Treatment and prevention of cancer using virus-specific immune cells expressing chimeric antigen receptors

Abstract

This invention provides CAR-modified virus-specific T cells (CAR-VSTs) to eliminate hematological malignancies without causing graft-versus-host disease (GVHD) and to avoid allogeneic rejection. [Solution] A virus-specific immune cell comprising a chimeric antigen receptor (CAR) or a nucleic acid encoding a CAR for use in a method of treating or preventing cancer is disclosed, wherein the CAR comprises (i) an antigen-binding domain that specifically binds to CD30, (ii) a transmembrane domain, and (iii) a signaling domain, the signaling domain comprising (a) an amino acid sequence derived from the intracellular domain of CD28, and (b) an amino acid sequence comprising an immune receptor tyrosine-based activation motif (ITAM).

Description

[Technical Field]

[0001] This application is U.S. Patent Application No. 63 / 015,769, filed on April 27, 2020. Priority is claimed, and its contents and elements are incorporated herein by reference for all purposes. It gets included.

[0002] (Technical field) This invention relates to molecular biology and cell biology, as well as to medical treatment and prevention. Regarding the method. [Background technology]

[0003] Despite the success of autochimeric antigen receptor (CAR) T cells in hematological malignancies, There are obstacles to the wider use of this curative treatment, including manufacturing failures and pre-injection disease. The progression of the disease and the exorbitant costs make it a contraindication for many people. The option of using CAR T cells is urgently needed.

[0004] "Ready-made" products that allow for the rapid administration of T cells collected from healthy donors are a form of adoptive cell immunotherapy. Access to and costs can be improved. However, "commercially available" CAR The development of T-cell therapy has been hampered by two major pitfalls: namely, non-hematological treatment. Polyclonal CAR T cells from marginal donors cause graft-versus-host disease (GVHD). The possibility of this occurring, and rejection of allogeneic CAR T cells by allogeneic reactive T cells of the recipient. Yes, that's correct. [Overview of the project]

[0005] In a first embodiment, the disclosure relates to a virus-specific immune cell that has a chimeric antigen receptor (CAR) or nucleic acid encoding CAR, wherein the CAR is (i) specifically bound to CD30 (ii) antigen-binding domain, (ii) transmembrane domain, and (iii) signal transduction domain. The signaling domain includes (a) an amino acid sequence derived from the intracellular domain of CD28. , and (b) amino acid sequences derived from immune receptor tyrosine-based activation motifs (ITAMs) It provides immune cells that are composed of the following:

[0006] In some embodiments, the above signal transduction domain is located at SEQ ID number 26. In contrast, it contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0007] In some embodiments, the transmembrane domain originates from the transmembrane domain of CD28. ru.

[0008] In some embodiments, the transmembrane domain is less than SEQ ID number 20 It contains amino acid sequences with at least 80% amino acid sequence identity.

[0009] In some embodiments, the antigen-binding domain is configured for SEQ ID number 14. an amino acid sequence having at least 80% amino acid sequence identity, and a SEQ ID number It contains an amino acid sequence that has at least 80% amino acid sequence identity with no. 15.

[0010] In some embodiments, the antigen-binding domain is small relative to SEQ ID number 18. It contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0011] In some embodiments, the above signaling domain is (a) an intracellular domain of CD3ζ Contains amino acid sequences derived from yl.

[0012] In some embodiments, the above signal transduction domain is located at SEQ ID number 25. In contrast, it contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0013] In some embodiments, the CAR is located between the antigen-binding domain and the transmembrane domain. Further includes the provided hinge area.

[0014] In some embodiments, the hinge region is less than SEQ ID number 33 It contains amino acid sequences with at least 80% amino acid sequence identity.

[0015] In some embodiments, the CAR corresponds to SEQ ID number 35 or 36 It contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0016] In some embodiments, the virus-specific immune cells target antibodies other than CD30. A CAR containing an antigen-binding domain that specifically binds to the source, or a target antigen other than CD30. It contains nucleic acids encoding CARs that include antigen-binding domains that bind heterologously.

[0017] This disclosure also relates to viruses comprising chimeric antigen receptors (CARs) or nucleic acids that encode CARs. CAR provides specific immune cells, and (i) antigen-binding plasmids that specifically bind to CD30. (ii) transmembrane domain, and (iii) immunoreceptor tyrosine-based activation molecule It contains a signal transduction domain including -f (ITAM), The aforementioned virus-specific immune cells exhibit antigen binding, specifically binding to target antigens other than CD30. Antigen-binding domains that specifically bind to CARs containing domains, or to target antigens other than CD30. It contains nucleic acids that code for CAR, which includes n.

[0018] In some embodiments, the virus-specific immune cells are one or more non-identical CARs. or comprising nucleic acids that encode one or more non-identical CARs.

[0019] In some embodiments, the target antigen other than CD30 is a cancer cell antigen.

[0020] In some embodiments, target antigens other than CD30 are CD19, CD20, CD22, ROR1R, CD4, CD7, CD38, BCMA, Mesotheline, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA It will be selected.

[0021] In some embodiments, the target antigen other than CD30 is CD19.

[0022] In some embodiments, the virus-specific immune cells are virus-specific T cells. That is the case.

[0023] In some embodiments, the virus-specific immune cells are Epstein-Barr It is specific to the Epstein-Barr virus (EBV).

[0024] This disclosure also relates to a method for producing virus-specific immune cells: Virus-specific immune cells are identified by chimeric antigen receptors (CARs) or nucleic acids encoding CARs. A step of modifying to include, wherein the CAR specifically binds to CD30. It includes (ii) an antigen-binding domain, a transmembrane domain, and (iii) a signal transduction domain. The signal transduction domain is (a) an amino acid sequence derived from the intracellular domain of CD28. , and (b) an amino acid mixture containing an immune receptor tyrosine-based activation motif (ITAM) A method that includes a sequence, a process, or a step.

[0025] In some embodiments, the above signal transduction domain is located at SEQ ID number 26. In contrast, it contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0026] In some embodiments, the transmembrane domain originates from the transmembrane domain of CD28. ru.

[0027] In some embodiments, the transmembrane domain is less than SEQ ID number 20 Both contain amino acid sequences with 80% amino acid sequence identity.

[0028] In some embodiments, the antigen-binding domain is configured for SEQ ID number 14. It contains an amino acid sequence having at least 80% amino acid sequence identity, and SEQ It contains an amino acid sequence that has at least 80% amino acid sequence identity with ID number 15. nothing.

[0029] In some embodiments, the antigen-binding domain is small relative to SEQ ID number 18. It contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0030] In some embodiments, the above signaling domain is (a) an intracellular domain of CD3ζ Contains amino acid sequences derived from yl.

[0031] In some embodiments, the above signal transduction domain is located at SEQ ID number 25. In contrast, it contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0032] In some embodiments, the CAR is located between the antigen-binding domain and the transmembrane domain. Further includes the provided hinge area.

[0033] In some embodiments, the hinge region is less than SEQ ID number 33 It contains amino acid sequences with at least 80% amino acid sequence identity.

[0034] In some embodiments, the CAR corresponds to SEQ ID number 35 or 36 It contains an amino acid sequence that has at least 80% amino acid sequence identity.

[0035] In some embodiments, the virus-specific immune cells target antibodies other than CD30. A CAR containing an antigen-binding domain that specifically binds to the source, or a target antigen other than CD30. It contains nucleic acids encoding CARs that include antigen-binding domains that specifically bind to antigens.

[0036] The present invention also includes administering a compound of formula (I) to a subject that requires it. This provides a method for producing virus-specific immune cells: Virus-specific to include a chimeric antigen receptor (CAR) or nucleic acid encoding a CAR. A process for modifying immune cells, wherein the CAR (i) contains an antigen that specifically binds to CD30. (ii) the synthetic domain, (ii) the transmembrane domain, and (iii) the activity of the immunoreceptor tyrosine base A process comprising a signaling domain containing a chemical motif (ITAM); Here, the virus-specific immune cells specifically bind to target antigens other than CD30. CARs containing an antigen-binding domain, or antigen-binding domains that specifically bind to target antigens other than CD30. Contains nucleic acids that encode CARs, including the syntactic domain.

[0037] In some embodiments, this method: Virus-specific to include a chimeric antigen receptor (CAR) or nucleic acid encoding a CAR. A step of modifying immune cells, wherein the CAR specifically binds to target antigens other than CD30. A method comprising an antigen-binding domain.

[0038] In some embodiments, the virus-specific immune cells are one or more non-identical CARs. or comprising nucleic acids that encode one or more non-identical CARs.

[0039] In some embodiments, the target antigen other than CD30 is a cancer cell antigen.

[0040] In some embodiments, target antigens other than CD30 are CD19, CD20, CD22, ROR1R, CD4, CD7, CD38, BCMA, Mesotheline, EGFR, From GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY and PSCA Selected.

[0041] In some embodiments, the target antigen other than CD30 is CD19.

[0042] In some embodiments, the virus-specific immune cells are virus-specific T cells. That is the case.

[0043] In some embodiments, the virus-specific immune cells are Epstein-Barrvirus It is specific to EBV.

[0044] This disclosure also concerns viruses that can be obtained or obtained by the methods described herein. It provides specific immune cells.

[0045] This disclosure also relates to virus-specific immune cells and pharmaceutically acceptable carriers. The present invention provides a pharmaceutical composition comprising a juvant, an excipient, or a diluent.

[0046] This disclosure also includes the use of the virus as described herein for use in methods of treatment or prevention. The present invention provides specific immune cells or pharmaceutical compositions.

[0047] This disclosure also includes the use of the following methods for treating or preventing cancer: The present invention provides Rus-specific immune cells or a pharmaceutical composition.

[0048] This disclosure also relates to the manufacture of pharmaceuticals for the treatment or prevention of cancer, and the virus caused by this disclosure. The use of S-specific immune cells or pharmaceutical compositions is provided.

[0049] This disclosure also relates to the therapeutically effective amount of virus-specific immune cells or pharmaceutical compositions provided by this disclosure. The present invention provides a method for treating or preventing cancer, which includes administering a prophylactic effective dose.

[0050] In some embodiments, the above cancers include CD30-positive cancers, EBV-related cancers, hematological cancers, and myeloid cancers. Malignant tumors of the blood vessels, malignant tumors of the hematopoietic system, lymphoblastic hematological malignancies, myelodysplastic syndrome, leukemia, T-cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, diffuse large B cell lymphoma Bolar lymphoma, primary mediastinal B-cell lymphoma, EBV-associated lymphoma, EBV-positive B-cell lymphoma Tumor, EBV-positive diffuse large B-cell lymphoma, EBV-positive with X-linked lymphoproliferative disorder Sexual lymphoma. EBV-positive lymphoma associated with HIV infection / AIDS, oral leukoderma, - Kitt lymphoma, post-transplant lymphoproliferative disorder, central nervous system lymphoma, anaplastic large cell lymphoma tumor, T-cell lymphoma, ALK-positive undifferentiated T-cell lymphoma, ALK-negative undifferentiated T-cell lymphoma peripheral T-cell lymphoma, cutaneous T-cell lymphoma, NK-T-cell lymphoma, extranodal NK- T-cell lymphoma, thymoma, multiple myeloma, solid tumor, epithelial cell carcinoma, gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma Gastrointestinal cancers, liver cancer, hepatocellular carcinoma, bile duct cancer, head and neck cancer, head and neck squamous cell carcinoma, oral cancer, oropharyngeal cancer , oral cavity cancer, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, colorectal cancer, colorectal cancer, colon cancer, cervical cancer, prostate cancer, Lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, urothelial cancer, skin cancer , melanoma. Progressive melanoma, renal cell carcinoma, renal cell carcinoma, ovarian cancer, ovarian cancer, mesothelioma, breast cancer, brain Tumors, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, progressive systemic mastocytosis, The disease is selected from the group consisting of germ cell tumors or testicular germinal carcinomas.

[0051] This disclosure also relates to the treatment or prevention of diseases or conditions characterized by alloreactive immune responses. Virus-specific immune cells or pharmaceutically active ingredients according to this disclosure for use in methods of prevention To provide finished products.

[0052] This disclosure also relates to the treatment or prevention of diseases or conditions characterized by alloreactive immune responses. The use of virus-specific immune cells or pharmaceutical compositions as described herein in the manufacture of pharmaceuticals is proposed. To provide.

[0053] This disclosure also relates to methods for treating or preventing diseases or conditions characterized by alloreactive immune responses. and, in a therapeutically or prophylactically effective amount of virus-specific immune cells or, according to this disclosure, The present invention provides a method comprising administering a pharmaceutical composition.

[0054] In some embodiments, the disease or condition characterized by the above-mentioned alloreactive immune response is an allotransferable disease. It is a disease or condition related to plants.

[0055] In some embodiments, the above disease or condition is graft-versus-host disease (GVHD). ru.

[0056] In some embodiments, the disease or condition is graft rejection.

[0057] In some embodiments, the above method involves taking allografts before harvesting the donor of the allograft. - Administer a therapeutically or prophylactically effective amount of virus-specific immune cells or a pharmaceutical composition to the target. This includes doing so.

[0058] In some embodiments, the above method provides a therapeutically or prophylactically effective amount of virus-specific immunosuppressant. This includes administering disease cells or a pharmaceutical composition to a recipient for allogeneic transplantation.

[0059] In some embodiments, the above method involves distributing an allograft in a therapeutically or prophylactically effective amount. This includes contacting virus-specific immune cells or a composition.

[0060] This disclosure also relates to methods of treating or preventing diseases or conditions by allografting. This disclosure provides virus-specific immune cells or pharmaceutical compositions for the purpose of [doing so].

[0061] This disclosure also relates to the manufacture of pharmaceuticals for treating or preventing diseases or conditions through allografting. This disclosure provides for the use of virus-specific immune cells or pharmaceutical compositions.

[0062] This disclosure also relates to a therapeutically or prophylactically effective amount of virus-specific immune cells or by this disclosure. Treatment or prevention of disease or condition by allograft, including administration of a pharmaceutical composition to a target To provide a method.

[0063] In some embodiments, the above method involves taking allografts before harvesting the donor of the allograft. - Administer a therapeutically or prophylactically effective amount of virus-specific immune cells or a pharmaceutical composition to the target. This includes doing so.

[0064] In some embodiments, the above method provides a therapeutically or prophylactically effective amount of virus-specific immunosuppressant. This includes administering disease cells or a pharmaceutical composition to a recipient for allogeneic transplantation.

[0065] In some embodiments, the above method involves distributing an allograft in a therapeutically or prophylactically effective amount. This includes contacting virus-specific immune cells or a composition.

[0066] In some embodiments, the allogeneic transplantation includes adoptive transfer of allogeneic immune cells.

[0067] In some embodiments, the above disease or condition is T-cell dysfunction, cancer, or infectious disease. It is a disease.

[0068] This disclosure also relates alloreactive immune cells to virus-specific immune cells or pharmaceuticals as described herein. The present invention provides a method for killing alloreactive immune cells, which includes contacting them with a target composition. [Brief explanation of the drawing]

[0069] Embodiments and experiments illustrating the principle of the present invention will be described below with reference to the attached drawings.

[0070] [Figure 1]Figure 1. Scatter plot showing the expression of HLA-A2 and CD3 EBSTs in cells obtained after culturing HLA-A2 and CD3 cells for 7 days: HLA-A2-positive main cells (upper left) or CD30-CAR-constructed EBST obtained from HLA-A2-positive main cells; or Allorea T cells derived from HLA-A2-negative main cells and non-transduced EBST obtained from HLA-A2-positive main cells (lower left) and non-transduced EBST after 7 days of co-culture (lower right), or CD30-CAR-constructed EBST obtained from HLA-A2-positive main cells (lower right).

[0071] [Figure 2A-2B] Figures 2A and 2B. Bar graphs showing the number of (2A) EBVST (i.e., CD3+, HLA-A2 positive) and (2B) alloreactive T cells (i.e., CD3+, HLA-A2 negative) cells after 7 days.

[0072] [Figure 3] Figure 3. Scatter plot showing the expression of HLA-A2 and CD71 in cells obtained after 7 days in co-cultures containing HLA-A2 positive PBMCs and non-transduced cells (NT; upper left panel), CD30-CAR construct transducers (CD30.CAR; upper right panel), CD19-CAR construct transducers (CD19.CAR; lower left panel), or CD30-CAR and CD19-CAR construct transducers (CD30+CD19.CAR; lower right panel) EBVSTs derived from HLA-A2 negative targets.

[0073] [Figure 4] Figure 4. Graph showing the proliferation of CD30.CAR EBVST prepared from blood samples taken from four representative donors. The graph shows the cumulative magnification of cells that proliferated in the culture.

[0074] [Figure 5A-5B]Figures 5A and 5B. Graphs showing the cytotoxicity of CD30.CAR EBVST against CD30-negative BJAB Burkitt lymphoma cells (5A) and CD30-positive HDLM2 Hodgkin lymphoma cells, measured by a 51Cr release test after co-culture of CD30.CAR EBVST (effector) and 51Cr-labeled target cells (target).

[0075] [Figure 6A-6B] Figures 6A and 6B. Graphs showing the reactivity of CD30.CAR EBVST prepared from blood samples collected from four representative donors to EBV antigen, measured by ELISpot analysis. Cells were stimulated with EBV latent antigen (latent) peptide, EBV soluble antigen (lytic) peptide, or not stimulated with the antigen (negative), and the number of spot-forming units per 5 × 10⁴ cells was determined. (6A) shows the reactivity of EBVST that has not been transduced with the retrovirus encoding CD30.CAR. (6B) shows the reactivity of CD30.CAR EBVST (transduced with the retrovirus encoding CD30.CAR).

[0076] [Figure 7] Figure 7. Representative images showing the results of PET scans performed on patient #1 before and 6 weeks after CD30.CAR EBVST injection.

[0077] [Figure 8] Figure 8. Representative images showing the results of PET and CT scans performed on patient #2 before and 6 weeks after CD30.CAR EBVST injection.

[0078] [Figure 9] Figure 9. The table shows the vector copy numbers in peripheral blood cells determined by qRT-PCR in blood samples obtained before (pre-infusion) and after (post-infusion) injection of CD30.CAR EBVST, as indicated by the period shown.

[0079] [Figure 10]Figure 10. Bar graph showing the results of the analysis of cell specificity to different antigens in the peripheral blood of patient #1 during the indicated period before lymphocyte depletion (pre-LD) and after CD30.CAR EBVST infusion, as determined by ELISpot analysis. PBMCs isolated from blood samples at the indicated time points were stimulated with EBV latent antigen (latent) peptide, EBV soluble antigen (lytic) peptide, other viral antigen peptide, tumor-associated antigen (TAA) antigen peptide, or unstimulated antigen (no pepmix), and the number of spot-forming units per 3 × 10⁵ cells was determined. [Modes for carrying out the invention]

[0080] The inventors have developed a method to eliminate hematological malignancies without causing GVHD and to reverse allogeneic rejection. We developed a CAR-modified virus-specific T cell (CAR-VST) approach to avoid it. .

[0081] This disclosure is intended to protect allogeneic tissues, including pre-made cell therapies, from graft rejection, or GV. To treat HD, we provide strategies to eliminate alloreactive T cells.

[0082] CD30 has been identified as a marker for alloreactive T cells, and therefore, the present invention These individuals express a chimeric antigen receptor (CAR) for CD30 (CD30.CAR). These were targeted by manipulating T cells used for sea urchin therapy. CD30.CAR expression V ST is used to reduce the alloreactive immune response in recipients. It can be used in methods of providing medical treatment.

[0083] Administering allogeneic T cells to HLA mismatch recipients means that some of the T cells are essential Because it exhibits alloreactivity, it carries the risk of alloreactive immune responses such as GVHD. Akira et al. used virus-specific cells as platform cells for expressing CD30.CAR. T cells (VSTs) were used, but this rarely causes GVHD in allogeneic recipients. This is because it has been shown to cause this, and this is probably due to their restricted TCR This is the result of the repertoire. In particular, Epstein-Barr virus-specific T cells (EBVS) T) has been administered to over 300 allogeneic recipients without evidence of GVHD.

[0084] Furthermore, CD30.CAR-expressing VST itself exhibits alloreactivity in recipients. Protected from cellular rejection, and therefore, ready-made for the treatment of, for example, CD30+ cancer It can be used directly as a therapy.

[0085] Therefore, CD30.CAR VST (i) induces allogeneic reactions in allogeneic hosts. (ii) Eliminate responsive T cells and eliminate CD30-positive cancer without causing GVHD. It sustains for a sufficient amount of time with the necessary activity.

[0086] CD30.CAR expression VST is also specific to other target antigens besides CD30, for example. By manipulating the expression of specific CARs, further target antigens can be targeted. Such cells can be manipulated to kill cells that express the target antigen. It is possible to eliminate CD30-expressing allogeneic T cells, thus eliminating the expression of the relevant target antigen. It is useful as a ready-made treatment method for cancer and other diseases.

[0087] Virus-specific immune cells This disclosure relates to virus-specific immune cells, particularly those specific to Epstein-Barr virus (EBV). This relates to immune cells. In this specification, "cell" is referred to in the singular form (i.e., "a / cell"). If applicable, it will be understood that multiple / groups of such cells are also intended.

[0088] As used herein, "virus-specific immune cells" refers to immune cells that are specific to a virus. It refers to receptors that can recognize the peptides of viral antigens in virus-specific immune cells. The body expresses / contains (preferably T cell receptors) (e.g., sites presented by MHC molecules) (Integrated). Virus-specific immune cells express endogenous nucleic acids that encode such antigen receptors. As a result of, or as a result of being manipulated to express such receptors, It can express / contain receptors. Virus-specific immune cells are preferably viral It expresses / contains a TCR specific to the antigen peptide.

[0089] Immune cells are cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, and lymphocytes. They may be globules or monocytes. Lymphocytes include, for example, T cells, B cells, NK cells, NKT cells, Alternatively, they may be innate lymphoid cells (ILCs) or their precursors. Immune cells are an example. For example, CD3 polypeptides (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), T CR polypeptide (TCRα or TCRβ), CD27, CD28, CD4 or CD8 It can be expressed. In some embodiments, immune cells are T cells, for example, CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In this embodiment, T cells are T helper cells (T H It is a cell. Several implementation forms In this state, T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)). be.

[0090] Virus-specific T cells are T cells that respond to specific viral antigens, or viruses In response to cells containing / expressing the antigen, T cells may exhibit specific functional characteristics. In some embodiments, the properties are related to effector T cells, such as cytotoxic T cells. This is a functional characteristic.

[0091] In some embodiments, virus-specific T cells have one or more of the following characteristics It can be displayed. T cells contain / express unique viruses / viral antigens. Cyto-atics for cells; diffusion, expression of IFNγ, expression of CD107a, IL- Expression of 2, expression of TNFα, expression of perforin, expression of granzyme, granulidine The expression of and / or the ligand of FAS (FASL) is unique to the T cell. When stimulated with a virus / viral antigen, or when a T cell is intrinsically linked to a virus / viral antigen, This corresponds to exposure to cells containing / expressing viral antigens.

[0092] Virus-specific T cells, when presented with the appropriate MHC molecules, are particularly... It expresses / contains a TCR capable of recognizing peptides of heterologous viral antigens. S-specific T cells may be CD4+ T cells and / or CD8+ T cells.

[0093] Virus-specific immune cells are specific to any virus. For example, viruses are dsDNA viruses (for example, adenoviruses, herpesviruses, Poxviruses, ssRNA viruses (e.g., parvovirus), dsRNA viruses Rus (e.g., reovirus), (+) ssRNA virus (e.g., picornavirus) (Togavirus), (-)ssRNA virus (e.g., orthomyxovirus, rhabdomyl virus) Viruses), ssRNA-RT viruses (e.g., retroviruses), or dsDNA-RT viruses. This disclosure may be a T virus (e.g., hepadnavirus). In particular, this disclosure is for adenovirus. Family, Herpesviridae, Papillomaviridae, Polioviridae, Poxvirus Sciidae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae Picornaviridae, Coronaviruses, Coronaviruses, Flaviviridae, Toga Viridae, Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaui Rusidae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdovirus The virus is intended to be of the family and the family Reoviridae. In some embodiments, the virus is Pusstein-Barr virus, adenovirus, herpes simplex virus type 1, herpes simplex virus type 2 Type Virus, Varicella-zoster virus, Human cytomegalovirus, Human herpesvirus Hepatitis B virus, type 8, human papillomavirus, BK virus, JC virus, smallpox, hepatitis B virus Parvovirus B19, Human Astrovirus, Norwalk Virus, Coxsat Key viruses, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory disease Syndrome viruses, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza Nza virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantan virus, Ebola virus, Marburg virus, measles virus, mumps virus, paravirus Luenza virus, picornavirus, respiratory-gated virus, rabies virus, hepatitis D virus Select from viruses such as Virus, Rotavirus, Orbivirus, Cortivirus, and Bannavirus. It will be selected.

[0094] In some embodiments, the above virus is the Epstein-Barr virus (EBV). Adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), Influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus Human immunodeficiency virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCV) Selected from MV (herpes simplex virus) or herpes simplex virus (HSV).

[0095] In some embodiments, the virus-specific immune cells are, for example, Epstein-B. - Epidemic virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus Hepatitis B virus (HPV), influenza virus, measles virus, hepatitis B virus HBV), Hepatitis C virus (HCV), Human immunodeficiency virus (HIV), Lymphocytic Selected from choriomeningitis virus (LCMV) or herpes simplex virus (HSV). It may be specific to viral peptides / polypeptides.

[0096] T cells specific to viral antigens are referred to herein as virus-specific T cells (VST). ) can be called a T cell that is specific to the antigen of a particular virus. It may be stated that there are; for example, EBV antigen-specific T cells, EBV-specific T cells, Alternatively, it may be referred to as "EBVST".

[0097] Therefore, in some embodiments, the above virus-specific immune cells are Epstein • Barr virus-specific T cells (EBVST), adenovirus-specific T cells (AdVST) ), cytomegalovirus-specific T cells (CMVST), human papillomavirus (HP) VST), influenza virus-specific T cells, measles virus-specific T cells, hepatitis B Virus-specific T cells (HBVST), hepatitis C virus-specific T cells (HCVST), Human immunodeficiency virus-specific T cells (HIVST), lymphocytic choriomeningitis virus-specific These are either LCMVSTs or herpes simplex virus-specific T cells (HSVSTs). ru.

[0098] In some preferred embodiments, the virus-specific immune cells are subjected to EBV antigen peptide It is specific to cytoplasm / polypeptides. In a preferred embodiment, virus-specific immune cells are These are Epstein-Barr virus-specific T cells (EBVSTs).

[0099] Regarding the EBV virus, for example, Stanfield and Luftiq, F It is described in 1000Res. (2017)6:386 and Odumade et al., Cl See Microbiol Rev(2011) 24(1):193-209. I want to.

[0100] EBV involves the binding of the viral protein BMFR2 to β1 integrin, and the virus The binding of the protein gH / gL to integrins avβ6 and avβ8 in epithelial cells Infection occurs. EBV has viral glycoproteins gp350 and CD21 and / or CD35. The interaction, followed by the interaction between the virus gp42 and MHC class II, leads to B cells. Infection occurs. These interactions induce the fusion of the viral envelope with the cell membrane, and the virus The virus allows the cells to enter. Once inside, the viral capsid lyses, The viral genome is transported to the nucleus.

[0101] EBV has two replication modes: latent and soluble. The latent cycle is vilion It does not result in production and can occur in B cells and epithelial cells. EBV genomic circular DNA is They exist as episomes in the cell nucleus and are copied by the host cell's DNA polymerase. During the incubation period, only a portion of the EBV gene is expressed, known as the incubation program. One of three different patterns, with separate sets of viral proteins and RNA It produces. The latent cycle is, for example, Amon and Farrell, Reviews. In Medical Virology (2004) 15(3):149-56 It is included and its entirety is incorporated herein by reference.

[0102] EBNA1 protein and non-coding RNA EBER are involved in latent programs I-III. Each is manifested in its own way. Latent programs II and III are EBNALP, LMP1, L Further including the expression of MP2A and LMP2B proteins, latent program III is EB This further includes the expression of NA2, EBNA3A, EBNA3B, and EBNA3C.

[0103] EBNA1 is multifunctional and influences genetics through positive and negative regulation of the viral promoter. It plays a role in gene regulation, extrachromosomal replication, and maintenance of the EBV episomal genome. ellman et al., J Gen Virol. (2009);90(Pt9):2251- 2259). EBNA2 is involved in the regulation of latent viral transcription in cells infected with EBV. Contributes to immortalization (Kempkes and Ling, Curr Top Micro biol Immunol. (2015) 391:35-59). EBNA-LP is naturally occurring. It is necessary for the transformation of B cells and recruits transcription factors for viral replication (Szymu la et al., PLoS Pathog. (2018);14(2):e1006890). E BNA3A, 3B, and 3C interact with RBPJ to affect gene expression and infection cells. It contributes to the survival and proliferation of 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 mimic activated B cell receptors to function normally. Inhibits B cell signaling (Portis and Longnecker, On cogene(2004)23(53):8619-8628). EBER is a host cell ta It forms a complex with protein and ribonucleoprotein and plays a role in cell transformation. It has been proposed.

[0104] The latent cycle progresses according to one of the latent programs I-III in B cells. It can progress to stages III, II, and I. It infects naive B cells in a resting state. Then, EBV enters latent program III. The expression of the latent phase III gene activates B cells. Upon sexualization, B cells become proliferative blasts. Subsequently, EBV is typically expressed in memory B cells. By restricting to a subset of genes that cause blast differentiation, during latent phase II... It progresses. Further restriction of gene expression causes EBV to enter latent phase I. EBNA1 expression This allows EBV to replicate when memory B cells divide. In epithelial cells, latency Only Phase II occurs.

[0105] In primary infection, EBV replicates in oral and pharyngeal epithelial cells and undergoes a latent period in B lymphocytes. III, II, and I infections are established. Latent EBV infection in B lymphocytes indicates viral survival. This is necessary for subsequent replication in epithelial cells and for the release of infectious viruses into saliva. EBV infection of B lymphocytes during latent phases III and II, latent phase II infection of oral epithelial cells, and In NK cell or T cell latent phase II infection, the presence of a uniform EBV genome and gene expression are due to the presence of a uniform EBV genome. It can lead to malignant tumors characterized in that way.

[0106] Latent EBV in B cells can be reactivated and switched to lytic replication. The lytic cycle leads to the production of infectious virions and can occur in B cells and epithelial cells. For example, Kenney, Arvin et al., Human Herpesviruses: B iology,Therapy and Immunoprophylaxis;Cam Outlined in Chapter 25 of bridge University Press (2007) Therefore, this entirety is incorporated herein by reference.

[0107] Lysolytic replication requires the EBV genome to be linear. The potential EBV genome is It is a bisome and therefore must be linearized for soluble reactivation. In cells, lytic replication usually occurs only after reactivation from the incubation period.

[0108] Immediate lytic gene products such as BZFL1 and BRLF1 are transactivators. It acts in a way that enhances the expression of those genes themselves, as well as the expression of late-stage lytic cycle genes.

