Compositions Comprising Polyinosinic-Polycytidylic Acid, Epstein-Barr (EBV) Virus-Like Particles, and EBV-Specific CAR-T Cells for Improving Immune Cell Therapy

Abstract

The present disclosure relates to immunogenic compositions comprising polyinosinic-polycytidylic acid (PIC), a stabilizer comprising an aminoglycoside or non-aminoglycoside antibiotic, at least one cation, and optionally an immunogen. The present disclosure also relates to immunogenic compositions comprising Epstein-Barr (EBV) virus-like particles. The present disclosure further describes the use of EBV-specific CAR-T cells and immunogenic compositions comprising PICs, alone or in combination with VLPs or CAR-T cells, for the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to compositions for improving immune cell therapy, as well as CARs and immune cells comprising said CARs for cell therapy. The present invention also relates to the use of said compositions or said CAR-containing immune cells as a medicament, alone or in combination therapy, particularly for treating cancer. [Background technology]

[0002] In recent years, advances have been made in the treatment of cancer. Among these advances is the use of immune cell therapy, in which a patient's immune response is harnessed to treat cancer. Such immune cell therapy treatment methods include the use of cell-based immunotherapy, in which cells of the immune system are utilized for therapeutic treatment. Immune system cells, such as T cells and other immune cells, can be engineered to target tumor antigens.

[0003] Recent advances in the use of chimeric antigen receptor (CAR)-modified T cell (CART) therapy, which relies on retargeting T cells to appropriate cell surface molecules on cancer cells, have shown promising results in harnessing the power of the immune system to treat cancer (e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)). Most notably, CD19-specific CAR (CD19CAR) T cell therapy has yielded impressive results, including long-term remissions, in B-cell malignancies (e.g., Maude, Frey et al., 2014). Despite the success of CAR therapy in hematological cancers such as B-cell leukemia and lymphoma, only limited success has been achieved in solid tumors.

[0004] Cell-based therapies using immune cells such as T cells have provided a new modality for cancer treatment, but limitations to the effectiveness of such therapies have been found, particularly for solid tumors. For example, cancer cells can adapt to create an immunosuppressive microenvironment that protects them from immune recognition and elimination. This microenvironment poses challenges for therapeutic methods involving the stimulation of an immune response, including immunotherapeutic methods such as targeted T cell therapy. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,703,199 [Patent Document 2] U.S. Patent No. 5,399,346 [Patent Document 3] U.S. Patent No. 5,580,859 [Patent Document 4] U.S. Patent No. 5,589,466 [Patent Document 5] WO01 / 96584 issue [Patent Document 6] WO01 / 29058 issue [Patent Document 7] U.S. Patent No. 6,326,193 [Patent Document 8] U.S. Patent No. 5,350,674 [Patent Document 9] U.S. Patent No. 5,585,362 [Patent Document 10] U.S. Patent Application Publication No. 2015 / 0210769 [Patent Document 11] U.S. Patent No. 8,008,449 [Patent Document 12] WO2006 / 121168 [Patent Document 13] U.S. Patent No. 8,354,509 [Patent Document 14] WO2009 / 114335 [Patent Document 15] U.S. Patent No. 7,695,715 [Patent Document 16] U.S. Patent No. 7,332,582 [Patent Document 17] U.S. Patent No. 8,686,119 [Patent Document 18] U.S. Patent No. 9,205,148 [Patent Document 19] WO2012 / 145493 [Patent Document 20] WO2015 / 112800 [Patent Document 21] WO2016 / 092419 issue [Patent Document 22] WO2015 / 085847 issue [Patent Document 23] WO2014 / 179664 [Patent Document 24] WO2014 / 194302 issue [Patent Document 25] WO2014 / 209804 [Patent Document 26] WO2015 / 200119 issue [Patent Document 27] U.S. Patent No. 8,735,553 [Patent Document 28] U.S. Patent No. 7,488,802 [Patent Document 29] U.S. Patent No. 8,927,697 [Patent Document 30] U.S. Patent No. 8,993,731 [Patent Document 31] U.S. Patent No. 9,102,727 [Patent Document 32] U.S. Patent No. 8,907,053 [Patent Document 33] WO2010 / 027827 issue [Patent Document 34] WO2011 / 066342 issue [Patent Document 35] U.S. Patent Application Publication No. 2016 / 0108123 [Patent Document 36] U.S. Patent No. 8,217,149 [Patent Document 37] WO2013 / 079174 [Patent Document 38] U.S. Patent No. 8,779,108 [Patent Document 39] U.S. Patent No. 7,943,743 [Patent Document 40] WO2015 / 081158 issue [Patent Document 41] WO2015 / 181342 [Patent Document 42] WO2014 / 100079 [Patent Document 43] WO2016 / 000619 issue [Patent Document 44] WO2014 / 022758 issue [Patent Document 45] WO2014 / 055897 issue [Patent Document 46] WO2015 / 061668 issue [Patent Document 47] WO2015 / 112805 [Patent Document 48] WO2015 / 109124 issue [Patent Document 49] WO2015 / 195163 [Patent Document 50] U.S. Patent No. 8,168,179 [Patent Document 51] U.S. Patent No. 8,552,154 [Patent Document 52] U.S. Patent No. 8,460,927 [Patent Document 53] U.S. Patent No. 9,175,082 [Patent Document 54] U.S. Patent Application Publication No. 2015 / 0259420 [Patent Document 55] WO2015 / 116539 [Patent Document 56] U.S. Patent No. 9,505,839 [Patent Document 57] WO2008 / 132601 [Patent Document 58] U.S. Patent No. 9,244,059 [Patent Document 59] WO2010 / 019570 issue [Patent Document 60] WO2014 / 140180 issue [Patent Document 61] WO2016 / 028672 issue [Patent Document 62] WO2009 / 044273 [Patent Document 63] U.S. Patent Application Publication No. 2015 / 0218274 [Patent Document 64] WO2016 / 161270 issue [Patent Document 65] WO2016 / 111947 issue [Patent Document 66] WO2016 / 071448 issue [Patent Document 67] WO2016 / 144803 [Patent Document 68] U.S. Patent No. 8,552,156 [Patent Document 69] U.S. Patent No. 8,841,418 [Patent Document 70] U.S. Patent No. 9,163,087 [Patent Document 71] U.S. Patent No. 5,554,512 [Patent Document 72] U.S. Patent No. 6,291,661 [Patent Document 73] U.S. Patent No. 7,294,331 [Patent Document 74] U.S. Patent No. 7,361,330 [Patent Document 75] U.S. Patent No. 9,486,519 [Patent Document 76] WO03 / 064383

