cells are APCs that can internalize antigens on the MHC class I. Cross-presentation is described, for example, in Alloatti et al., Immunological Review s (2016), 272(1):97-108, which is incorporated by reference in its entirety. APCs capable of cross-presentation include, for example, dendritic cells. (DC), macrophages, B cells and sinusoidal endothelial cells.

[0115] As described herein, in some embodiments, EBV antigens (one or more APCs for stimulating immune cells specific for EBV antigens (one or more) A population of cells (e.g., PBMCs) containing immune cells specific for a plurality of Therefore, such a population of cells specific for EBV antigen(s) is the subject of the present invention. In such an embodiment, the APCs are expanded according to the methods described above. For example, dendritic cells, macrophages, B cells, or whatever cell type EBV A cell capable of presenting an antigen to immune cells specific for that antigen or antigens. There may be other cell types present in the population.

[0116] In some embodiments, the method comprises administering to the patient an EBV antigen(s) / peptide(s) thereof. APCs that have been modified to express / contain a gene(s) are used. In some embodiments, the APCs are administered peptides corresponding to EBV antigen(s). (one or more) has been contacted with said peptide(s), and Presentation may occur as a result of internalization of the peptide(s). In some embodiments, the APCs are "pulsed" with such peptide(s). This generally means that the APC is "treated" with the peptide ( of the peptide(s) for a period of time sufficient to internalize the peptide(s). This involves culturing in vitro in the presence of

[0117] In some embodiments, the APCs are peptides that respond to an EBV antigen(s). The peptide(s) are expressed in a cell as a result of expression from a nucleic acid encoding the antigen. APCs may present nucleic acid encoding the EBV antigen(s). , as a result of infection with EBV (e.g., in the case of B cells, APCs are those in which nucleic acid encoding an antigen(s) is / are present, e.g., trans. that have been introduced into cells by transfection, transduction, electroporation, etc. As a result, it may contain nucleic acid encoding an EBV antigen(s). When the nucleic acid encoding the antigen(s) is provided in a plasmid / vector, There is a match.

[0118] In some embodiments, the APCs used in the methods of the present disclosure are activated T cells. Cells (ATCs), dendritic cells, B cells (including, for example, LCLs), and artificial antigen presenting cells ( aAPC) (e.g., Neal et al., J Immunol Res Ther (2017) 2(1):68-79, and Turtle and Riddell, Cancer J. .(2010)16(4):374-381).

[0119] In some embodiments, the APCs are specific for an EBV antigen(s). The APCs are co-cultured to generate / expand a population of immune cells that contains specific immune cells. That is, in some embodiments, the APC is autologous with respect to the population of cells that comprises The APCs are derived from the same subject as the population of cells with which they are to be co-cultured ( Alternatively, the APCs may be obtained from the same subject as the population of cells with which they are to be co-cultured. (derived from cells that can

[0120] The use of polyclonal activated T cells (ATCs) as APCs, and Various methods for preparing IgG1 are described, for example, in Ngo et al., J Immunother. 14) 37(4):193-203, which is incorporated herein by reference. Briefly, ATCs were cultured in the presence of IL-2 and PBMCs. By stimulating with agonistic anti-CD3 and anti-CD28 antibodies in Thus, T cells can be generated by non-specific activation in vitro. .

[0121] Dendritic cells, for example, Ngo et al., J Immunother. (2014) 37(4) 193-203, according to methods well known in the art. Dendritic cells may be generated by CD14 selection from PBMCs. They may be prepared from monocytes that have been cultured to induce their differentiation into immature dendritic cells. The cells may be cultured in a cell culture medium that induces For example, it may contain IL-4 and GM-CSF. Immature dendritic cells may produce IL-6 By culturing in the presence of IL-1β, TNFα, PGE2, GM-CSF and IL-4, This can sometimes help them mature.

[0122] LCL may be prepared according to methods well known in the art, for example, by Hui-Y uen et al., J Vis Exp (2011) 57:3321, and Hussain et al. and Mulherkar, Int J Mol Cell Med(2012)1(2) :75-87, both of which are incorporated herein by reference in their entireties. Briefly, LCLs may be generated according to the method described in PBM using concentrated cell culture supernatant of V-producing cells (e.g., B95-8 cells). C can be produced by incubating it in the presence of cyclosporine A. can.

[0123] Artificial antigen presenting cells (aAPCs) contain, for example, the costimulatory molecules CD80, CD86, and CD87. These include K562cs cells engineered to express D83 and 4-1BBL ( The cells are, for example, as described in Suhoski et al., Mol Ther. (2007) 15(5):9 (described in 81-8).

[0124] In some embodiments, the APC is not an EBV-infected cell. In embodiments, the APC is not an EBV-infected B cell. APC is not EBV-LCL.

[0125] In some embodiments, the stimulating step comprises stimulating the PBMCs with EBV antigen(s). In some embodiments, the method comprises contacting the polypeptide with a peptide or peptides corresponding to In the present invention, the restimulation step comprises inducing immune cells specific for EBV antigen(s) into E APCs presenting peptide(s) corresponding to BV antigen(s); In some embodiments, the restimulation step comprises contacting with an EBV antigen (one or more of the EBV antigens). Immune cells specific for an EBV antigen(s) are transfected with peptides corresponding to the EBV antigen(s). The method includes contacting the peptide(s) with an ATC that displays the peptide(s).

[0126] In some embodiments, the method further comprises the step of: Further, agents for enhancing the expression of the antibody can be used. Such agents include, for example, costimulatory molecules. Cells expressing CD80, CD86, CD83 and / or 4-1BBL In some embodiments, the present invention includes a method for the production of a medicament for the treatment of a pulmonary artery disease, such as a pulmonary artery disease (PAD) or a pulmonary artery disease (PAD) cell. The cells expressing costimulatory molecules are HLA-negative and EBV replication-deficient LCLs, which Also referred to as "universal LCL" or "uLCL". As described in U.S. Patent Application Publication No. 2018 / 0250379(A1).

[0127] Other examples of agents for enhancing costimulation include, for example, costimulatory receptors expressed by T cells. Specific for stimulatory receptors (e.g., 4-1BB, CD28, OX40, ICOS, etc.) agonist antibodies and T cell-expressing costimulatory receptors. Costimulatory molecules that can Such agents include, for example, immobilized on beads. may be provided.

[0128] In some embodiments, the restimulation step comprises the step of: Specific immune cells responding to EBV antigen(s) in the presence of uLCL are identified. The method includes contacting the peptide(s) with an ATC that displays the peptide(s).

[0129] The immune cell population is subjected to a peptide (or peptides) corresponding to an EBV antigen (or antigens). ) and / or a peptide(s) corresponding to an EBV antigen(s). In order to promote the activation and proliferation of T cells, contacting the APCs with one or in the presence of multiple cytokines. , the stimulus is IL-7, IL-15, IL-6, IL-12, IL-4, IL-2 and / or or IL-21. These cytokines are Added exogenously to cytokines produced by cells in culture It will be understood that in some embodiments, the added cytokines The cytokines are recombinantly produced.

[0130] Thus, in some embodiments, the methods of the disclosure involve the use of EBV antigens (one or more PBMCs contacted with peptide(s) corresponding to the The presence of APCs presenting peptide(s) corresponding to V antigen(s). present, IL-7, IL-15, IL-6, IL-12, IL-4, IL-2 and / or involves culturing in the presence of one or more of IL-21.

[0131] In some embodiments, the culture comprises administering to the subject an IL-7, IL-15, IL-6, IL-12 In some cases, this is in the presence of IL-4, IL-2 and / or IL-21. In an embodiment, the culture comprises the addition of IL-7, IL-15, IL-6 and / or IL-12. In some embodiments, the culture is in the presence of IL-7 and / or In the presence of IL-15.

[0132] In some embodiments, the final concentration of IL-7 in the culture is 1 to 100 ng / m l, for example, 2 to 50 ng / ml, 5 to 20 ng / ml, or 7.5 to 15 ng / ml In some embodiments, the final concentration of IL-7 in the culture is one of 1 / ml. The concentration is approximately 10ng / ml.

[0133] In some embodiments, the final concentration of IL-15 in the culture is 1 to 100 ng / ml, for example, 2 to 50 ng / ml, 5 to 20 ng / ml, or 7.5 to 15 ng / ml. In some embodiments, the concentration of IL-15 in culture is one of 100 mg / ml. The final concentration is approximately 10 ng / ml.

[0134] In some embodiments, the final concentration of IL-15 in the culture is between 10 and 1000 nM. g / ml, for example, 20 to 500 ng / ml, 50 to 200 ng / ml, or 7 In some embodiments, the concentration of the IgG in the culture is one of 5 to 150 ng / ml. The final concentration of IL-15 is approximately 100 ng / ml.

[0135] In some embodiments, the final concentration of IL-6 in the culture is 10 to 1000 ng. / ml, for example, 20 to 500ng / ml, 50 to 200ng / ml, or 75 In some embodiments, the I The final concentration of L-6 is approximately 100 ng / ml.

[0136] In some embodiments, the final concentration of IL-12 in the culture is 1 to 100 ng / ml, for example, 2 to 50 ng / ml, 5 to 20 ng / ml, or 7.5 to 15 ng / ml. In some embodiments, the concentration of IL-12 in culture is one of 100 mg / ml. The final concentration is 10ng / ml.

[0137] In some embodiments, the final concentration of IL-7 is 1 to 100 ng / ml (e.g., One of the following: 2-50ng / ml, 5-20ng / ml, or 7.5-15ng / ml , for example, 10 ng / ml), and the final concentration of IL-15 is 10 to 1000 ng / ml (e.g., 20-500ng / ml, 50-200ng / ml, or 75-15 0 ng / ml, for example, about 100 ng / ml).

[0138] In some embodiments, the final concentration of IL-7 is 1 to 100 ng / ml (e.g., One of the following: 2-50ng / ml, 5-20ng / ml, or 7.5-15ng / ml , for example, 10 ng / ml), and the final concentration of IL-15 is 10 to 1000 ng / ml (e.g., 20-500ng / ml, 50-200ng / ml, or 75-15 0 ng / ml, for example, about 100 ng / ml).