[0109] The initial lytic gene product is a catalytic component of viral replication (e.g., EBV DNA polymerase). BALF5; DNA polymerase processing factor BMRF1, DNA binding protein Calcium BALF2, helicase BBLF4, primase BSLF1, and primase-related (Trivalent protein BBLF2 / 3) and deoxyribonucleotide metabolism (e.g., thymidine kinase) It plays a role in the ze BXLF1 and dUTPase BORF2. Other early lysis genes The product acts on transcription factors (e.g., BMRF1, BRRF1) to improve RNA stability and process It plays a role in Sing (e.g., BMLF1) or immune evasion (e.g., Apot) It is involved in BHRF1, which inhibits systolic function.

[0110] Late-lysed gene products are traditionally classified as those expressed after the initiation of viral replication. These are generally structural components of virions, such as nucleocapsid proteins. Furthermore, glycoproteins that mediate EBV binding and fusion (e.g., gp350 / 220, gp It encodes 85, gp42, gp25). Other late-lysing gene products are involved in immune evasion. It has a role; BCLF1 encodes a viral homolog of IL-10, and BALF1, It encodes a protein homologous to the anti-apoptotic protein Bcl2.

[0111] As used herein, "EBV-specific immune cells" refers to Epstein-Barr virus (E) cells. This refers to immune cells specific to EBV. EBV-specific immune cells recognize the peptide of the EBV antigen. It expresses / contains a receptor that can recognize (preferably a T cell receptor) (for example, MHC (When presented by the child). EBV-specific immune cells are preferably MHC class I. It expresses / contains a TCR specific to the EBV antigen peptide presented as such.

[0112] In some embodiments, EBV-specific immune cells are T cells, e.g., CD3+ T cells. These are cells. In some embodiments, T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In one embodiment, T cells are T helper cells (T H It is a cell. In terms of morphology, T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)). )

[0113] EBV-specific T cells are T cells that respond to the EBV antigen to which they are specific, or that contain EBV. Cells that express / respond to (e.g., EBV-infected cells) or related EBV antigens This can then reveal specific functional properties of T cells. In some embodiments, the properties are effervescent. These are functional characteristics associated with T cells, such as cytotoxic T lymphocytes (CTLs).

[0114] In some embodiments, the EBV special T cell is unique to EBV / E May display cytotics for cells containing / expressing BV antigen; Dispersion, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perf Expression of orin, expression of granzyme, expression of granuridine, and / or ligament of FAS The expression of FASL; T cells respond to stimulation with their specific EBV / EBV antigen. Or depending on exposure to cells containing / expressing EBV / EBV antigen, which is unique to the T cell. EBV special T cells may exhibit one or more characteristics.

[0115] EBV-specific T cells, preferably when presented by appropriate MHC molecules, The cells express / contain a TCR capable of recognizing the EBV antigen peptide, which is specific to the cells. BV-specific T cells may be CD4+ T cells and / or CD8+ T cells.

[0116] EBV-specific immune cells can detect any EBV antigen, for example, the EBV antigen described herein. It may be specific to the source. A population of immune cells specific to EBV, or multiple EBV-specific immune cells. The composition containing immune cells may contain immune cells specific to one or more EBV antigens.

[0117] In some embodiments, the EBV antigen is an EBV latent antigen, for example, type III latent antigen. Antigens (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BA) RF1, EBNA2, EBNA3A, EBNA3B, or EBNA3C), Type II latent antigen (For example, EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B or BAR) F1), or type I latent antigen (e.g., EBNA1 or BARF1). In the application method, EBV antigen is EBV lytic antigen, for example, immediate early lytic antigen (for example, BZLF1, BRLF1, or BMRF1), early lytic antigen (e.g., BMLF1, BMR) F1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, B NLF2A, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BM RF2, FU, or EBNA1-FUK), or late-lysis antigens (e.g., BALF4, BI) LF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3 or It is a GP350.

[0118] Chimeric antigen receptor This disclosure relates to virus-specific immune cells containing / expressing chimeric antigen receptors (CARs). do.

[0119] Chimeric antigen receptors (CARs) provide both antigen-binding and T-cell activation functions. It is a recombinant receptor molecule. The CAR structure and engineering are described in their entirety by reference, for example, in this specification. It is incorporated into Dotti et al., Immunol Rev (2014) 257(1) as outlined. It is being done.

[0120] CARs are antigen-binding domains linked to the signaling domain via a transmembrane domain. It includes. Any hinge or spacer domain contains an antigen-binding domain and a transmembrane domain. It can provide separation between cells and can act as a flexible linker. When expressed by [unclear], the antigen-binding domain is provided to the extracellular space, and the signaling domain Insulin is located inside cells.

[0121] The antigen-binding domain of a CAR mediates the binding of the CAR to a specific target antigen. The antigen-binding domain of a CAR is based on the antigen-binding region of an antibody that is specific to the target antigen. For example, the antigen-binding domain of a CAR is the phase of an antibody that specifically binds to a target antigen. It may contain the amino acid sequence of the complementarity-determining region (CDR). The antigen-binding domain of the CAR is the target It contains, or is not, the light chain and heavy chain variable region amino acid sequence of an antibody that specifically binds to an antigen. It can be. The antigen-binding domain contains the amino acid sequences of the variable regions of the antibody's light and heavy chains. It may be provided as a single-chain variable fragment (scFv). The antigen-binding domain of the CAR is Ligand: Targets antigens based on other proteins such as receptor binding and protein-protein interactions. For example, IL-13Rα2 target CARs can be antigen-binding based on IL-13. Developed using domains (e.g., Kahlon et al. 2004 Cancer) See Res 64(24):9160-9166.

[0122] The transmembrane domain is provided between the antigen-binding domain and the CAR signaling domain. The transmembrane domain fixes the CAR to the cell membrane of cells expressing the CAR, and the antigen-binding domain The CAR provides the signaling domain to the extracellular space and the signaling domain to the cell. The nucleotides originate from the transmembrane domain sequences of cell membrane-bound proteins (e.g., CD28, CD8, etc.). obtain.

[0123] Throughout this specification, the reference polypeptide / domain / amino acid sequence "derived" The cytoplasm, domain, and amino acid sequence are the reference polypeptide / domain / amino acid sequence. At least 60%, preferably 70%, 75%, 80%, 85%, 90% of the no-acid sequence. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% amino acid sequence identity. Reference polypeptide / domain / amino acid sequence " The resulting polypeptide, domain, and amino acid sequence are preferably a reference polypeptide / domain. It retains the functional and / or structural properties of the main amino acid sequence.

[0124] For example, the amino acid sequence derived from the intracellular domain of CD28 is, for instance, SEQ ID As shown in number 26, 60%, preferably 70%, of the intracellular domain of CD28 %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% Amino acid sequences having %, 97%, 98%, 99%, or 100% amino acid sequence identity It may include. Furthermore, the amino acid sequence derived from the intracellular domain of CD28 is preferably SE The functional characteristics of the amino acid sequence with Q ID number 26, namely CD28-mediated signal transduction. It retains the ability to activate.

[0125] The amino acid sequence of a given polypeptide or its domain is from a database known to those skilled in the art. It can be searched from or determined from the retrieved nucleic acid sequence. This includes GenBank, EMBL, and UniProt.

[0126] The signaling domain contains amino acid sequences necessary for activating immune cell function. CAR The signaling domain is responsible for the phosphorylation and activation of CAR-expressing cells by the immune receptor tyrosine. The intracellular domain of CD3-ζ provides the synbase activation motif (ITAM). May contain mino acid sequences. Signaling domains containing sequences of other ITAM-containing proteins. For example, the domain containing the ITAM-containing region of FcγRI is also used in CAR. (Haynes et al., 2001 J Immunol 166(1):182-187) )。CARs containing a signaling domain derived from the intracellular domain of CD3-ζ are often referred to as first generation CARs.

[0127] The signaling domain of a CAR typically also includes the signaling domain of a costimulatory protein (e.g., CD2 8, 4-1BB, etc.) necessary to provide costimulatory signals that enhance immune cell activation and effector function. CARs having a signaling domain that includes additional costimulatory sequences are often referred to as second generation CARs. In some cases, CARs are engineered to provide costimulation of different intracellular signaling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, while 4-1BB costimulation induces signaling through TNF receptor-associated factor (TRAF) adapter proteins. Thus, the signaling domain of a CAR sometimes contains costimulatory sequences derived from the signaling domains of one or more costimulatory molecules. CARs containing a signaling domain with multiple costimulatory sequences are often referred to as third generation CARs. Any hinge or spacer region can provide separation between the antigen-binding domain and the transmembrane domain and can act as a flexible linker. Such a region can be, or can include, a flexible domain that allows the binding moieties to be oriented in different directions, such as being derived from the CH1-CH2 hinge region of IgG.

[0128] By engineering a CAR to express specifically for a particular target antigen, immune cells ( can be, for example, derived from the CH1-CH2 hinge region of IgG, a flexible domain that allows the binding moieties to be oriented in different directions, or can include it.

[0129] By engineering a CAR to express specifically for a particular target antigen, immune cells ( Typically, not only T cells but also other immune cells such as NK cells can be directed to kill cells expressing the target antigen. The binding of CAR-expressing T cells (CAR-T cells) to the target antigen to which it is specific induces intracellular signaling and, as a result, activation of the T cells. Activated CAR-T cells divide and are stimulated to produce factors that result in the death of cells expressing the target antigen. The "antigen-binding domain" refers to a domain capable of binding to a target antigen. The target antigen can be, for example, a peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid,

[0130] antigen-binding domain lipid, or a fragment thereof. The antigen-binding domain according to the present disclosure can be an antibody / antibody fragment (e.g., Fv, scFv, Fab, single-chain Fab (scFab), single-domain antibody (e.g., VhH), etc.) against a target antigen or another target antigen-binding molecule (e.g., a target antigen-binding peptide or nucleic acid aptamer, ligand or other molecule). In some embodiments, the antigen-binding domain includes the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an antibody capable of specifically binding to the target antigen. In some embodiments, the domain capable of binding to the target antigen is an antigen-binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, kunitz domain, avimer, knottin, finomer, atrimer, DARPin,

[0131] affibody, nanobody (i.e., single-domain antibody (sdAb)), affilin, aphidro repeat protein (ArmRP), OBody, or fibronectin-repeat In some embodiments, the domain capable of binding to the target antigen is an antigen-binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, kunitz domain, avimer, knottin, finomer, atrimer, DARPin, affibody, nanobody (i.e., single-domain antibody (sdAb)), affilin, aphidro repeat protein (ArmRP), OBody, or fibronectin-repeat protein. - Completed, for example, Reverdatto et al., Curr Top Med Chem. 20 It is included in 15;15(12):1082-1101, the whole of which is incorporated herein by reference. It is included (for example, Boersma et al., J Biol Chem (2011) 28 See also 6:41273-85 and Emanuel et al., Mabs (2011) 3:38-48. (Referring to Teru).

[0132] The antigen-binding domain of this disclosure is generally an antibody capable of specifically binding to a target antigen. It contains VH and VL. Antibodies generally have six complementarity-determining regions (CDR); heavy chain variable region (V H) has three: HC-CDR1, HC-CDR2 and HC-CDR3, as well as light chain variable region. The VL region includes three: LC-CDR1, LC-CDR2, and LC-CDR3. Six Together, CDRs define the paratope of the antibody, which is part of the antibody that binds to the target antigen. The VH and VL regions include a framework region (FR) on both sides of each CDR. This provides a scaffold for the CDR. The VH from the N-terminus to the C-terminus includes the following structure: N-terminus - [ HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC -FR3]-[HC-CDR3]-[HC-FR4]-C; and VL include the following structure: N stage-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2] -[LC-FR3]-[LC-CDR3]-[LC-FR4]-C.

[0133] The VH and VL sequences indicate that the antigen-binding domain can be linked to other domains of the CAR. It may be provided in any appropriate format, subject to the conditions. In relation to the antigen-binding domain of this disclosure The formats contemplated include Carter, Nat.Rev.Immunol ( 2006), 6:343-357, for example, scFv, dsFV, (scFv)2 diabody, tribody, tetrabody, Fab, minibody, and F(ab)2 formats.

[0134] In some embodiments, the antigen-binding domain comprises the CDRs of an antibody / antibody fragment capable of binding to a target antigen. In some embodiments, the antigen-binding domain comprises the VH and VL regions of an antibody / antibody fragment capable of binding to a target antigen. The portion composed of VH and VL of an antibody may also be referred to herein as a variable fragment (Fv). VH and VL may be provided on the same polypeptide chain and linked via a linker sequence; such a portion is called a single-chain variable fragment (scFv). Linker sequences suitable for the preparation of scFv are known to those skilled in the art and may include serine and glycine residues.

[0135] In some embodiments, the antigen-binding domain comprises or consists of an Fv capable of binding to a target antigen. In some embodiments, the antigen-binding domain comprises or consists of an scFv capable of binding to a target antigen.

[0136] The antigen-binding domain (and thus the CAR) is specific for a target antigen that can be any target antigen. In some embodiments, the target antigen is an antigen whose expression / activity, or the overregulated expression / activity thereof, is positively associated with a disease or disorder (e.g., cancer, infectious disease or autoimmune disease). The target antigen is preferably expressed on the cell surface of cells expressing the target antigen. C AR is a type of CAR that targets cells / tissues that express a target antigen containing a specific antigen-binding domain. Therefore, it is understood that it targets the effector activity of cells that express CAR.

[0137] In some embodiments, the target antigen may be a cancer cell antigen. Cancer cell antigens are cancer Cancer cell antigens are antigens that are expressed or overexpressed by cells. Cancer cell antigens are any peptide / poly Peptides, glycoproteins, lipoproteins, glycans, glycolipids, lipids, or fragments thereof It is possible that the expression of cancer cell antigens may be associated with cancer. Cancer cell antigens are abnormally expressed by cancer cells. It may be expressed (for example, cancer cell antigens may be expressed with abnormal localization), or cancer cells Cancer cell antigens can be expressed with abnormal structures depending on the cell. It may be possible. In some embodiments, the antigen is expressed on the cell surface of cancer cells. In other words, cancer cell antigens are cancer cell surface antigens. In some embodiments, the present invention Some of the antigens bound by the antigen-binding molecules described in the document are presented on the outer surface of cancer cells. It is (i.e., extracellular). Cancer cell antigens can be cancer-associated antigens. In the embodiment, the cancer cell antigen, whose expression is related to the development, progression, or severity of cancer symptoms. These are antigens. Cancer-related antigens may be associated with the cause or pathology of cancer, or as a result of cancer. It can be abnormally expressed. In some embodiments, the cancer cell antigen is, for example, equivalent to that of a non-cancerous cell antigen. Compared to the expression levels of cells (e.g., non-cancerous cells derived from the same tissue / cell type), Expression is upregulated by cancer cells (e.g., at the RNA and / or protein level). ) is an antigen. In some embodiments, cancer-associated antigens are preferentially expressed by cancer cells. This can be done, and comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type) can be used. And it is not expressed. In some embodiments, cancer-associated antigens are mutant oncogenes or mutants It may also be a product of a tumor suppressor gene. In some embodiments, cancer-associated antigens are overproduced. Current cell proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, or cells It may be a product of surface glycolipids or glycoproteins.

[0138] Cancer cell antigens are tumor antigens, according to Zarour HM, DeLeo A, Finn OJ, et al. Category: In: Kufe DW, Pollock RE, Weichselbau m RR, et al., editors. Holland Frey Cancer Medicine. 6th edition. Hamilton ( ON): BC Decker; 2003. Cancer cell antigens are carcinoembryonic antigens: CEA, immature Mature laminin receptor, TAG-72; overexpressed protein: BING-4, calcium activity Chloride channel 2, cyclin-B1, 9D7, EphA3, HER2 / neu, Telomerase, SAP-1, Survivin; Cancer testicular antigens: BAGE, GAGE, MAGE, SAGE, XAGE, CT9, NY-ESO-1, PRAME, SSX-2; System-limited inhibitors Original: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate-specific Antigens; Mutant antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectam Chin, MART-2, p53, translated antigen: MUC1, idiotype antigen: Ig, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat Shock protein 90 (HSP90), glucose regulatory protein 78 (GRP78) Vimentin, nucleolin, feto-acinel pancreatic protein (FAPP), alkaline phosphate Fatase placental-like 2 (ALPPL-2), Siglec-5, stress-induced lymph protein Protein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophyllin B is mentioned.

[0139] In some embodiments, cancer cell antigens are Zhao and Cao, Front Im It is a cancer cell antigen described in munol. (2019)10:2250 (The whole thing is referenced.) (As incorporated herein by reference). In some embodiments, the cancer cell antigen is CD30 , CD19, CD20, CD22, ROR1R, CD4, CD7, CD38, BCMA, Mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, Selected from LeY and PSCA.

[0140] In some embodiments, the cancer cell antigen is expressed by cells of hematological malignancies. It is an antigen. In some embodiments, the cancer cell antigen is CD30, CD19, CD20, Selected from CD22, ROR1R, CD4, CD7, CD38, and BCMA.

[0141] In some embodiments, the cancer cell antigen is an antigen expressed by cells of a solid tumor. In some embodiments, the cancer cell antigens are mesothelin, EGFR, GPC3, M The selection is made from UC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

[0142] In some embodiments, the cancer cell antigen is CD19. CD19 is a metabolite of B cells. It is a maker, for example, B-cell lymphoma, acute lymphoblastic leukemia (ALL), and chronic It is a useful target for the treatment of lymphocytic leukemia (CLL) (e.g., Wang et al., Ex p Hematol Oncol. (2012)1:36).

[0143] In some embodiments, the antigen-binding domain (and therefore CAR) is multispecific. Yes. "Multispecificity" means that the antigen-binding domain exhibits specific binding to one or more targets. This means that, in some embodiments, the antigen-binding domain is bispecific antigen-binding. It is a domain. In some embodiments, the antigen-binding molecule contains at least two different anti- The proto-binding site (i.e., at least two antigen-binding sites, e.g., non-identical VH and VL) (including) includes. The individual antigen-binding portions of the multispecific antigen-binding domain are, for example, linkers. - They can be concatenated via arrays.

[0144] In some embodiments, the antigen-binding domain is configured to target at least two non-identical target antigens. It binds to the original and is therefore at least bispecific. The term "bispecific" refers to the anti- The primordial binding domain can specifically bind to at least two different antigenic determinants. This means that, in some embodiments, the multispecific antigen-binding domain / CAR is targeted anti At least one of the originals is CD30.

[0145] Each of the target antigens may independently be one of the target antigens described herein. In the embodiment, each target antigen is independently a cancer cell antigen as described herein.

[0146] The antigen-binding domain (e.g., a multispecific antigen-binding domain) provided in this disclosure is for antigen binding. It is understood that the domain contains an antigen-binding portion that can bind to a specific target. For example, antigenic binding to CD30 and other antigens. The combined domain consists of (i) an antigen-binding portion that can bind to CD30, and (ii) CD It may contain an antigen-binding moiety capable of binding to target antigens other than 30.

[0147] In the embodiments and aspects of this disclosure, the target antigen is CD30. Therefore, In some aspects and embodiments of this disclosure, the antigen-binding domain is a CD30-binding domain. be.

[0148] CD30 (also known as TNFRSF8) is described in UniProt:P28908 It is a protein that has been identified as such. CD30 belongs to the tumor necrosis factor receptor superfamily. It is a one-pass type I transmembrane glycoprotein. The structure and function of CD30 are, for example, van der Weyden et al., Blood Cancer Journal (2017) 7: e603 and Muta and Podack Immunol. Study. (2013)5 See 7(1-3):151-8.

[0149] Alternative splicing of mRNA encoded by the human TNFRSF8 gene is Isoform 1 (long isoform; UniProt: P28908-1, v1; S EQ ID number: 1), Isoform 2 ("Cytoplasmic", "Short", or "C30V" isoform) Form, UniProt:P28908-2; SEQ ID number:2), and SEQ Isoform 3, which has a missing amino acid sequence corresponding to positions 1-463 of ID number 1. The three isoforms of UniProt:P28908-3;SEQ ID number:3) This occurs. This isoform corresponds to positions 1-111 and 446 of SEQ ID number 1. The amino acid sequence is missing. The N-terminal 18 amino acids of SEQ ID number 1 are signal It forms a peptide (SEQ ID number 4), followed by an extracellular plasmid of 367 amino acids. IN (positions 19-385 of SEQ ID number 1, indicated in SEQ ID number 5), 21 Amino acid transmembrane domain (positions 386-406 of SEQ ID number 1, SEQ ID number (shown in No. 6), and the cytoplasmic domain of 189 amino acids (position 4 of SEQ ID No. 1) Formed by 07-595 (as indicated by SEQ ID number 7).

[0150] In this specification, "CD30" refers to CD30 derived from any species. Includes CD30 isoforms, fragments, variants, or homologs. When used herein, A "fragment," "mutant," or "homologous" of a reference protein is a reference protein (for example, a reference At least 60%, preferably 70%, 75% of the amino acid sequence of the isoform 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% As having 98%, 99%, or 100% amino acid sequence identity, in some cases, special It can be marked. In some embodiments, a fragment of a reference protein, a mutant, or an isoform. The protein and its homolog are characterized by their ability to perform the function performed by the reference protein. It can be kicked.

[0151] In some embodiments, the above CD30 is a mammal (for example, a primate (rhesus monkey, Crynomolgus macaques, or humans, and / or rodents (e.g., rats or mice) CD30) This is the origin. In a preferred embodiment, CD30 is human CD30. Isoform Fragments, mutants, or homologs are, at will, derived from a given species, e.g., immature or from humans. At least 70%, preferably 8, of the amino acid sequence of the mature CD30 isoform. 0%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 It can be characterized as having 8%, 99%, or 100% amino acid sequence identity. The fragments of CD30 are 10, 20, 30, 40, 50, 100, 200, 300, 400, It may have one minimum length among 500 or 590 amino acids, and 10, 20, 30, 40, 5 The maximum length of one of 0, 100, 200, 300, 400, 500, or 595 amino acids It may have.

[0152] In some embodiments, the CD30 corresponds to SEQ ID number 1, 2, or 3. at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 9 It has 4%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. It contains the following amino acid sequence.

[0153] In some embodiments, the above CD30 is at least the same as SEQ ID number 5. Also 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% Amino acids having %, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity It contains or consists of an acid sequence.

[0154] In some embodiments, the fragment of CD30 corresponds to SEQ ID number 5 or 19. At least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94% It has amino acid sequence identity of %, 95%, 96%, 97%, 98%, 99%, or 100%. It contains or consists of an amino acid sequence.

[0155] The CD30 binding domain of the CAR of this disclosure is preferably specific to CD30 or a fragment thereof. It exhibits specific binding. The CD30 binding domain of the CAR of this disclosure preferably binds to extracellular CD30. It shows specific binding to the domain. The CD30 binding domain is an anti-CD30 antibody or other CD3 The binding agent may be derived from, for example, a CD30-binding peptide or a CD30-binding small molecule.

[0156] The CD30-binding domain may originate from the antigen-binding portion of an anti-CD30 antibody.

[0157] Anti-CD30 antibodies include HRS3 and HRS4 (e.g., Hombach et al., Scan d J Immunol (1998) 48(5):497-501), Schlapsc. hy et al., Protein Engineering, Design and Select tion(2004)17(12):847-860, BerH2(MBL Inter national Cat# K145-3, RRID:AB_590975), SGN -30 (For example, Forero-Torres et al., Br J Haematol (200 9) As described in 146:171-9, MDX-060 (for example, Ansell et al.) This is described in J Clin Oncol (2007) 25:2764-9; 5F1 1. Also known as iratumumab), MDX-1401 (e.g., Car Darelli et al., Clin Cancer Res. (2009)15(10):3 (as described on 376-83) and CD30 antibody (WO2020 / 068764A1 , WO2003 / 059282A2, WO2006 / 089232A2, WO2007 / 084672A2, WO2007 / 04616A2, WO2005 / 001038A2, US2007 / 166309A1, US2007 / 258987A1, WO2004 / 0 This includes the provisions described in 10957A2 and US2005 / 009769A1.

[0158] In some embodiments, the CD30 binding domain according to this disclosure is used for anti-CD30 antibodies. Includes R. In some embodiments, the CD30 binding domain according to this disclosure is anti-CD30 anti Includes the VH and VL regions of the body. In some embodiments, the CD30 binding domain according to the disclosure The index contains scFv, which includes the VH and VL regions of the anti-CD30 antibody.

[0159] There are several different conventions for defining antibody CDR and FR, e.g., Kabat et al. Sequences of Proteins of Immunological I nterest, 5th Ed.Public Health Service, Nat. ional Institutes of Health, Bethesda, MD ( 1991), Chothia et al., J. Mol. Biol. 196:901-917(19 87), and VBASE2 are described by Retter et al., Nucl. Acids This is described in Res.(2005)33(suppl 1)D671-D674. The CDR and FR of the VH and VL regions of the antibodies described in the specification conform to VBASE2. That's how it's defined.

[0160] In some embodiments, the antigen-binding domain of this disclosure includes: VH with the following CDR incorporated: HC-CDR1 with amino acid sequence SEQ ID number 8 HC-CDR2 with amino acid sequence SEQ ID number 9 HC-CDR3 having the amino acid sequence of SEQ ID number 10, or a variant thereof in which one, two, or three amino acids in one or more HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids; and The following VL incorporates the following CDR: LC-CDR1 having amino acid sequence with SEQ ID number 11 LC-CDR2 with the amino acid sequence of SAS LC-CDR3 having the amino acid sequence of SEQ ID number 13, or a variant thereof in which one, two, or three amino acids in one or more LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0161] In some embodiments, the antigen-binding domain includes the following: For the amino acid sequence of SEQ ID number 14, at least 80% sequence identity (for example) , at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% Amino acids containing %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) A VH containing or consisting of an array; and For the amino acid sequence of SEQ ID number 15, at least 80% sequence identity (for example) , at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% Amino acids containing %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) A VL containing or consisting of an array.

[0162] In some embodiments, the CD30 binding domain is the VH sequence described herein and It contains, or may contain, a single-stranded variable fragment (scFv) containing a VL sequence. VH sequence The VH and VL sequences can be covalently bonded. In some embodiments, the VH and VL sequences are flexible. The linkers are connected by a flexible linker array, for example, the flexible linker array described herein. A flexible linker sequence can be attached to the ends of the VH sequence and VL sequence, thereby the VH sequence The column and VL sequence are concatenated. In some embodiments, VH and VL are SEQ I It contains or is linked via a linker sequence comprising amino acid sequence D16 or D17. They are connected.

[0163] In some embodiments, the above CD30 binding domain is the amino acid with SEQ ID number 18. At least 80%, 85%, 86%, 87%, 88%, 89%, and 90% relative to the acid sequence. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 It contains, or consists of, an amino acid sequence having 0% sequence identity.

[0164] In some embodiments, the CD30-binding domain is, for example, a cell binding to CD30. In the external domain, it can be bound to CD30. In some embodiments, CD The 30-binding domain is bound to the CD30 epitope by the antibody HRS3, for example, Human CD3 numbered according to SEQ ID number 1, as shown in SEQ ID number 19 It can bind within the region of amino acid positions 185-335 (Schlapsch y et al., Protein Engineering, Design and Select ion (2004) 17(12):847-860, the entire article is incorporated herein by reference. (To be absorbed).

[0165] In some embodiments, the target antigen is CD19. Therefore, In some embodiments and designs, the antigen-binding domain is a CD19-binding domain.

[0166] CD19 is a protein identified by UniProt P15391-1, v6. In this specification, "CD19" refers to CD19 of any species origin. CD19 isoforms (e.g., P15391-2), fragments, and mutants (including mutants) (This includes mu) or homologous forms.

[0167] The CD19 binding domain may originate from the antigen-binding portion of the anti-CD19 antibody. For example, in the body, Zola et al., Immunology and Cell Biolog This includes FMC63, as described in y(1991)69:411-422.

[0168] In some embodiments, the CD19-binding domain according to this disclosure is the C of an anti-CD19 antibody. Includes DR. In some embodiments, the CD19 binding domain according to this disclosure is anti-CD1 9 includes the VH and VL regions of the antibody. In some embodiments, CD19 binding according to the present disclosure. The domain includes scFv containing the VH and VL regions of the anti-CD19 antibody.

[0169] In some embodiments, the antigen-binding domain of this disclosure includes: VH with the following CDR incorporated: HC-CDR1 with amino acid sequence SEQ ID number 37 HC-CDR2 with amino acid sequence SEQ ID number 38 HC-CDR3 having the amino acid sequence of SEQ ID number 39, or a variant thereof in which one, two, or three amino acids in one or more HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids; and The following VL incorporates the following CDR: LC-CDR1 with amino acid sequence SEQ ID number 40 LC-CDR2 with HTS amino acid sequence LC-CDR3 having the amino acid sequence of SEQ ID number 42, or a variant thereof in which one, two, or three amino acids in one or more LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0170] In some embodiments, the antigen-binding domain includes: For the amino acid sequence of SEQ ID number 43, at least 80% sequence identity (for example) , at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% Amino acids containing %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) A VH containing or consisting of an array; and For the amino acid sequence of SEQ ID number 44, at least 80% sequence identity (for example) , at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% Amino acids containing %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) A VL containing or consisting of an array.

[0171] In some embodiments, the CD19 binding domain is the VH sequence and VL sequence described herein. It contains, or may consist of, a single-strand variable fragment (scFv) containing a sequence. VH sequence and V The L sequence can be covalently bonded. In some embodiments, the VH and VL sequences are flexible phosphorus Linked by a linker array, for example, a flexible linker array as described herein. The linker sequence may be appended to the ends of the VH sequence and the VL sequence, thereby the VH sequence and The VL sequence is concatenated. In some embodiments, VH and VL are SEQ ID numbers. Linked via a linker sequence containing or consisting of 16 or 45 amino acids ru.

[0172] In some embodiments, the CD19 binding domain is an amicin with SEQ ID number 46. At least 80%, 85%, 86%, 87%, 88%, 89%, and 90% relative to the no-acid sequence. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 It contains, or consists of, an amino acid sequence having 00% sequence identity.

[0173] In some embodiments, the CD19-binding domain is, for example, an extracellular domain of CD19. In the main, it can be coupled to CD19. In some embodiments, CD The 19-binding domain binds to the CD19 epitope, which is conjugated by the antibody FMC63. It is possible.

[0174] transmembrane domain The CARs of this disclosure include a transmembrane domain. The transmembrane domain is located in a biological membrane, for example, a cell. An arbitrary three-dimensional structure formed by a sequence of thermodynamically stable amino acids in a membrane. In relation to this disclosure, the transmembrane domain extends to the cell membrane of cells expressing CAR. A non-acid sequence is also acceptable.