Patent document 77

Patent document 78

Non-licensed literature

[0006] [Non-licensed document 1] Sadelain, Cancer Discovery 3: 388-398 (2013) [Non-licensed document 2] Cell Mol Immunol. April 2007; 4(2): 113-20 pp. [Non-licensed document 3] VIROLOGICA SINICA, April 2011, 26(2): 81-94

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0007] Given the continuing need for improved strategies for cancer treatment, novel compositions and methods for improving cell-based immunotherapy, particularly CART therapy, are highly desirable. [Means for solving the problem]

[0008] The present disclosure provides compositions for improving immune cell therapy. The compositions are useful for generating, inducing, strengthening, and / or enhancing an immune response, which may be an innate and / or adaptive immune response mediated by immune cell therapy. In some embodiments, the immune cell is an immune effector cell (e.g., a T cell or an NK cell) that expresses a chimeric antigen receptor (CAR) molecule, e.g., a CAR molecule that binds to a tumor antigen, e.g., an antigen expressed on the surface of a solid tumor or a hematological tumor.

[0009] In some aspects, the present disclosure relates to an immunogenic composition comprising (a) polyinosinic-polycytidylic acid (PIC), (b) a stabilizer that is an aminoglycoside antibiotic (preferably kanamycin) or a non-aminoglycoside amine, and (c) at least one cation, such as calcium ion. In some embodiments, the composition further comprises at least one immunogen or antigen, where the immunogen or antigen is a recombinant protein, virus-like particle (VLP), peptide, mRNA, or vaccine. The immunogenic composition may be for use in inducing and / or enhancing activation of an immune response in an individual, such as the immune response of immune cell therapy (preferably CAR-T cell therapy) for cancer treatment. The immunogenic composition may be used in combination with genetically engineered immune cells (e.g., CAR-T cells or genetically engineered TCR-T cells) in cancer treatment. In some examples, the immunogenic composition comprises polyinosinic-polycytidylic acid, kanamycin, calcium, and an Epstein-Barr virus (EBV) VLP vaccine. In some cases, the immunogenic composition is administered in combination (concurrently or sequentially) with engineered immune cells, preferably CAR-T cells, more preferably CAR-T cells that target EBV-associated cancer.

[0010] In another aspect, the disclosure relates to immunogenic compositions comprising an Epstein-Barr virus (EBV) VLP. In some examples, the VLP comprises glycoprotein 350 / 220 (gp350) protein or a fragment thereof, and optionally, the VLP comprises one, two, three, or more polypeptide sequences that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 102-117. In one preferred embodiment, the VLP comprises one, two, three, four or more EBV proteins selected from the group consisting of gp350, BKRF4, BVRF1, BDLF3, BZLF2, BXLF2, BNRF1, BALF4 and BZLF1; or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to a polypeptide sequence described herein. In another preferred embodiment, the VLP comprises EBV proteins including gp350, BKRF4, BVRF1, BDLF3, BZLF2, BXLF2, BNRF1, BALF4, and BZLF1; or functional variants thereof that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to the polypeptide sequences described herein. In some preferred embodiments, the VLP does not comprise LMP1, EBNA2, EBNA3a, EBNA3b, or EBNA3c. The immunogenic composition may be for use in inducing and / or enhancing activation of an immune response in an individual, such as the immune response of immune cell therapy (preferably CAR-T cell therapy) for cancer treatment. The immunogenic composition may be used in combination with genetically engineered immune cells (e.g., CAR-T cells or genetically engineered TCR-T cells) in cancer treatment.In some specific examples, the immunogenic composition is to be administered in combination (concurrently or sequentially) with genetically engineered immune cells, preferably CAR-T cells, more preferably CAR-T cells that target EBV-associated cancer, even more preferably CAR-T cells that bind to glycoprotein 350 / 220 (gp350) protein.

[0011] In another aspect, the disclosure provides a CAR, or an immune cell (preferably a CAR-T cell) comprising a CAR, wherein the CAR comprises an EBV glycoprotein 350 / 220 antigen-binding domain, wherein the antigen-binding domain comprises a heavy chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 138, 139, or 140, and / or a light chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 141, 142, or 143. In some examples, the antigen-binding domain that binds to EBV gp350 / 220 comprises: (1) SEQ ID NOs: 11, 12, 13, 26, 27, and 28, respectively; (2) SEQ ID NOs: 14, 15, 16, 29, 30, and 31, respectively; (3) SEQ ID NOs: 17, 18, 19, 32, 33, and 34, respectively; (4) SEQ ID NOs: 20, 21, 22, 35, 36, and 37, respectively; or (5) SEQ ID NOs: 23, 24, 25, 38, 39 and 40, respectively a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequence of It comprises an EBV glycoprotein 350 / 220 antigen-binding domain that competes with or binds to the same epitope in EBV glycoprotein 350 / 220 as bound by any one of antigen-binding domains (1) to (5) of the CAR as described herein.

[0012] In some instances, the EBV glycoprotein 350 / 220 antigen binding domain comprises: (1) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 1 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 6 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (2) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 2 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (3) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 3 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (4) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 4 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 9 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (5) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 5 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 10 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (6) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (7) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (8) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (9) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (10) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (12) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (13) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (14) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; or (15) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence. Contains one of the following:

[0013] In some aspects, the present disclosure provides: [1] (a) Polyinosinic-polycytidylic acid (PIC); (b) a stabilizer that is an aminoglycoside antibiotic or a non-aminoglycoside amine; (c) at least one cation; and (d) optionally, an immunogen that is a recombinant protein, virus-like particle (VLP), peptide, mRNA, or vaccine. An immunogenic composition comprising:

[0014] [1A] An immunogenic composition comprising a VLP of Epstein-Barr virus (EBV), preferably wherein the VLP comprises glycoprotein 350 / 220 (gp350) protein or a fragment thereof, and optionally wherein the VLP comprises one, two, three or more polypeptide sequences that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 102-117.