[0139] In some embodiments, the final concentration of IL-7 is 1 to 100 ng / ml (e.g., One of the following: 2-50ng / ml, 5-20ng / ml, or 7.5-15ng / ml , for example, 10 ng / ml), and the final concentration of IL-6 is 10 to 1000 ng / ml ( For example, 20-500ng / ml, 50-200ng / ml, or 75-150ng / ml, for example, about 100 ng / ml), and the final concentration of IL-12 is 1 to 20 ng / ml. 100ng / ml (e.g., 2–50ng / ml, 5–20ng / ml, or 7.5– 10 ng / ml) and the highest concentration of IL-15. Final concentration is 1-100ng / ml (e.g., 2-50ng / ml, 5-20ng / ml, or or one of 7.5 to 15 ng / ml, for example, 10 ng / ml.

[0140] In some embodiments, the final concentration of IL-7 in the stimulated culture is 1 to 100 ng. / ml (e.g., 2–50 ng / ml, 5–20 ng / ml, or 7.5–15 ng / ml ml, e.g., 10 ng / ml), and IL-1 in stimulated cultures. 5 final concentration is 10-1000ng / ml (e.g., 20-500ng / ml, 50-2 00ng / ml, or one of 75-150ng / ml, e.g., about 100ng / ml).

[0141] In some embodiments, the final concentration of IL-7 in the stimulated culture is 1 to 100 ng. / ml (e.g., 2–50 ng / ml, 5–20 ng / ml, or 7.5–15 ng / ml ml, e.g., 10 ng / ml), and the final concentration of IL-6 in stimulated cultures Concentration is 10-1000ng / ml (e.g., 20-500ng / ml, 50-200ng / ml, or 75-150ng / ml, e.g., about 100ng / ml) and the final concentration of IL-12 in stimulated cultures is 1–100 ng / ml (e.g., 2–5 One of 0ng / ml, 5-20ng / ml, or 7.5-15ng / ml, e.g. For example, 10 ng / ml) and the final concentration of IL-15 in the stimulation culture is 1 to 10 0ng / ml (e.g., 2-50ng / ml, 5-20ng / ml, or 7.5-15 ng / ml, e.g., 10 ng / ml).

[0142] In some embodiments, the final concentration of IL-7 in the restimulation culture is 1 to 100 nm. g / ml (e.g., 2–50 ng / ml, 5–20 ng / ml, or 7.5–15 ng / ml) / ml, e.g., 10 ng / ml), and IL-1 in restimulation cultures -15 final concentration is 10-1000ng / ml (e.g., 20-500ng / ml, 50 ~200ng / ml, or 75-150ng / ml, e.g., about 100ng g / ml).

[0143] Stimulation and restimulation according to the methods of the present invention typically involve co-culture of T cells with APCs, for a period of time sufficient for the PC to stimulate the T cells and for the T cells to undergo cell division. This involves carrying out the process over a period of time.

[0144] In some embodiments, the methods of the present disclosure involve the use of a method for detecting EBV antigen(s) PBMCs contacted with a peptide or peptides that bind to EBV antigens or In the presence of APCs presenting a peptide(s) corresponding to at least 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days The method involves culturing the cells for one of three or more days, or for at least seven days. In some embodiments, the culture is for a period of 24 hours to 20 days, e.g., 4 8 hours to 14 days, 3 days to 12 days, 4 days to 11 days, 6 days to 10 days, or 7 Over a period of ~9 days.

[0145] In some embodiments, the methods of the present disclosure involve the use of a method for detecting EBV antigen(s) The stimulation step described herein involves contacting the cells with a peptide or peptides that or a peptide ( In the presence of APCs expressing one or more 4 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, or at least 7 days In some embodiments, the culturing involves culturing for a period of 24 For example, over a period of 48 hours to 14 days, 3 days to 12 days for one of the following periods: 4-11 days, 6-10 days, or 7-9 days That is it.

[0146] Stimulation and restimulation are achieved by separating cells in culture from the medium in which they were cultured. or the culture is terminated by dilution, e.g., by the addition of cell culture medium. In some embodiments, the method includes culturing or restimulating the cells. At the end of the intense incubation, the cells are harvested.

[0147] In some embodiments, the restimulation step according to the present disclosure comprises using cell culture medium (and and any other additives as described herein) to the cell culture medium for the restimulation step. in appropriate amounts to achieve the desired ratio / concentration of conditioned medium (and any additives). This may be established by adding

[0148] At the end of the culture period for a given stimulation or restimulation step, the cells are harvested and cultured. The cells may be harvested by centrifugation and separated from the supernatant. The culture supernatant may be separated from the cell pellet. The cell pellet may then be resuspended in, for example, For the stimulation step, the cells may be resuspended in cell culture medium. Therefore, the cells may be subjected to a washing step after harvesting. The washing step involves washing the cell pellet with isotonic buffer. Resuspend the cells in buffer (e.g., phosphate-buffered saline (PBS)) and centrifuge to remove the This may include harvesting the cells by centrifugation and discarding the supernatant.

[0149] A population of immune cells specific for EBV antigen(s) is generated according to the present disclosure Methods for inducing and / or amplifying typically involve more than one stimulation step. There is no upper limit to the number of stimulation steps that may be performed in the methods according to the present disclosure. In some embodiments, the method comprises more than 2, 3, 4 or 5 stimulation steps. In some embodiments, the method comprises the steps of: The method according to the present disclosure includes one of 12, 13, 14 or 15 stimulation steps. These stimulation steps may differ from each other.

[0150] In some embodiments, the PBMCs used in the methods of the present disclosure are CD45 RA positive cells are depleted. That is, in some embodiments, the PBMCs are "depleted" from RA positive cells. "CD45RA-positive cell-depleted PBMCs" or "CD45RA-negative PBMCs" Depletion of CD45RA positive cells is achieved by generating / expanding cells according to the methods of the present disclosure. The aim of the present invention is to reduce the number of NK cells and / or regulatory T cells in the population of cells that are Illustrated.

[0151] In some embodiments, the method comprises isolating CD45RA positive cells from PBMCs, e.g. For example, a depletion step prior to a stimulation step according to the present disclosure. The method further comprises extracting CD45RA positive cells from the cells obtained by the stimulation step according to the present disclosure. For example, a depletion step prior to a restimulation step according to the present disclosure. Cell depletion is carried out by any suitable method, for example by MACS. For example, a Miltenyi Biotec column and an anti-CD45RA antibody This can be achieved by MACS using magnetic beads coated with IgG.

[0152] In some embodiments, to derive APCs for use in the methods of the present disclosure, The cell population used is depleted of CD45RA positive cells. In one embodiment, the population of cells used to derive the APCs is a "CD45RA positive For example, ATCs are a “cell-depleted” or “CD45RA-negative” population. In an embodiment used as a marker, ATCs are a population of CD45RA positive cell depleted PBMCs. or may be derived from a population of CD45RA negative PBMCs.

[0153] In some embodiments, the method further comprises: Specific immune cells are modified to increase IL-7-mediated signaling in the cells. IL-7-mediated signaling promotes survival and antitumor activity of tumor-specific T cells. It has been shown to increase: e.g., Shum et al., Cancer Discov. (2017)7(11):1238-1247, and International Publication WO2018 / 0389 See paragraph 45(A1).

[0154] Specific Exemplary Embodiments of Method Steps The following specific exemplary method steps are specifically contemplated in connection with the present disclosure.

[0155] (A) PBMCs were cultured using BZLF1 pepmix, BRLF1 pepmix, or BMRF1 pepmix. pepmix, BMLF1 pepmix, BALF2 ​​pepmix, BNLF2A pepmix contacting the cells with the BMRF2 pepmix, BNLF2B pepmix and / or BMRF2 pepmix; and stimulating immune cells specific for EBV lytic antigens.

[0156] (B) PBMCs were cultured using BZLF1 pepmix, BRLF1 pepmix, or BMRF1 pepmix. Pepmix, BMLF1 Pepmix, BXLF1 Pepmix, BALF1 Pepmix BLLF2 pepmix, BALF2 ​​pepmix and / or BNLF2A pepmix By contacting the mixture with the EBV antibody, immune cells specific for EBV lytic antigens are stimulated. To do so.

[0157] (C) PBMCs were cultured with BZLF1 pepmix, BRLF1 pepmix and / or By contacting the cells with the BMRF1 pepmix, specific immunity against EBV lytic antigens was induced. Stimulating immune cells.

[0158] (D) PBMCs were cultured using BMRF1 pepmix, BMLF1 pepmix, or BALF2 ​​pepmix. Pepmix, BNLF2A pepmix, BNLF2B pepmix and / or BM By contacting the cells with the RF2 pepmix, immune cells specific for EBV lytic antigens were produced. Stimulating the cells.

[0159] (E) PBMCs. BZLF1 pepmix, BRLF1 pepmix, BMRF1 pepmix, BML F1 pepmix, BALF2 ​​pepmix, BNLF2A pepmix, BNLF2B pepmix and / or BMRF2 pepmix; and EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix By contacting the infected cells with EBV, immune cells specific for EBV lytic antigens and EBV latency are recruited. Stimulating immune cells specific for an antigen.

[0160] (F) PBMCs. BZLF1 pepmix, BRLF1 pepmix, BMRF1 pepmix, BML F1 pepmix, BXLF1 pepmix, BALF1 pepmix, BLLF2 pepmix mix, BALF2 ​​pepmix and / or BNLF2A pepmix; and EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix By contacting the infected cells with EBV, immune cells specific for EBV lytic antigens and EBV latency are recruited. Stimulating immune cells specific for an antigen.

[0161] (G) PBMCs. BZLF1 pepmix, BRLF1 pepmix and / or BMRF1 pepmix cus; and EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix By contacting the infected cells with EBV, immune cells specific for EBV lytic antigens and EBV latency are recruited. Stimulating immune cells specific for an antigen.