[0175] The transmembrane domain is an amino acid that forms a hydrophobic alpha-helix or beta-barrel. The sequence may include or consist of the amino acid sequence of the transmembrane domain of the CAR of this disclosure. This may also be the amino acid sequence of the transmembrane domain of a protein that includes a transmembrane domain. Or it may be derived from there. The transmembrane domain is used in GenBank, UniProt, and Swivel. ss-Prot, TrEMBL, Protein Information Resou rce, Protein Data Bank, Ensembl, and InterPro It is recorded in databases such as and / or amino acid sequence analysis such as TMHMM. It can be identified / predicted using tools (Krogh et al., 2001 J Mol). Biol 305:567-580).

[0176] In some embodiments, the amino acid sequence of the transmembrane domain of the CAR of this disclosure is on the cell surface It may also be the amino acid sequence of the transmembrane domain of the protein expressed, or based thereon. It may occur. In some embodiments, the protein expressed on the cell surface is a receptor or is a ligand, for example, an immune receptor or ligand. In some embodiments, transmembrane The amino acid sequence of the domain is ICOS, ICOSL, CD86, CTLA-4, CD80 MHC class Iα, MHC class IIα, MHC class IIβ, CD3ε, CD3 γ, CD3ζ, TCRα TCRβ, CD4, CD8α, CD8β, CD40L, PD- 1, PD-L1, PD-L2, 4-1BB, 4-1BB, OX40, OX40L, GIT R, TIM-3, Galectin 9, LAG3, CD27, CD70, LIGHT, HVEM , TIM-4, TIM-1, ICAM1, LFA-3, CD2, BTLA, CD160, LILRB2, VTCN1, CD2, CD48, 2B4, SLAM, CD30L, DR3 , amino acids of one transmembrane domain of TL1A, CD226, CD112 and CD276 The sequence may be or derived therefrom. In some embodiments, the transmembrane These are amino acids in the transmembrane domains of CD28, CD3-ζ, CD8α, CD8β, or CD4. It is an array or derived therefrom. In some embodiments, the transmembrane is CD28 It is either the amino acid sequence of the transmembrane domain or derived therefrom.

[0177] In some embodiments, the transmembrane domain is an amino acid with SEQ ID number 20 or 48. For the array, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 It contains, or consists of, an amino acid sequence having % sequence identity.

[0178] In some embodiments, the transmembrane domain corresponds to the amino acid sequence of SEQ ID number 21. and at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9 A sequence of 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. It contains, or consists of, an identical amino acid sequence.

[0179] In some embodiments, the transmembrane domain corresponds to the amino acid sequence of SEQ ID number 22. and at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9 A sequence of 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. It contains, or consists of, an identical amino acid sequence.

[0180] Signal transduction domains The chimeric antigen receptor disclosed herein includes a signaling domain. The signaling domain is This provides a sequence for initiating intracellular signal transduction in cells expressing CAR.

[0181] ITAM-containing sequences The signaling domain contains an ITAM-containing sequence. The ITAM-containing sequence contains one or more immune receptor tyrosine-based activation motifs (ITAMs). An ITAM consists of the amino acid sequence YXXL / I, where "X" represents any amino acid. In ITAM-containing proteins, the above sequence often consists of 6-8 amino acids; YXXL / I(X) 6-8 It is separated by YXXL / I. When a phosphate group is added to the tyrosine residue of ITAM by tyrosine kinase, a signaling cascade is initiated within the cell.

[0182] In some embodiments, the signal transduction domain is the amino acid sequence YXXL / I or the amino acid sequence YXXL / I(X) 6-8 It contains one or more copies of YXXL / I. In some embodiments, the signaling domain contains at least 1, 2, 3, 4, 5, or 6 copies of the amino acid sequence YXXL / I. In some embodiments, the signaling domain contains the amino acid sequence YXXL / I(X) 6-8 It contains at least one, two, or three copies of the amino acid sequence according to YXXL / I.

[0183] In some embodiments, the signal transduction domain comprises an ITAM-containing amino acid sequence. The amino acid sequence of the ITAM-containing sequence of the protein, or an amino acid sequence derived therefrom. It contains an acid sequence. In some embodiments, the signaling domain is CD3-ζ, FcγRI, CD3ε, CD3δ, CD3γ, CD79α, CD79β, FcγRIIA FcγRIIC, FcγRIIIA, FcγRIIIA, FcγRIV or DAP12 It is either the amino acid sequence of one intracellular domain of or contains an amino acid sequence derived therefrom. In some embodiments, the signaling domain is the intracellular domain of CD3-ζ. It contains an amino acid sequence that is or is derived from it.

[0184] In some embodiments, the above signaling domain is the ami of SEQ ID number 25. At least 80%, 85%, 86%, 87%, 88%, 89%, and 90% relative to the no-acid sequence. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 It contains an amino acid sequence having 00% sequence identity, or an amino acid sequence consisting of such a sequence. nothing.

[0185] Co-stimulatory array The signaling domain may further contain one or more co-stimulatory sequences. The disclosed amino acid sequence provides co-stimulation to cells expressing the CAR. The co-stimulation targets the antigen. When bound to CAR, it promotes the proliferation and survival of CAR-expressing cells, and also promotes the growth of CAR-expressing cells. It can promote tokine production, differentiation, cytotoxic function, and memory formation. Molecular mechanism of T cell costimulation. The structure is from Chen and Flies, (2013) Nat Rev Immunol This is outlined in 13(4):227-242.

[0186] The co-stimulatory sequence may be the amino acid sequence of the co-stimulatory protein, or derived therefrom. This is also good. In some embodiments, the co-stimulatory sequence is the intracellular domain of the co-stimulatory protein. It is either an amino acid sequence or an amino acid sequence derived therefrom.

[0187] When a CAR binds to a target antigen, the co-stimulating sequence undergoes ligation by an alloligon. During this process, the type of CAR provided by the co-stimulating protein from which the co-stimulating sequence originates is generated. It provides simultaneous stimulation to the cells present. For example, a SIG containing a co-stimulatory sequence derived from CD28. In the case of CARs containing a signaling domain, binding to the target antigen is via CD80 and / or CD8 Signaling in cells expressing a type of CAR induced by the binding of 6 to CD28 It induces transmission. Therefore, the co-stimulatory sequence is the co-stimulatory protein from which the co-stimulatory sequence originates. It can deliver co-stimulatory signals.

[0188] In some embodiments, the co-stimulatory protein is a member of the B7-CD28 superfamily. A receptor (e.g., CD28, ICOS), or a member of the TNF receptor superfamily. —(For example, 4-1BB, OX40, CD27, DR3, GITR, CD30, HVEM) ) may be. In some embodiments, the co-stimulatory sequence is CD28, 4-1BB, ICOS , CD27, OX40, HVEM, CD2, SLAM, TIM-1, CD30, GITR , DR3, CD226, and one of the intracellular domains of light, or derived therefrom In some embodiments, the co-stimulatory sequence is the intracellular domain of CD28, or It originates from this.

[0189] In some embodiments, the above signaling domains are one or more non-overlapping co-stimulators. Includes a sequence. In some embodiments, the above signaling domains are 1, 2, 3, 4, 5 Alternatively, it may include six co-stimulatory sequences. Multiple co-stimulatory sequences may be provided in series.

[0190] A given amino acid sequence initiates signal transduction mediated by a given co-stimulatory protein. Whether this is possible depends, for example, on the signal transmission mediated by co-stimulatory proteins. Correlation between proteins (for example, signal transduction whose expression / activity is mediated by a co-stimulatory protein) By analyzing the expression / activity of factors that are upregulated or downregulated as a result, adjustments can be made. It can be investigated.

[0191] Co-stimulatory proteins, through several transduction pathways, contribute to cell proliferation and effector functions. and upregulates the expression of genes that promote survival. For example, CD28 and ICOS are phosphatidyl Signal transduction is mediated via phatidylinositol 3-kinase (PI3K) and AKT, N Cell proliferation, effector function and It upregulates the expression of genes that promote survival. CD28 also mediated through RAS to CDC AP1 / 2 is activated via 42 / RAC1 and ERK1 / 2, and ICOS is C-MAF Activate 4-1BB, OX40, and CD27 via the MAPK path It recruits TNF receptor-related factors (TRAFs) and signaling pathways via PI3K.

[0192] In some embodiments, the signal transduction domain is CD28 or It includes a co-stimulatory sequence derived from this.

[0193] In some embodiments, the above signaling domain is the ami of SEQ ID number 26. At least 80%, 85%, 86%, 87%, 88%, 89%, and 90% relative to the no-acid sequence. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 It contains an amino acid sequence having 00% sequence identity, or a co-stimulatory sequence consisting of such an amino acid sequence. .

[0194] Kofler et al. MOL.THER.(2011) 19:760~767 is CAR- To minimize regulatory T cell-mediated suppression of T cell activity, CAR ligation To reduce the induction of IL-2 production, the LCK kinase binding site has been mutated. This describes the intracellular domain of a variant CD28. The amino acids of the mutant CD28 intracellular domain are listed. The sequence is shown with SEQ ID number 27.

[0195] In some embodiments, the above signaling domain is the ami of SEQ ID number 27. At least 80%, 85%, 86%, 87%, 88%, 89%, and 90% relative to the no-acid sequence. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 It contains an amino acid sequence having 00% sequence identity, or a co-stimulatory sequence consisting of such an amino acid sequence. .

[0196] In some embodiments, the above signal transduction domain is SEQ ID number 28 At least 80%, 85%, 86%, 87%, 88%, 89%, 9% of the amino acid sequence 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or It contains or consists of an amino acid sequence having 100% sequence identity.

[0197] In some embodiments, the signal transduction domain is 4-1BB or It includes a co-stimulatory sequence derived from it.

[0198] In some embodiments, the above signaling domain is the amino acid with SEQ ID number 49. At least 80%, 85%, 86%, 87%, 88%, 89%, and 90% relative to the acid sequence. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 It contains an amino acid sequence having 0% sequence identity, or a co-stimulatory sequence consisting of such an amino acid sequence.

[0199] In some embodiments, the above signal transduction domain is SEQ ID number 50 At least 80%, 85%, 86%, 87%, 88%, 89%, 9% of the amino acid sequence 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or It contains or consists of an amino acid sequence having 100% sequence identity.

[0200] Hinge area The CAR may further include a hinge region. The hinge region comprises an antigen-binding domain and a transmembrane domain. It can be provided between domains. The hinge region is sometimes called the spacer region. The hinge region provides flexible linkage between the antigen-binding domain and the transmembrane domain of the CAR. It is a mino acid sequence.

[0201] The presence, absence, and length of the hinge region have been shown to affect CAR function. (For example, Dotti et al., Immunol Rev (2014) 257(1) supra .).

[0202] In some embodiments, the above CAR is the CH1-CH2 hinge region of human IgG1. Region, for example, the one derived from CD8α as described in WO2012 / 031744A1 In the range area, or CD28 as described in WO2011 / 041093A1, for example. It is either a hinge region from which it originates, or contains an amino acid sequence derived therefrom, or is derived from It includes a hinge region. In some embodiments, the above CAR is human IgG1 CH1- Includes a hinge region originating from the CH2 hinge region.

[0203] In some embodiments, the hinge region is the amino acid with SEQ ID number 29 or 30. For the array, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 It contains, or consists of, an amino acid sequence having % sequence identity.

[0204] In some embodiments, the CAR is located in the CH1-CH2 hinge region of human IgG4. Includes the hinge region.

[0205] In some embodiments, the hinge region is the amino acid distribution of SEQ ID number 47 For each column, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% It contains, or consists of, an amino acid sequence having sequence identity.

[0206] In some embodiments, the CAR is the CH2-CH3 region of human IgG1. In other words, it is an amino acid sequence that is or is derived from the Fc region, or consists of such a sequence. Includes the hinge area.

[0207] In some embodiments, the hinge region is the amino acid distribution of SEQ ID number 31. For each column, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% It contains, or consists of, an amino acid sequence having sequence identity.

[0208] Hombach et al., Gene Therapy (2010) 17:1206-1213 This is a variant CH2 that reduces the activation of FcγR-expressing cells such as monocytes and NK cells. -The CH3 region is described. The amino acid sequence of the mutant CH2-CH3 region is given by SEQ ID. This is shown in number 32.

[0209] In some embodiments, the hinge region is the amino acid distribution of SEQ ID number 32 For each column, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% It contains, or consists of, an amino acid sequence having sequence identity.

[0210] In some embodiments, the hinge region is the CH1-CH2 hinge of human IgG1 The di region or an amino acid sequence derived therefrom, and the CH2-CH3 region of human IgG1 It is a region (i.e., an Fc region) or contains or derived from an amino acid sequence. Yes.

[0211] In some embodiments, the hinge region is aligned with the amino acid sequence of SEQ ID number 33. and at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9 A sequence of 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. It contains, or consists of, an identical amino acid sequence.

[0212] additional array Signal peptide CARs are signal peptides (also known as leader sequences or signal sequences) They may also be included. Signal peptides typically form a single alpha-helix. It consists of a sequence of approximately 30 hydrophobic amino acids. It is a secreted protein and a protein expressed on the cell surface. Proteins often contain signal peptides. Signal peptides are found in many proteins. This is publicly known, and databases such as GenBank, UniProt and Ensembl Recorded in and / or, for example, SignalP (Petersen et al., 2011) Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl,2008 Bioinformatics 24: It can be identified / predicted using amino acid sequence analysis tools such as 2172-2176. Cut.

[0213] The signal peptide may be located at the N-terminus of the CAR, and may also be present in newly synthesized CARs. It may be present. Signal peptides provide efficient transport of CAR to the cell surface. The granulocyte peptide is removed by cleavage and therefore expressed on the cell surface. Not included in the JukuCAR.

[0214] In some embodiments, the signal peptide is the amino acid sequence of SEQ ID NO: 34 At least 80%, 85%, 86%, 87%, 88%, 89%, 90%, and 91% of those. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Includes or consists of sequence identity. In some embodiments, signal peptide D is at least 80%, 85%, 86%, and 87% of the amino acid sequence of SEQ ID NO: 51. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , containing an amino acid sequence having 98%, 99%, or 100% sequence identity, or It consists of.

[0215] Linker sequence and further functional sequence In some embodiments, the CAR has different domains (i.e., antigen-binding domains) One or more phosphorus molecules between the in-line, hinge region, transmembrane domain, and signaling domain. Includes a CAR sequence. In some embodiments, CARs are between subsequences of a domain (e.g., It contains one or more linker sequences (between the VH and VL of the antigen-binding domain).

[0216] The linker sequence is known to those skilled in the art, for example, Chen et al., Adv Drug Del iv Rev(2013)65(10):1357-1369 (The entire text is available by reference.) (As described in the specification) In some embodiments, linker array The linker array may be flexible. A flexible linker array is a linker array that It enables relative movement of linked amino acid sequences. Flexible linkers are known to those skilled in the art. Some of them are Chen et al., Adv Drug Deliv Rev (2013) 65(1 0): Identified at 1357-1369. Flexible linker sequences are often found at a high rate. It contains glycine and / or serine residues. In some embodiments, the linker sequence is small. It contains at least one glycine residue and / or at least one serine residue. In the application form, the linker sequence consists of glycine and serine residues. In some embodiments, The linker sequence is 1-2, 1-3, 1-4, 1-5, 1-10, 1-20, 1-30, 1 It has a length of approximately 40 or 1-50 amino acids.

[0217] In some embodiments, the linker sequence is SEQ ID number: 16 or 45 It contains or consists of the amino acid sequence shown in [the image]. In some embodiments, The marker sequence is amino acid sequence 1, 2, and 3 shown in SEQ ID number: 16 or 45. It includes or consists of four or five tandem copies.

[0218] CAR may further contain additional amino acids or sequences of amino acids. For example, antigens. The conjugated molecules and polypeptides undergo expression, folding, transport, processing, purification, or It may include an amino acid sequence to facilitate detection. For example, CAR may have His(e.g., 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin Tag The sequence may include the C-terminus or C-terminus, depending on the case. In some embodiments, CA R is the detectable portion, for example, fluorescent labeling, luminescence labeling, immunodetectable labeling, radioactive labeling, chemical Includes labels, nucleic acid labels, or enzyme labels.

[0219] A specific example of CAR In some embodiments of this disclosure, CAR is: At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 18 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% An antigen-binding domain containing or consisting of a minoic acid sequence; At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 33 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% A hinge region containing or consisting of a mino acid sequence; At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 20 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% A transmembrane domain containing or consisting of a mino acid sequence; and, At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 28. 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% Mino Acid It includes or consists of.

[0220] In some embodiments of this disclosure, the CAR corresponds to the amino acid sequence of SEQ ID NO: 35 or 36 and at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 8 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 It contains, or is derived from, an amino acid sequence having 8%, 99%, or 100% sequence identity. Yes.

[0221] In some embodiments, CAR is selected from the following embodiments; Hombach E. Cancer Res. (1998) 58(6):1116-9, Hombach et al. Gene Therapy (2000) 7:1067-1075, Hombach et al. J Immunother. (1999) 22(6):473-80, Hombach et al. Cancer Res. (2001) 61:1976-1982, Hombach et al. J Immunol (2001) 167:6123-6131, Savoldo et al. Bl ood (2007) 110(7):2620-30, Koehler et al. Cancer Res.(2007)67(5):2265-2273, Di Stasi et al.Bloo d(2009)113(25):6392-402, Hombach et al. Gene Th erapy(2010)17:1206-1213,Chmielewski et al.Gen e Therapy (2011) 18:62-72, Kofler et al. Mol. Ther (2011)19(4):760-767, Gilham, Abken and Pu. le.Trends in Mol.Med.(2012)18(7):377-384 ,Chmielewski et al.Gene Therapy(2013)20:177-1 86, Hombach et al. Mol.Ther. (2016) 24(8):1423-14 34. Ramos et al. J. Clin. Invest. (2017) 127(9):346 2-3471, WO2015 / 028444A1, or WO 2016 / 008973 A1 (All of these are incorporated herein by reference in their entirety).

[0222] In some embodiments of this disclosure, CAR is: At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 46 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% An antigen-binding domain containing or consisting of a minoic acid sequence; At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 47 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% A hinge region containing or consisting of a mino acid sequence; At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 48 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% A transmembrane domain containing or consisting of a mino acid sequence, and At least 60%, 65%, 70%, 75%, and 8% of the amino acid sequence of SEQ ID NO: 50. 0%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 A having sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100% It contains or consists of amino acids.

[0223] In some embodiments of this disclosure, the CAR corresponds to the amino acid sequence of SEQ ID NO: 52 or 53 and at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 8 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 It contains, or is derived from, an amino acid sequence having 8%, 99%, or 100% sequence identity. Yes.

[0224] CAR-expressing virus-specific immune cells This disclosure relates to virus-specific immune cells containing / expressing chimeric antigen receptors (CARs). do.

[0225] CAR-expressing virus-specific immune cells may express or contain the CARs according to this disclosure. CAR-expressing virus-specific immune cells contain the nucleic acid encoding CAR according to this disclosure. However, it is also acceptable, or it may be expressed. CAR-expressing cells may contain the CAR that they express. It will be understood. Cells that express nucleic acids encoding CAR are encoded by nucleic acids. It is understood that CARs that are present also appear and that they are included.

[0226] Virus-specific immune cells containing CARs / nucleic acids encoding CARs, according to this disclosure, are cells It can be characterized by reference to its functional characteristics.

[0227] In some embodiments, a virus containing a CAR / nucleic acid encoding a CAR according to the Disclosure S-specific immune cells exhibit one or more of the following characteristics: (a) IFNγ, granuzyme, perforin, granisin, CD107a, TNFα , one or more cytotoxic / effector factors such as FASL (e.g., IFNγ, Granisi (N, perforin, CD107a, TNFα, FASL), diffusion / population expansion, and / or In cells expressing target antigens that are specific to CARs, growth factors (e.g., IL-2) are expressed. Expression in accordance with, and / or specific to immune cells and / or specific to immune cells Expression of an immune cell in response to the antigen-presenting cell; (b) Cells that express target antigens that are specific to CAR, virus-specific immune cells Cells infected with the virus and / or virus-specific immune cells are specific to the virus. Cytotoxicity against cells that present peptides of the virus antigen; (c) Cells that do not express the target antigen that CAR is specific to, virus-specific immune cells Cells that are not infected with the heterogeneous virus, and / or virus-specific immune cells are specific The virus does not cause cytotoxicity to cells that do not present the antigen peptide of the virus (i.e.) , above the baseline); (d) Cancer cells, virus-specific immunohistochemistry cells, and cells expressing target antigens that are specific to CAR. Cancer cells containing virus-infected cells that are specific to the virus, and / or virus-specific immune cells. Anticancer activity against cancer cells that present peptides of viral antigens that are specific to (e.g.) For example, cytotoxicity against cancer cells, inhibition of tumor growth, reduction of metastasis, etc.; and (e) Alloreactive immune cells, e.g., alloreactive immune cells expressing target antigens that are specific to CAR. Cytotoxicity against responsive immune cells.

[0228] Cell proliferation / population growth can be determined by analyzing cell division or cell count over a period of time. It can be investigated. Cell division is, for example, 3 In vitro analysis of H-thymidine uptake Therefore, or by CFSE dilution assay, for example, the whole is provided herein for reference. Referenced from Fulcher and Wong, Imnollselbail (1999) 77(6) Proliferating cells can be analyzed as described in 559-564. For example, Buc As described in Biotechniques, 5- It can also be identified by analyzing the uptake of ethinyl-2'-deoxyuridine (EdU). Yes. 2008 Jun;44(7):927-9, and Saliand Mitch ison,PNAS USA 2008 Feb 19;105(7):2415-24 See section 20.

[0229] As used herein, “expression” may refer to gene or protein expression. DNA expression encompasses the transcription of DNA into RNA, and can be performed by various means known to those skilled in the art, for example, , by quantitative real-time PCR (qRT-PCR), or by reporter-based methods It can be measured by measuring the level of mRNA. Similarly, Protein expression can be performed by various methods well known in the art, for example, by antibody-based methods. For example, Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry - It can be measured by ELISA, ELISPOT, or reporter-based methods. can.

[0230] Cytotoxicity and cell death are described, for example, by Zaritskaya et al., Expert Rev. Vaccines (2011), 9(6):601-616 (the entire article is explained by reference) The investigation can be conducted using one of the methods outlined in the detailed document. Examples of in vitro assays for cytotoxicity / cell death assays include, 51 Chromium release Lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthio Azole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release Release assays such as those for calcein-acetoxymethyl (calcein-AM) release. Assays are one example. These assays are based on the detection of factors released from lysed cells. Then, cell death is measured. Cell death by a given cell type is, for example, when test cells are subjected to a given cell type. By co-culturing with cellular types and measuring the number / percentage of viable / dead test cells after an appropriate period. It can be analyzed.

[0231] The cells were found to be anti-cancer by analysis in appropriate in vitro assays or in vivo models. It can be evaluated for its cancer activity.

[0232] In some embodiments, CD30-specific CAR-expressing EBV-specific immunohistochemicals of the present disclosure The cell exhibits one or more of the following characteristics: (a) IFNγ, granzyme, pefrin, granulite, and granulite are used against cells expressing CD30. Expression of one or more site-Mexico factors such as din, CD107a, TNFα, and FASL. Response to EBV-infected cells, and / or cells that present EBV antigen peptides. The response to; (b) Cells expressing CD30, cells infected with EBV, and / or cells containing EBV antigen Cytotoxicity against cells that present cytotoxicity; (c) Cells that do not express CD30, cells that are not infected with EBV, and / or cells that do not express EBV It does not cause cytotoxicity to cells that do not present the original peptide (i.e., above baseline) (to rotate); (d) Cancers containing cells expressing CD30, cancers containing cells infected with EBV, and / or Anticancer activity against cancers, including cells that present EBV antigen peptides (for example, against cancer cells) This includes cytotoxicity, inhibition of tumor growth, and reduction of metastasis; as well (e) Alloreactive immune cells, for example, alloreactive immune cells expressing CD30 Cytotoxic.

[0233] In some embodiments according to various aspects of this disclosure, the virus-specific immune cells are: It may contain / express one or more CARs (e.g., 2, 3, 4, etc.).

[0234] In some embodiments, the virus-specific immune cells acquire one or more non-identical CARs May contain / express one or more non-identical CARs. Virus-specific immune cells that contain / express one or more non-identical CARs. can include / express CARs specific for non-identical target antigens. For example, Example 4 of the present specification describes virus-specific immune cells that include / express a CD30-specific CAR and a CD19-specific CAR. Each of the non-identical target antigens can independently be a target antigen as described herein. In some embodiments, each non-identical target antigen is independently a cancer cell antigen as described herein. In some embodiments, one of the non-identical target antigens is CD30. In some embodiments, the virus-specific immune cells that include / express one or more non-identical CARs include a CD30-specific CAR and a CAR specific for a target antigen other than CD30.

[0235] In some embodiments, one of the non-identical target antigens is CD30. In some embodiments, the virus-specific immune cells that include / express one or more non-identical CARs include a CD30-specific CAR and a CAR specific for a target antigen other than CD30. CD30-specific CAR, and a CAR specific for a target antigen other than CD30.

[0236] Production of CAR-expressing virus-specific immune cells Methods for generating / expanding a population of virus-specific immune cells in vitro / ex vivo are well known to those skilled in the art. Typical culture conditions (i.e., cell culture medium, additives, temperature, gas atmosphere), cell number, culture period, etc. can be determined, for example, by referring to Ngo et al., J Immunother . (2014) 37(4):193-203, which is hereby incorporated herein by reference in its entirety. . (2014) 37(4):193-203, which is hereby incorporated herein by reference in its entirety. Preferably, the cell cultures according to the invention can be maintained at 37

[0237] °C in a humidified atmosphere containing 5% CO2. The cells of the cell cultures can be established and / or maintained at any suitable density, as can be readily determined by those skilled in the art. For example, the culture can be established at an initial density of about 0 .5×10 cells to about 5×10 cells per ml of culture (e.g., about 1×10 6 cells to about 5×10 6 cells per ml of culture (e.g., about 1×10 6 cells / ml). established.

[0238] Cell culture is performed using wells in, for example, cell culture plates, cell culture flasks, and bioreactors. This can be done in any container suitable for the culture volume. In some embodiments, the process can be carried out in detail. Cells are bioreactors, for example, Somerville and Dudley, Onc. The following is described in oimmunology(2012)1(8):1435-1437 It is cultured in an io reactor, which is incorporated herein by reference in its entirety. In some embodiments, cells are cultured in a GRex cell culture vessel, for example, a GRex flask or It is cultured in a GRex 100 bioreactor.

[0239] This method generally involves antigen-presenting cells (A) that present viral antigen peptide:MHC complexes. In the presence of PCs, a population of immune cells (e.g., immune cells) containing cells with antigen-specific receptors This process causes the activation and proliferation of a heterogeneous population of cells, such as peripheral blood mononuclear cells (PBMCs). This includes culturing under conditions that provide appropriate co-stimulation and signal amplification. APC is They may be infected with a virus that codes for viral antigens / peptides (multiple), or they may be infected with a virus that codes for viral antigens / peptides. It may contain / express, and may present a viral antigen peptide in the context of an MHC molecule. Good. The stimulation triggers T cell activation, which promotes cell division (proliferation) and specifically targets viral antigens. This results in the generation and / or expansion of heterogeneous T cell populations. The process of T cell activation is as described by those skilled in the art. This is well known, for example, Immunobiology, 5th Edn. Janeway CA Jr, Travers P, Walport M et al., New York: Garl and Science (2001), Chapter 8 (the entire text is referenced in this specification). See (which is incorporated into the book).

[0240] The cell population obtained after stimulation showed a greater number of virus-specific T cells compared to the pre-stimulation population. It is concentrated in relation to (i.e., virus-specific T cells increase in the population after stimulation) (They exist at a certain frequency). In this way, virus-specific populations of T cells are different specific They are generated / proliferated from heterogeneous populations of sexually active T cells. It can be generated from a single T cell by stimulation and subsequent cell division. Virus-specific The existing population of T cells is stimulated by the virus-specific T cell population and as a result It can be expanded by cell division.

[0241] The aspects and embodiments of this disclosure relate particularly to EBV-specific immune cells. Therefore, In some embodiments, the virus may be EBV, and the viral antigen may be EBV antigen. Methods for generating / expanding populations of EBV-specific immune cells include, for example, WO2013 / 088114A1,Lapteva and Vera,Stem Cells Int (2011):434392, Straathof et al., Blood (2005) 105 (5):1898-1904, WO2017 / 202478A1, WO2018 / 052 947A1 and WO2020 / 214479A1 (all of these are by reference in their entirety) (This is incorporated herein).

[0242] This method targets the T cell receptor (TCR) specific to the EBV antigen peptide:MHC complex. The T cells containing the APC present an EBV antigen peptide:MHC complex that is specific to the TCR. The step includes being stimulated by APC, which encodes the EBV antigen / peptide, or Infection with a virus containing / expressing it, and the EBV antigen peptide in association with MHC molecules. This indicates that stimulation triggers T cell activation, promoting cell division (proliferation) and specifically targeting the EBV antigen. This leads to the generation and / or expansion of heterogeneous T cell populations.

[0243] This method typically involves a population of immune cells that corresponds to the EBV antigen or AP antigen of the viral antigen. By contacting the peptide corresponding to the C-presented peptide, the virus / viral antigen This includes stimulating immune cells specific to [the target group]. Such method steps are described herein as " This is sometimes called a "stimulation" or "stimulation step." The steps in such a method are typically This includes the maintenance of cells in cultures in vitro / ex vivo, and may be referred to as "stimulated cultures." .

[0244] In some embodiments, the above method includes one or more additional stimulation steps. In other words, in some embodiments, the method restimulates the cells obtained by the stimulation step. This includes one or more further steps. Such further stimulation steps are described in this specification. In books, this is sometimes called "restimulation" or "restimulation step." This typically involves maintaining cells in culture in vitro / ex vivo, and is called "restimation culture." It could be called "..."

[0245] PBMC obtained by the stimulation step (for re-stimulation) described herein (for stimulation) ) or "contacting" a cell population with a peptide corresponding to a viral antigen is generally considered to be, Culturing PBMCs / cell populations in vitro / ex vivo in cell culture medium containing peptides. It will be understood that this includes the following. Similarly, PBMCs / cell populations against viral antigens "Bringing" the corresponding APC-presenting peptide into contact with the APC and PBMC / cells generally involves the interaction between the APC and the PBMC / cell. It is understood that this includes co-culturing the population in cell culture medium in vitro / ex vivo. Hey.

[0246] In some embodiments, the above method involves using PBMCs to obtain viral antigens (e.g., EBV antigens). This includes contacting the peptide(s) corresponding to ). In such embodiments, P APCs within a BMC population (e.g., dendritic cells, macrophages, and B cells) are (e.g., Internalized (through phagocytosis and ingestion), MHC class I molecules (cross-presentation) and / or MHC Antigens on class II molecules are treated and presented, and then CD8+ and / or in the PBMC population. Alternatively, it activates CD4+ T cells.