[0015] [1B] The composition of [1A], wherein the VLP comprises one, two, three, four or more EBV proteins selected from the group consisting of gp350, BKRF4, BVRF1, BDLF3, BZLF2, BXLF2, BNRF1, BALF4 and BZLF1; or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to a polypeptide sequence described herein.

[0016] [1C] The composition of [1A], wherein the VLP comprises EBV proteins gp350, BKRF4, BVRF1, BDLF3, BZLF2, BXLF2, BNRF1, BALF4 and BZLF1; or functional variants thereof that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0017] [1D] Any one of the compositions of [1A] to [1C], wherein the VLP does not contain LMP1, EBNA2, EBNA3a, EBNA3b, and EBNA3c.

[0018] [1E] A VLP comprising (i) at least one EBV structural polypeptide, (ii) at least one EBV lytic polypeptide, and (iii) membrane lipids, preferably said VLP has the following properties: (a) lacking EBV DNA; (b) one or more B-cell transforming abilities of EBV required for B-cell transformation are disabled or eliminated, while their immunogenicity is maintained; and / or (c) the EBV does not contain one or more EBV polypeptides required for B cell transformation selected from the group consisting of LMP1, EBNA2, EBNA3a, EBNA3b, and EBNA3c. The composition of [1A], having one or more of:

[0019] [2] A composition according to any one of [1] to [1E] for use in treating cancer.

[0020] [2A] A composition according to any one of [1] to [1E] for use in inducing an immune response, preferably wherein the immune response is an immune response of CAR-T cells against tumor cells.

[0021] [3] Use of a composition according to any one of [1] to [2A] in the manufacture of a medicament for the treatment of cancer.

[0022] [4] The composition or use according to any one of [2] to [3], wherein the cancer treatment is immune cell therapy (preferably CAR-T cell therapy), and optionally, the composition enhances the anti-cancer effect of the cell therapy.

[0023] [5] The composition or use according to any one of [2] to [4] for use in combination with engineered immune cells (preferably CAR-T cells or engineered TCR-T cells) for the treatment of cancer, optionally wherein the immunogenic composition is administered to the subject before, after or simultaneously with the immune cells.

[0024] [6] The composition or use according to any one of [1] to [5], wherein the immunogen is a tumor-associated antigen, an allergen, an antigen associated with a virus, or a fragment thereof.

[0025] [6A] The composition or use according to any one of [1] to [5], wherein the immunogen is a recombinant protein, a virus-like particle (VLP), a peptide, an mRNA, a DNA, a vaccine or a dendritic cell vaccine.

[0026] [6B] The composition or use according to any one of [1] to [5], wherein the immunogen is a virus-like particle (VLP).

[0027] [6C] The composition or use according to [6B], wherein the immunogen is a VLP of Epstein-Barr virus (EBV), preferably said VLP comprising glycoprotein 350 / 220 (gp350) protein or a fragment thereof.

[0028] [6D] The composition or use according to [6B], wherein the VLP further comprises one, two, three or more protein sequences selected from the group consisting of SEQ ID NOs: 102 to 117.

[0029] [7] The composition or use according to any one of [1] to [6D], wherein the stabilizer is an aminoglycoside antibiotic selected from kanamycin, streptomycin, dihydrostreptomycin, mannoside streptomycin, amikacin, amikacin, dibekacin, vietomycin, gentamicin, and any combination thereof; and preferably, the stabilizer is kanamycin.

[0030] [7A] The composition or use according to any one of [1] to [7], wherein the composition contains 0.5 mg / ml to 10 mg / ml of PIC.

[0031] [7B] The composition or use according to any one of [1] to [7A], wherein the PIC has a molecular weight range of about 66,000 to 2,000,000 daltons.

[0032] [7C] The composition or use according to any one of [1] to [7A], wherein the PIC has a molecular weight range of about 300,000 to 1,200,000 daltons or a Svedberg size of about 6.4 to 24.0.

[0033] [7D] The composition or use according to any one of [1] to [7A], wherein the PIC has a molecular weight range of about 66,000 to 660,000 daltons or a molecular size range of about 6.4 to 18.3 Svedberg.

[0034] [7E] The composition or use according to any one of [1] to [7A], wherein the PIC has a molecular weight range of about 300,000 to 660,000 Daltons or a molecular size range of about 12.8 to 18.3 Svedberg.

[0035] [7F] The composition or use according to any one of [1] to [7A], wherein said PIC has an average molecular weight of 150,000 Daltons or more or an average molecular size of 9.3 Svedberg or more.

[0036] [7G] The composition or use according to any one of [1] to [7A], wherein the PIC has an average molecular weight of 250,000 Daltons or more or an average molecular size of 11.8 Svedberg or more.

[0037] [7H] The composition or use according to any one of [1] to [7A], wherein the PIC has an average molecular weight of 350,000 Daltons or more or an average molecular size of 15.3 Svedberg or more.

[0038] [8] The composition or use according to any one of [1] to [6B], wherein the stabilizing agent is a non-aminoglycoside amine selected from the group consisting of polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, galactose, polylysine, protamine, shell oligosaccharide, chitosan, spermine, glucosamine, and any combination thereof; preferably, the stabilizing agent is polylysine, chitin, chitosan, or glucosamine; more preferably, the stabilizing agent is ε-polylysine, hexylglucosamine, or acetylglucosamine.

[0039] [9] The composition or use according to any one of [1] to [8], wherein the cation is selected from the group consisting of calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, zinc, and any combination thereof; and preferably, the cation is calcium.

[0040]

[10] The composition or use according to any one of [1] to [9], wherein the immune cell is a T cell expressing a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding domain that specifically binds to an antigen expressed on the surface of a cancer, and preferably the antigen-binding domain is an scFv domain.

[0041]

[11] The composition or use according to [1], wherein the intracellular domain comprises at least one costimulatory domain.

[0042]

[12] The costimulatory domains of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death 1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen 1 (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, I L-7R Alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX , CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile) , CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0043]

[13] The composition or use according to

[12] , wherein the costimulatory domain comprises the signal transduction region of 4-1BB / CD137.

[0044]

[14] The composition or use according to

[12] , wherein the costimulatory domain comprises the signal transduction region of CD28.

[0045]

[15] The composition or use according to any one of

[11] to

[14] , wherein the CAR comprises two or more costimulatory domains.