[0162] (H) PBMCs. BMRF1 pepmix, BMLF1 pepmix, BALF2 ​​pepmix, BNL F2A pepmix, BNLF2B pepmix and / or BMRF2 pepmix and EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix By contacting the infected cells with EBV, immune cells specific for EBV lytic antigens and EBV latency are recruited. Stimulating immune cells specific for an antigen.

[0163] (I) Immune cells induced by any one of (A) to (H) were treated with IL-7, IL-15, or IL Stimulation in the presence of IL-6 and / or IL-12.

[0164] (J) Inhibition of immune cells by any one of (A) to (I) with IL-7 and / or IL- Stimulate in the presence of 15.

[0165] (K) The PBMCs are depleted of CD45RA positive cells. or stimulating immune cells.

[0166] (L) BZLF1 pepmix, BRLF1 pepmix, BMRF1 pepmix, BMLF1 pepmix, BALF2 ​​pepmix, BNLF2A pepmix, BNL APs pulsed with F2B pepmix and / or BMRF2 pepmix To stimulate immune cells specific for EBV lytic antigens by coculture with C .

[0167] (M) BZLF1 pepmix, BRLF1 pepmix, BMRF1 pepmix, BMLF1 Pepmix, BXLF1 Pepmix, BALF1 Pepmix, BLLF 2 pepmix, BALF2 ​​pepmix and / or BNLF2A pepmix Co-culture with repulsed APCs produced immune cells specific for EBV lytic antigens. Stimulating the cells.

[0168] (N) BZLF1 pepmix, BRLF1 and / or BMRF1 pepmix Co-culture with more pulsed APCs resulted in specific immunity to EBV lytic antigens. Stimulating cells.

[0169] (O) BMRF1 pepmix, BMLF1 pepmix, BALF2 ​​pepmix, BNLF2A pepmix, BNLF2B pepmix and / or BMRF2 pepmix Co-culture with APCs pulsed with EBV resulted in specific expression of EBV lytic antigens. Stimulating targeted immune cells.

[0170] (P) EBV lytic antigen-specific immune cells and EBV latent antigen-specific immune cells Immune cells, BZLF1 pepmix, BRLF1 pepmix, BMRF1 pepmix, BML F1 pepmix, BALF2 ​​pepmix, BNLF2A pepmix, BNLF2B pepmix and / or BMRF2 pepmix; and EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix Stimulation by co-culture with APCs pulsed with IgG.

[0171] (Q) What are the immune cells specific for EBV lytic antigens and those specific for EBV latent antigens? Immune cells, BZLF1 pepmix, BRLF1 pepmix, BMRF1 pepmix, BML F1 pepmix, BXLF1 pepmix, BALF1 pepmix, BLLF2 pepmix mix, BALF2 ​​pepmix and / or BNLF2A pepmix; and EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix Stimulation by co-culture with APCs pulsed with IgG.

[0172] (R) EBV lytic antigen-specific immune cells and EBV latent antigen-specific immune cells Immune cells, BZLF1 pepmix, BRLF1 pepmix and / or BMRF1; and To EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix Stimulation by co-culture with APCs pulsed with IgG.

[0173] (S) EBV lytic antigen-specific immune cells and EBV latent antigen-specific immune cells Immune cells, BMRF1 pepmix, BMLF1 pepmix, BALF2 ​​pepmix, BNL F2A pepmix, BNLF2B pepmix and / or BMRF2 pepmix and EBNA1 pepmix, LMP1 pepmix and / or LMP2 pepmix Stimulation by co-culture with APCs pulsed with IgG.

[0174] (T) The APC is an ATC. To become excited.

[0175] (U) The ATCs are derived from PBMCs that have been depleted of CD45RA positive cells. ) to stimulate immune cells.

[0176] (V) Stimulation of immune cells using any one of (L) to (U) in the presence of uLCL and.

[0177] (W) Inhibition of immune cells by any one of (L) to (V) with IL-7 and / or IL- Stimulate in the presence of 15.

[0178] (X) Stimulating immune cells with any one of (A) to (K), followed by (L) to (W ) to stimulate immune cells.

[0179] Characteristics of the disclosed methods / populations of immune cells generated / expanded by the disclosed methods sex The methods of the disclosure optionally include a method for determining the properties of the method and / or a compound produced by the method. Characterized by reference to the properties of the population of immune cells that are acquired / expanded There is.

[0180] In some embodiments, the immune system generated / expanded according to the methods of the present disclosure The population of cells has one or more of the following characteristics: a) stimulation with a peptide(s) corresponding to one or more EBV lytic antigens containing cells that produce IFNγ in response to stimulation; b) stimulation with a peptide(s) corresponding to one or more EBV lytic antigens; The cells contain cells that produce IFNγ in response to stimulation and that are capable of binding to one or more EBV latent antigens. The cells produce IFNγ in response to stimulation with the corresponding peptide(s). To c) contain cells that produce IFNγ in response to stimulation by EBV-infected cells; d) cytolytic activity against autologous EBV-infected cells; e) Anticancer activity against EBV-positive cancers in vivo; f) inhibition of EBV-positive tumor growth in vivo; and g) Reduction of metastasis of EBV-positive cancer in vivo.

[0181] The population of immune cells is subjected to a peptide(s) corresponding to an EBV antigen and / or Contains cells that produce IFNγ in response to stimulation by EBV-infected cells (e.g., LCLs) The immune cell population is assessed, for example, by ELISPOT analysis, to see whether In this case, the ELISPOT assay may be performed, for example, as described in Example 2. It may be said that.

[0182] A population of immune cells was assayed for cytolytic activity against autologous EBV-infected cells by Zarits Kaya et al., Expert Rev Vaccines (2011), 9(6):601 616, which is incorporated herein by reference in its entirety. Using, for example, 51 This may be assessed using a Cr release assay. is performed using autologous EBV-LCL, e.g., as described in Example 3. There is.

[0183] A population of immune cells has anti-cancer activity against EBV-positive cancers and / or EBV-positive tumors. Inhibition of tumor growth of the compound may be assessed in vivo by assay in an appropriate model. Suitable models and analyses include the EBV-LCL xenotransplantation model used in Example 4. Metastasis can be measured by localization of cancer cells in animals in such models. The presence of the serotype can be assessed by monitoring, for example, as performed in Example 4. This can be done.

[0184] In some embodiments, the immune system generated / expanded according to the methods of the present disclosure The population of cells was grown according to a reference method for generating / expanding EBV-specific immune cells. Compared to the population of immune cells generated / expanded, one or more of the following: May have: h) upon stimulation with a peptide(s) corresponding to one or more EBV antigens containing a greater number / proportion of cells that produce IFNγ in response; i) stimulation with a peptide(s) corresponding to one or more EBV lytic antigens; containing a greater number / proportion of cells that produce IFNγ in response to stimulation; j) A larger number of different EBV antigens (i.e., a wider variety of EBV antigens) containing specific cells; k) A higher number / percentage of cells producing IFNγ in response to stimulation by EBV-infected cells Contains; L) greater anticancer activity against EBV-positive cancers in vivo; m) greater inhibition of EBV-positive tumor growth in vivo; n) greater reduction in metastasis of EBV-positive cancers in vivo; o) greater persistence in vivo; and p) inhibiting one or more pro-inflammatory sites in the subject to which the population of immune cells is administered. Increased amounts of cytokines (e.g., GM-CSF and / or IFNγ) are produced. To induce. p) administering to the subject one or more anti-inflammatory cytokines Inducing reduced production of cytokines (e.g., IL-10) results.

[0185] The survival / persistence of a given population of immune cells in vivo can be assessed, for example, by detecting whether the cells express a detectable marker. using methods in which cells are labeled with a reporter or a marker and their survival monitored over time. Such methods can include, for example, probing cells with firefly luciferase. This includes labelling the target protein and measuring activity at different time points.

[0186] The production of cytokines by a subject to which a given population of immune cells is administered can be monitored, e.g., by measuring the production of cytokines from the subject. This can be assessed by analysis of available blood-derived samples (e.g., whole blood, plasma, serum). Cytokine levels can be measured, for example, by ELISA, e.g., as described in Example 4. This can be determined by ELISA as described in.

[0187] As a reference method to generate / expand EBV-specific immune cells, the latent pepmid Examples of such methods include those described in Example 1 herein, in which cox (only) is used in stimulation. This may be the case.

[0188] Methods of using populations of immune cells generated / expanded by the methods of the present disclosure The present invention relates to a method for treating EBV-related diseases, comprising: The populations of immune cells comprising such antibodies find use in therapeutic and / or prophylactic methods.

[0189] A method for treating / preventing a disease / condition in a subject, comprising administering to a subject a therapeutically effective amount of a compound according to the methods of the present disclosure. A population of EBV-specific immune cells generated / expanded by The present invention provides a method for use in a method of medical treatment / prevention comprising administering to said subject a subject a therapeutically effective amount of said compound. Populations of EBV-specific immune cells generated / expanded according to the disclosed methods Also provided are compositions for use in methods for treating / preventing a disease / condition. Also provided are populations of immune cells generated / expanded according to the methods of the present disclosure. In the manufacture of a medicament for use in a method for treating / preventing a disease / condition. EBV-specific immune responses generated / expanded according to the methods of the present disclosure in Uses of the population of cells are also provided.

[0190] In particular, methods for treating / preventing diseases / conditions by adoptive cell transfer (ACT) EBV-specific immune cells generated / expanded according to the present disclosure The use of populations of

[0191] Adoptive cell transfer generally involves transferring cells (e.g., immune cells) to a host, typically from a cell line from which it was isolated. This refers to the process by which cells are obtained from a subject by drawing a blood sample containing the cells. , typically modified and / or expanded, and then subsequently transferred to the same subject (adoptive transfer of autologous / autologous cells) The cells are administered to either a different subject (in the case of adoptive transfer of allogeneic cells) or to a different subject (in the case of adoptive transfer of allogeneic cells). The treatment typically involves providing a population of cells to a subject that have certain desired properties, or To increase the frequency of such cells having such properties in the subject. Adoptive transfer refers to the introduction of a cell or cell population into a subject and / or the transfer of a cell or cell population to a subject. Alternatively, the procedure may be performed with the aim of increasing the frequency of a particular cell population in a subject.