[0247] A peptide that "corresponds" to a reference antigen contains the amino acid sequence of the reference antigen, or it does not. For example, the peptide "corresponding" to EBNA1 in EBV has the same amino acid composition as EBNA1. The amino acid sequence found within the column (i.e., a partial sequence of the amino acid sequence of EBNA1) ) contains or consists of. The peptides used herein are typically 5-30 a The length of amino acids, for example, 5-25 amino acids, 10-20 amino acids, or 12-18 amino acids. It has one of the acids. In some embodiments, the peptide is 5, 6, 7, 8, 9, 1 Of the 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids It has one length. In some embodiments, the peptide has a length of about 15 amino acids. As used herein, "peptide" may refer to a group of non-identical peptides.

[0248] In some embodiments, the above method uses peptides corresponding to one or more antigens. In such embodiments, there is at least one peptide corresponding to each antigen. For example, if the method uses peptides corresponding to EBNA1 and LMP1, the peptide The drug contains at least one peptide corresponding to EBNA1, and at least one peptide corresponding to LMP1. Both contain one peptide.

[0249] In some embodiments, the above method involves a peptide corresponding to all or part of the reference antigen. A peptide is used. The peptide corresponding to all of the given antigens covers the entire amino acid sequence of the antigen. —In other words, together, the peptide is composed of amino acids from the amino acid sequence of a given antigen. It contains all of them. A peptide corresponding to a part of a given antigen contains a part of the amino acid sequence of the antigen. Cover. In some embodiments, the peptide covers a portion of the amino acid sequence of the antigen. Tides together make up, for example, more than 10% of the amino acid sequence of the antigen, for example, 15%, 20%, 25%. %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 It can cover percentages of 80%, 85%, 90%, or more than 95%.

[0250] In some embodiments, the above method uses duplicate peptides. The "duplicate" peptides are They share a common amino acid sequence, or more typically, an amino acid sequence. For example, the first peptide The do consists of amino acid sequences corresponding to positions 1-15 of the EBNA1 amino acid sequence, and the second p Ptido consists of amino acid sequences corresponding to positions 5-20 of the EBNA1 amino acid sequence. Peptides 1 and 2 are duplicate peptides corresponding to EBNA1, and consist of 11 amino acids. There is duplication. In some embodiments, there are 1-20, 5-20, and 8-15 duplicate peptides. Or it is duplicated by one of 10 to 12 amino acids. In some embodiments, The duplicated peptides are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or It is duplicated by one of the 15 amino acids. In some embodiments, the duplicated peptide The cydorhizome has 11 duplicate amino acids.

[0251] In some embodiments, the method involves 1 to 1, which correspond to all or part of a predetermined reference antigen. We use peptides with a length of 5 to 30 amino acids, where 20 amino acids are duplicated.

[0252] In some embodiments, the method involves 11 amino acids corresponding to all of a given reference antigen. A peptide having a length of 15 amino acids with overlapping acids is used. A mixture of such peptides is then used. In this specification, the substance is referred to as "pepmix peptide" in relation to a given antigen. It may be referred to as "pool" or "pepmix". For example, in the implementation of this specification The "EBNA1 pepmix" used in Example 1 is from UniProt: The entire amino acid sequence of EBNA1 shown in P03211-1, v1, consists of 11 amino acids. This refers to a pool of 158-mer and 15-mer peptides with overlapping acids.

[0253] In some embodiments of various aspects of this disclosure, a "corresponding" to a given viral antigen is described. The "peptide" may also be a pepmix for the antigen.

[0254] In certain embodiments, this method uses peptides corresponding to one or more EBV antigens. In certain embodiments, this method uses Pepmix for one or more EBV antigens. In some embodiments, one or more EBV antigens are used as EBV latent antigens, for example. Type III latent antigens (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LM) P2B, BARF1, EBNA2, EBNA3A, EBNA3B, or EBNA3C), Type II latent antigens (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2) B or BARF1), or type I latent antigen (e.g., EBNA1 or BARF1), E BV lytic antigens, e.g., immediate-early lytic antigens (e.g., BZLF1, BRLF1, or BM) RF1), early soluble antigens (e.g., BMLF1, BMRF1, BXLF1, BALF1) , BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, B HLF1, BLLF2, BKRF4, BMRF2, FU or EBNA1-FUK), and Late hemolytic antigens (e.g., BALF4, BILF1, BILF2, BNFR1, BV) Select from RF2, BALF3, BALF5, BDLF3, or gp350.

[0255] In some embodiments according to various aspects of this disclosure, one or more EBV antigens are BZ LF1, BRLF1, BMLF1, BMRF1, BXLF1, BALF1, BALF2, BGLF5, BHRF1, BNLF2A, BNLF2A, BNLF2B, BHLF1, B LLF2, BKRF4, BMRF2, BALF4, BILF1, BILF2, BNFR1 EBV lytic antigen selected from BVRF2, BALF3, BALF5, and BDLF3 It is or includes the same. In some embodiments, one or more EBV antigens are , BZLF1, BRLF1, BMLF1, BMRF1, BALF2, BNLF2A, BN It is an EBV lytic antigen selected from LF2B, BMRF2, and BDLF3, or Includes.

[0256] In some embodiments, one or more EBV antigens are EBNA1, EBNA- LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1, LMP1 , or containing an EBV latent antigen selected from LMP2A and LMP2B. In some embodiments, one or more EBV antigens are EBNA1, LMP1, L An EBV latent antigen selected from MP2A and LMP2B, or containing one thereof.

[0257] In some embodiments, one or more EBV antigens are EBNA1, LMP1, LMP 2, BARF1, BZLF1, BRLF1, BMLF1, BMRF1, BMRF2, BA Selected from LF2, BNLF2A, and BNLF2B.

[0258] In some embodiments, the above method is used with EBNA1, LMP1, LMP2, BAR F1, BZLF1, BRLF1, BMLF1, BMRF1, BMRF2, BALF2, B Peptides corresponding to NLF2A and BNLF2B are used in some embodiments. The methods are EBNA1, LMP1, LMP2, BARF1, BZLF1, BRLF1, B For MLF1, BMRF1, BMRF2, BALF2, BNLF2A, and BNLF2B Use the Pepmix.

[0259] In some embodiments, the above method involves PBMCs (e.g., CD45RA-positive cells) PBMC, which has been depleted, EBNA1, LMP1, LMP2, BARF1, BZLF1 BRLF1, BMLF1, BMRF1, BMRF2, BALF2, BNLF2A and B The process includes contacting the peptide corresponding to NLF2B. In some embodiments, The above method involves using PBMCs (for example, PBMCs from which CD45RA-positive cells have been depleted) in an EB environment. NA1, LMP1, LMP2, BARF1, BZLF1, BRLF1, BMLF1, BM Pepmix for RF1, BMRF2, BALF2, BNLF2A, and BNLF2B This includes the step of bringing it into contact with the other.

[0260] In some embodiments, the method is used to obtain the fine particles obtained by the stimulation step described herein. This involves contacting a population of cells with a peptide corresponding to a viral antigen. In this state, APCs within a cell population (e.g., dendritic cells, macrophages, and B cells) are (for example) If so, it moves internally (by phagocytosis and phagocytosis), and MHC class I molecules (cross-presentation) and / or The antigen on the MHC class II molecule is treated and presented, and then CD8+ and / Alternatively, restimulate CD4+ T cells.

[0261] In some embodiments, the above method involves the PBMC being converted into a peptide corresponding to the viral antigen. This includes bringing the presented APC into contact with the present APC. In some embodiments, this method is described herein. The cell population obtained by the stimulation steps described is then subjected to an APC displaying page corresponding to the viral antigen. This includes contact with Petit.

[0262] In some embodiments, the above method includes bringing the PBMC into contact with the EBV-LCL. Hmm. The production of EBV-specific immune cells by stimulating PBMCs with EBV-LCL is, for example... See, Straathof et al., Blood (2005) 105(5):1898-1904. This is described in [the relevant section], which is incorporated herein by reference.

[0263] EBV-LCL is, for example, seen in Hui-Yuen et al., J Vis Exp (2011) 57. :3321, and Hussain and Mulherkar, Int J Mol. Cell Med (2012) 1(2):75-87 (both entire articles are referenced in this book) Infect the PBMC with EBV as described in the specification. Specific T cells were isolated from PBMCs (Primary Bacterial Cells) from blood samples of healthy donors and autologous gamma-ray irradiation. It can be prepared by co-culturing with injected EBV-LCL.

[0264] The co-culture of T cells and APCs during stimulation and re-stimulation is carried out in cell culture medium. The cell culture medium allows T cells and APCs to be maintained in the culture in vitro / ex vivo according to this disclosure. It can be any cell culture medium that can be used. A culture medium suitable for use in lymphocyte culture. The medium is well known to those skilled in the art, for example, RPMI-1640 medium, AIM-V medium, Isco Includes VES medium, etc.

[0265] In some embodiments, the cell culture medium is RPMI-1640 medium (for example, A Advanced RPMI-1640 medium) and / or Click medium (Eagle Hamami These media may contain (also known as EHAA) media. The compositions of these media are as described by those skilled in the art. This is well known. For example, preparations of RPMI-1640 medium include Moore et al., JAMA (19 67) 199:519-524 describes the preparation of Click's medium, Cli This is described in ck et al., Cell Immunol (1972) 3:264-276. RPMI-1640 medium is, for example, from ThermoFisher Scientific. It can be obtained from, and click medium is, for example, Sigma-Aldrich (catalog number It can be obtained from C5572). Advanced RPMI-1640 medium is, for example ThermoFisher Scientific (catalog number 12633012) ) can be obtained from.

[0266] In some embodiments, the above method includes RPMI-1640 medium and click medium. In cell culture medium, peptides (multiple peptides) corresponding to viral antigens (e.g., EBV antigen) The presence of PBMCs that have come into contact with the viral antigen, or APC-presenting peptides (multiple peptides) corresponding to the viral antigen. This includes culturing PBMCs under certain conditions. In some embodiments, the method involves RPMI-1 In cell culture media including 640 medium and click medium, peptides corresponding to viral antigens are used. The cell population obtained by the stimulation step described herein, which has been brought into contact with the cell population, is to be cultured. include.

[0267] In some embodiments, the cell culture medium is 25-65% (by volume) of RPMI-164 It includes 0 medium and 25-65% click medium. In some embodiments, it is used for cell culture. The medium contains 30-60% RPMI-1640 medium and 30-60% click medium. In some embodiments, the cell culture medium is RPMI-1640 medium containing 35-55%, and It contains 35-55% click medium. In some embodiments, the cell culture medium is 40-5 Contains 0% RPMI-1640 medium and 40-50% click medium. Several actual In the application method, the cell culture medium consisted of 45% RPMI-1640 medium and 45% cri Includes a culture medium. In certain embodiments, the cell culture medium is 47.5% RPMI-1640 Includes culture medium and 47.5% click medium.

[0268] In some embodiments, the cell culture medium comprises one or more cell culture medium additives. This is also fine. Cell culture medium additives are well known to those skilled in the art, and antibiotics (e.g., penicillin, s) are also good. Treptomycin), serum (e.g., human serum, fetal bovine serum (FBS), bovine serum alcohol) Bumin (BSA), L-glutamine, cytokines / growth factors, and other growth factor-rich additives Contains additives.

[0269] In some embodiments, the cell culture medium is 2.5-20% (by volume) (for example, Additives rich in growth factors (5%), e.g., 5-20% FBS, e.g., 7.5-15% The FBS contains 0.5% or 10% FBS. In some embodiments, the cell culture medium is 0.5% ~5% GlutaMax, for example, including 1% GlutaMax. Several implementations In this state, the cell culture medium is 0.5-5% Pen / Strep, for example, 1% Pen Includes / Strep

[0270] In certain embodiments, the cell culture medium includes human platelet lysate. Several embodiments Morphologically, the cell culture medium contains 1-20% (e.g., 5%) human platelet lysates (by volume). For example, 2.5-20% human platelet lysate, for example, 2.5-15%, 2.5-10% , or containing 5% human platelet lysate. Human platelet lysate is, for example, Sexton Bi It can be obtained from otechnologies.

[0271] In certain embodiments, the cell culture medium contains L-glutamine. This refers to cell culture media containing 0.5-10 mM L-glutamine, for example, 1-5 mM L-glutamine. It contains glutamines, for example, 2 mM L-glutamine.

[0272] The APC under this disclosure may be a professional APC. PCs are specialized to present antigens to T cells, and they have MHC- It is efficient for processing and presenting peptide complexes and high levels of co-stimulatory molecules. It is expressed. Professional APCs include dendritic cells (DCs), macrophages, and B Cells are included. Non-specialized APCs transfer MHC-peptide complexes to T cells, especially MHC cells. These are other cells that can present the I-peptide complex to CD8+ T cells.

[0273] In some embodiments, the APC is internally transferred by the APC (for example, Cross-presentation of antigens on MHC class I (taken up by docytosis / phagocytosis) This is an APC that can perform the cross-relationship of internalized antigens to CD8+ T cells on MHC class I. Examples of differential presentations include Alloatti et al., Immunological Reviews. This is described in (2016), 272(1):97-108, which is the entire reference. This is incorporated herein by reference. Cross-presentable APCs include, for example, dendritic cells (DCs). ), macrophages, B cells, and sinusoidal endothelial cells are included.

[0274] As described herein, in some embodiments, a virus antigen specific to the above viral antigen is APCs that stimulate immune cells include a population of cells that contain immune cells specific to the viral antigen (e.g.) For example, it is contained within PBMCs, from which a cell population specific to the viral antigen expands. In such embodiments, the APC is, for example, a dendritic cell, a macrophage, a B cell, or Any other cells within a population of cells that can present antigens to immune cells specific to the Virus antigen. It can also be cytoplasmic.

[0275] In some embodiments, the above method expresses / includes the viral antigen / its peptide. Modified APC is used. In some embodiments, the APC comes into contact with the peptide. As a result of internalizing these peptides, they present peptides corresponding to the viral antigen. Obtain. In some embodiments, APCs generally transport peptides internally. This includes culturing APCs in vitro in the presence of peptides for a sufficient period of time. It may be "pulsed".

[0276] In some embodiments, the APC is a result of the expression of nucleic acids encoding antigens within cells. APC can then present peptides corresponding to viral antigens. APC is a result of viral infection. (For example, in the case of EBV-infected B cells, e.g., LCL), nucleic acids encoding viral antigens. APC may include, for example, transfection, transduction, electroporation. As a result of nucleic acids encoding antigens introduced into cells via means such as iontophoresis, viral antibodies It may contain nucleic acids that encode the progenitor. Nucleic acids that encode viral antigens are plasmids / vectors. - May be provided inside.

[0277] In some embodiments, the APC is activated T cell (ATC), dendritic cell, B cell Cells (e.g., including LCL), and artificial antigen-presenting cells (aAPCs), e.g., Neal et al. ,J Immunol Res Ther(2017)2(1):68-79 and Tur tle and Riddell Cancer J. (2010)16(4):374 Select from those listed in -381.

[0278] In some embodiments, the APC includes immune cells specific to the viral antigen. The population of cells co-cultured for the generation / expansion of a cell population is of its own origin. In some embodiments, the population of cells co-cultured with APC is the same as the target population. Derived from the subject (or cells obtained from it).

[0279] To use polyclonal activated T cells (ATCs) as APCs and to prepare ATCs For example, see Ngo et al., J Immunother. (2014) 37(4): This is described in pp. 193-203 and is incorporated herein by reference. In short, ATC, in the presence of IL-2, agonist anti-CD3 antibody and agonist anti-CD28 antibody Stimulating PBMCs in the body to nonspecifically activate T cells in vitro. It can be generated by [this method].

[0280] Dendritic cells are, for example, Ngo et al., J Immunother. (2014) 37(4): Prepare according to methods well known in the art, as described in 193-203. This can be done. Dendritic cells can be obtained from monocytes by CD14 selection from PBMCs. It can be prepared. Monocytes may contain, for example, IL-4 and GM-CSF. They can be cultured in a cell culture medium that induces differentiation into dendritic cells. Immature dendritic cells are IL- 6. By culturing in the presence of IL-1β, TNFα, PGE2, GM-CSF, and IL-4 And that allows it to mature.

[0281] LCL is, for example, seen in Hui-Yuen et al., J Vis Exp (2011) 57:33. 21, and Hussain and Mulherkar, Int J Mol Cel. As described in l Med(2012)1(2):75-87, in this art, It can be generated according to the methods of knowledge, and both are incorporated herein by reference in their entirety. In short, LCL is a cell that produces EBV in the presence of cyclosporine A, for example. For example, by incubating concentrated cell culture supernatant of B95-8 cells with PBMCs, It can produce life.

[0282] Examples of artificial antigens (aAPCs) that cells present include cost estimation molecules CD80 and CD8 6. K562cs, engineered to represent CD83 and 4-1BBL. Lu (for example, Suhoski et al., Mol. (2007) Ther. (15) 5:981- (As described in 8) is included.

[0283] In some embodiments, the above stimulation step involves stimulating the PBMC to the corresponding PEP of the viral antigen. This includes contacting with a cydoid. In some embodiments, the restimulation step involves the viral antigen The process involves bringing immune cells specific to the virus into contact with APC-presenting peptides that correspond to the viral antigen. Includes. In some embodiments, the restimulation step involves immune cells specific to the viral antigen. This includes contacting the ATC-presenting peptide corresponding to the viral antigen.

[0284] In some embodiments, the method enhances co-stimulation in stimulation and / or re-stimulation. Further agents are used. Such agents may, for example, represent a cost estimator. The cells that represent (for example, cells like LCL or K562cs, such as CD80, CD86, CD) This includes 83 and / or 4-1BBL). In some embodiments, a co-stimulatory molecule is expressed. The cells that perform this are HLA-negative EBV replication, also known as "universal LCL" or "uLCL". It is a non-LCL. uLCL is described, for example, in US2018 / 0250379A1. Yes, they are.

[0285] Other examples of drugs to enhance co-stimulation include, for example, co-expression by T cells. Agonist antibodies specific to the stimulatory receptor (e.g., 4-1BB, CD28, OX40, IC) Costimulatory receptors (such as OS) and those expressed by T cells can activate costimulatory receptors. Exciting molecules (e.g., CD80, CD86, CD83, 4-1BBL, OX40L, ICOS) Examples include L, etc. Such drugs may be provided, for example, immobilized on beads.

[0286] In some embodiments, the stimulation and / or restimulation according to the present disclosure uses uLCL. LCL expresses the EBV antigen and also expresses CD30 along with other costimulatory molecules. Therefore, uLCL is stimulated by EBV antigen, and CD30 is transmitted via CAR. CAR EB For VST stimulation and in vitro / ex vivo expansion of CD30.CAR EBVST It is useful for providing co-stimulation.

[0287] In some embodiments, uLCL is stimulated by antigens (e.g., EBV and / or CD30). It is used as a cell that provides co-stimulation. In some embodiments, uLCL provides co-stimulation. It is used as a cell to be supplied. In some embodiments, uLCL is antigen-stimulated and It is used as a cell that provides co-stimulation. In some embodiments, uLCL is irradiated (For example, with 100 Gray). In some embodiments, the corresponding viral antigen The APC-presenting peptide is uLCL.

[0288] In certain embodiments, the method of the present disclosure provides a viral antigen-specific immune response in the presence of uLCL. This includes culturing cells. In certain embodiments, the method of the present disclosure is performed in the presence of uLCL. The process includes a restimulation step, which involves culturing immune cells specific to the virulence antigen. In this embodiment, uLCL (e.g., irradiated uLCL) provides immunity specific to the viral antigen. In co-culture with cells, uLCL can be used in a ratio between 1:1 and 1:10, for example, 1:1 0.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5 It could be one of 0.5, 1:6, 1:6.5, 1:7, 1:7.5, or 1:8. In several embodiments, uLCL (e.g., irradiated uLCL) is specific to the viral antigen. The ratio of immune cells to uLCL is 1:2 to 1:5, for example, 1:2, 1:2.5, 1: 3, 1:3.5, 1:4, 1:4.5, or 1:5, which are specific to the viral antigen. It can be used in co-culture with immune cells. In some embodiments, it is used against uLCL The ratio of immune cells specific to viral antigens is approximately 1:3.

[0289] In certain embodiments, the method disclosed herein includes / expresses the CAR described herein (or uLC (contains / expresses nucleic acids encoding such CARs) virus-specific immune cells This includes culturing in the presence of L. In certain embodiments, the methods described herein are described herein. Contains / expresses CARs (or contains / expresses nucleic acids that encode such CARs) The process includes a restimulation step, which involves culturing virus-specific immune cells in the presence of uLCL. In some embodiments, uLCL (e.g., irradiated uLCL) is described herein. Virus-specific immune cells containing / expressing CAR (or CA as described herein) (Virus-specific immune cells containing / expressing R) and uLCL in a 1:1 to 1:10 ratio. For example, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, One of the following ratios: 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, or 1:8 Virus-specific substances containing / expressing nucleic acids that encode such CARs Target immune cells. In some embodiments, uLCL (e.g., irradiated uLCL) Virus-specific immune cells containing / expressing CARs as described herein (or similar) (Contains / expresses nucleic acids encoding CAR) and between 1:2 and 1:5 for uLCL, For example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5 The ratio of virus-specific immune cells containing / expressing one of the CAs described herein. It can be used in co-culture with virus-specific immune cells containing / expressing R. In this embodiment, the CARs described herein are included in / express (or such CARs are expressed in) The ratio of virus-specific immune cells (containing / expressing nucleic acids) to uLCL is ~1:3. be.

[0290] In some embodiments, the above restimulation step is performed in the presence of uLCL to the viral antigen. This involves contacting specific immune cells with ATC-presenting peptides corresponding to viral antigens. nothing.

[0291] A population of immune cells is identified by a peptide corresponding to the viral antigen, or by an A corresponding to the viral antigen. Contacting PC-presenting peptides promotes T cell activation and proliferation. Alternatively, it can be carried out in the presence of multiple cytokines. In some embodiments, Intense is IL-7, IL-15, IL-6, IL-12, IL-4, IL-2 and / or I The procedure is performed in the presence of one or more L-21 molecules. Cytokines are added exogenously to the culture. It is understood that this is added to cytokines produced by cells in the culture. In some embodiments, the added cytokine is generated by recombination at sites This is Cain.

[0292] Therefore, in some applications, the above method is used for IL-7, IL-15, IL-6, In the presence of one or more of IL-12, IL-4, IL-2 and / or IL-21, the virus PBMCs that have been in contact with a peptide corresponding to the antigen, or peptides corresponding to the viral antigen In the presence of the APC being presented.

[0293] In some embodiments, the culture contains IL-7, IL-15, IL-6, and IL-1 2. In the presence of IL-4, IL-2, and / or IL-21. In some embodiments, The culture is in the presence 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 IL-15.

[0294] In some embodiments, the final concentration of IL-7 in the culture is 1 to 100 ng. / ml, for example, 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml It is one of them. In some embodiments, the final concentration of IL-7 in the culture is It is approximately 10 ng / ml.

[0295] In some embodiments, the final concentration of IL-15 in the culture is 1 to 100 nucleotides. g / ml, for example, 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml It is one of l. In some embodiments, the final concentration of IL-15 in the culture The final concentration of IL-15 in the culture is approximately 10 ng / ml. In some embodiments, the final concentration of IL-15 in the culture is approximately 10 ng / ml. The concentration ranges from 10 to 1000 ng / ml, for example, 20 to 500 ng / ml, 50 to 200 ng / It is one of ml or 75-150 ng / ml. In some embodiments, The final concentration of IL-15 in the nutrient solution is approximately 100 ng / ml.

[0296] In some embodiments, the final concentration of IL-6 in the culture is 10 to 1000 ng. / ml, for example, 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml It is one of the g / ml values. In some embodiments, the final IL-6 in the culture The concentration is approximately 100 ng / ml.

[0297] In some embodiments, the final concentration of IL-12 in the culture is 1 to 100 nucleotides. g / ml, for example, 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml It is one of l. In some embodiments, the final concentration of IL-12 in the culture is 1 The value is 0 ng / ml.

[0298] In some embodiments, the final concentration of IL-7 is 1 to 100 ng / ml (e.g., For example, one of the following: 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml For example, 10 ng / ml, and the final concentration of IL-15 is 1-100 ng / ml. For example, among 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml For example, one value is approximately 10 ng / ml.

[0299] In some embodiments, the final concentration of IL-7 is 1 to 100 ng / ml (e.g., For example, one of the following: 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml For example, the concentration is 10 ng / ml, and the final concentration of IL-15 is 10-1000 ng / ml. l (for example, 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml) It is one of the ml values ​​(for example, approximately 100 ng / ml).

[0300] In some embodiments, the final concentration of IL-7 is 1 to 100 ng / ml (for example) , one of the following: 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml For example, 10 ng / ml), and the final concentration of IL-6 is 10-1000 ng / ml (example For example, 20-500 ng / ml, 50-200 ng / ml, or 75-150 ng / ml One example is approximately 100 ng / ml, and the final concentration of IL-12 is 1-100 ng / ml (for example, 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml) One of the ml (for example, 10 ng / ml), and the final concentration of IL-15 is 1-10 0 ng / ml (for example, 2-50 ng / 7.5-15 ng / ml, for example, 10 ng / ml) l).

[0301] In some embodiments, the final concentration of IL-7 in the stimulated culture is 1 to 100 ng / ml (for example, 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml) One of the ml (for example, 10 ng / ml), and the final IL-15 in the stimulated culture. The concentration is 10-1000 ng / ml (for example, 20-500 ng / ml, 50-200 ng) It is either / ml or one of the following: 75-150 ng / ml (for example, about 100 ng / ml). .

[0302] In some embodiments, the final concentration of IL-7 in the stimulated culture is 1 to 100 ng / ml (for example, 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml) ml (for example, one of 10 ng / ml), and the final concentration of IL-6 in the stimulated culture is 1 0-1000 ng / ml (for example, 20-500 ng / ml, 50-200 ng / ml or It is one of 75-150 ng / ml, for example, about 100 ng / ml, in the stimulated culture. The final concentration of IL-12 is 1-100 ng / ml (for example, 2-50 ng / ml, 5-2 It is either 0 ng / ml or one of 7.5-15 ng / ml, for example, 10 ng / ml, and The final concentrations of IL-15 in the culture were 1-100 ng / ml, 2-50 ng / ml, and 5 (One of the following: ~20 ng / ml or 7.5~15 ng / ml, for example, 10 ng / ml) .

[0303] In some embodiments, the final concentration of IL-7 in the restimulated culture is 1 to 100 nucleotides. g / ml (for example, 2-50 ng / ml, 5-20 ng / ml, or 7.5-15 ng / ml) One of the l values ​​(for example, 10 ng / ml), and the final IL-15 level in the restimulated culture. The concentration is 10-1000 ng / ml (for example, 20-500 ng / ml, 50-200 ng) It is either / ml or one of the following: 75-150 ng / ml (for example, about 100 ng / ml). .

[0304] The stimulation and restimulation described herein typically last for a period sufficient for APCs to stimulate T cells. This involves co-culturing T cells and APCs for a sufficient period of time for T cells to undergo cell division.

[0305] In some embodiments, the above method involves contacting a peptide corresponding to a viral antigen. In the presence of PBMCs or APC-presenting peptides corresponding to viral antigens, at least 1 Hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, or less This includes culturing for a period of at least 7 days. In some embodiments, the culturing is 24 hours to 20 For example, days, such as 48 hours to 14 days, 3 to 12 days, 4 to 11 days, or 6 to 10 days. , or a period of 7 to 9 days.

[0306] In some embodiments, this method involves contacting a peptide corresponding to a viral antigen with the product. The population of cells obtained by the stimulation step described in the specification corresponds to the A viral antigen. In the presence of the PC-presenting peptide, at least 1 hour, 6 hours, 12 hours, 24 hours, and 48 hours This includes culturing for a period of time of 72 hours, 4 days, 5 days, 6 days, or at least 7 days. In some embodiments, the culture period is 24 hours to 20 days, for example, 48 hours to 14 days, 3 days to The duration is 12 days, 4-11 days, 6-10 days, or 7-9 days.

[0307] Stimulation and re-stimulation are performed by separating cells in the culture from the culture medium in which they were cultured. Or, for example, by diluting the culture by adding cell culture medium. Obtain. In some embodiments, the method involves collecting cells at the end of the stimulated or restimulated culture. Includes a step. In some embodiments, the restimulation step is used for the cells in the restimulation step. To achieve the desired percentage / concentration of culture medium, acclimatization medium (and any additives) Add the appropriate amount of cell culture medium (and any other additives described herein). It can be established by and

[0308] At the end of the culture period of the given stimulation or restimulation step, the cells are harvested and separated from the cell culture supernatant. They can be separated. The cells may be collected by centrifugation, and the cell culture supernatant is the cell The cells may be separated from the pellet. The cell pellet is then, for example, used for a restimulation step. The cells can be resuspended in the cell culture medium. In some embodiments, the cells are washed after collection. It may undergo a wash. The washing process involves rinsing the product in an isotonic buffer such as phosphate-buffered saline (PBS). This may include resuspending the cellular pellet, recovering the cells by centrifugation, and discarding the supernatant. good.

[0309] A method for generating and / or expanding a population of immune cells specific to viral antigens (multiple antigens may be present). The method typically involves more than one stimulation step. There are no limits. In some embodiments, the method involves more than two, three, four, or five stimulation steps. This includes methods 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1. The method includes one of the stimulation steps 1, 12, 13, 14, or 15. The steps may be different from each other.

[0310] In some embodiments, the PBMCs used in this method are CD45RA-positive cells that are depleted. It is thirsty. That is, in some embodiments, PBMCs are "CD45RA positive". It is either a "cell-depleted PBMC" or a "CD45RA-negative PBMC". CD45RA Depletion of positive cells affects NK cells and / or regulatory T cells in the generated / proliferated cell population. The intention is to reduce the number of [the specified number].

[0311] In some embodiments, the above method, for example, involves the CD45RA positive step before the stimulation step. The method includes the step of depleting the PBMCs of sex cells. In some embodiments, the method is, for example However, before the restimulation step, the stimulation step of CD45RA-positive cells by the present disclosure The process includes a step of depleting the obtained cells. Depletion of CD45RA-positive cells is, for example, M iltenyi® Biotec columns and magnetic anti-CD45RA antibody coated beads This can be achieved by any suitable method, such as magnetically activated cell sorting (MACS), which uses It is possible.

[0312] In some embodiments, the cells used to induce APCs in this method In this population, CD45RA-positive cells are depleted. That is, in some embodiments, The population of cells used to induce APC is "CD45RA-positive cell depletion" or This is the "CD45RA-negative" population. For example, in embodiments where ATC is used as APC. ATC is a population of CD45RA-positive cell-depleted PBMCs, or CD45RA-negative PBMs. It may originate from group C.

[0313] In some embodiments, the above method relates to IL-7-mediated signaling in cells. Further includes modifications of immune cells specific to viral antigens to increase IL-7 pathway. Intercellular signaling has been shown to increase the survival and antitumor activity of tumor-specific T cells. (For example, Shum et al., Cancer Discov. (2017) 7(11):12 See 38-1247 and WO2018 / 038945A1.