[0046]

[16] The composition or use according to

[15] , wherein the CAR comprises two costimulatory domains, one of the two costimulatory domains being CD28, and the other costimulatory domain being selected from 4-1BB / CD137 or OX40.

[0047]

[17] The composition or use according to any one of

[10] to

[16] , wherein the intracellular domain of the CAR comprises at least one activation domain.

[0048]

[18] The composition or use according to

[17] , wherein the activation domain comprises CD3, preferably said CD3 is CD3 zeta.

[0049]

[19] The composition or use according to any one of [4] to [9], wherein the immune cells are T cells, natural killer (NK) cells, TCR-expressing cells, dendritic cells, gamma delta T cells, or NK-T cells.

[0050]

[20] The composition or use according to any one of [1] to

[19] , wherein the cells are autologous T cells.

[0051]

[21] The composition or use according to any one of [4] to

[19] , wherein the cells are allogeneic T cells.

[0052]

[22] The composition or use according to any one of [4] to

[19] , wherein the cells are genetically engineered to express at least one cytokine.

[0053]

[23] The composition or use according to

[22] , wherein the at least one cytokine is at least one selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-15 / IL-15sushi, IL-15 / IL-15sushi anchor, IL-18, IL-21, GM-CSF, and TGF-β.

[0054]

[24] The composition or use according to any one of [4] to

[19] , wherein the cells are genetically engineered to further express at least one chemokine.

[0055]

[25] The composition or use according to

[24] , wherein the at least one chemokine is selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL19, CXCL1, CXCL2, CXCL9, CXCL10, CCL21, and CXCL12.

[0056] [25A] The composition, use, or method according to any one of [5] to

[25] , wherein the cells comprise a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, the antigen-binding domain comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) as provided in Table 1 (Table 2) and Table 2 (Table 3), or a specific VH and VL combination as provided in Table 1 (Table 2) and Table 2 (Table 3).

[0057] [25B] The composition, use, or method according to any one of [5] to

[25] , wherein the cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, the antigen-binding domain comprising one, two, three or more HCDRs and / or one, two, three or more LCDRs as provided in Table 3 and Table 4, or a specific HCDR1-3 and LCDR1-3 combination as provided in Table 3 and Table 4.

[0058] [25C] The composition, use, or method according to any one of [5] to

[25] , wherein the cells contain a CAR comprising any one of the CAR amino acid sequences as provided in Table 5.

[0059] [25D] Cells (1) SEQ ID NOs: 11, 12, 13, 26, 27, and 28, respectively; (2) SEQ ID NOs: 14, 15, 16, 29, 30, and 31, respectively; (3) SEQ ID NOs: 17, 18, 19, 32, 33, and 34, respectively; (4) SEQ ID NOs: 20, 21, 22, 35, 36, and 37, respectively; (5) SEQ ID NOs: 23, 24, 25, 38, 39, and 40, respectively; (6) SEQ ID NOs: 118, 119, 120, 121, 122, and 123, respectively; or (7) SEQ ID NOs: 124, 125, 126, 127, 128, and 129, respectively. The composition, use, or method according to any one of [5] to

[25] , comprising a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, the antigen-binding domain comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequences:

[0060] [25E] A composition, use, or method according to any one of [5] to

[25] , wherein the cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, and the antigen-binding domain competes with or binds to the same epitope in Epstein-Barr virus (EBV) glycoprotein 350 / 220 as that bound by any one of the antigen-binding domains (1) to (5) of the CAR described in [25D].

[0061] [25F] The composition, use, or method according to any one of [5] to

[25] , wherein the cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, and the antigen-binding domain comprises a heavy chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 138, 139, or 140, and / or a light chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 141, 142, or 143.

[0062] [25G] The cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, wherein the antigen-binding domain is (1) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 1 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 6 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (2) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 2 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (3) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 3 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (4) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 4 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 9 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (5) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 5 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 10 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (6) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (7) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (8) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (9) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (10) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (12) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (13) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (14) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (15) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (16) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 130 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 131 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; or (17) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 132 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 133 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence. A composition, use, or method according to any one of [5] to

[25] , comprising any one of:

[0063] In some aspects, the present disclosure provides:

[26] A method for enhancing an anti-cancer response of immune cell therapy in an individual, comprising administering to the individual an effective amount of any one of the immunogenic compositions [1] to [25G] above. Regarding.

[0064] In some aspects, the present disclosure provides:

[27] (a) administering to an individual an effective amount of the immunogenic composition described in any one of [1] to [9] above; and (b) administering to an individual an effective amount of the cells described in any one of

[10] to [25G] above. 1. A method of treating an individual with cancer, comprising: optionally administering the immunogenic composition to the subject before, after, or simultaneously with the administration of immune cells. Regarding.

[0065] In some further aspects, the present disclosure relates to:

[28] An Epstein-Barr virus (EBV) VLP for use in the treatment of cancer, wherein the VLP is to be administered to a subject in combination with engineered immune cells (preferably CAR-T cells or engineered TCR-T cells) in the treatment of cancer, optionally wherein the VLP is administered to the subject before, after or simultaneously with the immune cells.

[0066]

[29] A VLP for use according to

[28] , wherein the VLP comprises glycoprotein 350 / 220 (gp350) protein or a fragment thereof, and preferably the VLP further comprises one, two, three or more protein sequences selected from the group consisting of SEQ ID NOs: 102 to 117.

[0067]

[30] The VLP for use in

[28] or

[29] , wherein the immune cells are T cells expressing a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising an antigen-binding domain that specifically binds to an antigen expressed on the surface of a cancer, and preferably the antigen-binding domain is an scFv domain.

[0068]

[31] A VLP for use in

[30] , wherein the intracellular domain of the CAR comprises at least one costimulatory domain.

[0069]

[32] Co-stimulatory domains are expressed in CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death 1 (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen 1 (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, and TNF receptor. Body proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7 R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEAC A VLP for use in

[30] or

[31] , which is a signaling region of AM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0070]

[33] VLPs for use in

[32] , in which the costimulatory domain contains the signaling region of 4-1BB / CD137.

[0071]

[34] A VLP for use according to

[32] , wherein the costimulatory domain comprises the signaling region of CD28.

[0072]

[35] A VLP for use according to any one of

[30] to

[34] , wherein the CAR comprises two or more costimulatory domains.