[0192] Adoptive transfer of immune cells has been reported, for example, in Kalos and June, 2013, Immuni ty 39(1):49-60, and Davis et al., 2015, Cancer J.2 1(6):486-491, both of which are incorporated by reference in their entireties. Those of skill in the art will recognize suitable reagents for adoptive transfer of cells in accordance with the present disclosure. and procedures as described, for example, in Dai et al., 2016, J Nat Cancer Inst, 1 08(7):djv439, which is incorporated by reference in its entirety. It can be determined by:

[0193] In some embodiments, the method further comprises: (a) A population of immune cells specific for EBV is generated / expanded according to the methods of the present disclosure. to do great things; and (b) subjecting said generated / expanded population of immune cells specific for EBV to To be administered to Includes.

[0194] In some embodiments, the method further comprises: (a) isolating / obtaining a population of immune cells (e.g., PBMCs) from a subject; (b) A population of immune cells specific for EBV is generated / expanded according to the methods of the present disclosure. to do great things; and (c) subjecting said generated / expanded population of immune cells specific for EBV to To be administered to Includes.

[0195] In some embodiments, the subject from which immune cells (e.g., PBMCs) are isolated is The same subjects to whom the generated / expanded population of immune cells specific for BV is administered. (i.e., the adoptive transfer may be autologous / autologous cells). In the method, the subject from which immune cells (e.g., PBMCs) are isolated is subjected to a specific The generated / expanded population of immune cells may be administered to a subject other than the subject to which it is administered (e.g., i.e., adoptive transfer may be of allogeneic cells).

[0196] In some embodiments, the method may include one or more of the following: R: Obtaining a blood sample from the subject; isolating immune cells (e.g., PBMCs) from said blood sample; EBV-specific immune cell populations are generated / expanded according to the methods of the present disclosure thing; harvesting / isolating said population of immune cells specific for EBV; The population of immune cells specific for EBV is combined with an adjuvant, diluent or carrier. To mix; administering to the subject said population of immune cells specific for EBV.

[0197] The method includes attenuating the onset / progression of a disease / condition, alleviating symptoms of a disease / condition, or The method may be effective for reducing the pathology of the disease / condition. to prevent progression of a disease / condition, e.g. to prevent worsening of a disease / condition or to It may be effective to slow the onset of the condition. The method may provide an improvement in a disease / condition, e.g., a reduction in the symptoms of a disease / condition, or may cause a decrease in some other correlate of the severity / activity of the disease / condition. In some embodiments, the methods involve treating a disease / condition at a later stage (e.g., For example, it may prevent the progression of the disease (e.g., chronic stage or metastasis).

[0198] Therapeutic and prophylactic utilities of populations of cells generated / expanded according to the present disclosure However, the cure may not be evident from a reduction in EBV load and / or number / activity of EBV-infected cells. What diseases / conditions might benefit from treatment or prophylactic benefit? It will be understood that the present invention extends to the treatment / prevention thereof.

[0199] For example, the disease / condition may be a disease / condition in which EBV or cells infected with EBV are pathologically involved. The condition, e.g., EBV infection, may be involved in the initiation, development or progression of a disease / condition, and / or the progression of a disease. A disease / condition that is positively associated with the severity of one or more symptoms of the disease / condition, or Diseases / conditions in which BV infection is a risk factor for the initiation, development or progression of the disease / condition It may be the case.

[0200] Treatment is aimed at reducing the EBV load, decreasing the number / percentage of EBV-positive cells, Reducing the activity of EBV positive cells, delaying the onset / progression of symptoms of the disease / condition / to prevent, reduce the severity of symptoms of the disease / condition, and inhibit the survival / development of EBV positive cells; and / or increasing survival of the subject. There are cases.

[0201] In some embodiments, the disease to be treated / prevented according to the present disclosure The disease / condition is a disease / condition characterized by EBV infection.

[0202] In some embodiments, the subject is diagnosed with EBV / EBV-infected cells, e.g., For example, its detection 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 EBV Detection of positive cancer cells (e.g., EBV-positive cells in tumors) allows the detection of the EBV-positive cells described herein. The disease / condition may affect any tissue or organ. In some embodiments, the disease may affect any organ system. The disease / condition may affect several tissues / organs / organ systems.

[0203] In some embodiments, the subject is infected with EBV, or in other words, infected with EBV. Based on the determination that the subject contains infected cells, the subject is selected for treatment / prophylaxis according to the present disclosure. This may be the case.

[0204] EBV, for example, Jha et al., Front Microbiol. (2016) 7:1 As outlined in US Pat. No. 602, which is incorporated herein by reference in its entirety, , and has been implicated in several cancers.

[0205] Thus, in some embodiments, the subject matter that is treated / prevented according to the present disclosure is The disease that causes this is cancer.

[0206] Cancer is caused by unwanted cell proliferation (or any disease caused by unwanted cell proliferation). It can refer to any type of cancer, neoplasm, or tumor. or malignant, and may be primary or secondary (metastatic). A neoplasm or tumor is any growth or proliferation of cells that is characterized by an abnormal growth or proliferation of cells. Cancer can be a growth of any organ and can be located in any tissue. For example, the adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bones, bone marrow, brain, breasts, appendix, central nervous system System (including or excluding brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells alveoli (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx , liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx , omentum, oral cavity, ovaries, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon , skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva and / or may be of tissue / cell origin from white blood cells.

[0207] Tumors may be of the nervous system or non-nervous system. Nervous system tumors include those of the central nervous system or the peripheral nervous system: e.g., gliomas, medulloblasts tumor, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-neural cancers / tumors are tumors of any tissue that is involved with other non-neural tissues. Examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-small cell lung cancer, and pulmonary fibrosis. Hodgkin's lymphoma (NHL), chronic myeloid leukemia (CML), acute bone marrow leukemia Myelodysplastic leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL) , chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer Cancers include colon, ovarian, pancreatic, thymic, NSCLC, hematological cancers and sarcomas. can be.

[0208] In some embodiments, the cancer is colon cancer, colon carcinoma, 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 Cancer, bladder cancer, urothelial cancer, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer or The tumor is selected from the group consisting of myeloma, ovarian tumors, and thyroid tumors.

[0209] In some embodiments, the cancer to be treated / prevented is EBV-associated gastroenteritis. "EBV-associated" cancers are cancers that are caused by infection with EBV or The onset, development, or progression of cancers that worsen, cancers for which infection is a risk factor, and / or infections , may be cancer that is positively associated with severity or metastasis.

[0210] EBV-associated cancers that may be treated / prevented according to the present disclosure include B cell-associated Cancer, e.g. Burkitt's lymphoma, post-transplant lymphoproliferative disorder (PTLD), central nervous system Lymphoma (CNS lymphoma), Hodgkin's lymphoma, non-Hodgkin's lymphoma, and immunodeficiency EBV-associated lymphoma associated with (e.g., EBV-positive lymphoma associated with X-linked lymphoproliferative disease) , EBV-positive lymphoma associated with HIV infection / AIDS, and oral hairy leukoplakia) , and cancers associated with epithelial cells, such as nasopharyngeal carcinoma (NPC) and gastric cancer. (GC) and others.

[0211] In some embodiments, the cancer is lymphoma (e.g., EBV positive lymphoma), cervical squamous cell carcinoma (HNSCC; e.g., EBV-positive HNSCC), nasopharyngeal carcinoma (NPC EBV-positive NPC), and gastric cancer (GC; e.g., EBV-positive GC). It is selected.

[0212] EBV infection has also been implicated in multiple sclerosis and systemic lupus erythematosus (SLE; e.g., A Scherio and Munger, Curr Top Microbiol Immunol. Nol. (2015);390(Pt 1):365-85) It is also involved in the onset and progression of autoimmune diseases, and the EBV antigen EBNA2 is involved in SLE, Multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, juvenile idiopathic arthritis and sepsis In recent years, it has been shown to associate with a gene region that is associated with risk factors for the onset of Riac disease. As shown (Harley et al., Nat Genet. (2018) 50(5):699 -707).

[0213] Thus, in some embodiments, the subject matter that is treated / prevented according to the present disclosure is The diseases / conditions that may be affected are SLE, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and type 1 diabetes. The arthritis is selected from chronic arthritis, juvenile idiopathic arthritis and celiac disease.

[0214] The population of cells generated / expanded according to the present disclosure also has the potential to be EBV-infected independent. find use in the treatment / prevention of diseases / conditions.

[0215] Cells generated / expanded according to the present disclosure as described hereinabove. The population finds use in methods for treating cancer.

[0216] In some embodiments, the cancer may express a cancer antigen. , which are antigens expressed or overexpressed by cancer cells. Peptides / polypeptides, glycoproteins, lipoproteins, glycans, glycolipids, lipids or may include any of their fragments. may be associated with cancer. Cancer antigens may be aberrantly expressed by cancer cells. (e.g., cancer antigens are expressed with abnormal localization) or are expressed differently by cancer cells. Cancer antigens may be expressed with normal structures and can elicit an immune response. In some embodiments, the antigen is expressed on the cell surface of a cancer cell. That is, the cancer antigen may be a cancer cell surface antigen. The portion of the antigen that is bound by the antigen-binding molecule described herein is present on the outer surface of the cancer cell. In some embodiments, the cancer antigen is exposed (i.e., extracellular). Cancer-associated antigens are antigens whose expression is associated with the onset, progression, or severity of cancer symptoms. may be involved in the causation or pathology of cancer, or may be abnormally expressed as a result of cancer In some embodiments, the cancer antigen may be, for example, a comparable non-cancerous cell. compared to expression levels by cells (e.g., non-cancerous cells from the same tissue / cell type). If a gene is expressed by a cancer cell, its expression (e.g., at the RNA level and / or protein level) In some embodiments, the antigen is upregulated (at a qualitative level). Cancer antigens are preferentially expressed by cancer cells and are not expressed by comparable non-cancerous cells (e.g. For example, non-cancerous cells derived from the same tissue / cell type. In an embodiment, the cancer antigen is a gene that is a product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, the cancer antigen is an overexpressed cellular protein, a gene Cancer antigens produced by oncogenic viruses, oncofetal antigens, cell surface molecules (e.g. In some embodiments, the IL-16 polypeptide is a product of a polypeptide that is a cell surface protein or glycoprotein. Cancer antigens are expressed by cell signaling molecules, such as cytokines, chemokines, and interferons. In some embodiments, the antibody is a galactamycin, an interleukin, or a lymphokine. The antigens are growth factors or hormones.