[0314] In some embodiments, this method is referred to in WO2018 / 038945A1 (the whole document is referenced). Nucleic acids according to embodiments described herein (as incorporated herein) are specific to viral antigens The method further includes introducing it into PBMCs or immune cells. In some embodiments, the method is This method involves introducing nucleic acids into PBMCs or immune cells specific to the viral antigen, wherein the nucleic acids are fine Encodes a polypeptide for increasing intracellular STAT5-mediated signaling.

[0315] In some embodiments, the nucleic acids (i) promote homodimerization of polypeptides. (ii) Encoding a polypeptide containing the intracellular domain of IL-7Rα do.

[0316] In some embodiments, the domain that promotes homodimerization of the polypeptide is It contains an amino acid sequence that provides for the formation of disulfide bonds between monomers of a polypeptide, or consisting thereof. In some embodiments, promoting the homodimerization of polypeptides The domain is one of the SEQ ID numbers 1-24 of WO2018 / 038945A1. It contains or consists of the amino acid sequence (for example, WO2018 / 038945A1) See paragraphs

[0074] to

[0076] .

[0317] The intracellular domain of IL-7Rα is UniProt:P16871-1, v1, 265 It contains, or may contain, an amino acid sequence corresponding to position ~459.

[0318] Nucleic acids are used in this technology field, including transduction, transfection, and electroporation. It can be introduced into cells by well known methods. In some embodiments, nucleic acids are introduced into the nucleus Transduction using an acid-containing viral vector (e.g., a retroviral vector) It is then introduced into the cells.

[0319] In some embodiments, the above method promotes (i) homodimerization of polypeptides. (ii) encoding a polypeptide containing the intracellular domain of IL-7Rα. Using a viral vector containing nucleic acids, EBV antigen-specific PBMCs or immune cells are formed This includes introducing quality improvements.

[0320] The embodiments and examples of the methods described herein express / contain the CAR described herein. sea ​​urchin, immune cells as described herein (e.g., virus-specific immune cells as described herein) This includes modifying it.

[0321] The aspects and embodiments of the methods described herein are the nuclei encoding the CARs described herein. Immune cells as described herein (e.g., viruses as described herein) express / contain acid This includes modifying specific immune cells.

[0322] Such methods typically involve introducing nucleic acids encoding CARs into immune cells. nothing.

[0323] Immune cells (e.g., virus-specific immune cells) are used in accordance with methods well known to those skilled in the art. May contain / may be modified to express a CAR or nucleic acid encoding a CAR as described in the details. This method is generally used for nucleic acid transfer for the permanent (stable) or transient expression of the transferred nucleic acid. Includes.

[0324] By using any suitable genetic engineering platform, you can modify the cells according to this disclosure. This can be done. Appropriate methods for modifying cells include, for example, the whole thing is made clear by reference. Incorporated into the detailed report: Maus et al., Annu Rev Immunol (2014) 32: Gamma retroviral vectors, lenticular viruses, as described in 189-225 Adenovirus vectors, DNA transfection, transposable vectors Genetic engineering platforms such as gene delivery and RNA transfection. Use is included. In some embodiments, this includes CAR or nucleic acids that encode CAR. Modifying sea urchin cells involves using a viral vector containing nucleic acid that codes for CARs to alter the cell's characteristics. This includes implementing it.

[0325] In some embodiments, the method disclosed herein codes a retrocoded CAR described herein. Use a virus.

[0326] Another method is, for example, Wang and Riviere Mol Ther Onc This includes data listed in olytics. See (2016)3:16015.

[0327] This method generally encodes nucleic acids / vectors containing such nucleic acids / multiple vectors. The method includes introducing multiple nucleic acids into cells. In some embodiments, the method involves the cells This further includes culturing cells under conditions suitable for the expression of nucleic acids or vectors. In this embodiment, the method is carried out in vitro. To introduce nucleic acids / vectors into cells... Appropriate methods include transduction, transfection, and electroporation. Born.

[0328] In some embodiments, the introduction of nucleic acids / vectors into the cells is transduction, for example, This includes the introduction of a torovirus. Therefore, in some embodiments, the nucleic acid is a virus The vector is either contained in a viral vector or the vector itself is a viral vector. Transduction of immune cells by methods such as Simmons and Alberola-Il a, See Methods Mol Biol. (2016) 1323:99-108. I want to.

[0329] The agent may be used in the method disclosed herein to enhance the efficiency of transduction. Sadimetrin (polybren) is used in the interaction between virions and sialic acid residues expressed on the cell surface. By neutralizing the charge repulsion, cations are commonly used to improve transduction. It is a polymer. Other agents commonly used to enhance transduction include, for example, LentiBOOST (Sirion Biotech), Retronectin (T Akara), Vectofusin (Miltenyi Biotech), and SureENTRY (Qiagen) and ViraDuctin (Cell Biola This includes poloxamer-type drugs such as bs.

[0330] In certain embodiments, the method disclosed herein is a vector encoding a CAR as described herein. In cell transduction using nucleic acids, vector fusin-1 (Miltenyi (Biotec catalog number 170-076-165) is used. In some embodiments... The method involves vectorizing the retrovirus encoding the CAR described herein. The process of bringing the cells into contact with 1, and the cells transduced by the retrovirus, and The process includes contacting a mixture containing vectorfucin-1.

[0331] In some embodiments, it is desirable that the above method introduce a nucleic acid encoding CAR. The cells are centrifuged in the presence of a cell culture medium containing a viral vector that contains nucleic acids. This includes (referred to as "spication" in this technical field).

[0332] In some embodiments, the entire method is incorporated herein by reference, for example. Koh et al., Molecular Therapy - Nucleic Acids (2 As described in 013)2,e114, electroporation is used in this disclosure This includes introducing nucleic acids or vectors.

[0333] In some embodiments, the above method is used to express CAR and / or virus-specific immune cells For example, other cells (e.g., cells that are not specific to the virus, and / or cells that express CAR) Further includes purifying / isolating from (non-existent) cells. Purifying immune cells from a heterogeneous population of cells / Methods for isolation are well known in the art, for example, the generation of immune cell markers. FACS or MACS-based methods can be used to select cell populations based on the current situation. In some embodiments, the method is specific to a particular type of cell, for example, a virus-specific cell. T cells (e.g., virus-specific CD8+ T cells, virus-specific CTLs), or CA R-expressing virus-specific T cells (e.g., CAR-expressing virus-specific CD8+ T cells, C This is for purifying / isolating AR-expressing virus-specific CTLs.

[0334] This disclosure also relates to cells obtained or obtainable by the methods described herein. And provide such groups.

[0335] composition This disclosure relates to one or more CAR-expressing virus-specific immune cells (e.g., a population) as a result of this disclosure. Further, compositions containing the above are provided.

[0336] The cells described herein may be formulated as a pharmaceutical composition or pharmaceutical for clinical use. It may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The composition is local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, subarachnoid, intraocular, Transdermal administration routes may include intraconjunctival, intratumoral, subcutaneous, intradermal, subarachnoid, oral, or injectable. It can be formulated for this purpose.

[0337] Appropriate formulations may contain cells in sterile or isotonic media. Drugs and pharmaceutical compositions It can be formulated in the form of a fluid containing a gel. The fluid formulation can be used in a selected area of ​​the human or animal body. It can be formulated for administration by injection or infusion into the region (e.g., via a catheter). .

[0338] In some embodiments, the above composition is used for, for example, injection or infusion into blood vessels or tumors. It is formulated for that purpose.

[0339] This disclosure also provides a method for producing pharmaceutically useful compositions, and the production of such compositions The method includes the steps of generating the cells described herein; and the steps of isolating the cells described herein. and / or the cells described herein may be pharmaceutically acceptable carriers, adjuvants, or excipients. This may include one or more steps selected from the steps of mixing with a diluent.

[0340] For example, in a further embodiment, the present disclosure may be used in the treatment of a disease / condition (e.g., cancer). The present invention relates to a method for formulating or manufacturing a pharmaceutical or pharmaceutical composition for the purpose of [the use of pharmaceuticals], and the method is described herein. Mix the cells with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent. This includes formulating a pharmaceutical composition or pharmaceutical by means of.

[0341] MHC mutation and matching MHC class I molecules are non-α(α) chains and β(β)2-microglobulin (B2M) It is a covalent heterodimer. The α chain has three domains called α1, α2, and α3. The α1 and α2 domains together are present to the MHC class I molecule. Peptides form a groove for binding, creating a peptide:MHC complex. In humans, MH Class C Iα chains are encoded by human leukocyte antigen (HLA) genes. There are three main types. The main HLA loci (HLA-A, HLA-B, and HLA-C) and three sub-loci (H LA-E, HLA-F, and HLA-G exist.

[0342] MHC class I α chains are polymorphic, and different α chains bind to and present different peptides. This is possible. The genes encoding MHC class Iα polypeptides are extremely diverse and different Cells derived from the target often express different MHC class I molecules.

[0343] This variability is involved in organ transplantation and the adoption of cells between individuals. The recipient's immune system recognizes the non-self MHC molecules as exogenous and does not transplant or nutrient the cells. This can trigger an immune response against the transplanted cells, which may lead to graft rejection. This refers to the process where cells within a population of transplanted cells / tissues / organs acquire exogenous MHC molecules from the recipient. It may contain immune cells that recognize it as such, and it triggers an immune response against the recipient tissue, which It can lead to graft-versus-host disease (GVHD).

[0344] Alloreactive T cells recognize non-self MHC molecules (i.e., allogeneic MHC) and react to them. It contains a TCR that can initiate an immune response. Alea functional T cells are non-self MH Corresponding to cells that express the C molecule, cell diffusion, growth factor (e.g., IL-2) expression, cytoplasm Xyco / effector factors (e.g., IFNγ, granzyme, pefrin, granulidine, C) D107a, TNFα, FASL) expression and / or cyto-mexican activity) one or more properties It can display quality.

[0345] As used herein, "allore reactivity" and "allore reactive immune response" refer to effector immune responses. This refers to an immune response against cells / tissues / organs that are not genetically identical to the diseased cells. Effector immune cells are encoded by non-self MHC / HLA molecules (i.e., effector immune cells) Cells expressing MHC / HLA molecules that are not identical to the MHC / HLA molecules expressed, or cells It may exhibit alloreactivity or alloreactive immune response to tissues / organs containing cells.

[0346] Subjects referred to as "MHC mismatch" and "HLA mismatch" in this specification are The subject is one who has MHC / HLA genes encoding non-identical MHC / HLA molecules. In some embodiments, the target of the MHC mismatch or HLA mismatch is a non-identical M Possesses MHC / HLA genes encoding HC class Iα and / or MHC class II molecules The "MHC compliant" and "HLA compliant" subjects referred to herein are the same M These are subjects that possess MHC / HLA genes encoding HC / HLA molecules. In the application configuration, the target of MHC compliance or HLA compliance is the same MHC class Iα and / or It possesses MHC / HLA genes that encode MHC class II molecules.

[0347] Where cells / tissues / organs are referred to herein as homogeneous with respect to the reference object / treatment In combination, cells / tissues / organs are obtained / derived from cells / tissues / organs other than the reference subject. In some embodiments, the allogeneic material is based on the reference MHC / HLA gene. MHC / HLA molecules that are coded as such (for example, MHC class Iα and / or MHC class MHC / HLA molecules that are not identical to MHC class II molecules (e.g., MHC class Iα and / or Includes MHC / HLA genes that encode MHC class II molecules.

[0348] Where cells / tissues / organs are referred to herein as allogeneic with respect to treatment, cells / Tissues / organs are obtained / derived from cells / tissues / organs of a subject other than the one being treated. In some embodiments, the allogeneic material is used in relation to the MHC / HLA genes of the subject being treated. MHC / HLA molecules that are coded as such (for example, MHC class Iα and / or MHC class MHC / HLA molecules that are not identical to MHC class II molecules (e.g., MHC class Iα and / or Includes MHC / HLA genes that encode MHC class II molecules.

[0349] When a cell / tissue / organ is referred to as itself in this specification with respect to a reference, Cells / tissues / organs are obtained / derived from reference cells / tissues / organs. Where an organ is referred to herein as autogenetic with respect to the reference, cells / tissues / The organ is either genetically identical to the reference object or derived from / from a genetically identical object. Cells / tissues / organs are referred to herein as subjects of treatment (e.g., autologous cells to subjects). When referred to as self in the context of treatment by administration of (a substance), cells / tissues / organs are considered self. Obtained from / derived from the cells / tissues / organs to which they are placed. In the context of the treatment of the subject, when referred to as autoheritable, cells / tissues / The organ is either genetically identical to the subject being treated, or originates from a genetically identical subject. Obtained from / from. Autologous cells / tissues / organs are referenced to the MHC / HLA genes. Therefore, the encoded MHC / HLA molecules (e.g., MHC class Iα and / or MHC class Iα) MHC / HLA molecules (e.g., MHC class Iα and / or) that are identical to the WLAS II molecule. Includes MHC / HLA genes that encode MHC class II molecules.

[0350] Where cells / tissues / organs are referred to herein as homogeneous with respect to a reference, Cells / tissues / organs are either not genetically identical to the reference object, or are derived from a reference object that is not genetically identical. To come from / obtain from. Cells / tissues / organs in this specification in the context of the treatment of a subject. When cells / tissues / organs are referred to as homogeneous, they are genetically related to the subject being treated. Derived from / from subjects that are not identical or genetically identical. Cells / tissues / organs are encoded by reference MHC / HL genes. M is not identical to molecule A (e.g., MHC class Iα and / or MHC class II molecules). HC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) It may contain MHC / HLA genes that cause this.

[0351] In some embodiments, the subject is administered according to the method described herein. CAR expresses / contains (or expresses / contains nucleic acids that encode such CARs) Virus-specific immune cells are selected based on the HLA / MHC profile of the target being treated. It will be selected.

[0352] In some embodiments, the cells administered to the target are HLA / MH cells that are compatible with the target. The cells are selected based on the fact that they are C. In some embodiments, the cells administered to the subject are Selection is based on near or complete HLA / MHC matching to the target.

[0353] As used herein, HLA / MHC alleles are defined as having the same amino acid sequence. When encoding a polypeptide having a specific characteristic, it may be determined to be a "match". That is, "one "Synonymous difference and / or non-synonymous" refers to a synonymous difference in the nucleotide sequence encoding a polypeptide. Regardless of the possibility of differences in the pedicle region, the determination is made at the protein level.

[0354] Cells that are "HLA-compatible" with respect to the reference are (i) HLA-A, -B, -C, and -8 / 8 match across DRB1; or (ii) HLA-A, -B, -C, -DRB1, and (iii) HLA-A, -B, -C, -DR A 12 / 12 match is possible across B1, -DQB1, and -DPB1. Regarding the references... Cells that are "almost perfectly HLA-matched" are (i) HLA-A, -B, -C, and -DR Matching B1 with ≥4 / 8 (i.e., 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8) , or (ii) ≥ 5 / 1 across HLA-A, -B, -C, -DRB1, and -DQB1 0 (i.e., 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10) - (iii) HLA-A, -B, -C, -DRB1, -DQB1 and -DPB ≥ 6 / 12 (i.e., 6 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 1) 2. (11 / 12, or 12 / 12) may coincide.

[0355] (Regardless of whether they are of the same species origin) For subjects with nearly or perfect HLA matching The administration of cells is particularly caused by viruses in which immune cells are specific, or For the treatment of diseases / conditions related to the present invention, expressing / containing the CARs described herein. or virus-specific immune cells that express / contain nucleic acids encoding such CARs It may be advantageous in the context of administration. In such cases, the host cells to the administered cells The presentation of viral antigens by (via their natural TCRs) is crucial for their activity in vivo. It is expected to increase sexualization, proliferation, and survival, and as a result improve the therapeutic effectiveness of these organisms. It can be done.

[0356] Method using CAR-expressing virus-specific immune cells CAR-expressing virus-specific immune cells as described herein (for example, as described herein) CD30-specific CAR-expressing EBV-specific T cells (CD30.CAR EBVST) It finds use in therapeutic and / or preventive methods.

[0357] This disclosure includes administering the CAR-expressing virus-specific immune cells to the target of the present disclosure. Methods for treating / preventing diseases / conditions in the subject are provided.

[0358] A virus expressing the CAR according to this disclosure for use in medical procedures / preventive methods. S-specific immune cells are also provided. These are used in methods to treat / prevent diseases / conditions. Virus-specific immune cells expressing the CAR according to this disclosure are also provided for the purpose of treating diseases / In the manufacture of a pharmaceutical product for use in a method for treating / preventing a condition, the present disclosure The use of virus-specific immune cells expressing CAR is also provided.

[0359] This method generally targets a population of virus-specific immune cells that express CAR according to this disclosure. It will be understood that this includes administering to elephants. In some embodiments, this disclosure Virus-specific immune cells expressing CAR by such cells in the form of a pharmaceutical composition containing such cells It can be administered in a controlled manner.

[0360] In particular, in methods of treating / preventing diseases / conditions by adoptive cell transfer (ACT), The use of virus-specific immune cells expressing CAR is intended as a result of the disclosure.

[0361] Virus-specific immune cells expressing CAR according to this disclosure can be used to treat disease / condition through allogeneic transplantation. It is particularly useful in methods for treating conditions.

[0362] As used herein, "allotransplantation" refers to a transplant that is not genetically identical to the recipient. This refers to the transplantation of cells, tissues, or organs into a recipient. The cells, tissues, or organs may be derived from a donor who is not genetically identical to the pient subject. or may be derived therefrom. Allografting differs from autotransplantation in that it involves the recipient versus This refers to the transplantation of cells, tissues, or organs derived from / originating from a donor who is genetically identical to an elephant.

[0363] Adoptive transfer of allogeneic immune cells is understood to be a form of allotransplantation. In one embodiment, CAR-expressing virus-specific immune cells cure diseases / conditions through allogeneic transplantation. It is used as a therapeutic / preventive agent in methods for treatment / prevention.

[0364] The administration of CAR-expressing virus-specific immune cells and compositions of this disclosure is preferably "therapeutic." This is an "effective" or "preventively effective" dose, which is sufficient to show therapeutic or preventive benefit to the subject. It is a matter of minutes. The actual amount administered, as well as the rate and time course of administration, depends on the disease / condition and administration. It depends on the nature and severity of the specific item administered. It also depends on the prescription of treatment, such as the dosage. The treatment of a disease / disorder is typically the responsibility of a general practitioner and other physicians. The individual patient's condition, delivery site, administration method, and other factors known to the practicing physician should be taken into consideration. Examples of the above technologies and protocols are found in Remington's Pharmaceut. ical Sciences,20th Edition,2000,pub.Lipp Incott, Williams & Wilkins.

[0365] Multiple doses may be provided. Multiple doses may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2 More than 3 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, On the 28th, 29th, 30th, or 31st day, or in one of the following 1, 2, 3, 4, 5, or 6 months. For example, the dose may be administered once every 7, 14, 21, or 28 days (±3, 2, or 1 day). ru.

[0366] In some embodiments, the treatment may involve other therapeutic or prophylactic interventions, such as chemotherapy or immunotherapy. This may further include therapy, radiation therapy, surgery, vaccination and / or hormone therapy. Other therapeutic or preventive interventions such as those described herein may occur before, during, and / or after the treatments included in this disclosure. Therefore, delivery of other therapeutic or prophylactic interventions may be via different routes of administration as a treatment of this disclosure. It's possible.

[0367] Depending on the condition being treated, the administration may be done alone, in combination with other treatments, simultaneously, or It may be either consecutive. CAR-expressing virus-specific immunohistochemistry as described herein. The cells and compositions may be administered simultaneously with or in conjunction with another therapeutic intervention.

[0368] Simultaneous administration refers to administering two or more therapeutic interventions together, for example, a combination of both active ingredients. That is, as a pharmaceutical composition in a combined preparation, or immediately after each other, and depending on the circumstances This refers to administering the drug via the same route of administration, for example, to the same artery, vein, or other blood vessel.

[0369] Sequential administration involves separate administrations of one or more further therapeutic interventions, following a predetermined time interval. This refers to the administration of one therapeutic intervention following the treatment. In some embodiments, the treatment follows the same pathway. It is not necessary for it to be administered by [a specific method]. The time interval may be any time interval.

[0370] Adoptive cell transfer generally refers to the process by which cells (e.g., immune cells) are obtained from a target. And typically, it is obtained by taking a blood sample from which the cells are isolated. Then, Cells are typically modified and / or proliferated, and then adopted into the same subject (autologous / autologous cell adoption). It is administered to either a different target (in the case of transfer) or a different target (in the case of adoptive transfer of allogeneic cells). Therapy typically involves providing treatment to a population of cells having certain desired characteristics, or similar treatments. The aim is to increase the frequency of such cells with certain characteristics in the target population. Transplantation is the introduction of cells or a population of cells into a target, and / or cells or a population of cells in a target. This may be done with the aim of increasing the frequency of the group.

[0371] Adoptive transfer of immune cells is, for example, Kalos and June (2013), Imm Unity 39(1):49-60, and Davis et al. (2015), Cance These are described in r J.21(6): 486-491, and all of them are by reference. The whole is incorporated herein. Those skilled in the art will, for example, incorporate the whole by example. Dai et al., 2016 J Nat Cancer Inst 108(7):djv43 By taking example 9, we can determine appropriate reagents and procedures for adoptive cell transfer according to this disclosure. It is possible.

[0372] This disclosure includes / expresses virus-specific immune cells containing CARs as disclosed, or this disclosure Administering to virus-specific immune cells that contain / express nucleic acids encoding CARs. To provide a method that includes doing so.

[0373] In some embodiments, the above method generates virus-specific immune cells, and This includes generating / expanding a population of virus-specific immune cells. Several embodiments Therefore, the method is to make virus-specific immune cells contain / express CARs according to this disclosure. This includes modifying the method to include virus-specific immune cells. In some embodiments, the method involves modifying the immune cells to include virus-specific immune cells. This includes including / modifying nucleic acids that encode CARs as disclosed herein.

[0374] In some embodiments, the Method may be modified to express / include the CAR according to the Disclosure. (or modified to express / contain nucleic acids that encode such CARs) This includes administering immune cells specific to Russ.

[0375] In some embodiments, the above method includes: (a) Modifying virus-specific immune cells that express or contain CARs as a result of this disclosure. , or expressing or including nucleic acids encoding CARs as disclosed herein, and (b) A modified or otherwise modified device that expresses the CARs of this disclosure. or modified to express nucleic acids encoding CAR as disclosed herein, or including The treatment should target immune cells specific to the virus.

[0376] In some embodiments, the above method includes: (a) Isolation or acquisition of virus-specific immune cells; (b) Modifying virus-specific immune cells that express or contain CARs as a result of this disclosure. , or expressing or including nucleic acids encoding CARs as disclosed herein, and (c) Modified to express the CARs of this disclosure, or containing the CARs of this disclosure or modified to express nucleic acids encoding CAR as disclosed herein, or including The treatment should target immune cells specific to the virus.

[0377] In some embodiments, the above method includes: (a) Isolation of immune cells (e.g., PBMCs) from the subject; (b) Generation / expansion of virus-specific immune cell populations; (c) Modifying virus-specific immune cells that express or contain CARs as a result of this disclosure. , or expressing or including nucleic acids encoding CARs as disclosed herein, and (d) Modified to express the CARs of this disclosure, or containing the CARs of this disclosure or modified to express nucleic acids encoding CAR as disclosed herein, or including The treatment should target immune cells specific to the virus.

[0378] In some embodiments, the method expresses CD30-specific CARs according to the disclosure. Modified EBV-specific immune cells, or CD30-specific CARs according to this disclosure This includes administering to immune cells that have been modified to express or constitute nucleic acids. ru.

[0379] In some embodiments, the above method includes: a) To express or include CD30-specific CARs according to the Disclosure, or CD according to the Disclosure Modify EBV-specific immune cells to express or contain nucleic acids encoding 30-specific CARs. to do, and (b) Modified to express CD30-specific CARs as per the Disclosure, or as per the Disclosure A nucleus containing a CD30-specific CAR or encoding a CD30-specific CAR according to this disclosure Administer to EBV-specific immune cells that have been modified to express or contain acid.

[0380] In some embodiments, the above method includes: (a) Isolation or acquisition of EBV-specific immune cells; (b) expressing or containing CD30-specific CARs as described herein, or C as described herein Modify EBV-specific immune cells to express or contain nucleic acids encoding D30-specific CARs. To change, and (c) Modified to express CD30-specific CARs as described herein, or as described herein This includes CD30-specific CARs, or codes for CD30-specific CARs according to this disclosure. Administering to EBV-specific immune cells that have been modified to express or contain nucleic acids. .

[0381] In some embodiments, the above method includes: (a) Isolation of immune cells (e.g., PBMCs) from the subject; (b) Generation / expansion of EBV-specific immune cell populations; (c) expressing or containing CD30-specific CARs as described herein, or C as described herein Modify EBV-specific immune cells to express or contain nucleic acids encoding D30-specific CARs. To change, and (d) Modified to express CD30-specific CARs as disclosed herein, or as disclosed herein This includes CD30-specific CARs, or codes for CD30-specific CARs according to this disclosure. Administering to EBV-specific immune cells that have been modified to express or contain nucleic acids. .

[0382] In some embodiments, immune cells (e.g., PBMCs) are isolated from the target cells. It is the same object being subjected to (i.e., adoption can be of autologous / autologous cells). In one embodiment, immune cells (e.g., PBMCs) are isolated from a target to which the cells are administered. The target is different from the target (i.e., the adoptive transfer may be from the same species of cell).

[0383] In some embodiments, the above method includes: Obtaining blood samples from the subject; Isolation of immune cells (e.g., PBMCs) from blood samples obtained from subjects; (For example, in the presence of cells containing / expressing viral antigens / peptides (e.g., APCs)) By culturing PBMCs, or by using virus-infected cells (e.g., APCs) By culturing PBMCs in the presence of [unspecified substance], a population of virus-specific immune cells is generated. To expand; In vitro or ex vivo cell culture of virus-specific immune cells; Modifying virus-specific immune cells that express or contain CAR as described in this disclosure, or expressing or including nucleic acids encoding CARs as disclosed herein (for example, such Using a viral vector encoding CAR, or a viral vector containing such nucleic acid This was achieved through the introduction of certain traits; This disclosure allows for the cultivation of virus-specific immune cells that express / contain CAR, and In vitro or ex vivo cell cultures generate nucleic acids encoding CARs according to this disclosure. To represent / include; Nucleic acids that express / contain the CARs of this disclosure, or that encode the CARs of this disclosure Collection / isolation of immune cells specific to viruses that manifest / contain; This disclosure describes how to formulate virus-specific immune cells that express / contain CARs. or, for example, cells with a pharmaceutically acceptable adjuvant, diluent, or carrier By mixing, the nucleic acids encoding CAR according to this disclosure are formulated into a pharmaceutical composition. to do; Virus-specific immune cells expressing / containing CARs according to this disclosure, or according to this disclosure Immune cells expressing / containing nucleic acids encoding CAR, or pharmaceutical compositions containing such cells To administer a substance to a target.

[0384] In some embodiments, the above method treats cells or subjects to induce CAR expression. / enhancement, and / or proliferation of virus-specific immune cells containing / expressing CAR or This may further include inducing / enhancing survival.

[0385] Treatment and / or preventive measures aim to reduce the onset / progression of the disease / condition, and to alleviate the symptoms of the disease / condition. This method may be effective in alleviating or reducing the pathological condition of the disease / condition. To prevent progression, for example, to prevent the worsening of a disease / condition, or to prevent the onset of a disease / condition. It may be effective in slowing down the speed. In some embodiments, the method modifies the disease / condition Benefits include, for example, a reduction in the severity of symptoms of a disease / condition, or a reduction in the severity / activity of some of the symptoms of a disease / condition. This may result in a reduction of other correlations. In some embodiments, the method may lead to the onset of disease / condition. It can prevent the disease from progressing to later stages (e.g., the chronic stage or metastasis).

[0386] The therapeutic and prophylactic usefulness of CAR-expressing virus-specific immune cells as disclosed herein is due to CAR The number / activity of cells expressing / overexpressing the target antigen, and / or the number of virus-infected cells. Treatment / prevention of any disease / condition from which a therapeutic or preventive benefit would be obtained from a reduction in the number / activity It will likely be understood that this extends to defense as well.

[0387] In some embodiments, the disease / condition treated / prevented according to this disclosure is characterized by immune cells. A disease / condition in which a different virus is pathologically involved. That is, several implementations In terms of form, a disease / condition is a disease caused by or exacerbated by a viral infection. / condition, disease / condition in which viral infection is a risk factor, and / or viral infection A disease / condition is a disease / condition that is positively associated with the onset, onset, progression, and / or severity of the disease / condition. .

[0388] In some embodiments, the target antigen of the CAR is pathologically involved, in accordance with this disclosure. A disease / condition that is treated / prevented. In other words, in some embodiments, the disease / condition is targeted. Diseases / conditions caused or exacerbated by antigen expression / overexpression, target antigen expression Diseases / conditions in which current / overexpression is a risk factor, and / or disease in which the expression / overexpression of a target antigen is a risk factor. This refers to a disease / condition that is positively associated with the onset, progression, and severity of the condition.

[0389] Diseases / conditions are pathologically related to cells that express / overexpress CD30 or CD30. Diseases / conditions, for example, cells that express / overexpress CD30, the onset of a disease / condition, Positively related to the severity of one or more symptoms of the disease / condition, or the progression of the disease / condition. Diseases / conditions in which CD30 expression / overexpression is at risk of the onset, onset, or progression of a disease / condition. It may be a disease / condition that is a risk factor.

[0390] The diseases / conditions treated / prevented in accordance with this disclosure are diseases / conditions characterized by EBV infection. It is possible. For example, a disease / condition in which EBV or EBV-infected cells are pathologically involved. A disease / condition, such as EBV infection, is positively associated with the onset, development, or progression of the disease / condition. Disease / condition, and / or the severity of one or more symptoms of the disease / condition, or EBV infection It may be a disease / condition in which the onset, onset, or progression of the disease / condition is a risk factor.

[0391] Treatment may aim to achieve one or more of the following: reducing the viral load, Reducing the number / percentage of virus-positive cells (e.g., EBV-positive cells), target antigens of CAR By reducing the number / percentage of cells expressing / overexpressing (e.g., CD30-expressing cells) Furthermore, it reduces the activity of virus-positive cells (e.g., EBV-positive cells) and targets CARs. Decreasing the activity of cells that express / overexpress antigens (e.g., CD30-expressing cells) to delay / prevent the onset / progression of symptoms of a disease / condition, and to reduce the severity of symptoms of a disease / condition. To reduce the degree of infection, and to decrease the survival / proliferation of virus-positive cells (e.g., EBV-positive cells). This involves cells that express / overexpress the target antigen of CAR (for example, CD30-expressing cells). To reduce the survival / proliferation of ) or to increase the survival of the subject.