[0073]

[36] A VLP for use according to

[35] , wherein the CAR comprises two costimulatory domains, one of the two costimulatory domains being CD28 and the other costimulatory domain being selected from 4-1BB / CD137 or OX40.

[0074]

[37] A VLP for use according to any one of

[30] to

[36] , wherein the intracellular domain of the CAR comprises at least one activation domain.

[0075]

[38] A VLP for use according to

[37] , wherein the activation domain comprises CD3, preferably said CD3 being CD3 zeta.

[0076]

[39] The VLP for use according to any one of

[34] to

[38] , wherein the immune cell is a T cell, a natural killer (NK) cell, a TCR-expressing cell, a dendritic cell, a gamma delta T cell, or an NK-T cell, preferably the immune cell is a CAR-T cell.

[0077]

[40] The VLP for use according to any one of

[28] to

[39] , wherein the cancer is a lymphoproliferative disorder (LPD) such as Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), B-cell lymphoma including diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma, NK / T-cell lymphoma, or post-transplant lymphoproliferative disorder (PTLD), or an EBV-associated cancer selected from epithelial carcinoma (nasopharyngeal, lung, breast), lymphoepithelioma, carcinoma with lymphocytic infiltration (GCLS, e.g., gastric cancer) or glioma.

[0078]

[41] A VLP for use according to any one of

[28] to

[40] , wherein the antigen recognized by the CAR is an Epstein-Barr virus antigen (EBV antigen), preferably the EBV antigen is EBV glycoprotein 350 / 220 (gp350 / 220).

[0079]

[42] A VLP for use according to any one of

[28] to

[41] , wherein the cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, the antigen-binding domain comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) as provided in Table 1 (Table 2) and Table 2 (Table 3), or a specific VH and VL combination as provided in Table 1 (Table 2) and Table 2 (Table 3).

[0080]

[43] A VLP for use according to any one of

[28] to

[42] , wherein the cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, the antigen-binding domain comprising one, two, three or more HCDRs and / or one, two, three or more LCDRs as provided in Table 3 (Table 4) and Table 4 (Table 5), or a specific HCDR1-3 and LCDR1-3 combination as provided in Table 3 (Table 4) and Table 4 (Table 5).

[0081]

[44] A VLP for use according to any one of

[28] to

[43] , wherein the cell contains a CAR comprising any one of the CAR amino acid sequences as provided in Table 5.

[0082]

[45] Cells (1) SEQ ID NOs: 11, 12, 13, 26, 27, and 28, respectively; (2) SEQ ID NOs: 14, 15, 16, 29, 30, and 31, respectively; (3) SEQ ID NOs: 17, 18, 19, 32, 33, and 34, respectively; (4) SEQ ID NOs: 20, 21, 22, 35, 36, and 37, respectively; (5) SEQ ID NOs: 23, 24, 25, 38, 39, and 40, respectively; (6) SEQ ID NOs: 118, 119, 120, 121, 122, and 123, respectively; or (7) SEQ ID NOs: 124, 125, 126, 127, 128, and 129, respectively. A VLP for use according to any one of

[28] to

[43] , comprising a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220 protein, the VLP comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequences:

[0083]

[46] A VLP for use according to any one of

[28] to

[43] , wherein the cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, and the antigen-binding domain competes with or binds to the same epitope in Epstein-Barr virus (EBV) antigen glycoprotein 350 / 220 as that bound by any one of the antigen-binding domains (1) to (5) of the CAR described in

[45] .

[0084]

[47] The VLP for use according to any one of

[28] to

[43] , wherein the cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220, and the antigen-binding domain comprises a heavy chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 138, 139, or 140, and / or a light chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 141, 142, or 143.

[0085]

[48] ​​The cell comprises a CAR comprising an antigen-binding domain that binds to EBV gp350 / 220 protein, wherein the antigen-binding domain is (1) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 1 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 6 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (2) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 2 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (3) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 3 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (4) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 4 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 9 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (5) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 5 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 10 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (6) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (7) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (8) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (9) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (10) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (12) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (13) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (14) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (15) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (16) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 130 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 131 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; or (17) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 132 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 133 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence. A VLP for use according to any one of

[28] to

[43] , comprising any one of:

[0086] In some further aspects, the present disclosure provides a CAR, or an immune cell (preferably a CAR-T cell) comprising a CAR, wherein the CAR comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as provided in Table 1 and Table 2, or an EBV glycoprotein 350 / 220 antigen-binding domain comprising a specific VH and VL combination as provided in Table 1 and Table 2.

[0087] In some further aspects, the disclosure provides a CAR, or a CAR-T cell comprising a CAR, wherein the CAR comprises an EBV glycoprotein 350 / 220 antigen-binding domain comprising one, two, three or more HCDRs and / or one, two, three or more LCDRs as provided in Table 3 and Table 4, or specific HCDR1-3 and LCDR1-3 combinations as provided in Table 3 and Table 4.

[0088] In some further aspects, the present disclosure provides immune cells (preferably CAR-T cells) comprising a CAR, or a CAR comprising any one of the CAR amino acid sequences as provided in Table 5.

[0089] In some further aspects, the present disclosure provides a CAR, or an immune cell (preferably a CAR-T cell) comprising a CAR, wherein the CAR comprises: (1) SEQ ID NOs: 11, 12, 13, 26, 27, and 28, respectively; (2) SEQ ID NOs: 14, 15, 16, 29, 30, and 31, respectively; (3) SEQ ID NOs: 17, 18, 19, 32, 33, and 34, respectively; (4) SEQ ID NOs: 20, 21, 22, 35, 36, and 37, respectively; (5) SEQ ID NOs: 23, 24, 25, 38, 39, and 40, respectively; (6) SEQ ID NOs: 118, 119, 120, 121, 122, and 123, respectively; or (7) SEQ ID NOs: 124, 125, 126, 127, 128, and 129, respectively. an EBV glycoprotein 350 / 220 binding domain comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequence of: or It comprises an EBV glycoprotein 350 / 220 antigen-binding domain that competes with or binds to the same epitope in EBV glycoprotein 350 / 220 as bound by any one of the antigen-binding domains (1) to (7) of the CAR as described herein.