[0217] In some embodiments, the cancer is "positive" for the cancer antigen, which means The cancer comprises cells that express the cancer antigen (e.g., cells that express the cancer antigen on their cell surface). This means that cancer antigen-positive cancers may overexpress cancer antigens. Overexpression of a cancer antigen is greater than the expression level by comparable non-cancerous cells / non-tumor tissues. It can be revealed by detecting gene expression levels or protein expression levels. Cancer antigen gene / protein expression is known to affect the onset, development, progression or progression of cancer symptoms. may be a risk factor for severity and / or metastasis; and / or There are cases where they are positively related.

[0218] subject The subject according to various aspects of the disclosure described herein may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be a patient. The subject may be a person suffering from a disease requiring treatment. You may have been diagnosed with or are suspected of having such a disease / condition or at risk of developing or suffering from such a disease / condition There may be some sexual orientation.

[0219] In embodiments according to the present disclosure, the subject is preferably a human subject. In certain embodiments, the patient is treated according to the therapeutic or prophylactic methods disclosed herein. The subject may be a subject who has a disease / condition or who is at risk for developing a disease / condition. In an embodiment according to the present disclosure, a subject is diagnosed with a particular marker for such a disease / condition. The cells may be selected for treatment by a method based on the characterization of the cells.

[0220] Sequence identity Determining the percent identity between two or more amino acid or nucleic acid sequences Pairwise and multiple sequence alignments for this purpose can be performed using, for example, published Using computer software available from the University of California, San Jose, for example the ClustalOmega software, Soding, J., 2005, Bioinformatics 21, 95 1-960), T-coffee (Notredame et al., 2000, J.Mol.Bi ol.(2000)302, 205-217), Kalign software (Lassm Ann and Sonnhammer, 2005, BMC Bioinformatics , 6(298)), and the MAFFT software (Katoh and Standley , 2013, Molecular Biology and Evolution, 30 (4) 772-780) and the like, and can be achieved in a variety of ways known to those skilled in the art. When using such software, for example, gap penalties can be set. For the elongation penalty, the default parameters are preferably used. do.

[0221] Numbered paragraphs The following numbered paragraphs (para) describe features and feature combinations contemplated in connection with this disclosure. Further description of the alignment is provided:

[0222] 1. Contains immune cells specific for Epstein-Barr virus (EBV) lytic antigens A method for generating or expanding a population of immune cells, comprising: (i) culture medium containing EBV lytic antigens that are specific for all or part of one or more EBV lytic antigens; or (ii) one or more peptides, or the whole of one or more EBV lytic antigens. or a portion thereof. The method includes stimulating immune cells specific for EBV lytic antigens by contacting the method.

[0223] 2. The immune cells specific for EBV lytic antigens are treated with one or more EBV lytic antigens. The antigen is contacted with APCs presenting one or more peptides corresponding to all or a portion of the antigen. and further restimulating said immune cells specific for EBV lytic antigens by The method of claim 1 , comprising:

[0224] 3. The one or more EBV lytic antigens are BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF 2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5 and BD 2. The method of claim 1, wherein the LF1 is selected from LF2 and LF3.

[0225] 4. The one or more EBV lytic antigens are BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2, and BDLF3 The method of claim 1, wherein the .alpha.-amino acid is selected from the group consisting of .alpha.-amino acid and .beta.-amino acid.

[0226] 5. The method of claim 1, wherein the PBMCs are depleted of CD45RA positive cells. The method according to

[0227] 6. Immune cells specific for Epstein-Barr virus (EBV) lytic antigen and E and to generate a population of immune cells comprising immune cells specific for BV latent antigens. A method for the culture or expansion of peripheral blood mononuclear cells (PBMCs), comprising: (i) culturing one or more one or more peptides corresponding to all or part of a number of EBV lytic antigens, or one or more peptides corresponding to all or part of a plurality of EBV latent antigens; or (ii) one or more antibodies corresponding to all or part of one or more EBV lytic antigens. corresponds to a plurality of peptides and to all or part of one or more EBV latent antigens by contacting the cells with antigen-presenting cells (APCs) that present one or more peptides. EBV lytic antigen-specific immune cells and EBV latent antigen-specific immune cells The method includes stimulating the cells.

[0228] 7. The immune cells specific for EBV lytic antigens and the immune cells specific for EBV latent antigens and a antibody that responds to all or a portion of one or more EBV lytic antigens. or peptides, and one or more corresponding to all or part of an EBV latent antigen. By contacting the cells with APCs presenting a number of peptides, the cells were specifically targeted to EBV lytic antigens. and restimulating said immune cells specific for EBV latent antigens. 7. The method of claim 6, further comprising:

[0229] 8. The one or more EBV lytic antigens are BZLF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF 2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5 and BD 7. The method of claim 6, wherein the LF1 is selected from LF2 and LF3.

[0230] 9. The one or more EBV lytic antigens are BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BNLF2B, BMRF2, and BDLF3 The method of claim 6, wherein the .alpha.-amino acid is selected from the group consisting of .alpha.-amino acid and .beta.-amino acid.

[0231] 10. The one or more EBV latent antigens are EBNA1, EBNA-LP, EBN A2, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1, LMP2A and LMP2B.

[0232] 11. The one or more EBV latent antigens are EBNA1, LMP1, LMP2A and and LMP2B.

[0233] 12. The method of claim 1, wherein the PBMCs are depleted of CD45RA positive cells. 6. The method according to claim 6.

[0234] 13. A method for the isolation of immune cells obtained or obtainable by the method of claim 1. A separated group.

[0235] 14. A method for treating or preventing a disease or disorder comprising the steps of: 14. A method comprising administering to a subject the isolated immune cell population described in 13.

[0236] 15. The method of claim 14, wherein the disease or disorder is cancer.

[0237] 16. The disease or disorder is selected from the group consisting of EBV-positive lymphoma, EBV-positive nasopharyngeal carcinoma and EBV-positive lymphoma. The method of claim 14, wherein the cancer is an EBV-associated cancer selected from EBV-positive gastric cancer.

[0238] The present invention encompasses a variety of combinations of the described aspects and preferred features. Combinations are included unless they are clearly unacceptable or clearly avoided.

[0239] The section headings used herein are for organizational purposes only and are not intended to be limiting. Nothing in this document should be construed as limiting the subject matter covered herein.

[0240] Various aspects and embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings, in which: Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in the text are incorporated herein by reference.

[0241] Throughout this specification, including the following claims, unless the context otherwise requires, , the words "comprise", and "comprises" and Conjugations such as "comprising" are used with the specified integer ) or a step or a group of whole or steps, but Exclude any other whole or process or group of whole or processes It will be understood that this does not mean that

[0242] As used in this specification and the appended claims, "a," "an," and " The singular form with "the" is plural unless the context clearly indicates otherwise. It should be noted that the range includes one particular Expressed herein as from a value and / or to another particular value with "about" When such a range is expressed, in another embodiment, the range is from the one particular value. and / or up to said other particular value. Similarly, values ​​may be expressed using the antecedent "about." When expressed as approximations, the specific values ​​form alternative embodiments. It will be understood.

[0243] When a nucleic acid sequence is disclosed herein, the reverse complement thereof is also specifically contemplated.

[0244] The various methods described herein may be performed in vitro or in vivo. In some embodiments, the various methods described herein are carried out in vitro. The term "in vitro" is intended to encompass experiments in which cells are cultured. In contrast, the term "in vivo" can include experiments using intact multicellular organisms. As intended. [Brief description of the drawings]