[0392] In some embodiments, the above target is, for example, peripherally, or in a disease / condition. Therefore, in the affected organs / tissues (for example, organs / tissues), for example, a virus (for example) For example, cells infected with the virus (e.g., EBV), or target antigens of CAR (e.g., EBV). For example, detection of cells expressing / overexpressing CD30, or virus-positive cancer cells (e.g.) For example, detection of EBV-positive cancer cells, or detection of CAR target antigens (e.g., CD3 Based on the detection of cancer cells expressing / overexpressing 0), for the treatment described herein It can be selected. The disease / condition may affect any tissue, organ, or organ system. In some embodiments, a disease / condition may affect several tissues / organs / organ systems.

[0393] In some embodiments, the subject is infected with EBV or has been infected with EBV Based on the decision to include cells, they may be selected for therapeutic / preventive purposes according to this disclosure. In several embodiments, the target is cells that express / overexpress CD30, for example, cells that express CD30 Based on the determination that it contains current / overexpressing cancer cells, for the treatment / prevention described herein It can be chosen.

[0394] In some embodiments, the subject expresses / contains the CAR described herein. Immunity specific to viruses (or those expressing / containing nucleic acids that encode such CARs) Before administering the cells, lymphocyte apheresis chemotherapy is administered.

[0395] In other words, in some embodiments, the method for treating / preventing a disease / condition according to this disclosure is (i) administered as a target for lymphocyte apheresis chemotherapy, and (ii) subsequently, according to this disclosure Expressing / containing a CAR, or expressing / containing a nucleic acid encoding a CAR as disclosed herein. This includes administering virus-specific immune cells.

[0396] As used herein, "lymphocyte apheresis chemotherapy" refers to the lymphocytes within the patient being treated. Lymphocytes (for example, T cells, B cells, NK cells, NKT cells, or innate lymphocytes (IL)) This refers to treatment with chemotherapeutic agents that cause depletion of lymphocytes (C) or their precursors. A "depletion-causing chemotherapy agent" refers to a chemotherapy agent that causes depletion of lymphocytes.

[0397] Its use in lymphocyte apheresis chemotherapy and adoptive cell transfer therapy methods is, for example, K lebanoff et al., Trends Immunol. (2005) 26(2):111 -7 and Muranski et al., Nat Clin Pract Oncol. (2006) See (12)668-81 (these are all incorporated herein by reference). (It can be inserted). The purpose of lymphocyte depletion chemotherapy is to reduce the endogenous lymphocyte population of the recipient. The goal is to deplete the group's resources.

[0398] In the context of treating diseases through adoptive transfer of immune cells, lymphocyte depletion chemotherapy is typical. It is administered before adoptive transfer to the recipient subject to receive the adopted cells. Conditioning the body. Lymphocyte depletion chemotherapy, for example, involves immunosuppressive cytokines. Eliminate cells that express this gene and create the "lymphocyte space" necessary for the proliferation and activity of adoptive lymphocytes. By creating a tolerant environment, the sustainability and activity of adoptive cells are enhanced. It is thought to promote sexual activity.

[0399] Chemotherapy agents commonly used in lymphocyte apheresis include, for example, fludara. It contains vin, cyclophosphamide, bedamustine, and pentostatin.

[0400] Aspects and embodiments of this disclosure particularly concern the administration of fludarabine and / or cyclophosphamide. This relates to lymphocyte depletion chemotherapy, including the administration of certain agents. In certain embodiments, lymphocyte depletion by the present disclosure The chemotherapy regimen includes the administration of fludarabine and cyclophosphamide.

[0401] Fludarabine interferes with ribonucleotide reductase and DNA polymerase. It is a purine analog that inhibits DNA synthesis. It is used in leukemia (especially chronic phosphorus (Passive leukemia, acute myeloid leukemia, acute lymphoblastic leukemia) and lymphoma (especially non-Hodgkin's It is often used as a chemotherapy agent for the treatment of lymphoma. Fludarabine is administered intravenously. It can be administered orally.

[0402] Cyclophosphamide causes irreversible intra- and inter-strand crosslinks between DNA bases. It is a killing agent. It is often used in the treatment of cancers, including lymphoma, leukemia, and multiple myeloma. It is used as a chemotherapeutic agent for this purpose. Cyclophosphamide is administered intravenously or orally. It is possible.

[0403] The course of lymphocyte depletion chemotherapy described herein involves multiple administrations of one or more chemotherapy agents. May include. A course of lymphocyte depletion chemotherapy is administered in the doses described herein. This may include administering fludarabine and cyclophosphamide for several days as described in the book. As an example, the process of lymphocyte depletion chemotherapy involves administering fludarabine at 30 mg / m². 2 / daily errands Administer the drug at the specified dose for three consecutive days, and cyclophosphamide at 500 mg / m². 2 / daily errands This may include administering the drug in a specific dose for three consecutive days.

[0404] On the day of administration of the final dose of the chemotherapy agent according to the lymphocyte depletion chemotherapy process, lymphocyte depletion This could be considered the day of completion of the chemotherapy course.

[0405] In some embodiments, fludarabine is 5-100 mg / m² 2 Daily dose, for example For example, 15-90 mg / m² 2 / day, 15~80mg / m 2 / day, 15~70mg / m 2 / day , 15-60 mg / m² 2 / day, 15~50mg / m 2 / day, 10~40mg / m 2 / day, 5-60 mg / m² 2 / day, 10~60mg / m 2 / day, 15~60mg / m 2 / day, 20 ~60mg / m 2 / day or 25-60 mg / m² 2 It is administered at a rate of / day. So, is fludarabine administered at a dose of 20-40 mg / m²? 2 For example, 25-35 mg / m² per day. 2 / day For example, approximately 30 mg / m² 2 It is administered daily.

[0406] In some embodiments, the fludarabine is used for a continuous period of more than 1 day but less than 14 days. During this time, it is administered in the dose specified in the previous paragraph. In some embodiments, the above fludarabine is 2 ~14 days, for example, 2-13 days, 2-12 days, 2-11 days, 2-10 days, 2- For 9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, or 2-4 consecutive days, It is administered in the dose specified in the previous paragraph. In some embodiments, the above fludarabine is 2- For 6 consecutive days, for example, 2 to 4 consecutive days, for example, 3 consecutive days, the dose specified in the previous paragraph is administered. It can be done.

[0407] In some embodiments, the fludarabine is 15-60 mg / m². 2 At a daily dose, For 2 to 6 consecutive days, for example, 30 mg / m² 2 The drug is administered at a daily dose for three consecutive days.

[0408] In some embodiments, the cyclophosphamide is 50-1000 mg / m². 2 For example, 100-900 mg / m³ per day 2 150-850 mg / m³ per day 2 / day 200-800 mg / m² 2 250-750 mg / m² per day 2 300-700m per day g / m 2 350-650 mg / m³ per day 2 400-600 mg / m³ per day 2 / day Or 450-550 mg / m² 2 It is administered at a daily dose. The recommended dosage of cyclophosphamide is 400-600 mg / m². 2 For example, 450-550 mg per day. / m 2 For example, approximately 500 mg / m² per day. 2 It is administered at a daily dose.

[0409] In some embodiments, the cyclophosphamide is administered continuously for 1 to 14 days. It is administered in the dose specified in the previous paragraph. In some embodiments, cyclophosphamide is 2- 14 days, for example, 2-13 days, 2-12 days, 2-11 days, 2-10 days, 2-9 For days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, or 2-4 consecutive days prior It is administered in the doses specified in the paragraph. In some embodiments, cyclophosphamide is 2- For 6 consecutive days, for example, 2 to 4 consecutive days, for example, 3 consecutive days, the dose specified in the previous paragraph is administered. It can be done.

[0410] In some embodiments, the above cyclophosphamide is 400-600 mg / m² 2 A daily dose of 500 mg / m² for 2 to 6 consecutive days.2 At a daily dose, administered for 3 consecutive days. To be given.

[0411] In some embodiments, the above-mentioned fludarabine and cyclophosphamide are simultaneously and They may be administered consecutively. Co-administration means, for example, that both drugs are contained in a combination (i.e., a combination). (In the combined preparation), or immediately after each other, and possibly via the same route of administration, e.g. For example, it refers to administering the drug together into the same artery, vein, or other blood vessel. Continuous administration means one This refers to administering one drug followed by the other drug separately after a predetermined time interval. In some embodiments, however, it is not necessary for the drugs to be administered via the same route. .

[0412] In some embodiments of the series of lymphocyte-depleting chemotherapy described herein, fludarabine and Cyclophosphamide is administered on the same day or on the same day. For example, lymphocyte apheresis chemotherapy During the legal process, fludarabine was administered at 30 mg / m². 2 Administer at the daily dose for three consecutive days. This, and cyclophosphamide 500 mg / m² 2 Administer at the daily dose for three consecutive days. Fludarabine and cyclophosphamide, including the above, can be administered for the same three consecutive days. In such cases, the process of lymphocyte apheresis chemotherapy involves fludarabine and cyclophosph It can be said that the treatment was completed on the final day of the three consecutive days that Famide was administered to the target patient.

[0413] In some embodiments, the CAR described herein is expressed / contains (or such) Virus-specific immune cells (expressing / containing nucleic acids encoding CAR) eliminate lymphocytes It is administered to the patient within a specific period after the completion of the chemotherapy course.

[0414] In some embodiments, the CAR described herein is expressed / contains (or such) Virus-specific immune cells (expressing / containing nucleic acids encoding CAR) are described herein. Within 1 to 28 days of completing the course of lymphocyte apheresis chemotherapy, for example, 1 to 21 days, 1 to It is administered to the target within 14 days, 1-7 days, 2-7 days, 2-5 days, or 3-5 days. In one embodiment, the CAR described herein is expressed / contains (or such CA) Virus-specific immune cells expressing / containing nucleic acids encoding R are described herein. Within 2 to 14 days (for example, within 3 to 5 days) of the completion of the lymphocyte apheresis chemotherapy course. It is administered to elephants.

[0415] In some embodiments, 1 × 10 7 individual cells / m 2 ~1 × 10 9 individual cells / m 2 ,example For example, 2 x 10 7 individual cells / m 2 ~1 × 10 9 individual cells / m 2 , 2.5×10 7 individual cells / m 2 ~8×10 8 individual cells / m 2 , 3 x 10 2 individual cells / m 2 ~6×10 8 individual cells / m 2 , or 4×10 7 individual cells / m 2 ~4×10 8 pieces / m 2 It is administered as follows.

[0416] In some embodiments, the CAR described herein is expressed / contains (or such) Virus-specific immune cells expressing / containing nucleic acids that encode CAR are 4 × 10 7 pieces cells / m 2 , 1 x 10 8 individual cells / m 2 , or 4×10 8 individual cells / m 2 It is administered .

[0417] Expressing / containing (or encoding such CARs) the CARs described herein The administration of virus-specific immune cells (that express / contain) is administered by intravenous infusion. This can be done. The administration volume may be between 1 and 50 ml, and the administration period may be between 1 and 10 minutes. It is acceptable to do so.

[0418] In some embodiments, the disease to be treated / prevented according to this disclosure is cancer.

[0419] Cancer is any unwanted cell proliferation (or any cell proliferation that appears as a result of unwanted cell proliferation). It can refer to a disease, neoplasm, or tumor. Cancer can be benign or malignant, and primary or secondary. (It may be metastatic.) A neoplasm or tumor can be any abnormal growth or proliferation of cells, Cancer can be located in the following tissues: for example, the adrenal gland, anus, appendix, bladder, blood, bone, bone marrow, brain, Breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, intrauterine Membranes, epithelial cells (such as renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidneys, tears Glands, larynx, liver, lungs, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, uterine muscle nasopharynx, greater omentum, oral cavity, ovaries, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands , sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, It may be tissue / cells derived from the uterus, vulva, and / or leukocytes.

[0420] The tumor may be a neurological tumor or a non-neurological tumor. Neurological tumors may be central or peripheral nervous system tumors. For example, glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, stellate It can originate from either a syringoma or an oligodendroglioma. Non-neurological cancers / tumors are any other non-neurological cancers / tumors. It can originate from tissue, for example, melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-holic Dikin lymphoma, Hodgkin lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia ( AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphoma sexual leukemia (CLL), liver cancer, epidermoid cancer, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, These include thymic carcinoma, NSCLC, hematological carcinoma, and sarcoma.

[0421] In some embodiments, the cancer is selected from the group consisting of: solid tumors, hematological malignancies. , gastric cancer (e.g., gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma), liver cancer (hepatocellular carcinoma, cholangiocarcinoma), head and neck cancer ( For example, squamous cell carcinoma of the head and neck, oral cancer (for example, oropharyngeal cancer), oral cancer, laryngeal cancer, nasopharynx Cancer, esophageal cancer), colorectal cancer (e.g., colon cancer), cervical cancer, prostate cancer, lung cancer (e.g., N SCLC, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), bladder cancer, urothelial cancer, skin cancer (e.g. , melanoma, progressive melanoma), renal cell carcinoma (e.g., kidney cancer), ovarian cancer (e.g., egg Focal cancer, mesothelioma, breast cancer, brain tumors (e.g., glioblastoma), prostate cancer, pancreatic cancer, myeloid hematological malignancies Tumors, lymphoblastic hematological malignancies, myelodysplastic syndrome (MDS), acute myeloid leukemia (A) ML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphoma, Non-Hodgkin lymphoma (NHL), thymoma, or multiple myeloma (MM).

[0422] In some embodiments, the cancer is pathologically related to a virus that is specific to immune cells. It is a cancer that is related to a viral infection. In other words, in some embodiments, cancer is caused by a viral infection. Cancers caused by or worsened by, cancers in which viral infection is a risk factor, and / or This refers to cancers in which viral infection is positively associated with the onset, development, progression, severity, or metastasis of the cancer. .

[0423] EBV infection has been reviewed, for example, by Jha et al. in Front Microbiol. It is involved in several types of cancer. See (2016)7:1602.

[0424] In some embodiments, the cancer being treated / prevented is EBV-related cancer. In this embodiment, cancer is cancer caused by or worsened by EBV infection, E Cancers in which BV infection is a risk factor, and / or cancers in which EBV infection is a risk factor for the onset, development, or progression of cancer. Cancers positively associated with disease, severity, or metastasis. Cancers can be characterized by EBV infection. For example, cancer can contain cells infected with EBV. Such cancer is called EBV-positive cancer. It is possible.

[0425] EBV-related cancers that can be treated / prevented in accordance with this disclosure include B-cell cancers such as Burkitt lymphoma. Related cancers, post-transplant lymphoproliferative disorders (PTLD), central nervous system lymphoma (CNS lymphoma) ), Hodgkin lymphoma, non-Hodgkin lymphoma, and EBV-associated lymphoma associated with immunodeficiency ( For example, this includes: EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, HI EBV-positive lymphoma associated with V infection / AIDS, oral hair follicle leukoplakia, and nasopharyngeal carcinoma (NPC) ), including gastric cancer (GC).

[0426] In some embodiments, the above cancer is lymphoma (e.g., EBV-positive lymphoma), head and neck squamous cell carcinoma (HNSCC; e.g., EBV-positive HNSCC), nasopharyngeal carcinoma (NPC; e.g. The cancer is selected from EBV-positive NPCs and gastric cancer (GC; for example, EBV-positive GCs).

[0427] In some embodiments, the cancer is a cancer in which the target antigen of the CAR is pathologically involved. In other words, in some embodiments, cancer is caused by the expression of a target antigen. or worsening cancer, cancer in which the expression of the target antigen is a risk factor, and / or cancer in which the expression of the target antigen is a risk factor Cancer is positively associated with the onset, development, progression, severity, or metastasis of cancer. Cancer can be characterized in such a way that, for example, cancer may contain cells that express a target antigen. This can be described as being positive for the target antigen.

[0428] Cancers that are "positive" for a target antigen are cells that express the target antigen (for example, on the cell surface). It may be a cancer containing cells. Cancers that are "positive" for the target antigen may overexpress the target antigen. Overexpression of the target antigen is higher than the expression level in equivalent non-cancerous cells / non-tumor tissues. This can be determined by detecting the gene or protein expression levels of the target antigen.

[0429] In some embodiments, the target antigen is the cancer cell antigen described herein. In several embodiments, the target antigen is CD30.

[0430] In some embodiments, the above-mentioned cancer is a cancer in which CD30 is pathologically involved. In some embodiments, the above cancer is caused or exacerbated by CD30 expression. Cancers in which CD30 expression is a risk factor, cancers in which CD30 expression is a risk factor, and / or cancers in which CD30 expression is a risk factor for the onset of cancer. Cancer is positively associated with progression, severity, or metastasis. Cancer is characterized by CD30 expression. For example, cancer may contain cells that express CD30. Such cancer is CD30 positive. It can be called sexual cancer.

[0431] CD30-positive cancer is characterized by cells that express CD30 (for example, the CD30 protein on the cell surface). It may be a cancer that includes cells expressing CD30. CD30-positive cancers may overexpress CD30. CD30 overexpression is higher than the expression levels in equivalent non-cancerous cells / non-tumor tissues. This can be determined by detecting the expression levels of 30 genes or proteins.

[0432] CD30-positive cancers are, for example, described by van der Weyden et al., Blood Can cer Journal (2017) 7:e603 and Muta and Podack It is described in Immunol Res (2013), 57(1-3):151-8. Both of these are incorporated herein in their entirety by reference. CD30 is activated On small subsets of T and B lymphocytes, as well as classical Hodgkin lymphoma and anaplastic large cells. It is expressed by various lymphoid neoplasms, including follicular lymphoma. Variable expression of CD30 is also Peripheral T-cell lymphoma, other unspecified (PTCL-NOS), adult T-cell leukemia / liary Lymphoma, cutaneous T-cell lymphoma (CTCL), extranodal NK-T cell lymphoma, various B-cell non- Hodgkin lymphoma (diffuse large B-cell lymphoma, especially EBV-positive diffuse large B-cell lymphoma) CD3 Expression of 0 is also observed in several non-hematopoietic malignancies, including germ cell tumors and testicular germinal carcinomas. This has also been observed.

[0433] Transmembrane glycoprotein CD30 is a member of the tumor necrosis factor receptor superfamily. Yes (Falini et al., Blood (1995) 85(1):1-14). TNF / TN Members of the TNF-R (F receptor) superfamily modulate the immune response at multiple levels. CD30 plays a role in regulating the function or proliferation of normal lymphoid cells. It was originally described as an antigen recognized by the monoclonal antibody Ki-1, and this is (Muta and Po) duck, Immunol Res (2013)57:151-158). CD30 antigen Expression is used to identify ALCL and Reed-Sternberg cells in Hodgkin's disease. It is used (Falini et al., Blood (1995) 85(1):1-14). Therefore, due to its widespread expression in lymphoma malignant cells, CD30 is used in antibody-based immune responses. It is a potential target for developing both therapeutic and cell therapies. Importantly, CD30 It is typically not expressed on normal tissues under physiological conditions, and therefore, resting, maturing, or pre-maturation. (Younes and Ansell, Semin) Hematol (2016) 53:186-189). Antibodies targeting CD30. Brentuximab vedotin, a substance conjugate, is used for the treatment of CD30-positive HL. Originally approved (Adcetris® US Package Insert 2018) Data from the brentuximab vedotin trial suggests it is a therapeutic target for CD30-positive lymphoma. While I support CD30 as a viable option, I am concerned about the potential harm associated with its use.

[0434] Hodgkin lymphoma (HL) is a rare malignant tumor that involves the lymph nodes and lymphatic system. The incidence of L is bimodal, with the majority of patients diagnosed between the ages of 15 and 30, and those over 55. Another peak follows in adults. In 2019, there were 8,110 new cases in the US (3 in women). 540 people (4,570 men), and 1,000 deaths (4 women) due to this disease. It is estimated that there are 10 cases (590 men) (American Cancer). Society 2019). 20 in the SEER database of the National Cancer Institute, USA. Based on cases from 2012 to 2016, the incidence of HL in pediatric HL patients in the United States is as follows: The ratio is: Age 1-4: 0.1; Age 5-9: 0.3; Age 10-14: 1.3; Age 1 5-19: 3.3 per 100,000 years (SEER Cancer Statistics) [CS Review, 1975-2016]. The World Health Organization (WHO) classification is HL. Two main types: classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant Hodgkin lymphoma. It is divided into quinine lymphoma (NLPHL). In Western countries, cHL accounts for 95%, and NLPH L accounts for 5% of all HL (National Comprehensive Canc er Network Guidelines 2019).

[0435] First-line chemotherapy for cHL patients with advanced disease has a 70-75% cure rate. Related to the healing rate (Karantanos et al., Blood Lymphat Cancer) (2017) 7:37-52). Salvage chemotherapy and subsequent autologous stem cell transplantation (ASC). T) is commonly used for patients who have relapsed after initial treatment. Unfortunately, 5 in cHL patients Up to 0% of patients experience disease relapse after ASCT. Overall survival of patients who relapsed after ASCT. The median is approximately 2 years (Alinari Blood (2016) 127:287-2 95). Despite aggressive multi-drug chemotherapy, 10% to 40% of patients still receive rescue chemotherapy. Randomized clinical trials supporting ASCT in non-responders who did not achieve a response to therapy There are no data points. Patients who do not respond to salvage chemotherapy, relapse after ASCT, or this approach For patients who are not candidates for treatment, the prognosis remains critical, and new treatment approaches are urgently needed. (Keudell British Journal of Haemat) ology(2019)184:105-112).

[0436] The majority of the pediatric population (children, adolescents, and young adults) can be cured with currently available treatments, A small number of patients may have refractory or recurrent disease, with acceptable safety with improved efficacy benefits. Novel therapies with a complete sexual profile may be required (Flerlage et al., Blood) (2018)132:376-384;Kelly,Blood(2015) 126: 2452-2458;McClain and Kamdar,in UpToDate 2019;Moskowitz,ASCO Educational Book(20 19)477-486). HL patients treated with high-dose chemotherapy in childhood generally have cardiac problems. , long-term sequelae associated with the treatment of lung, gonad, and endocrine toxicity, as well as secondary malignant neoplasms. To experience (Castellino et al., Blood (2011) 117(6):1806- 1816).

[0437] In some embodiments, the CD30-positive cancers include solid tumors, hematological malignancies, and hematopoietic malignancies. Scars, Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic T-cell lymphoma, ALK-negative anaplastic T-cell lymphoma, peripheral T-cell lymphoma (e.g., PT) CL-NOS), T-cell leukemia, T-cell lymphoma, cutaneous T-cell lymphoma (CTCL), N KT cell lymphoma (e.g., extranodal NK-T cell lymphoma), non-Hodgkin lymphoma (NH L), B-cell non-Hodgkin lymphoma, diffuse large B-cell lymphoma (e.g., diffuse large B-cell lymphoma (NOS), primary mediastinal B-cell lymphoma, EBV-positive B-cell lymphoma, E BV-positive diffuse large B-cell lymphoma, progressive systemic mastocytosis, germ cell tumors and sperm It can be selected from follicular embryonic carcinomas.

[0438] In some embodiments, the above cancers include CD30-positive cancers, EBV-related cancers, hematological cancers, and myeloid cancers. Hematological malignancies, hematopoietic malignancies, lymphoblastic hematological malignancies, myelodysplastic syndromes, leukemia T-cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, phosphorus Parkinson's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, diffuse Large B-cell lymphoma, primary mediastinal B-cell lymphoma, EBV-associated lymphoma, EBV-positive B-cell lymphoma Lymphoma, EBV-positive diffuse large B-cell lymphoma, E associated with X-linked lymphoproliferative disorder BV-positive lymphoma, EBV-positive lymphoma associated with HIV infection, oral leukoderma, Burke Lymphoma, post-transplant lymphoproliferative disorders, central nervous system lymphoma, anaplastic large cell lymphoma, T-cell lymphoma, ALK-positive undifferentiated T-cell lymphoma, ALK-negative undifferentiated T-cell lymphoma. Peripheral T-cell lymphoma, cutaneous T-cell lymphoma, NK-T-cell lymphoma, extranodal NK-T cells Alveolar lymphoma, thymoma, multiple myeloma, solid tumors, epithelial cell carcinoma, gastric cancer, gastric cancer, gastrointestinal adenocarcinoma, digestive Duct adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cavity cancer, oropharyngeal cancer, mouth Cavity cancer, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, colorectal cancer, colorectal cancer, colorectal cancer, colorectal cancer, cervical cancer, previous Adenocarcinoma, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, urothelial cancer Skin cancer, melanoma, progressive melanoma. Renal cell carcinoma, kidney cancer, ovarian cancer, mesothelioma, Breast cancer, brain tumor, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, progressive systemic obesity The following are selected: cytosis, germ cell tumor, or testicular germ carcinoma.

[0439] In some embodiments, the cancer may be a recurrent cancer. As used herein, "Recurrent" cancer responded to treatment (e.g., first-line therapy for cancer), but then, for example This refers to cancer that reappears or progresses after a period of remission. For example, recurrent cancer is cancer that has grown or progressed after treatment. Cancer that has been inhibited by (for example, first-line therapy for cancer) and subsequently grown / progressed. obtain.

[0440] In some embodiments, the above-mentioned cancer may be a refractory cancer. "Refractory" cancer refers to cancer that does not respond to treatment (for example, first-line cancer therapy). For example, intractable cancers are those whose growth / progression is controlled by treatment (e.g., first-line therapy for cancer). It may be cancer that was not inhibited. In some embodiments, refractory cancer is treated with cancer treatment. The cancer may be one in which the patient has not shown a partial or complete response to treatment.

[0441] In embodiments where the cancer is anaplastic large cell lymphoma, the cancer is treated with chemotherapy and brentuximab. The cancer may be recurrent or refractory to treatment with buvedotin or crizotinib. In embodiments where peripheral T-cell lymphoma is present, the cancer is treated with chemotherapy or brentuximab vedotin. The condition may be recurrent or refractory to treatment. The cancer is extranodal NK-T cell lymphoma. In the embodiment, cancer is treated with chemotherapy (with or without asparaginase) or Brentzk The cancer may be relapsed or refractory with treatment using simab vedotin. The cancer is diffuse large cell type. In embodiments where the cancer is B-cell lymphoma, the cancer is treated with chemotherapy (with or without rituximab). ) or treatment with CD19 CAR-T therapy may be recurrent or refractory. In embodiments of primary mediastinal B-cell lymphoma, the cancer is treated with chemotherapy and immune checkpoint therapy. Regarding treatment with inhibitors (e.g., PD-1 inhibitors) or CD19 CAR-T therapy, It may be recurrent or refractory.

[0442] The treatment of cancer by the method disclosed herein achieves one or more of the following therapeutic effects: To reduce the number of cancer cells, to reduce the size of cancerous tumors / lesions in the subject, To inhibit (e.g., prevent or delay) the proliferation of cancer cells in the target area. Inhibits the growth of ulcers / lesions (e.g., prevents or delays growth), inhibits the onset / progression of cancer. (For example, to prevent or delay), to reduce the severity of cancer symptoms in the subject, To increase survival (e.g., progression-free survival or overall survival), the number or activity of cancer cells in the subject. To reduce the correlation and / or reduce the cancer burden in the subject.

[0443] The subjects were used to determine their response to the treatment. ia for Response Assessment:The Lugano Cl Assification (for example, Cheson et al., J Clin Oncol (20 14) It can be evaluated according to the following (as described in 32:3059-3068). In embodiments, treatment of the subject by the method of this disclosure achieves one of the following: complete response Effective, partial response, or stable disease.

[0444] In some embodiments, the treatment of the cancer further includes chemotherapy and / or radiotherapy. nothing.

[0445] Chemotherapy and radiotherapy involve the use of drugs or ionizing radiation (e.g., X-rays or gamma rays), respectively. This refers to the treatment of cancer using radiation therapy. Drugs are chemical substances, such as small molecule drugs. Antibiotics, DNA intercalators, protein inhibitors (e.g., kinase inhibitors) , or biological agents, such as antibodies, antibody fragments, aptamers, nucleic acids (e.g., DNA, R The drug may be a peptide, polypeptide, or protein. Alternatively, it may be formulated as a pharmaceutical. The formulation may contain one or more pharmaceutically acceptable diluents, excipients or It may contain one or more drugs (e.g., one or more activators) along with the carrier.

[0446] Chemotherapy may involve the administration of one or more drugs. The drugs may be administered individually, depending on the condition being treated. It may be administered alone or in combination with other treatments, simultaneously or sequentially.

[0447] Chemotherapy can be administered via one or more routes, such as parenteral, intravenous injection, oral, subcutaneous, or cutaneous. It may be administered intra- or intratumorally.

[0448] Chemotherapy can be administered according to the treatment plan. The treatment regimen may be determined by a physician or doctor. Chemotherapy drugs can be prepared and adjusted to suit patients who require treatment. This may be a predetermined schedule, plan, scheme, or schedule. Dimen specifies the type of chemotherapy administered to the patient, the dosage of each drug or radiation, and the time interval between administrations. This may indicate one or more of the following: the length of each treatment, the number and nature of any treatment holidays. Regarding combination therapy, a single treatment regimen should be provided that indicates how each drug should be administered. N may be provided.