[0090] In some further aspects, the present disclosure provides a CAR, or an immune cell (preferably a CAR-T cell) comprising a CAR, wherein the CAR comprises an EBV glycoprotein 350 / 220 antigen-binding domain, wherein the antigen-binding domain comprises a heavy chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 138, 139, or 140, and / or a light chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 141, 142, or 143.

[0091] In some further aspects, the present disclosure provides a CAR, or an immune cell (preferably a CAR-T cell) comprising a CAR, wherein the CAR comprises an EBV glycoprotein 350 / 220 antigen-binding domain, wherein the antigen-binding domain is (1) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 1 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 6 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (2) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 2 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (3) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 3 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (4) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 4 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 9 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (5) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 5 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 10 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (6) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (7) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (8) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (9) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (10) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (12) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (13) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (14) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (15) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (16) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 130 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 131 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; or (17) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 132 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 133 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence. Any one of; or It comprises an EBV glycoprotein 350 / 220 antigen-binding domain that competes with or binds to the same epitope in EBV glycoprotein 350 / 220 as bound by any one of the antigen-binding domains (1) to (17) of the CAR as described herein.

[0092] In some further aspects, the present disclosure provides an immune cell or CAR-T cell as described herein for use in treating cancer, preferably wherein the cancer is a B-cell lymphoma, including Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma, NK / T-cell lymphoma, or lymphoproliferative disorder (LPD), such as post-transplant lymphoproliferative disorder (PTLD), or an EBV-associated cancer selected from epithelial carcinoma (nasopharyngeal, lung, breast), lymphoepithelioma, carcinoma with lymphocytic infiltration (GCLS, e.g., gastric cancer), or glioma.

[0093] In some embodiments, with respect to a composition, use, or method as described in the present disclosure, the cancer is selected from the group consisting of B-cell lymphoma, T-cell lymphoma, multiple myeloma, chronic myelogenous leukemia (CML), acute myeloma leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloproliferative neoplasm (MPN), B-cell acute lymphoblastic leukemia (B-ALL), a solid tumor, carcinoma, or sarcoma; and preferably, the cancer is a solid tumor. In another embodiment, the cancer is a virus-specific cancer.

[0094] In some embodiments, the cancer is a lymphoproliferative disorder (LPD) such as Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma, NK / T-cell lymphoma, or post-transplant lymphoproliferative disorder (PTLD), or an EBV-associated cancer selected from epithelial carcinoma (nasopharyngeal, lung, breast), lymphoepithelioma, carcinoma with lymphocytic infiltration (GCLS, e.g., gastric cancer), or glioma.

[0095] In some embodiments, with respect to the composition, use, or method as described in any one of

[10] to

[28] above, the antigen recognized by the CAR is selected from the group consisting of CD38, GD2, CD123, CLL-1, CD19, CD33, BCMA, CS1, CD4, CD5, CD7, CD20, DLL3, GPC3, GPC2, EpCAM, NY-ESO-1, alpha-fetoprotein (AFP), Flt3 receptor, transmembrane activating factor, and CAR. The present invention is directed to a method for treating rheumatoid arthritis, comprising administering to the patient a therapeutically effective amount of a therapeutically effective amount of a rheumatoid arthritis inhibitor, selected from the group consisting of tetracycline-regulated protein and cyclophilin ligand interactor (TACI), CEA, ERBB2, EGFR, GD2, MSCA, mesothelin, MUC1, PSMA, HER2, claudin 6, Trop2, MUC3A, claudin 18.2, gp100, MAGE-A1 / 3 / 4, LMP1, nectin-4 / FAP, CD171, MUC16, CD20, CD80 / 86, c-MET, DR5, EpHA2, and FR-α.

[0096] In some embodiments, the antigen recognized by the CAR is a viral antigen.

[0097] In some embodiments, the antigen recognized by the CAR is an Epstein-Barr virus antigen (EBV antigen).

[0098] In some embodiments, the EBV antigen may be present on the surface of an EBV-infected cell, preferably an EBV-infected cancer cell, an EBV-infected B cell, or an EBV-infected epithelial cell. The EBV antigen may be an EBV virion envelope protein or a protein of the EBV envelope complex (such as gB, gL, or gH). The EBV viral antigen is preferably EBV glycoprotein 350 / 220 (gp350 / 220). In preferred embodiments, the present disclosure focuses on targeting EBV antigens and treating EBV-associated medical conditions. An exemplary EBV gp350 protein is shown in the UniProt database, entry P03200-1, version 1, dated July 21, 1986. An exemplary EBV gp220 is shown in the same database entry, but is missing positions 502-750.

[0099] In some important embodiments, the immune cells or CAR-T cells comprise a CAR that comprises an EBV glycoprotein 350 / 220 antigen-binding domain that comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as provided in Table 1 and Table 2, or a specific VH and VL combination as provided in Table 1 and Table 2. In another embodiment, the CAR comprises an EBV glycoprotein 350 / 220 antigen-binding domain that comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) that has at least 60%, 70%, 80%, 85%, 90%, 95%, 99% identity to the VH and / or VL amino acid sequences as provided in Table 1 and Table 2.

[0100] In some important embodiments, the CAR comprises an EBV glycoprotein 350 / 220 antigen-binding domain comprising one, two, three or more HCDRs and / or one, two, three or more LCDRs as provided in Table 3 and Table 4, or specific HCDR1-3 and LCDR1-3 combinations as provided in Table 3 and Table 4.

[0101] In some important embodiments, the cells comprise an EBV glycoprotein 350 / 220-binding CAR comprising an amino acid sequence as provided in Table 5, preferably, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144 to 161. In other embodiments, the CAR comprises an amino acid sequence at least 60%, 70%, 80%, 85%, 90%, 95%, 99% identical to any one of the CAR sequences as provided in Table 5, preferably, the CAR is selected from the group consisting of SEQ ID NOs: 144 to 161.

[0102] In some embodiments, with respect to a composition, use, or method as described in any one of [2] to

[29] above, the composition and the cells are (to be) administered simultaneously or sequentially, for example, the composition is (to be) administered prior to or subsequent to administration of the cells.