[0245] Next, various embodiments and experiments illustrating the principles of the disclosed methods and compositions are provided in the accompanying drawings. The discussion will be made with reference to the accompanying drawings. [Figure 1A] Bar graph showing the number of spot forming cells (SFC) per 100,000 cells specific for the indicated EBV antigens in populations of cells expanded from PBMCs obtained from four different EBV-positive lymphoma patients. Shown are the numbers of SFC per 100,000 cells in response to stimulation with the indicated antigens in EBVST generated by stimulation with latent pepmix or latent + lytic pepmix (T2 EBVST, AII-EBVST, respectively). [Figure 1B] Bar graph showing the number of spot forming cells (SFC) per 100,000 cells specific for the indicated EBV antigens in populations of cells expanded from PBMCs obtained from four different EBV-positive lymphoma patients. The number of SFC per 100,000 cells is shown in response to stimulation with the indicated antigens in EBVST generated by stimulation with lytic pepmix or latent + lytic pepmix (lytic EBVST, AII-EBVST, respectively). [Diagram 2]Bar graph showing the number of spot-forming cells (SFC) per 100,000 cells specific for the indicated EBV antigens in EBVST (T2-EBVST, lytic EBVST, BR-EBVST, respectively) expanded from PBMCs from two EBV-positive lymphoma patients by stimulation with latent pepmix, lytic pepmix, or latent + lytic pepmix. Total number of cells in the bars is indicated. [Diagram 3] Bar graph showing the number of spot-forming cells (SFC) per 100,000 cells specific for the indicated EBV antigens in EBVST expanded from PBMCs from three healthy donor subjects by stimulation with latent pepmix, immediate early lysed pepmix, early lysed pepmix, or latent + lysed pepmix (T2-EBVST, IE-EBVST, E-EBVST, BR-EBVST, respectively). [Figure 4] Bar graph showing the percentage of autologous EBV-LCL lysed in an in vitro assay of cytolytic activity by EBVST (T2-EBVST, IE-EBVST, E-EBVST, BR-EBVST, respectively) expanded from PBMCs from three healthy donor subjects by stimulation with latent pepmix, immediate early lysis pepmix, early lysis pepmix, or latent + lysis pepmix. [Diagram 5] Bar graphs showing the number of spot-forming cells (SFC) per 100,000 cells specific for the indicated EBV antigens or LCLs in EBVST expanded from PBMCs by stimulation with the latency pepmix or the latency + lysis pepmix (T2-EBVST, BR-EBVST, respectively). [Figure 6A] Graphs and images showing the in vivo anti-cancer activity of EBVST expanded from PBMCs by stimulation with latent pepmix or latent + lytic pepmix (T2-EBVST, BR-EBVST, respectively) against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer. The control group received PBS instead of EBVST. Tumor volume (mm3) over time is shown. [Figure 6B]Graphs and images showing the in vivo anti-cancer activity of EBVST expanded from PBMCs by stimulation with latent pepmix or latent + lytic pepmix (T2-EBVST, BR-EBVST, respectively) against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer. Control groups received PBS instead of EBVST. Tumor burden over time as measured by luciferase activity of firefly luciferase-expressing EBV-LCL is shown. [Figure 6C] Tumor burden over time as measured by luciferase activity of firefly luciferase-expressing EBV-LCL is shown. [Figure 7] Images showing the load and location of firefly luciferase-expressing EBV-LCL in vivo in mice on the indicated days of an experiment to examine the anti-cancer activity of EBVST expanded from PBMCs by stimulation with latent pepmix or latent + lytic pepmix (T2-EBVST and BR-EBVST, respectively) against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer. The control group received PBS instead of EBVST. [Figure 8A] Graphs and images showing the in vivo anti-cancer activity of EBVST expanded from PBMCs by stimulation with latent pepmix, lysed(2) pepmix, or lysed(2)+latent pepmix (T2-EBVST, lysed(2)-EBVST, BR(2)-EBVST, respectively) against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer. The control group received PBS instead of EBVST. Dorsal aspects are shown on the indicated days relative to injection of EBVST. [Figure 8B]Graphs and images showing the in vivo anti-cancer activity of EBVST expanded from PBMCs by stimulation with latent pepmix, lysed(2) pepmix, or lysed(2)+latent pepmix (T2-EBVST, lysed(2)-EBVST, BR(2)-EBVST, respectively) against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer. Control groups received PBS instead of EBVST. Ventral aspects are shown on the indicated days relative to injection of EBVST. [Figure 8C] Graphs and images showing the in vivo anti-cancer activity of EBVST expanded from PBMCs by stimulation with latent pepmix, lytic(2) pepmix, or lytic(2)+latent pepmix (T2-EBVST, lytic(2)-EBVST, and BR(2)-EBVST, respectively) against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer. The control group received PBS instead of EBVST. Total tumor burden over time is shown. [Figure 8D] Graphs and images showing the in vivo anti-cancer activity of EBVST expanded from PBMCs by stimulation with latent pepmix, lytic(2) pepmix, or lytic(2)+latent pepmix (T2-EBVST, lytic(2)-EBVST, and BR(2)-EBVST, respectively) against autologous EBV-LCL in a mouse xenograft model of EBV-positive cancer. The control group received PBS instead of EBVST. Tumor volume (mm3) over time is shown. [Figure 9A] Box plots showing the levels of cytokines detected in the serum of mice treated with EBVST expanded from PBMCs by stimulation with the latent pepmix, the lytic(2) pepmix, or the lytic(2)+latent pepmix (T2-EBVST, lytic(2)-EBVST, and BR(2)-EBVST, respectively) in a mouse xenograft model of EBV-positive cancer at the indicated days after EBV injection. Levels of GM-CSF are shown. ***P=0.0001. [Figure 9B]Box plots showing the levels of cytokines detected in the serum of mice treated with EBVST expanded from PBMCs by stimulation with the latent pepmix, the lytic(2) pepmix, or the lytic(2)+latent pepmix (T2-EBVST, lytic(2)-EBVST, and BR(2)-EBVST, respectively) in a mouse xenograft model of EBV-positive cancer at the indicated days after EBV injection. Levels of IFNγ are shown. ***P=0.0001. [Figure 9C] Box plots showing the levels of cytokines detected in the serum of mice treated with EBVST expanded from PBMCs by stimulation with the latent pepmix, the lytic(2) pepmix, or the lytic(2)+latent pepmix (T2-EBVST, lytic(2)-EBVST, and BR(2)-EBVST, respectively) in a mouse xenograft model of EBV-positive cancer at the indicated days after EBV injection. Levels of IL-10 are shown. ***P=0.0001. [Figure 10] Bar graph showing the number of spot forming cells (SFC) per 100,000 cells specific for EBV antigens in populations of cells expanded by stimulation with a latency pepmix from PBMCs obtained from four different healthy donor subjects (D#1-D#4). WW=EBVST expanded from whole PBMCs and restimulated with ATCs derived from whole PBMCs; WD=EBVST expanded from whole PBMCs and restimulated with ATCs derived from PBMCs depleted for CD45RA positive cells; DW=EBVST expanded from PBMCs depleted for CD45RA positive cells and restimulated with ATCs derived from whole PBMCs; and DD=EBVST expanded from PBMCs depleted for CD45RA positive cells and restimulated with ATCs derived from PBMCs depleted for CD45RA positive cells. [Figure 11] Histograms showing CD80 and HLA-DR expression in ATCs derived from four different healthy donor subjects (D#1-D#4), total PBMCs, or PBMCs depleted of CD45RA positive cells, as determined by flow cytometry. EXAMPLES

[0246] In the following examples, the present inventors have demonstrated that a population of cells containing EBV-specific T cells can be obtained by EBV antigens were generated from PBMC populations by stimulation with peptides of different EBV antigens. The inventors have characterized the expanded cell population by its EBV reactivity, EBV-infected cells, and in vivo effector activity against EBV-positive cancer. We will characterize its anti-cancer activity in expanded populations. Increase the proportion of BV-reactive cells and their anti-cancer activity and persistence in vivo I'm researching ways to make it bigger.

[0247] Example 1: Generation of EBV-specific T cells Peripheral blood mononuclear cells (PBMCs) were cultured using standard Ficoll-Paque density gradient centrifugation. were isolated from blood samples obtained from healthy donors or lymphoma patients according to the method described above.

[0248] Generation of ATC Anti-CD3 agonist antibody (clone OKT3) and anti-CD28 agonist antibody were administered at 0 Add 0.5 ml of 1:1000 diluted 1 mg / ml antibody and incubate at 37°C for 2-4 hours or was coated onto wells of tissue culture plates by overnight incubation at 4°C. . 1×10 6 PBMCs (in 2 ml of cell culture medium) were incubated with 10 ng / ml of IL-1 CTL cell culture medium (RPMI-16) supplemented with 7 and 5 ng / ml IL-15 40 medium, 50% Click medium, 10% FBS, 1% GlutaMax, 1% P Anti-CD3 / CD28 agonist antibody coated plates in The cells were stimulated by culturing in 5% CO2 at 37°C. The next day 1 ml of cell culture medium was added with 20 ng / ml IL-7 and 200 ng / ml IL- The ATCs were maintained in culture and subsequently collected. , and were either used in experiments or stored frozen for between 5 and 7 days.

[0249] Universal LCL LCLs that lack surface expression of HLA class I and HLA class II (i.e., HLA-negative HLA class I and II molecules are encoded by the genes Targeted knockout in lymphoblastoid cell lines prepared by EBV transformation of cells These HLA-negative cells were obtained by cloning the genes necessary for EBV replication. The resulting cells obtained by this method were further modified to knock out the offspring. In the text it is referred to as universal LCL (uLCL).

[0250] Expansion of EBV-specific T cells EBV-specific T cells were cultured in 50% Advanced RPMI, 50% Click medium , 10% FBS, 1% GlutaMax, 1% Pen / Strep, IL Cell culture medium supplemented with IL-7 (10 ng / ml) and IL-15 (100 ng / ml) In the field, pepmix (a mixture of relevant antigens) obtained from JPT Technologies was used. Duplicate 15mer amino acids overlapping by 11 amino acids, spanning the entire amino acid sequence peptide library) in one of the following combinations: 6 PBMCs for 9 days The stimulation was expanded over a period of: (i) EBNA1 pepmix (JPT catalog number PM-EBV-EBNA1) + L MP1 pep mix (JPT Catalog No. PM-EBV-LMP1) + LMP2 pep mix (JPT Catalog No. PM-EBV-LMP2) - "Latency Pepmix" (ii) BZLF1 pepmix (JPT catalogue no. PM-EBV-BZLF1) + BRLF1 pepmix (JPT Catalog No. PM-EBV-BRLF1) + BMRF1 Pepmix (JPT Catalog No. PM-EBV-BMRF1) + BMLF1 pepmix vinegar * +BALF2 ​​Pepmix * +BNLF2A pepmix * +BNLF2B PepMit Kus * +BMRF2 pepmix * - "Soluble Pepmix" (iii) EBNA1 pepmix + LMP1 pepmix + LMP2 pepmix + BZLF1 Pep Mix + BRLF1 Pep Mix + BMRF1 Pep Mix + BMLF 1 Pepmix + BALF 2 Pepmix + BNLF 2A Pepmix + BNLF 2B Pepmix Pepmix + BMRF2 Pepmix - "Latent + Lytic Pepmix"

[0251] The pepmix combination (i.e., pepmix mixture) was diluted to 1 × 10 6 PBMs A final amount of 10 ng of pepmix mixture per C was used in the stimulation. * Peptides for BMLF1, BALF2, BNLF2A, BNLF2B, and BMRF2 The mix was created by combining the individual component peptides obtained from Genemed. It was prepared as follows. (iv) BZLF1 pepmix + BRLF1 pepmix + BMRF1 pepmix - "Early Dissolving Pepmix" (v) BMRF1 pepmix + BMLF1 pepmix + BALF2 ​​pepmix + BNLF2A pepmix + BNLF2B pepmix + BMRF2 pepmix (JP Catalog number PM-EBV-BMRF1) - "Early Lysis Pepmix"

[0252] Additional cell culture medium was added as needed over the course of 9 days, and cytokines were added at 5, Replenishment was performed on the sixth or seventh day.