[0449] The chemotherapy drugs are abemaciclib, abiraterone acetate, and abitrexate (methotol). Lexart, Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABV D, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Adcetris (br Lentuximab vedotin, ADE, adtrastuzumab emtansine, Adria Mycin (doxorubicin hydrochloride), afatinib dimaleate, afinitor (E Verolimus), Aquinzeo (Netupitant and Palonosetron hydrochloride), Aldara (I Mikimod, Aldesleukin, Alexensa (Alectinib), Alectinib, Alem Tuzumab, Alimta (pemetrexed disodium), Alicopa (copanlisib hydrochloride) Alkeran Injection (melphalan hydrochloride), Alkeran Tablets (melphalan), Aloxi (Palonosetron hydrochloride), Almbrik (brigatinib), Ambocline (chloram) Bucyl, amifostin, aminolevulinic acid, anastrozole, aprepitant, A Redia (pamidronate disodium), Arimidex (anastrozole), Allo Machine (exemestane), Alanone (nerarabin), arsenic trioxide, Alzera (offat) Mab), asparakinase Erwinia chrysanthemis, atezolizumab, avastin (Bevacizumab), Avelumab, Axicamatagenciloreucel, Axitinib, Azaci Tizine, Bavencio (avelumab), BEACOPP, Besenam (carmustine), Bere Odac (bellinostat), bellinostat, bendamustine hydrochloride, EP, Besponsa (Inotuzumab ozogamicin), bevacizumab, bexarotene, bexar (Toshitsumo Mab and iodine I131 (tositumomab), bicalutamide, BiCNU (carmustine), b Leomayci, Blinnatumomab, Blincyto (blinatumomab), Bortezomib, Bo Slif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, boomerang Busulfan, Busulfex (busulfan), Cabazitaxel, Cabametix (Cabozantinib smarate), Cabozantinib-S-marate, CAF, Calkens (acalabrutinib), Campus (alemtuzumab), Camptoser (irinotecan salt) (Salt), capecitabine, CAPOX, Carac (fluorouracil--Topical ), carboplatin, carboplatin-taxol, carfilzomib, Calmbris ( Carmustine, carmustine (carmustine), carmustine implant, casode Xx (bicalutamide), CEM, ceritinib, servidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil Chlorambucil-prednisone, CHOP, cisplatin, cladribine, clafen (Cyclophosphamide), Clopharabine, Clophalex (Clopharabine), Clo La (clofarabine), CMF, cobimetinib, cometric (cabozantinib-S-ma (rate), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmege (Dactinomycin), Cotelic (cobimetinib), Crizotinib, CVP, Cyclo Phosphamide, Cyphos (ifosfamide), Cyramza (ramucirumab), Cytaravi N, cytarabine liposome, cytosar U (cytarabine), cytoxane (cyclophosphamide) D), dabrafenib, dacarbazine, dacogen (decitabine), dactinomycin, da Rutzumab, Darzalex (Darzumab), Dasatinib, Donorubicin hydrochloride, Daunol Bicin hydrochloride and cytarabine liposomes, decitabine, defibrotide sodium, def Deterio (defibrotide sodium), Degarelix, Denilukin Defitox Dex, denosumab, Depotyte (cytarabine liposome). Dexamethasone, dexra Zoxane hydrochloride, dinutuximab, docetaxel, doxil (doxorubicin hydrochloride) (Liposome), Doxorubicin hydrochloride, Doxorubicin hydrochloride liposome, Dox-SL ( Doxorubicin hydrochloride liposome, DTIC-Dome (dacarbazine), Durvalma Bu, Efdex (fluorouracil - topical), Eliteoc (raspberry case), Elan (epirubicin hydrochloride), elotuzumab, eloxatin (oxaliplatin), Eltrombopag Oramine, Emend (Aprepitant), Emplity (Erots) Mab), Enacidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH Erbitux (cetuximab), eribulin mesylate, eribudge (bismodegive) ), erlotinib hydrochloride, elvinize (asparaginase elvinia chry Santhemi), Ethiol (Amifostin), Etopophos (Etoposide phosphate) There are salts such as etoposide, etoposide phosphate, and evaset (doxorubicin hydrochloride). Liposomes), everolimus, Evista (raloxifene hydrochloride), Evomela (melfa Lan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (flu Oloracil (topical agent), Fareston (toremifene), Faridac (panovinos TAT), Faslodex (fulvestrant), FEC, Femara (letrozole) ), filgrastim, fludarabine (fludarabine phosphate), fludarabine phosphate, flu Luoroplex (fluorouracil - for external use), fluorouracil injection, fluoro Lacil - for topical use, Flutamide, Forex (methotrexate), Forex PFS (Methotrexate), Forfiri, Forfiri bevacizumab, Forfiriset Ximab, FOLFIRINOX, FOLFOX, Forotin (Pralatrexate) ), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV pentavalent vaccine) Chin), Gaziva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine (Cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin) Gemzar (gemcitabine hydrochloride), Girotrif (afatinib dimaleate), G Livec (imatinib mesylate), Gliadel (carmustine implant), Glia Delwafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemandiol (propranolol hydrochloride), Ha - Septin (trastuzumab), HPV bivalent vaccine, recombinant, HPV monovalent vaccine, recombinant Replacement, HPV quadrivalent vaccine, recombinant, Hicamtin (topotecan hydrochloride), Hydrea (Hydro) Roxyurea, Hydroxyurea, HyperCVAD, Ibrance (Parbociclib) Ibrutumab, Chixetan, Ibrutinib, ICE, Iclusig (ponatinib salt) Idarubicin hydrochloride, idarubicin hydrochloride, idarubicin hydrochloride, idarubicin hydrochloride, idarubicin Zifa (enasidenib mesylate), Iffex (ifosfamide), ifosfamide Mid (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, I Mbruvica (ibrutinib), Infinzi (durvalumab), imiquimod, Imrit (Tarimogen laherparebeck), Inlyta (axitinib), Inotuzumab Ozogamicin, interferon alpha-2b, recombinant, interferon N-2 (Aldesleukin), Intron A (Recombinant Interferon Alpha) -2b, recombinant, interleukin-2 (aldesleukin), intron A ( Recombinant interferon alpha-2b), iodine I131 tositumomab and Tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, i Linotecan liposomes, istodax (romidepsin), ixabepyrone, citrate Xazomib, Ixempra (ixabepyrone), Jakafi (ruxolitinib phosphate), JEB, Jefftana (cabazitaxel), Cadsila (adtrastuzumab emtan) Syn), Keoxifen (raloxifene hydrochloride), Kepivans (palifermin), Ki Truda (pembrolizumab), Kiskari (ribociclib), Keemuria (tisagen) Lecroucel, Kyprolis (carfilzomib), Lanreotide acetate, Lapatinib Ditosylate, Latravo (olaratumab), lenalidomide, lenvatinib mesylate Salt, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Ikeran (chlorambucil), leuprolide acetate, leustatin (cladribine) Leblanc (aminolevulinic acid), Lymphoridine (chlorambucil), Lipodox ( Doxorubicin hydrochloride liposomes, Lomustine, Lonsurf (trifluridine / tipichloride) Lacil, Lupro (liuprolide acetate), Luprodepot (liuprolide acetate), Lupron Depo-Ped (leuprolide acetate), Lymphaz (olaparib), Marquivo (violet sulfate) Cristine liposomes, Maturan (procarbazine hydrochloride), mechloretamine hydrochloride, Megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride Salt, mercaptopurine, mesona, mesox (mesona), metazolastone (temozolomi (D). Methotrexate, methotrexate LPF (methotrexate), methyl methyl phosphate Lexon bromide, Mexart (methotrexate), Mexart AQ (methotrexate) ), midostaurin, mitomycin C. Mitoxantrone hydrochloride, mitoditrex (Mi Tomycin C), MOPP, Mozovir (Prelixa For), Mustagen (Mechloreta) (Mitomycin hydrochloride), Mutamycin (Mitomycin C), Mylan (Busulfan), Mylo Sarl (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticles Clitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (tartaric acid Vinorelbine, Necitumumab, Nelarabine, Neosar (cyclophosphamide), Nerachi Nib maleate, Nerinx (neratinib maleate), Netupitant and Palonose Thoron hydrochloride, Neurasta (pegfilgrastim), Neupogen (filgrastim) Nexavar (sorafenib tosylate), Nilandron (nilutamide), Nilotin Bu, Nirultamide Ninlaro (ixazomib citrate), niraparib tosylate hydrate Substances, nivolumab, nolvadex (tamoxifen citrate), N plate (romiplos Chim), Obinutuzumab, Odomo (Sonidegib), OEPA, Ofatumab, OFF, O Laparib, Olaratumab, Omasetaxin Mepesacinate, Oncaspar (Pegascar) ), ondansetron hydrochloride, onibaide (irinotecan hydrochloride liposome), ontaku (Denilkin Diftitox), Opdivo (Nivolumab), OPPA, diapers Rutinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticles Sub-formulations, PAD, palbociclib, parifermin, palonosetron hydrochloride, palonose Thoron-Netpitant, Pamidronate Disodium, Panitumumab Panobinostac Paraplatin (carboplatin), Paraplatin (carboplatin), Pazopanib salt Salts, PCV, PEB, Pegaspagaze, Pegfilgrastim, Peginterferon Alpha-2b, PEG-Intron (Peginterferon Alpha-2b), Pembron Lizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol AQ (cisplatin), Plerixa For Pomalidomide, Pomalist (Pomalidomide), Ponatinib hydrochloride, Portolazza (Nessinus) Tummumab, etc. Plalatrexate, prednisone, procarbazine hydrochloride, proloycete Kin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopagol) Lamin, propranolol hydrochloride, Provenz (Cypreucel T), Prinetol (Me Ru Captopurine, Prixan (mercaptopurine), radium-223 dichloride, raloxy Fen hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant rhizome Topapillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HP) V) Bivalent vaccine, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human Papillomavirus (HPV) quadrivalent vaccine, recombinant interferon α-2b, Regola Fenib, Relistol (methylnaltrexone bromide), R-EPOCH, Rebrimid (Lenalidomide), Rheumatrex (methotrexate), Ribociclib, R-ICE Rituxan (rituximab), Rituxan Hysera (rituximab and hyaluronider Zehit, Rituxan, Rituximab, and Hyaluronidasehiton. Lorapitant hydrochloride Romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), rubraca (Rucaparib camsilate), Rucaparib camsilate, Ruxolitinib phosphatidyl , Redapt (midostaurin), intrapleural aerosol (talc), siltoximab, siltoximab Preucel-T, Somatuline Depot (lanreotide acetate), Sonidecib, Sorafenib Tosylate, Sprycel (dasatinib), Stanford V, sterile talc powder (talc Talc, Steritalk (talc), Stibalga (regorafenib), Sunitinib malate , Sutent (sunitinib malate), Cilatron (pegylated interferon alpha-2) b) Silvant (siltoximab), Synribo (omacetaxin mepesacinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso ( Osimertinib, talc, tarimozine laherpalebbek, tamoxifen citrate Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin ( Bexarotene, Tasigna (nilotinib), Taxol (paclitaxel), Taxote Lu (docetaxel), Tecentriq (atezolizumab), Temodal (temozolomide) Temsirolimus, thalidomide, salomid (thalidomide), thioguanine, thiotepa , Tisagenlecleucel, Trac (fluorouracil - for external use), Topotecan hydrochloride, Toremifene, Tricel (Temsirolimus), Tositumomab and Iodine I131, Totec (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzma B, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Lysenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin ( Dinutuximab, Uridine triacetate, VAC, Barbicin, Valstar (Barbicin) Vandetanib, VAMP, Barbit (lorapitant hydrochloride), Vectibix (Pani tumumab), Vaip, Velban (vinblastine sulfate), Velcade ( Bortezomib, Velsar (vinblastine sulfate), vemurafenib, Benkle Kusta (Venetoclax), Venetoclax (Venetoclax), Belze Nio (abemaciclib), Weidl (leuprolide acetate), Vidaza (azacitid) Vinblastine sulfate, Vincasar PFS (vincristine sulfate), Vincri Stin sulfate, vincristine sulfate liposomes, vinorelvin tartrate, VIP, Bismodezib, Vistguard (uridine triacetate), Voraxaze (glucarpida) Vorinostat, Votrient (pazopanib hydrochloride), Vyxe os (daunorubicin hydrochloride and cytarabine liposomes), Wellcovorin ( Leucovorin calcium, Xalkori (crizotinib), Xeloda (capesitan) bottle), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (ipilimumab), Yescarta (acicabtadine / siloleucel) , Yondelis (trabectedin), Zaltrap (Ziv-Aflibercept) Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate) (Patent), Zelboraf (vemurafenib), Zevalin (ibritumomab / tiuxetate) (n), Ginecard (dexrazoxane hydrochloride), Ziv-Aflibercept, Zo fran (ondansetron hydrochloride), Zoladex (goserelin acetate), Zoledr onic acid, Zolinza (Vorinostat), Zometa (Zored (Lonic acid), Zydelig (Idelalisib), Zykadia (Ceritin You can choose between ib) and Zytiga (abiraterone acetate).

[0450] EBV infection also affects multiple sclerosis and systemic lupus erythematosus (SLE; for example, A Scherio and Munger Curr Top Microbiol Immun See ol.(2015);390(Pt 1):365-85), and EBV antigen EB NA2 is involved in the onset and progression of various autoimmune diseases, and recently it has been involved in SLE, multiple autoimmune diseases. Celiac sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, juvenile idiopathic arthritis, and celiac disease It has been shown to be associated with risk factors for the development of the disease (Harley et al., Nat G enet. (2018) 50(5):699-707).

[0451] Therefore, in some embodiments, the disease / condition treated / prevented in accordance with this disclosure is Autoimmune diseases, SLE, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, young people The diagnosis is selected from sexually idiopathic arthritis and celiac disease.

[0452] The aspects and embodiments of this disclosure relate to one or more non-identical target antigens specific to one or more non-identical target antigens. This relates to CAR-expressing virus-specific immune cells containing CAR. In some embodiments, C Virus-specific immune cells containing CARs specific to CD30 are specific to antigens other than CD30. Includes CAR. For example, Example 4 of this specification includes CD30-specific CAR and CD19-specific This document describes virus-specific immune cells, including target CARs.

[0453] In some embodiments, cancers treated / prevented according to the present invention are one or more non-identical lesions. It is a cancer that contains cells expressing a target antigen. In some embodiments, the cancer is of non-identical target antigens. This is a type of cancer that exhibits both of these characteristics.

[0454] Uses related to the treatment / prevention of alloreactive immune responses CAR-expressing virus-specific immune cells and compositions of this disclosure are used in methods including allografting. For example, it can be used to treat / prevent diseases / conditions in a subject.

[0455] CAR-expressing virus-specific immune cells and compositions disclosed herein are alloreactive immune responses (especially In methods for reducing / preventing T cell-mediated alloreactive immune responses and their adverse consequences. It is useful.

[0456] Alloreactive T cells express CD30. Chan et al., J Immunol (2002) )169(4):1784-91 describes how CD30-expressing T cells respond to CD30 alloimmune responses. Subsequences of activated T cells (which also express CD25 and CD45RO), which play an important role. It has been identified as a set. CD30 expression and proliferation of CD30-expressing T cells are in response to the same antigen. The answer is that it increases. Chen et al., Blood (2012) 120(3):691-6 is CD CD30 expression on 8+ T cell subsets was identified as a potential biomarker for GVHD. Furthermore, CD30 has been proposed as a therapeutic target for GVHD.

[0457] Furthermore, virus-specific T cells are more restricted than polyclonal activated T cells (ATCs). It has a well-developed TCR repertoire and therefore does not induce GVHD after administration to allogeneic subjects. It is unlikely to occur. This is because of the G in the study of allogeneic EBV-specific T cells (EBVSTs). This is reflected in the low incidence of VHDs.

[0458] The CAR-expressing virus-specific immune cells and compositions of this disclosure are used in methods including allografting. It is particularly useful in the processing / production of allogeneic transplants.

[0459] In particular, CAR-expressing virus-specific immune cells and compositions are used in treatments including the administration of allogeneic materials. The manufacture and use of "ready-made" materials for use in therapeutic and preventive methods It is planned.

[0460] As described above, the CAR-expressing virus-specific immune cells of this disclosure are used to treat diseases caused by adoptive cell transfer. Useful for the treatment / prevention of disease / condition. The CAR-expressing virus-specific immune cells of this disclosure are useful for nourishing The recipient has low sensitivity to T cell-mediated alloreactive immune responses after transplantation, Therefore, enhanced proliferation / survival in recipients after transplantation, and superior treatment / prevention. It demonstrates effectiveness.

[0461] The CAR-expressing virus-specific immune cells and compositions of this disclosure also express the CAR-expressing virus of this disclosure. It is also useful in methods that include allogeneic transplantation of allogeneic cells other than virus-specific immune cells. In particular, the CAR-expressing virus-specific immune cells and compositions of this disclosure are allografts (cells, groups) Depleting the targets of tissues and organ populations and alloreactive immune cells (e.g., alloreactive T cells) It is useful for that purpose.

[0462] In such a method, CAR-expressing virus-specific immune cells and compositions are provided by the donor and and / or conditioning of the recipient, and / or alloreceptive immunosuppression after allografting. It is useful in allogeneic transplantation procedures to reduce / prevent disease responses.

[0463] The cells, tissues, and organs transplanted are, for example, immune cells (e.g., adoptive cell transplantation). Heart, lungs, kidneys, liver, pancreas, intestines, face, cornea, skin, hematopoietic stem cells (bone marrow), blood, hands, This includes legs, penis, bones, uterus, thymus, islets of Langerhans, heart valves, and ovaries. Allograft A group of cells, tissues, or organs that are transplanted may be called an "allotransplant."

[0464] The diseases / conditions treated / prevented by allogeneic transplantation are those for which allogeneic transplantation offers therapeutic or preventive benefits. It could be any disease / condition that would result in allografting. In some embodiments, allografting The diseases / conditions that are treated / prevented include, for example, T-cell dysfunction, cancer, infections, or autoimmunity. It could be an infectious disease.

[0465] T cell dysfunction is a disorder in which normal T cell function is impaired in the immune response to pathogenic antigens. Diseases / conditions that cause regulation, such as microorganisms, bacteria, and viruses. It is produced by infection with exogenous factors such as spores, or by certain disease conditions, for example. , produced by the host in some forms of cancer (e.g., forms of tumor-associated antigens) T cell dysfunction may include T cell depletion or T cell anergy. T cell depletion is C D8+ T cells do not proliferate, or they do not respond to antigen stimulation with cytotoxicity and cytokines. For example, this includes a state in which T cells do not exert their effector functions, such as IFNγ secretion. The T cells also produce one or more markers of T cell depletion, such as PD-1, CTLA-4, T cell dysfunction can be characterized by the sustained expression of LAG-3 and TIM-3. This manifests as an infection, or as the inability to initiate an effective immune response to the infection. Infections can be chronic, persistent, latent, or slow, and can be caused by bacteria, viruses, fungi, or This can be the result of a parasitic infection. Therefore, patients with bacterial, viral, or fungal infections Treatment can be provided to the patient. An example of a bacterial infection is Helicobacter pylori. Infections can include HIV, hepatitis B, or hepatitis C. Infections are one example. T-cell dysfunction may be associated with cancer, such as evading tumor immunity. Many Human tumors can produce tumor-associated antigens that are recognized by T cells and can induce an immune response. It manifests.

[0466] Infections can be, for example, bacterial, viral, fungal, or parasitic infections. In the embodiment, chronic / persistent infections, for example, infections in which such infections are associated with T cell dysfunction or T In cases related to cell depletion, it may be particularly desirable to treat it. T cell depletion is multi T cell function in various chronic infections (including viruses, bacteria, and parasites) and cancer. It is well established that the state is incomplete (Wherry Nature Immunity) nology Vol.12, No.6, p492-499, June 2011). Osamu Examples of bacterial infections that can be treated include Bacillus species, Bordetella pertussis, and Clostridium. The genera include Corynebacterium, Vibriocloella, Staphylococcus, and Streptococcus. Infections caused by these diseases include Escherichia, Klebsiella, and Prote. us, Yersinia, Erwina, Salmonella, Listeria s p, Helicobacter pylori, Mycobacteria (e.g., Mycob acterium tuberculosis) and Pseudomonas aeru Ginosa can be mentioned. For example, bacterial infections can be sepsis or tuberculosis. Examples of treatable viral infections include influenza virus, measles virus, and type B influenza virus. Hepatitis virus (HBV), Hepatitis C virus (HCV), Human immunodeficiency virus (HIV) ), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus and human papillomavirus One example is infection caused by the fungal virus (HPV). Examples of treatable fungal infections include: This includes Alternaria sp, Aspergillus sp, and Candida. This includes infections caused by sp. and Histoplasma sp. Fungal infections are fungal It could be sepsis or histoplasmosis. Examples of treatable parasitic infections include malaria Infections caused by parasite species (e.g., Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale) , Plasmodium vivax, or Plasmodium chabaudi This includes chabaudi. Parasitic infections include malaria, leishmaniasis and crested ibis. It could be a disease such as soplasmosis.

[0467] In some embodiments, the above disease / condition is an autoimmune disease. In terms of treatment methods, therapy may aim to reduce the number of autoimmune effector cells. In some embodiments, the autoimmune disease is type 1 diabetes, celiac disease, Graves' disease, inflammation Selected from the following: ventriculointestinal disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. It can be done.

[0468] The CAR-expressing virus-specific immune cells and compositions of this disclosure also enable alloreactive immune responses. It is useful in the treatment / prevention of diseases / conditions characterized by alloreactive immune responses.

[0469] Diseases and conditions characterized by allogeneic reactive immune responses include allogeneic transplantation-related allogeneic reactions. This includes diseases / conditions caused or exacerbated by various reactive immune responses. These diseases / conditions include graft-versus-host disease (GVHD) and graft rejection, Perkey and M aillard Annu Rev Pathol.(2018)13:219-245 Please refer to the following.

[0470] Graft-versus-host disease (GVHD) can occur after allogeneic transplantation of multiple donor immune cells. Graft rejection involves the reactivity of donor-derived immune cells to recipient cells / tissues / organs. Graft rejection refers to the destruction of transplanted cells / tissues / organs by the recipient's immune system after transplantation. When the rejection is due to an allograft, it can be referred to as allograft rejection.

[0471] The CAR-expressing virus-specific immune cells and compositions disclosed herein respond to allogeneic reactions in allogeneic transplantation. It can be used to deplete sex T cells, otherwise, in allogeneic transplantation, the recipient This can lead to graft-versus-host disease (GVHD).

[0472] The CAR-expressing virus-specific immune cells and compositions of this disclosure are donors for allogeneic transplantation (e.g., For example, it is used to deplete allogeneic reactive T cells (before harvesting / collecting allogeneic transplant cells). If not, it may result in GVHD in the recipient during allograft.

[0473] The CAR-expressing virus-specific immune cells and compositions disclosed herein are otherwise ineffective in preventing graft rejection. Depletion of allogeneic-reactive T cells in recipients for allogeneic transplantation may be caused / promoted. It can be used to induce thirst.

[0474] This disclosure provides information on CAR-expressing virus-specific immunity for donors for allogeneic transplantation. Treatment of graft-versus-host disease (GVHD) after allografting, including administration of cells or compositions. This disclosure also provides methods for preventing graft-versus-host disease (GVHD) after allografting. A method for treating / preventing a CAR-expressing virus-specific graft, wherein the allograft is used according to the present disclosure. The present invention provides a method comprising contacting immune cells or a composition. The purpose of such a method is to Allogeneic Reactive Immune Response to Recipient Cells, Tissues and / or Organs for Allogeneic Transplantation The goal is to reduce / remove the ability of allogeneic reactive immune cells in the allogeneic graft to initiate immune response.

[0475] This disclosure relates to the use of CAR-expressing virus-specific immune cells or compositions in allogeneic transplantation. To treat / prevent graft rejection after allografting, including administering to recipients. The aim of such a method is to develop an allogeneic reactive immune response to allogeneic transplantation. The initial goal is to reduce / remove the ability of the target to receive the virus. CAR-expressing virus-specific immune cells Otherwise, it would result in an alloreactive immune response to donor cells, tissues, and / or organs. It is useful for eliminating immune cells in the recipient.

[0476] This disclosure depletes allogeneic transplantation of allogeneic reactive immune cells (e.g., allogeneic reactive T cells). The present invention provides a method that includes allogeneic transplantation (e.g., a population of cells, tissues, or organs to be transplanted) This includes contacting the CAR-expressing virus-specific immune cells or composition of the present disclosure. The law allows CAR-expressing virus-specific immune cells or the compositions of the present disclosure to be donors for allogeneic transplantation. - This may include administering to the subject. The purpose of such a method is to create a recipe for allograft. Initiating an allogeneic reactive immune response against the cells, tissues, and / or organs of the allograft in the allograft The goal is to reduce / eliminate the capabilities of alloreactive immune cells.

[0477] In some embodiments, the above method includes: Acquisition / collection of a population of cells, tissues, or organs from a subject; A population of cells, tissues, or organs, CAR-expressing virus-specific immune cells or groups according to this disclosure. A process of bringing the product into contact with the material; The present disclosure relates to the in vitro or ex vitro treatment in the presence of CAR-expressing virus-specific immune cells. Culture of a population of cells, tissues, or organs in a vein; Collecting / assigning populations of cells, tissues, or organs in which alloreactive immune cells have been depleted; and Transplantation / administration to populations of cells, tissues, or organs that have depleted alloreactive immune cells. .

[0478] This disclosure also applies to administering the CAR-expressing virus-specific immune cells or compositions of this disclosure to the subject. This includes depleting the target alloreactive immune cells (e.g., alloreactive T cells). The present invention provides a method including the following: The subject may be a donor subject for allogeneic transplantation, or the same They may be intended recipients for seed transplantation.

[0479] In some embodiments, the above method includes: In order to deplete alloreactive immune cells in the target, CAR expression viruses according to this disclosure To administer to specific immune cells or compositions; From subjects to whom CAR-expressing virus-specific immune cells or compositions according to this disclosure are administered, The process of obtaining / collecting a group of cells, tissues, or organs; and Transplantation / administration to populations of cells, tissues, or organs that have depleted alloreactive immune cells. .

[0480] In some embodiments, the above method includes: The CAR-expressing virus-specific immune cells or composition described herein are administered to the target, and the target A process of depleting alloreactive immune cells; and The CAR-expressing virus-specific immune cells or composition described herein are used in subjects who have been previously administered these cells or compositions. , transplanting / administering a population of cells, tissues, or organs.

[0481] Depletion of alloreactive immune cells is, for example, alloreactive immune cells in allografts or subjects. A decrease of 2, 10, 100, 1000, 10000 or more in the amount of cells It is possible.

[0482] The above method is performed in vitro or ex vivo, or in vivo in a subject. Methods and processes performed in vitro or ex vivo may be performed in vitro or ex vivo This may include cell culture.

[0483] This method is for the production of CAR-expressing virus-specific immune cells and compositions according to the present disclosure. The method may include further steps.

[0484] In some embodiments, allografting and CAR expression according to the Disclosure for allografting are described above. The administration of virus-specific immune cells or compositions to the recipient subject simultaneously (i.e., 1 (Within 1, 2, 3, 4, 5, 6, 8, 12, 24, 36, or 48 times) It will continue.

[0485] In some embodiments, CAR-expressing virus-specific immune cells or compositions according to the present disclosure Allogeneic transplantation and administration to recipients for allogeneic transplantation are performed sequentially. The time interval between the administration of AR-expressing virus-specific immune cells or compositions and allogeneic transplantation is several hours. This can be any time interval, including several days, weeks, months, or years. CAR-expressing virus Specific immune cells or compositions may be administered to the recipient before or after allogeneic transplantation. Yes, it is possible. CAR-expressing virus-specific immune cells or compositions are preferably used before allogeneic transplantation. It is administered to the patient.

[0486] In some embodiments, allografts and collection of allografts from the above-mentioned subjects (i.e., detailed CAR-expressing virus-specific immunohistochemistry for collection of cells, tissues and / or organs by the present disclosure The administration of cells or compositions to the donor subject may occur simultaneously (i.e., simultaneously, or, for example, once, twice). (This will be done within 1, 3, 4, 5, 6, 8, 12, 24, 36, or 48 times) In some embodiments, CAR-expressing virus-specific immune cells or composition according to the present disclosure The administration of substances to donor subjects for allogeneic transplantation and allogeneic transplantation from subjects (i.e., cells, The collection of tissues and / or organs is carried out sequentially. CAR-expressing virus-specific immune cells or The time interval between the administration of the composition and the collection of allogeneic transplants can be several hours, several days, several weeks, several months, or This can be any time interval including several years. CAR-expressing virus-specific immune cells or compositions It can be administered to donor subjects before or after the collection of allogeneic transplants. CAR-expressing virus-specific immunotherapy The disease cells or composition are preferably administered to the donor subject before the collection of allogeneic transplants.

[0487] In some embodiments, the above method is used for alloreactive immune responses, graft rejection and / or G This includes additional interventions to treat / prevent VHD.

[0488] In some embodiments, the above-mentioned alloreactivity, graft rejection, and / or GVHD are treated. Methods to prevent this include corticosteroids (e.g., prednisolone, hydrocortisone) ), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), antiproliferative agents (e.g.) For example, azathioprinem, mycophenolic acid) and / or mTOR inhibitors (e.g., siro Administration of immunosuppressive and / or lymphatic depletion therapies such as treatment with limus (everolimus, everolimus) include.

[0489] In some embodiments, the above-mentioned allogeneic reactivity and / or graft rejection is treated / prevented. The method for doing so is to use a monoclonal anti-IL-2Rα receptor antibody (e.g., basiliximab) , daclizumab), anti-T cell antibodies (e.g., anti-thymocyte globulin, anti-lymphocyte globulin) This includes antibody therapy such as treatment with an anti-CD20 antibody (e.g., rituximab) and / or other antibody therapies. nothing.

[0490] In some embodiments, a method for treating / preventing the above-mentioned allore reactivity and / or graft rejection. This includes blood transfusions and / or bone marrow transplants.

[0491] Where a method is disclosed herein, this disclosure also does not provide any information on how to use such a method. The present disclosure provides CAR-expressing virus-specific immune cells and compositions of the present disclosure. The CAR expression virus of this disclosure in the manufacture of products (e.g., pharmaceuticals) for use in the method The use of S-specific immune cells or compositions is also provided.

[0492] In some embodiments, methods of various aspects of this disclosure use immunosuppressants (may be multiple) Compared to methods that cause a decrease and / or increased survival of non-allogeneic reactive immune cells. For example, this method can be used in recipient subjects for allogeneic transplantation, or in allogeneic transplantation. Therefore, it is useful for preserving / maintaining non-allogeneic reactive immune cell compartments.

[0493] In some embodiments of the methods disclosed herein, including allografting, the method is used to treat immunosuppressants Compared to methods involving therapy, non-allogeneic reactive immunotherapy in recipients for allogeneic transplantation. Related to an increase in the number / percentage of cells. Some of the methods of this disclosure, including adoptive transfer of alloimmune cells. In that embodiment, the present method, compared to a method involving treatment with an immunosuppressant, is effective in alloimmune cells. This is related to an increase in the number / proportion of non-allogeneic reactive immune cells in the recipient group.

[0494] In some embodiments of the methods disclosed herein, including allografting, the method is used to treat immunosuppressants Compared to methods involving therapy, allogeneic transplantation is associated with an increase in the number / percentage of non-allogeneic reactive immune cells. To be connected.

[0495] This disclosure also relates to the CAR-expressing virus-specific immunofluorescence of this disclosure for use in the methods described below. Provides cells or compositions: Cells that express a target antigen that is specific to CAR (for example, cells that express CD30) Extinction; Virus-specific immune cells are specific to viruses (for example, cells infected with EBV, or Infection with the peptide of the antigen (which presents the EBV antigen peptide) or the peptide of that antigen To kill cells that present tides; and / or Death of alloreactive immune cells (e.g., T cells expressing CD30).

[0496] This disclosure also relates to such CAR-expressing virus-specific immune cells and such methods. Use of such compositions, as well as CAR-expressing virus-specific immune cells and for such purposes This provides a method for using the composition.

[0497] subject The subject in the embodiments of this disclosure may be any animal or human. Preferably, the subject is Mammals, or more preferably humans. The subject may be a non-human mammal, but more preferably humans. The subject is a human being. The subject may be male or female. The subject may also be a patient. The subjects may be diagnosed with a disease / condition described herein that requires treatment, and There may be suspicion of having such a disease / condition, or the onset of such a disease / condition. It's acceptable to have a risk of developing symptoms.

[0498] In the embodiments described herein, the subject is preferably a human subject. Therefore, the subjects treated according to the therapeutic or preventive methods disclosed herein are the diseases / conditions described herein. The subject has or is at risk of developing the condition. In the embodiment of the present invention, Treatment of the subject based on the characterization of specific markers for such diseases / conditions It can be chosen for medical treatment.