[0103] In some aspects, the present application has surprisingly and unexpectedly discovered that combination therapy of an immunogenic composition comprising a PIC and optionally an immunogen with an immune cell therapy, such as CAR-T cells, is particularly effective in treating solid tumors. It is contemplated that the immunogenic compositions described herein can improve, enhance, or augment the immune response and anti-cancer activity of the administered immune cells (e.g., CAR-T cells) in a subject with a solid tumor. Without wishing to be bound by theory, it is contemplated that PICs can promote T cell activation and proliferation (see Example 5). It is also contemplated that PICs can stimulate toll-like receptor 3 (TLR-3) and other cellular pathways to enhance antigen presentation by antigen-presenting cells (APCs) and induce the production of pro-inflammatory cytokines. Previous studies have demonstrated that PICs can enhance immune responses to non-cancer applications, such as rabies virus, hepatitis virus, and SAR-CoV (Lau et al., 2009; Lau et al., 2010 & Cell Mol Immunol. 2007 April; 4(2):113-20; VIROLOGICA SINICA, 2011 April; 26(2):81-94). Second, PICs can induce the secretion of cytokines, such as IFNγ and IL-2, which can increase the activation and proliferation and tumor infiltration of both B cells and NK cells, as well as the stimulation and expansion of CAR-T cells. In addition, the presence of an immunogen acting as a vaccine may further enhance the presentation of target antigens on antigen-presenting cells, which can be used to enhance the CAR-T therapy. Furthermore, the immunogenic compositions described herein may contribute to overcoming the immunosuppressive tumor microenvironment, a major challenge to the limited success of CAR-T therapy for solid tumors.

[0104] In another aspect, the present application successfully designed a specific CAR and generated CAR-T cells containing the CAR targeting EBV, which are particularly effective in treating cancers associated with EBV infection. Furthermore, the inventors unexpectedly discovered that a combination therapy of an immunogenic composition containing EBV VLPs with immune cell therapy using the CAR-T cells targeting EBV is particularly effective in treating cancers associated with EBV infection. The immunogenic composition containing EBV VLPs as described herein can improve, enhance, or increase the immune response and anti-cancer activity of administered immune cells (e.g., CAR-T cells) in subjects with such cancers. Without wishing to be bound by theory, it is hypothesized that EBV VLPs can promote the activation, proliferation, and killing activity of T cells (see Example 7). Additionally, the presence of the EBV-VLPs acting as a vaccine may further enhance the presentation of EBV antigens on antigen-presenting cells, which can be used to enhance the CAR-T therapy. Thus, the immunogenic compositions described herein may contribute to overcoming the immunosuppressive tumor microenvironment, a major challenge to the limited success of CAR-T therapy for solid tumors. [Brief explanation of the drawings]

[0105] [Figure 1] FIG. 1 shows that representative anti-EBV Gp350 CAR-T cells showed potent killing of PCI-gp350 cells but no detectable activity in PCI cells that lack gp350 expression. [Figure 2] FIG. 1 shows that co-incubation of a representative polyinosinic-polycytidylic acid-based adjuvant composition (designated ZNP) prepared in Example 1 with T cells and PBMCs promotes T cell activation and proliferation in a ZNP concentration-dependent manner, suggesting that the ZNP composition (or ZNP-EBV-VLP) may be useful in enhancing the efficacy of T cell therapies, including CAR-T cells. [Figure 3]Figure 1 shows that co-incubation of EBV VLPs with Raji cells significantly enhanced the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against the target cell line PCI-g (PCI-g) at different VLP concentrations (PCI-g + 1.25x107 VLPs, PCI-g + 1.25x108 VLPs compared to PCI-g + 0 VLPs). In contrast, EBV VLPs did not increase the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against control PCI cells. [Figure 4] 1 shows that the enhanced cytotoxicity of anti-EBV Gp350 CAR-T cells against the target cell PCI-gp350 cell line in the presence of EBV VLPs is dependent on co-incubation with VLP-targeted Raji cells. The cytotoxicity of the sample containing 1.25×10 VLPs co-incubated with Raji cells (Raji + 1.25×10 VLPs) is significantly higher than that of the sample containing 1.25×10 VLPs without co-incubation with Raji cells (Raji + 0 VLPs). [Figure 5] This figure shows that anti-EBV Gp350 CAR-T cells exhibit little or low cytotoxicity against PCI cell lines that do not express the gp350 antigen in the absence or presence of EBV VLPs. [Figure 6] Figure 1 shows that co-incubation of EBV VLPs with Raji cells significantly enhanced the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against the target cell line PCI-gp350 (PCI-g) at various VLP concentrations (CAR+VLPe7 or CAR+VLPe8 compared to CAR alone). In contrast, EBV VLPs do not increase the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against control PCI cells. EBV VLPs alone do not increase the cytotoxicity (% cytolysis) of mock T cells or PCI cells without gp350 expression. [Figure 7]Figure 7 shows that Gp350 CAR-T cells inhibited the growth of nasopharyngeal carcinoma C666-1 cells and suppressed tumor formation in vivo. C666-1 cells (genetically engineered with gp350 and luciferase expression) were subcutaneously injected into mice, followed by administration of 4×10 (4E6) or 2×10 (4E6) gp350 CAR-T or mock T cells. Figure 7A shows that tumor cell growth luminescence imaging results were recorded and compared on days 0, 7, 14, 18 (4×10 group), or 21 (2×10 group and PBS group), demonstrating specific tumor formation suppression by gp350 CAR-T cells. Figure 7B shows the mean tumor cell luminescence intensity changes on days 0, 7, 14, 18, and 21 after CAR-T injection for the 4×10 (4E6) CAR-T cell or mock T cell treatment groups. Figure 7C shows the mean tumor cell luminescence intensity change at days 0, 7, 14, 18, or 21 after CAR-T injection for 2×10 (2E6) CAR-T cells, mock T cells, or PBS treatment groups. Figure 7D shows the mean tumor volume change pattern after CAR-T injection at days 0, 7, 14, 18, or 21. Figure 7E shows dissection and tumor tissue mass assessment in mice across various experimental groups, demonstrating reduced tumor growth in the CAR-T treatment groups. [Figure 8] Figure 8A shows cell percentage and count comparison of human CD45+, CD8+, CAR+, and CAR+ / CD8+ cells in blood or spleen samples from the 4x106 (4E6) CAR-T and Mock T treatment group at day 18 (Figure 8A) and the 2x106 (2E6) CAR-T cell and Mock T cell treatment group at day 24 (Figure 8B). The results show that CAR-T cells were found to be present in the blood and spleen while suppressing tumor formation. [Figure 9]Figure 9 shows that gp350-targeted CAR-T cells inhibit T-cell lymphoma cell growth and suppress tumor formation in vivo. Figure 9A shows the tumor profile of a T-cell lymphoma mouse model injected with Jurkat-gp350-luc cells for 5 or 7 days to allow tumor formation. Mice were examined by IVIS imaging and administered 2x10 (2E6) or 1x10 (1E6) CAR-T cells or mock T cells, respectively. Tumor cell growth luminescence imaging was recorded and compared on days 0, 7, 14, 21, 28, and 32, demonstrating specific tumor formation suppression by gp350 CAR-T cells. Figures 9B and 9C show the change in mean tumor cell luminescence intensity over time in the 2x10 (2E6) or 1x10 (1E6) CAR-T or mock T groups, respectively, after CAR-T injection. [Figure 10]