[0253] At the end of the 9-day culture period, the cells were cultured in irradiated, peptide-pulsed The cells were restimulated by co-culture with autologous activated T cells (ATCs) in the presence of uLCL. In simple terms, 2×10 6 Add ATC to Pepmix (1×10 6 ATC Incubate with 10 ng of pepmix mixture per 100 μg of CTL medium at 37°C for 30 min. The cells were incubated, then irradiated with 30 Gy and collected. Lus-treated ATCs were treated with IL-7 (10 ng / ml) and IL-15 (100 ng / ml). Responder cells:peptide-pulsed A in a ratio of 1:1:5 in CTL medium containing TC: Ratio of irradiated uLCLs, cells in culture and uLCLs (irradiated at 100 Gy) Specifically, 1×10 5 responder cells and 1 × 10 5 pcs Peptide-pulsed ATC and 0.5 × 10 6 irradiated uLCLs and 24-well plates The cells were cultured in 2 mL of CTL medium in the wells of a tissue culture plate.

[0254] The cells were maintained at 37°C in a 5% CO2 atmosphere. After 3-4 days, IL-7 (10 Additional cell culture medium containing IL-15 (100 ng / ml) and IL-15 (100 ng / ml) was required. On the fifth or sixth day, IL-7 (10 ng / ml) and IL-1 Add additional cell culture medium containing 5 (100 ng / ml) as needed and incubate for 6 to 7 days. The expanded EBVSTs were then collected for analysis or for use in experiments.

[0255] In illustrative examples throughout the examples and figures: E produced by the method using the latent pepmix (i.e., (i) above) BVST is designated as "latent antigen type 2 (T2)-EBVST." Generated by a method using a soluble pepmix (i.e., (ii) above) EBVST is referred to as "soluble EBVST." Produced by the method using the incubation + lysis pepmix (i.e., (iii) above) The EBVSTs that are tested are referred to as "broad repertoire (BR)-EBVSTs." Generated by the method using the earliest soluble pepmix (i.e., (iv) above) The EBVST obtained is denoted as "immediately early (lE)-EBVST." Generated by the method using the initial lysis pepmix (i.e., (v) above) The EBVST is denoted as "early (E)-EBVST."

[0256] Example 2: Analysis of the specificity of EBVST for EBV antigens EBs prepared from PBMCs of EBV-positive lymphoma patients using different pepmixes To clarify whether the EBVST can recognize different EBV antigens, The results were analyzed by ELISPOT.

[0257] Briefly, EBVST was incubated on 96 well plates precoated with anti-IFNγ capture antibody. 1×10 5 Cells / well were plated and incubated with the corresponding EBV peptides indicated. After 18-20 hours of incubation, the plates were IFNγ+ spots were developed, dried overnight at room temperature in the dark, and quantified. The frequency of T cells specific to each antigen is calculated by the number of specific spots formed per number of input cells. Cells (SFC).

[0258] FIG. 1A and FIG. 1B show a method for isolating T cells specific for a lytic cycle antigen. We show that the latent What kind of antigens are produced by combining pepmixes of hidden and soluble antigens? The specificity for the single antigen is not compromised, and the total frequency of antigen-specific T cells increases by 100%. When the two were combined, they became larger.

[0259] A patient's T cells were either treated with the latent antigen pepmix or the lytic antigen pepmix alone. However, when used in combination with a latent antigen pepmix and a soluble antigen, When used in combination with pepmix, good specificity can be achieved. (See Donor 2.) This is due to the cytokines produced by activated T cells that help each other. This is thought to be the result of the production of

[0260] Figure 2 shows the results of EBV lysis in response to stimulation with a pepmix corresponding to EBV latent and lytic antigens. The number of cells secreting IFNγ was increased by the combination of the latent antigen pepmix and the lytic antigen pepmix. For EBVST generated using a combination of the latent antigen pep mix, only More EBVST was generated using either the lytic antigen pepmix alone or A superadditive increase in the number of IFN-γ producing cells was observed in response to the latent antigen pepmix. and lysed antigen pepmix.

[0261] In further experiments, different pepmixes were used to prepare PBMCs from healthy donors. The EBVSTs to be used are those that recognize different EBV antigens, as described above. To determine whether the antibody could be used to express the IL-11 mAb, we analyzed the antibody by ELISPOT.

[0262] The results are shown in Figure 3 and show that latent cycle antigens, immediate early lytic antigens and early lytic antigens are Antigen-specific T cells could be expanded from PBMCs of healthy donors. This makes it clear that:

[0263] Example 3: Stimulation of PBMCs with pepmixes corresponding to different EBV antigens Analysis of cell killing by EBVST obtained by EBVST obtained by stimulation of PBMCs with different pepmixes were We analyzed whether VST could kill autologous EBV-transformed B cell lines.

[0264] 10 μl Cr 51 10 ng of EBV latency + lysis pepmix (see Example 1 (ii) i)) 6 Add autologous LCL and incubate at 37 °C. After 1 hour of incubation, the LCLs were washed three times with CTL medium and incubated for 50 min at 4°C. EBVST was resuspended in CTL medium at 0.000 cells / ml. In 200 μl of CTL medium in a well of a plate, effector EBVST + 5,000 LCL:target cell ratio (100,000 EBVST + 5,000 LCL) The cells were then co-cultured with autologous pepmix-pulsed LCLs. The co-culture was incubated at 37°C for 4 hours. After incubation at 5% CO2, the supernatant was collected and the % specific Targeted lysis is determined by the release of Cr by the killed target cells. 51 To measure the gamma ray power The results were obtained using a counter.

[0265] The results are shown in Figure 4. EBV antigen-specific T cells can kill autologous EBV-transformed B cell lines. Ta.

[0266] Potent EBV-L of EBVST obtained using a method with lytic antigen pepmix CL killing capacity is due to the fact that only a small proportion of LCLs are in the lytic cycle (and therefore cannot detect the target antigen). This was surprising because one might expect that the phagocytosis of the cerebrospinal fluid would be expressed by the cerebrospinal fluid (CSF). This may be explained by the use of cytotoxic T cells and the presentation of antigens from dying cells during the lytic phase.

[0267] Example 4: Stimulation of PBMCs with pepmixes corresponding to different EBV antigens Analysis of in vivo anticancer activity of the EBVST obtained The present inventors have demonstrated that BR-EBVST and T2-EBVST can be used to treat EBV-positive cancers. The relative potency was examined in vivo using a mouse xenograft model.

[0268] Briefly, EBV-positive tumors were cultured at 3.5 × 10 6 Fireflies Luciferase-expressing autologous LCLs were established by subcutaneous implantation into the flanks of NSG mice. After 8 days, when the tumors were visible, the mice were treated with PBS (control group), 5 × 10 0 6 BR-EBVST, or 5 x 10 6 T2-EBVST was administered intravenously. The drug was administered.

[0269] Tumors were monitored throughout the experiment by bioluminescence imaging. The enzyme activity was measured by intraperitoneal injection of D-luciferin (1.5 mg / mouse) and IVIS. Monitored by imaging the mice after 10 minutes using an imaging device (Xenogen) Tumor volume was also monitored by measurement using calipers.

[0270] Prior to injection, BR-EBVST and T2-EBVST were isolated using different EBV antigens or examined their ability to produce IFNγ in response to stimulation with EBV-LCL using ELI The results are shown in Figure 5. Populations of immune cells expanded using peptides corresponding to EBV lytic antigens were shown to mediate EBV latency. It contains cells reactive to both EBV lytic antigens and EBV latent antigens, When compared with populations of immune cells expanded using peptides corresponding only to EBV- These results revealed that the IFN-γ-producing cells were significantly more abundant than the LCL-stimulated cells. To do.

[0271] Figures 6 and 7 show that BR-EBVST rapidly differentiated into EBV-positive tumors compared with T2-EBVST. Indicates that the control has been established.

[0272] FIG. 7 also shows that BR-EBVST-treated mice were significantly more potent than T2-EBVSTS-treated mice. These results show that the mice showed reduced metastasis compared to controls.

[0273] In further experiments, EBV-positive tumors were cultured at 3.5 × 10 6 Firefly lights The mice were then implanted subcutaneously with ferase-expressing autologous LCLs, and after 8 days, the mice were treated with PBS ( Control group), 1 × 10 6 BR(2)-EBVST, 1×10 6 Dissolution of Individuals (2) -EBVST, or 1×10 6 T2-EBVST was administered by intravenous infusion.

[0274] The lysed(2)-EVBST and BR(2)-EBVST used in this experiment are 1, except that the following combination of pepmixes was added to the EBVS: Used in stimulation to expand T: (vi) BZLF1 pepmix + BRLF1 pepmix + BMRF1 pepmix +BMLF1 pepmix +BXLF1 pepmix +BALF1 pepmix +BLL F2 pepmix + BALF2 ​​pepmix + BNLF2A pepmix - Lysis (2) -Pepmix" (vii) EBNA1 pepmix + LMP1 pepmix + LMP2 pepmix + BZLF1 Pep Mix + BRLF1 Pep Mix + BMRF1 Pep Mix + BMLF 1 Pepmix + BXLF 1 Pepmix + BALF 1 Pepmix + BLLF 2 Pepmix + BALF2 ​​pep mix + BNLF2A pep mix - Lytic (2) + Latent pep mix mix"

[0275] BMLF1, BXLF1, BALF1, BLLF2, BALF2, and BNLF2A The pepmix for this compound is a combination of individual peptides obtained from Genemed. EBNA1, LMP1, LMP2, BZLF1, BRLF1 and The pepmix for BMRF1 was prepared by JPT tech as shown in Example 1. Obtained from nologies.