[0499] The subjects may be homogeneous but heterogeneous subjects with respect to the intervention described herein. The targets of treatment / prevention are those from which CAR-expressing virus-specific immune cells originate, and are genetically related. They do not have to be the same. The targets treated / prevented in accordance with this disclosure are CAR-expressing virus-specific. Target immune cells may have an HLA mismatch with the target from which they originate. Treatment / The targets of prevention are those derived from CAR-expressing virus-specific immune cells, and HLA-based It can be checked.

[0500] Subjects to whom cells are administered in accordance with this disclosure shall have the same rights with respect to the source from which the cells originate. It can be species / non-self. The target to which the cells are administered is the cell that produces the cells being administered. The target may be different from the target obtained. The target to which the cells are administered is the target to which the cells are administered. The cells used for production do not need to be genetically identical to the target cells from which the product was obtained.

[0501] The target to which the cells are administered is the target from which the cells were obtained for the production of the administered cells. MHC / HLA molecules that are not identical to the MHC / HLA molecules encoded by the / HLA gene It may contain MHC / HLA genes that code for molecules. The target to which the cells are administered is Cells are produced by the target MHC / HLA genes that are encoded by the cells. MHC / HLA genes that encode MHC / HLA molecules identical to the MHC / HLA molecules themselves. It may include.

[0502] In some embodiments, the target to which the above cells are administered is for the production of the administered cells. The cells are HLA that are compatible with the target obtained. In some embodiments, the cells are The target being administered to has cells obtained for the production of the administered cells, approximately or It is a perfect HLA match.

[0503] In some embodiments, the target spans HLA-A, -B, -C, and -DRB1. 4 / 8 (i.e., 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8) is a match. How many? In that embodiment, the above targets are HLA-A, -B, -C, -DRB1 and -DQB1 Over the course of ≥5 / 10 (i.e., 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10) Or 10 / 10) match. In some embodiments, the above subject is HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 ≥ 6 / 12 (i.e., 6 (12 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 12, 11 / 12 or 12 / 12) Match In some embodiments, the target is HLA-A, -B, -C, and -DRB1. This is an 8 / 8 match across all. In some embodiments, the above subjects are HLA-A, -B, - This is a 10 / 10 agreement across C, -DRB1, and -DQB1. In some embodiments, The target group includes HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1. It's a 12 / 12 match.

[0504] Array ID For the purpose of determining the percentage identity between two or more amino acid or nucleic acid sequences, publicly available to those skilled in the art. In various methods of knowledge, for example, ClustalOmega (Soding, J.2005, Bioinformatics 21,951-960), T-coffee (Notr edame et al. 2000, J. Mol. Biol. (2000) 302, 205-217 ), Kalign (Lassmann and Sonnhammer 2005, BM C Bioinformatics,6(298)) and MAFFT(Katoh an d Standley 2013,Molecular Biology and Ev (olution, 30(4)772-780 software). Such software When using the default, for example, for gap penalties and extension penalties It is preferable that parameters be used.

[0505] array [Table 1] JPEG2026108797000003.jpg205170JPEG2026108797000004.jpg205170JPEG2026108797000005.jpg205170J PEG2026108797000006.jpg204170JPEG2026108797000007.jpg204170JPEG2026108797000008.jpg129170***

[0506] This invention relates to situations where such combinations are clearly unacceptable or clearly avoidable. This includes combinations of the described embodiments and preferred features, except for the combinations described.

[0507] The headings used herein are for structural purposes only and do not represent the subject matter being described. It should not be interpreted as limiting.

[0508] Herein, aspects and embodiments of the present invention will be described as examples with reference to the accompanying drawings. Other aspects and embodiments will be obvious to those skilled in the art. These documents are incorporated herein by reference.

[0509] Throughout this specification, including the following claims, unless otherwise required by context, use The words "comprise", "comprises", and "c Variations such as "omprising" are described by integers or steps or integers or steps It includes the base of but does not exclude any other integer or step or integer or step base. This is likely to be understood as meaning that.

[0510] As used herein and in the appended claims, the singular forms "a", "an", and The noun "the" refers to multiple objects unless the context clearly indicates otherwise. Please note that the range in this specification is "approximately" from one specific value and / or "approximately" from another specific value. It can be expressed as up to the value of . When such a range is expressed, there is one other embodiment. It includes from a specific value of and / or up to another specific value. Similarly, the use of the antecedent "about" When a value is expressed as an approximation, it is understood that a particular value may form a different embodiment. Hey.

[0511] Where nucleic acid sequences are disclosed herein, their reverse complements are also expressly intended.

[0512] The methods described herein can be performed in vitro or in vivo. In the embodiments described herein, the methods described herein are carried out in vitro. The term is intended to encompass experiments using cultured cells, while "in vivo" The term is intended to encompass experiments using intact multicellular organisms. [Examples]

[0513] In the following examples, the inventors have shown that CD30.CAR-expressing EBVST can be used in cancer cells. This section describes the effector activity and resistance to homogeneity rejection.

[0514] Example 1: Generation of a retrovirus encoding a CAR construct [Table 2]

[0515] A retrovirus encoding the CD30.CAR construct, and the cDNA encoding the CAR. pSFG-TGFbDNRII retrovirus skeleton (ATUM, Newark, CA) It was prepared by cloning.

[0516] A plasmid containing the CD30.CAR sequence, pSFG_CD30CAR, is subjected to polyethylene HEK 293 Vec-RD114 cells were transfected using PEI. Next, the cell culture supernatant from the transfected cells was used in a 6-well plate. 5 x 10 5 HEK 293Vec-Galv cells (Canadian Quebec) at cell / well density We transduce Biobeck Pharma (K State).

[0517] Trypsinized 293Vec-Galv_CD30-CAR cells ) and 2 × 10 6 The solution was reused in a 15 ml tube at a concentration of cells / ml. Dilution of 2 series. After performing the procedure, dilute 1.65 ml of the final cell suspension and add 220 ml of DMEM + 10%F Mixed with CS. Transfer 200 μl of this suspension to the wells of a 96-well plate. Approximately 30 cells were obtained per unit. Next, the best performing clone was selected, and retrovirus-containing cells were added. It was used to produce a supernatant. Then, the retrovirus-containing supernatant was collected and filtered. Stored at -80°C until use.

[0518] The retrovirus that codes for CD19.CAR, DN that codes for CD19.CAR A was created by cloning it into the pSFG retrovirus skeleton. CD19. Using a plasmid containing the CAR sequence, 85bCD19C, polyethyleneimine (PE HEK 293 Vec-RD114 cells were transfected using method I). The retrovirus-containing supernatant was collected, filtered, and stored at -80°C until use.

[0519] Example 2: Generation of CAR-expressing EBV-specific T cells Peripheral blood mononuclear cells (PBMCs) are subjected to standard Ficoll-Paque density gradient centrifugation. Therefore, it was isolated from blood samples obtained from healthy donors or lymphoma patients.

[0520] ATC generation Anti-CD3 (clone OKT3) and anti-CD28 agonist antibodies are administered at a concentration of 1 mg / ml. By adding 0.5 ml of a 1:1000 dilution, the wells of the tissue culture plate will be filled with Coat and incubate at 37°C for 2-4 hours, or overnight at 4°C, then 1 x 10 6 P BMC (in 2 ml of culture medium per well) RPMI medium, 44.5% Click's medium, 10% FBS and 1% Glutamate Culture on anti-CD3 / CD28 agonist antibody coated plates in (including x) The cells were stimulated in this way. The cells were maintained at 37°C in a 5% CO2 atmosphere. The next day, 1 ml of cell culture was performed. Prepare the culture medium by adding fresh fine-grained IL-7 at 20 ng / ml and IL-15 at 20 ng / ml. The cell culture medium was replaced. To maintain ATC during culture, the cell culture medium was changed every 2-4 days. And cytokines were supplemented as needed, or ATC was recovered and contained cytokines. The cells were re-seed in fresh cell culture medium. ATCs were collected and EBVST was administered between days 7 and 10. It was used in experiments for restimulation during the night.

[0521] General-purpose LCL LCL lacking surface expression of HLA class I and HLA class II (i.e., HLA-negative) LCL) is a lymphoblastoid cell line prepared by EBV transformation of B cells. Targeted knockout of genes encoding HLA class I and HLA class II molecules This was obtained by further modifying HLA-negative cells to add genes necessary for EBV replication. The cells obtained as a result of this method are referred to herein as universal. It is called ulcerative colitis (uLCL).

[0522] Proliferation and transduction of EBV-specific T cells (EBVSTs) PBMCs derived from healthy donors are used in CD45RA MACS microbeads (Milte CD45RA-expressing cells were depleted by magnetic cell separation using nyi Biotec. EBV-specific T cells are EBNA1 peptidone (JPT Cat No. PM). -EBV-EBNA1), LMP1 Pepmix (JPT Cat No. PM-EB V-LMP1), and LMP2 PEP obtained from JPT Technologies. Mix (JPT Cat No. PM-EBV-LMP2) (11 amino acid duplication 15 m - Using an amino acid peptide library (including the complete amino acid sequence of the related antigen), 44. 5-47% Advanced RPMI, 44.5-47% Click's medium, 10% FBS or 5% growth factor-rich additive, and IL-7 (10 ng / ml) and IL-15 In cell culture medium containing 1% GlutaMax with (10 ng / ml) added, 2 x 10 6 CD45RA-depleted PBMCs (2 ml of culture medium per well) can be stimulated to promote growth. The EBVST was maintained at 37°C in a 5% CO2 atmosphere.

[0523] After 4-6 days, the EBVST will be retrocoded with the CAR described in Example 1 as follows: The virus was introduced into the body.

[0524] In a non-tissue culture treated 24-well plate pre-coated with retronectin (Takara), Rovirus-containing supernatant (0.5-1 ml per well) was added. Plates were heated to 2000 x g. After centrifugation for 60-90 minutes, remove the retroviral supernatant and 0 cells per well. 0.25~0.5×10 6 The cells were replated.

[0525] After 8-10 days of culture, the irradiated peptide pulsed self-activated T4 was used in the presence of uLCL. The cells were re-stimulated by co-culturing them with cells (ATC). In short, 2 × 10 6 of ATC, Pepmix (1 x 10 6 (10 ng of Peptomix mixture per ATC) Both samples were incubated in CTL medium at 37°C for 30 minutes, followed by irradiation with 30 Gy, and then harvested. Next, the peptide processing ATC used IL-7 (10 ng / ml) and IL-15 (1 In culture in CTL media containing 00 ng / ml and in uLCL (irradiated with 100 Gy) The ratio of respondent cells was: peptide-modified ATC:1:1:5 (irradiated uLCL) and mixed. Physically, 1 x 10 5 Responding cells, 1 × 10¹ 5 Peptide pulse ATC and 0.5× 10 6 Irradiated uLCL was cultured in 2 mL of CTLs in a 24-well tissue culture plate. It was cultured underground.

[0526] To maintain EBVST during culture, the cell culture medium and cytoplasm should be changed every 2-4 days. The cytokines were replenished as needed, or EBVST was recovered and fresh cells containing cytokines were used. The cells were re-seeded in culture medium. EBVSTs were collected, and the mixed lymphocyte reaction (M) was observed on days 15-20. It was used in the LR assay.

[0527] Example 3: CD30 special cell that eliminates allogeneic reactive T cells and protects allogeneic VSTs from rejection. Evaluation of the capabilities of different CARs The inventors have demonstrated the ability of VST to resist homogeneous rejection in vitro with respect to CD30.C. The effect of AR expression was investigated.

[0528] Generation of primed alloreactive T cells 10 EBVSTs were produced using 1-2x EBVSTs derived from the same healthy donor. 6 PB Irradiate MC (per well) with 30 Gray, and 1x10 from a mismatched donor. 6 P BMC (per well) (with various HLA-A2 expression levels), 44.5% advanced RPMI, 44.5% click medium, 10% serum, and IL-7 (10 ng / ml) In cell culture medium containing 1% glutamax supplemented with IL-15 (10 ng / ml) Co-cultured. Primed alloreactive T cells grown from mismatched donor PBMCs. The cells are 0.5 × 10 6 Cells (in 2 ml of cell culture medium) are treated with anti-CD3 / CD28 agonists. The cells were re-stimulated by seeding on antibody-coated plates on days 6-10. To maintain the allore-reactive T cells, the cell culture medium and cytokines are changed every 2-4 days. Supplementation was performed as needed, or alloreactive T cells were collected and fresh cytokines were added. The cells were re-seeded in cell culture medium. Alloreactive T cells were collected and subjected to EBVS between 13 and 17 days. It was used in a mixed lymphocyte reaction (MLR) assay using T.

[0529] To evaluate allore rejection in vitro, 0.0% of the sample is derived from an HLA-A2-negative control. 2 x 10 OOBPBMC alloreactive T cells are mixed lymphocyte reactant (MLR) aggregate. In I: (i) HLA-A2 positive targets used to prime alloreactive T cells 0.2x10 generated from PBMC 4 EBVST, or (ii) HLA-A2 positive targets used to prime alloreactive T cells 0.2 × 10⁻¹⁰ PBMC generated from PBMC 4 EBVST is a CD30-specific CAR The structure was further modified to incorporate new traits.

[0530] Human IL-7 (10 ng / ml) and IL-15 (10 ng / ml) were subjected to MLR assay. Added.

[0531] Flow cytometry analysis was performed 7 days later, and the absolute number of cells was determined using counting beads. T cells derived from different sources were co-cultured and then aggregated based on HLA-A2 expression. It was possible to identify it within the group. Gallios Flow Cytometer (B Using eckman Coulter) to retrieve events, Kaluza Anal ysis Software (Beckman Coulter) for data analysis and graphics Used for displaying "F".

[0532] As shown in Figure 1, the number of non-transduced (NT) EBVSTs derived from HLA-A2 positive targets is After co-culturing alloreactive T cells derived from an HLA-A2-negative control for 7 days (lower left panel), Compared to when cultured in the absence of alloreactive T cells (upper left panel), the reduction was significant. In contrast, the number of CD30.CAR EBVSTs was increased in the absence of alloreactive T cells during culture. Compared to the case where (upper right panel), after 7 days of co-culture with alloreactive T cells (lower right panel) It increased to (ru).

[0533] Figure 2 shows the quantification of flow cytometry data. (Untransduced EBVST(NT)) It was largely eliminated in the presence of alloreactive T cells, but CD30.CAR-expressing EBVST It was resistant to elimination by alloreactive T cells (Figure 2A). In addition, alloreactive T Quantitative analysis of the cell population (CD3+, HLA-A2 negative) is performed using CD30.CAR EBVST. We revealed that the number of allogeneically reactive T cells is reduced compared to transduction EBVST conditions. (Figure 2B).

[0534] Therefore, EBVST expressing CD30.CAR reduces the number of alloreactive T cells. It has been shown to possess the ability to prevent allergic reactions and to be protected from allergic rejection.

[0535] Example 4: EBV-specific T19 expressing CD19-specific CAR and CD30-specific CAR Cell characterization The inventors manipulated the system to express both CD19.CAR and CD30.CAR. We generated and characterized virus-specific T cells, and observed that they exhibited allogeneic effects in mixed lymphocyte reactions. We investigated whether reactive T cells could be eliminated.

[0536] In short, HLA-A2 positive target cells depleted of CD19 and CD56 expression cells Next 1x10 5 The population of PBMCs was co-cultured in a mixed lymphocyte reaction (MLR) assay. Raised: (i) 0.1 x 10⁶ PBMCs generated from HLA-A2 negative subjects 5 EBV ST, or (ii)(a)HLA further transfected in a construct encoding CD30.CAR - 0.1 × 10⁶ generated from PBMCs of A2-negative subjects. 5 EBVST(b)CD19. CAR, or (c) both CD30.CAR and CD19.CAR (CD30+CD19 .CAR).

[0537] Human IL-2 was added to the MLR assay at a concentration of 20 IU / ml.

[0538] As shown in Figure 3, CD30.CAR EBVST (upper right panel) and CD30+CD1 9. EBVST (bottom right panel) with both CAR is not transduced (NT)E Compared to BVST (top left panel) and CD19.CAR EBVST (bottom left panel), By day 7, HLA-A2+ alloreactive T cells (distinguished by the activation marker CD71) This significantly reduced the rate of rejection (and thus avoided rejection).

[0539] Therefore, the present inventors have identified a CAR (CD19 in this example) that is specific to the target antigen and Using EBVST transduced with both CD30-specific CARs, a specific target antigen is targeted. This provides a novel approach to creating heterogeneous "ready-made" CAR T cells. The ability of dual CAR-EBVST to eliminate alloreactive T cells in vitro is that they The ability to avoid rejection with Nvivo and to sustain long-term effects in homogeneous, heterogeneous recipients. This suggests that...

[0540] Example 5: Cancer treatment using CD30.CAR EBVST 5.1 Preparation and Characterization of CD30.CAR EBVST from Health Provider Subjects price CD30.CAR EBVST was manufactured in a GMP facility. Informed Consent. After obtaining the blood sample, seven healthy individuals were selected according to the guidelines established by the Declaration of Helsinki. Approximately 250 - 400 mL of blood was collected from liquid - bank approved donors.

[0541] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation. PBM Cs were depleted of CD45RA - expressing cells using clinically - graded anti - CD45RA antibody conjugated to magnetic beads and magnetic cell separation, and by using a Miltenyi depletion column (Miltenyi Biotec, Bergisch Gladbach, Germany). Magnetic cell separation, and by using a Miltenyi depletion column (Miltenyi Biotec, Be rgisch Gladbach, Germany) to deplete CD45 RA - expressing cells.

[0542] 1.5 - 2.5x CD45RA - depleted cells of 10 s 7 PBMCs were seeded into 30 ml of culture medium containing 47.5% Advanced RPMI, 47.5% Click’s (EHAA) medium (Irvine S cientific), 2 mM L - glutamine (Thermo Fisher Sci entific) and 5% human platelet Lysate (HPL; Sexton Biote chnologies), supplemented with IL - 7 (10 ng / ml) and IL - 15 (10 ng / ml) in a G - Rex10 vessel, and activated by stimulation with a repetitive peptide library (pepmix) containing 11 - amino - acid - overlapping 15 - mer amino acids, covering the full protein sequences of relevant antigens. Pepmixes corresponding to EBNA1, LMP1, LMP2, BARF 1, BZLF1, BRLF1, BMLF1, BMRF1, BMRF2, BALF2, BN LF2a and BNLF2b were obtained from JPT Technologie s (Berlin, Germany). Stimulation was carried out with 5 ng of pepmix per 1×10 cells to be stimulated (i.e., for CD45RA - positive cells per antigen) (i.e., for CD45RA - positive cells 6 per antigen) (i.e., for CD45RA - positive cells [[ID=e43]]per antigen) (i.e., for CD45RA - positive cells Depleted 2x10 7 For stimulation using PBMC, each Pepmix 100 (ng was used). Stimulated culture was maintained at 37°C in a 5% CO2 atmosphere.

[0543] After 4-6 days, the EBVST produced by the stimulated culture described above is as follows: Sea urchin was transduced with the retrovirus encoding the CAR described in Example 1. 2 ml Mix the retrovirus-containing supernatant with 150 μg of Vectofucin-1 in a volume of 2 ml. A final volume of 4 ml was obtained and incubated at room temperature for 5-30 minutes. Then, retrow Add the Virus:Vectofucin-1 mixture to 8.5 ml of culture medium in a T75 container (as described in the previous section). (Included) 7~10×10 6 The solution was added to the cells. The culture was maintained at 37°C in a 5% CO2 atmosphere. Ta.

[0544] During the 8th to 10th day of culture, 1-2 × 10⁶ units produced by the transduction described in the previous section were obtained. 7 of CD30.CAR EBVST CD30.CAR EBVST in G-Rex100 container Transfer to C for irradiated uLCL in a range of 1:2 to 1:5 (typically about 1:3). D30.CAR EBVST ratio, irradiated (100 Gray) uLCL (Example) Restimulation was performed by co-culture with (as described in 2). ULCL was EBV antigen and CD30, and CD30.CAR expresses other co-stimulatory molecules, and therefore, antigen stimulation and co-stimulation are associated with CD30.CAR. It provides EBVST, and the potency of CD30.CAR EBVST without losing EBV specificity. It induces proliferation.

[0545] Restimulation culture is established using 200 ml of culture medium (as described in paragraph 3 of section 5.1), and if necessary... Additional culture medium was added accordingly. After 7-12 days, CD30.CAR EBVST was collected. It was then cryopreserved for subsequent injections.

[0546] CD30.CAR EBVST prepared from four representative healthy donors was used in Ability to proliferate in vitro, CD30 expression in vitro, and against CD30-negative cancer cell lines Cytotoxicity and specificity to different EBV antigens were evaluated.

[0547] Analysis of CD30.CAR EBVST proliferation The proliferation of CD30.CAR EBVST was observed at various time points during culture (0, 6, 10, 17, The number of cells is determined by counting them using a hemocytometer on days 18 and 19, and the cumulative number is then determined. The magnification was calculated.

[0548] Figure 4 shows CD30.CAR EBVS produced from four different healthy donor subjects. T is sufficient to achieve the therapeutic dose of CD30.CAR EBVST within 17-20 days. This indicates that the cells proliferated sufficiently in in vitro culture. The proliferated cells accounted for 77% to 99% of the total. % of cells expressed CD30.CAR (data not shown).

[0549] Analysis of CD30.CAR EBVST cytotoxicity The cytotoxic specificity of CD30.CAR EBVST was investigated using chromium-51. 51 Chromium The measurement was performed using a test assay. In short, CD30-negative BJAB Burkitt lymphoma cells Target cells, either cysts or CD30-positive HDLM2 Hodgkin lymphoma cells, 51 Cr They were incubated together for 1 hour. Non-transduced EBVST or CD30.CAR transduction. Using the EBVST as an effect pedal, in a 96-well plate, the wells were 40:1, 2 Incubated with the target at effector-to-target ratios of 0:1, 10:1, 5:1 and 2.5:1. After culturing for 4 - 6 hours, the co-culture supernatant was collected and the 51 chromium release was detected using a gamma counter. The percentage of specific lysis was determined from the average of 3 replicates using the following chemical formula: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] × 100.

[0550] Figure 5 shows that CD30.CAR EBVST was substantially non-cytotoxic to cells of the CD30-negative Burkitt lymphoma BJAB cell line, but showed high cytotoxicity to cells of the CD30-positive Hodgkin lymphoma HDLM2 cell line.

[0551] Analysis of the reactivity of CD30.CAR EBVST to EBV antigen IFN-γ ELISpot assays were performed to evaluate the response of CD30.CAR EBVST prepared from 4 different healthy donors to stimulation with EBV antigens.

[0552] For EBV latent cycle antigens (EBNA1, LMP1, LMP2 and BARF1) and EBV lytic cycle antigens (BZLF1, BRLF1, BMLF1, BMRF1, BMRF2, BALF2, BNLF2a and BNLF2b), IFN-γ production was measured in response to stimulation with Pepmix (obtained from JPT Technologies, Berlin, Germany). Briefly, CD30.CAR EBVST was plated in duplicate in wells of a 96-well multiscreen plate (Millipore Sigma) at 5 × 1 0 cells / well. Stimulation was performed using a total of 0.1 μg of peptide per well. 4 The process involved incubation in 5% CO2 at 37°C for 16-20 hours, followed by IFN-γ+ Plates were deployed for spot measurements, and Zernet Consulting (New Zealand) was used for quantification. It was sent to Fortley, Jersey. The frequency of antigen-specific reactions was 5 × 10⁻⁶. 4 per cell It is represented as a Spot Forming Unit (SFU).

[0553] Figure 6 shows CD30.CAR EBVS produced from four different healthy donor subjects. This indicates that T retained its specificity for the EBV antigen.

[0554] All four CD30.CAR EBVST strains possess both latent and soluble EBV antigens. 10 in response to stimulation 5 Spot-forming units exceeding 100 IFNv per cell ( It produces SFUs and exceeds 20% of the CD30-positive Hodgkin lymphoma cell line HDLM2. A functional release group that produces specific cell lysis in an effector-to-target ratio of 20:1. I passed the preliminary exam.

[0555] 5.2 CD30.CAR EB as allogeneic adoptive cell therapy for CD30+ lymphoma VST administration CD30+ refractory or relapsed Hodgkin lymphoma, non-Hodgkin lymphoma, ALK-positive undifferentiated Patients with T-cell lymphoma, ALK-negative anaplastic T-cell lymphoma, or other peripheral T-cell lymphomas Patients aged 12 to 75 years were eligible for treatment in this study.

[0556] Cyclophosphamide (Cy: 500 mg / m²) 2 ( / day) Fludarabine (Flu:30m) g / m 2 It was administered three times a day in combination with ( / day) and induced lymphopenia, but CD30.C Although it was completed 48 hours before the AR EBVST intravenous infusion, it was completed by 2 weeks before the intravenous infusion. Ta.

[0557] On day 0 of the trial, patients received intravenous infusions of 1-50 ml over approximately 1-10 minutes. Therefore, the patient received a single dose of the planned allogeneic CD30.CAR EBVST. Administer CD30.CAR EBVST with the best HLA class I and class II match. did.

[0558] In this study, allogeneic CD30.CAR EBVST cells were administered to a total of five patients. 3 For example, 4 x 10 7 A dose of CD30.CAR EBVST cells (DL1) was administered. For example, 1 × 10 8 A dose of 2 (DL2) of CD30.CAR EBVST cells was administered.

[0559] Monitoring was conducted for the administration of blood products, except that the injection was administered by a physician. The procedure was performed according to facility standards. Patients were monitored for at least 3 hours after injection. Clinical status Patients were evaluated for adverse events, including changes in clinical laboratory data. In particular, some CARs - Cytokine release syndrome (CRS) and neurotoxicity observed in T-cell immunotherapy We evaluated patients regarding the correlation with sex.

[0560] Blood samples were collected from patients at the following points in time: before the study, 3-4 hours after injection, and on day 0. 1, 2, 3, 4, and 6 weeks and 3 months after cytoplasmic injection. Sustained CD30.CAR EBVST. Samples were analyzed to evaluate their properties and effectiveness.

[0561] None of the patients experienced dose-limiting toxicity, nor did any cytokine release syndrome of any grade. No CRS (Chronic Surgical Condition) or graft-versus-host disease (GVHD) was observed.

[0562] Clinical responses in patients treated with allogeneic CD30.CAR EBVST Diagnostic imaging is performed to detect measurable disease before injection and at 6-8 weeks after injection on day 0. For patients and their treatment (via PET scan, CT scan, MRI, and nuclear imaging) The response was recorded.

[0563] Patient #1 received an intravenous injection of 11.9 mCi of FDG into the left cubital fossa (blood glucose level at the time of injection was 9 mCi). (The level was 9 mg / dL). PET and CT images were obtained from the middle cranial vault to the proximal femur, and images were taken. The fusion process was followed by polyhedral reconstruction in the axial, coronal, and sagittal planes, as well as three-dimensional reconstruction.

[0564] Patient number 2 received an intravenous injection of 7.29 mCi of FDG (blood glucose level at the time of injection was 99 mg / (It was dL). Approximately 60 minutes later, using a PET-CT scanner with CT attenuation correction technology... Images were obtained from the base of the skull to the proximal thigh. CT slices were obtained using a low-dose method, and multiple images were taken. A planar reformatted image was obtained.

[0565] Figures 7 and 8 show the clinical response in two patients treated with CD30.CAR EBVST. The answer is shown. The image of patient #1 shows the resolution of several areas of the disease, and the image of patient #2 shows the disease A significant decrease in the number of patients was observed, and in these patients, allogeneic CD30.CAR EBVST To demonstrate the therapeutic effectiveness of the treatment.

[0566] Analysis of the copy number of the CD30.CAR vector after administration Real-time qPC for the embedded genome of the retrovirus encoding CD30.CAR Quantitative analysis was performed using R. PBMCs were measured at several time points (before lymphocyte depletion, 3 hours, 1 week, Peripheral blood samples taken from patients at weeks 2, 3, 4, 6, and 3 months were used to obtain a single sample. I released it. I followed the manufacturer's instructions for the QIAamp DNA Blood Mini Kit. After extracting DNA from PBMCs using (Qiagen), the DNA was then transferred to a retroviral vector. Using primers and probes (applied biosystems) complementary to specific sequences The DNA was amplified. A standard curve was created using serial dilutions of the plasmid encoding the transgene. Established. Amplification was performed according to the manufacturer's instructions using the ABI7900HF real-time PCR system. The procedure was carried out using Applied Biosystems (M).

[0567] Figure 9 shows the vector copy number of the CD30.CAR transgene in patient #1 and patient #2. CD30.CAR EBVST does not proliferate in vivo, and these patients' end-stages This suggests that it becomes rapidly undetectable in peripheral blood.

[0568] Analysis of epitope diffusion in patients administered allogeneic CD30.CAR EBVST To assess epitope diffusion, immune cells were collected from patient #1 at several time points, and tumor Stimulation with ulcer-associated antigens and the reactions before and after infusion of allogeneic CD30.CAR EBVST I decided on my gender.

[0569] PBMC at several points in time (Pre-lymphodepletion, 3 hours, 1 Peripheral blood samples collected from patients at week 1, week 2, week 3, week 4, week 6, and month 3. Isolating from and essentially performing the ELISpot assembly as described in Example 5.1 above It was used in I, however, PBMC was 3 x 10 per well 5 Sowed and EBV latent In addition to evaluating soluble antigens, PBMCs were stimulated using two additional antigen groups; (1 ) A pool of peptide mix antigens derived from "other viruses" (adenovirus protein He xon and Penton, as well as CMV protein PP65), and (2) tumor-related TAA (Traditional Antigens) MAGE-A4, NY-ESO, PRAME, SSX2, and Survival A pool of Pepmix that corresponds to [the product / service].

[0570] Figure 10 shows that patient #1 did not show a response to tumor-associated antigens at any point in time. This indicates that there was no epitope diffusion in patient #1. Treatment with allogeneic CD30.CAR EBVST targets the patient's immune system and these other tumor antigens. This suggests that the individual was not sensitized to the substance.

[0571] 5.3 Conclusion The inventors have found that CD30.CAR EBVST produced from a healthy donor is a CD According to their use as ready-made treatments for patients with 30+ cancers, EBV specificity With the ability to retain and eliminate CD30-positive tumor cells, they are expanded to a sufficient number, and they This demonstrated that it is possible to save the functions of both TCR and CD30.CAR.

[0572] CD30.CAR EBVST is safe and in viv It was found to show therapeutic effect against CD30-positive lymphoma in peripheral blood CAR Despite the limited persistence of expression cells, and the spread of epidemiologically to other tumor-associated antigens... Despite the lack of evidence for the drug, a clinical response was observed.

Claims

[Claim 1] Virus-specific immune cells comprising a chimeric antigen receptor (CAR) or nucleic acid encoding a CAR for use in methods of treating or preventing cancer, The CAR comprises (i) an antigen-binding domain that specifically binds to CD30, (ii) a transmembrane domain, and (iii) a signal transduction domain. The aforementioned signal transduction domain is comprised of (a) an amino acid sequence derived from the intracellular domain of CD28, and (b) an amino acid sequence containing an immune receptor tyrosine-based activation motif (ITAM), and is a virus-specific immune cell.

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