[0276] Generated by a method using a soluble pepmix (i.e., (vi) above) EBVST was shown as "lytic (2)-EBVST" and lytic (2) + latent pep The EBVST produced by the method using the EBVST (i.e., (vii) above) is It is shown as "BR(2)-EBVST".

[0277] Tumors were monitored throughout the experiment by bioluminescence imaging as described above. Tumor volume was also monitored by measurement using calipers.

[0278] Plasma samples were also collected to determine levels of GM-CSF, IFNγ, and IL-10. Then, mice were harvested on days 3 and 8 after EBVST administration and analyzed by ELISA. Analyzed.

[0279] The results are shown in Figures 8 and 9.

[0280] EBV expanded by stimulation with peptides of EBV latent antigen plus EBV lytic antigen ST strongly inhibited tumor growth (Figures 8A-8D). Stimulation with EBV lytic antigen alone EBVST expanded by EBV can also inhibit tumor growth, and EBV latent antigens The degree of expansion was similar to or greater than that of the EBVST expanded by stimulation using only The compound inhibited tumor growth to a small extent.

[0281] EBV expands by stimulation with peptides of EBV latent antigen plus EBV lytic antigen Mice treated with ST were also expanded by stimulation with EBV latent antigens alone. Increased levels of proinflammatory cytokines were observed in mice treated with EBVST, compared to mice treated with EBVST. In contrast, stimulation with only a peptide of the EBV lytic antigen was Thus, mice treated with expanded EBVST only use EBV latent antigens. Similar levels of EBVST were observed in mice treated with EBVST compared to mice treated with EBVST. or increased levels of proinflammatory cytokines in their serum (Fig. 9A and and Figure 9B).

[0282] In contrast, stimulation with peptides of EBV latent antigens plus EBV lytic antigens expanded Mice treated with EBVST, which is a marker for EBV infection, were stimulated with EBV latent antigens alone. Reduced levels of IL-10 compared to mice treated with expanded EBVST in their serum, whereas stimulation with only peptides of EBV lytic antigens Mice treated with expanded EBVST showed no response to stimulation with EBV latent antigens alone. Compared to mice treated with EBVST, which is expanded by They showed reduced levels of IL-10 in their serum (Figure 9C).

[0283] Thus, BR-EBVST is able to kill a greater number of tumor cells and It was found that the immune system produces greater amounts of pro-inflammatory cytokines than the immune system. Without wishing to be bound by any particular theory, this may be due to the increased Changes in the tumor microenvironment that cause pitope spreading and non-viral tumor antigen-specific This may result in further tumor cell killing by T cells.

[0284] Example 5: Conclusion In summary, we show that:

[0285] A population of T cells that contains T cells specific for lytic and latent EBV antigens PBMCs from both healthy donors and lymphoma patients were lysed to express EBV antigens and latent E. coli. It can be obtained by stimulating with a pepmix corresponding to BV antigens (e.g. , see Figures 1 and 3);

[0286] PBMCs were stimulated with a pepmix corresponding to soluble and latent EBV antigens. By this, EBV antigen-reactive T cells react with peptidomimetics that only respond to lytic EBV antigens. EBV stimulation with either EBV β-lactam or a pepmix directed against only latent EBV antigens (see, e.g., FIG. 2 );

[0287] Pepmix corresponding to lytic + latent EBV antigens or pepmix corresponding to immediate early lytic antigens By stimulating PBMCs with a pep mix or a pep mix corresponding to early lysis antigens, The resulting T cell population was then cultured in PBMC with a pep mix directed against latent EBV antigens. These T cells killed EBV-LCL with a similar ability to the T cell population obtained by stimulating can be scratched (see, for example, FIG. 4);

[0288] By stimulating PBMCs with a pepmix corresponding to lytic + latent EBV antigens The resulting T cell population was generated by immunizing PBMCs with a pepmix directed against only latent EBV antigens. EBV-infected cells were more reactive than T cell populations obtained by stimulating contains a greater proportion of responsive cells (see, e.g., FIG. 5 );

[0289] By stimulating PBMCs with a pepmix corresponding to lytic + latent EBV antigens The resulting T cell population was stimulated by stimulating PBMCs with a pepmix corresponding to latent EBV antigens. Improved control of tumor growth and T cell proliferation when compared to populations of T cells obtained by and reduced metastasis for EBV-positive cancers (see, e.g., Figures 6 and 7). ).

[0290] This was obtained by stimulating PBMC with a pep mix corresponding to soluble EBV antigens. The population of T cells that express EBV was generated by stimulating PBMCs with a pep mix that corresponds to latent EBV antigens. The effect of EBV-positive cancers on tumor growth was significantly improved when compared with T cell populations obtained by showing similar suppression or improved regulation (see, for example, FIG. 8).

[0291] By stimulating PBMCs with a pepmix corresponding to lytic + latent EBV antigens Subjects with EBV-positive cancer treated with the resulting T cells respond to latent EBV antigens. The treatment is performed using T cells obtained by stimulating PBMC with a pepmix containing Elevated levels of proinflammatory cytokines (GM1, GM2, GM3, GM4, GM5) in peripheral blood compared with control subjects. -CSF, IFNγ) and reduced levels of anti-inflammatory cytokines (IL-10) (See, for example, FIG. 9.)

[0292] This was obtained by stimulating PBMC with a pep mix corresponding to soluble EBV antigens. Subjects with EBV-positive cancer treated with T cells expressing peptides corresponding to latent EBV antigens. Subjects to be treated with T cells obtained by stimulating PBMC with PUMIX Similar or elevated levels of proinflammatory cytokines in peripheral blood compared with tokine (GM-CSF, IFNγ) and similar or reduced levels of anti-inflammatory The immune system has a proinflammatory cytokine (IL-10) (see, for example, FIG. 9).

[0293] Example 6: EBV-specific T cell generation from PBMCs depleted of CD45RA positive cells Generate We next investigated the effect of depleting CD45RA positive cells from a PBMC population on E The effect on expanded populations of BV-specific T cells was examined.

[0294] Derivation of NK cells from PBMC populations allows for the expansion of EBVST from NK cell populations. The method may be problematic due to IL-15 mediated stimulation of NK cell proliferation. CD45RA is expressed on naive T cells, as well as on natural T regulatory cells and NK cells. Therefore, depletion of CD45RA+ cells may promote NK cell proliferation and proliferation in the PBM. It was speculated that depletion of CD45RA+ cells would result in the elimination of the anti-CD45RA+ cells from the C population. Regulatory T cells, which can inhibit the generation of antigen-specific T cells, are also involved in the elimination of T cells, particularly in cancer patients. Naive T cells that can grow as bystander cells and dilute antigen-specific T cells. The cells are also removed.

[0295] CD45RA expressing cells were isolated using Miltenyi® columns and CD45RA co-transfection. PBMCs were depleted using conjugated beads. PBMCs to be depleted of cells were incubated with the latent peptidomimetic vector essentially as described in Example 1. The antibody was used to expand EBV-specific T cells by stimulation with EBV.

[0296] PBMCs depleted of CD45RA positive cells were also used in the restimulation. The ATC was used as the starting population for the production of C. Produced as described in Example 1.

[0297] The following experimental conditions were compared: (i) Whole PBMCs (i.e., PBMCs not depleted of CD45RA positive cells) Restimulation using ATCs generated from whole EBVST+PBMCs expanded from In Figure 10, this is shown as "WW" (i.e., whole + whole). (ii) EBVST+CD45RA+ cells expanded from the whole PBMCs were depleted. Restimulation with ATCs produced from PBMCs that have been This is shown as (total + depletion). (iii) EBVST+ expanded from PBMCs depleted of CD45RA positive cells Restimulation with ATCs produced from whole PBMCs - this is referred to as "DW" (i.e. This is expressed as (i.e., depletion + total). (iv) EBVST+C expanded from PBMCs depleted of CD45RA positive cells Restimulation using ATC produced from PBMCs depleted of D45RA positive cells This is shown in FIG. 10 as "DD" (i.e., depleted + depleted).

[0298] The above (i) to (c) were analyzed using PBMCs obtained from four different healthy donors (D#1 to D#4). (iv) The EBVST prepared according to the method of claim 1, wherein the EBVST recognizes different EBV antigens. To clarify whether the ELISA kit can be used to detect the IL-1 expression, we performed ELISPOT analysis. The analysis was carried out essentially as described in Example 2.

[0299] The results are shown in Figure 10. For each donor, CD45RA positive cells were depleted. When the PBMCs collected from the EBVST were used as the starting population for expanding the EBVST, the PBMC collection In comparison with the method using the whole colony, IFN-α expression was significantly increased in response to stimulation with EBV pepmix. The proportion of cells in the expanded population secreting γ increased. Similarly, CD45RA positive cells were depleted. The PBMCs were used as the starting population to generate ATCs used in restimulation. When used in combination with EBV pepmix, it was found that the expanded population secreted IFNγ in response to stimulation with the EBV pepmix. The proportion of cells with

[0300] We used PBMCs or PBMCs in which CD45RA positive cells were depleted. The costimulatory molecules CD80 and HLA-DR (MHC class I) in ATCs generated from C. II) expression was analyzed by flow cytometry.

[0301] The results are shown in Figure 11. CD45RA positive cells were generated from depleted PBMCs. The ATCs generated from the PBMCs showed a higher expression of CD80 and and HLA-DR expression, thus improving antigen-presenting and costimulatory properties. It was.

Claims

[Claim 1] A method for generating or expanding a population of immune cells, including immune cells specific for Epstein-Barr Virus (EBV) lytic antigens, comprising stimulating the 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) that present one or more peptides corresponding to all or a portion of one or more EBV lytic antigens.

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