[0033] Figure 10 shows tumor shrinkage induced by CAR T cells containing anti-EBV Gp350 in a patient diagnosed with B-cell acute lymphoblastic leukemia with central nervous system involvement. Figure 10(A) shows MRI imaging results showing lesions in the brain before (upper panel) and after (lower panel) treatment, which nearly disappeared after treatment; Figure 10(B) shows that the percentage of abnormal blasts in the blood decreased after treatment. After treatment, the subject was discharged without CRS, ICANS, or significant abnormalities in cardiac, liver, and kidney function tests. DETAILED DESCRIPTION OF THE INVENTION

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0107] As used herein, the term "comprising" or "including" should be interpreted as specifying the presence of the specified feature, integer, step, or component as referenced, but does not exclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. However, in the context of the present disclosure, the term "comprising" or "including" also includes "consisting of." Variations of the word "comprise," such as "comprise" and "comprises," and variations of the word "including," such as "include" and "includes," have correspondingly varied meanings.

[0108] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0109] The term "about," when referring to a measurable value such as an amount, duration over time, etc., is intended to encompass variations of ±20%, or in some cases 10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as such variations are appropriate for performing the disclosed methods.

[0110] "Immunogenic composition," as used herein, means a substance that elicits an immune response when administered to a host.

[0111] The terms "poly I:C" or "PIC" refer to a composition containing polyriboinosinic and polyribocytidyl nucleic acids, which may also be referred to as polyinosinic-polycytidylic acid or polyinosinic-polycytidyl, respectively.

[0112] The terms "polypeptide," "peptide," "oligopeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0113] The term "immunogen" refers to an antigen that, upon exposure to a host organism, can elicit an immune response, such as a B cell (humoral / antibody) and / or T cell (cellular) adaptive immune response. The term "antigen" includes, but is not limited to, cells; cell extracts; proteins; lipoproteins; glycoproteins; nucleoproteins; polypeptides; peptides; polysaccharides; polysaccharide conjugates; polysaccharide peptidomimetics; lipids; glycolipids; carbohydrates; viruses; viral extracts; bacteria; bacterial extracts; fungi; fungal extracts; multicellular organisms such as parasites; and allergens. Antigens can be exogenous (e.g., from a source different from the individual to whom the antigen is administered, e.g., from a different species) or endogenous (e.g., derived within the host, e.g., diseased body components, cancer antigens, virus-infected cells that produce the antigen, etc.). Antigens can be native (e.g., naturally occurring); synthetic; or recombinant. Antigens include crude extracts; whole cells; and purified antigens, where "purified" indicates that the antigen is in a form that is enriched relative to the environment in which it normally resides and / or relative to a crude extract, e.g., a cultured form of the antigen.

[0114] In some embodiments, the immunogen is a recombinant protein, virus-like particle (VLP), peptide, mRNA, DNA, vaccine, or dendritic cell vaccine. In other embodiments, the immunogen can be one or more polypeptides / peptides derived from cancer cells or antigenic fragments or variants thereof. It will be understood that the immunogens described herein can further comprise additional components. For example, one or more immunogens can be contained in a lipid or liposome. In some embodiments, peptides or polypeptides corresponding to cancer antigens can generally be 10-20 amino acid residues in length and can include two or more peptide determinants, or up to about 30-50 residues, etc. In some embodiments, polypeptides are 10 to about 150 residues in length or longer. In some embodiments, longer peptides or polypeptides can also be prepared, e.g., by recombinant means. In certain embodiments, nucleic acids encoding the antigenic compositions and / or components described herein can be used, e.g., to generate antigenic compositions in vitro or in vivo for the various compositions and methods of the present disclosure. For example, in certain embodiments, a nucleic acid encoding an antigen is contained in a vector, e.g., in a recombinant cell. The nucleic acid can be expressed to produce a peptide or polypeptide comprising the antigenic sequence. The peptide or polypeptide can be secreted from the cell or can be contained as part of or within the cell.

[0115] In some embodiments, the immunogen is a "tumor-associated antigen" or "cancer antigen" and is selected from the group consisting of CTA, NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7, CT10, GAGE, PRAME; BAGE; RAGE, SAGE, HAGE, MPHOSPH1, DEPDC1, IMP3, and MAGE-A, and T antigens BK, p53, Ras, c-Myc, A-Raf, B-Raf, C-Raf, cyclin-dependent kinase, MAGE-A2, MAGE-A6, MAGE-A10, MAGE-A12, MART -1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK, PRAME, P15, RU1, RU2, SART-1, SART-3 receptor, Wilms tumor antigen (WT1), AFP, β-catenin / ш, caspase-8 / m, CE A, CDK-4 / m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCRABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RARa, tumor-associated calcium signaling factor 1 (TACSTD1), TACSTD2 receptor tyrosine kinase, epidermal growth factor receptor (EGFR) , EGFRvIII, platelet growth factor receptor (PDGFR), vascular endothelial growth factor receptor ia (VEGFR), cytoplasmic tyrosine kinase, src family, syk-ZAP70, integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STAT5 and STAT6, hypoxia-induced factors, HIF-1a and HIF-2a, nuclear factor kappa in (NF-kB), Notch, Notch1-4, c-Met receptor, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), PMSA,PR-3, MDM2, mesothelin, carcinoma kidney cells-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation control point, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, zclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GloboH, NYBR-1, RG The antigens may be selected from SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, and fos-related antigen 1.

[0116] In another embodiment, the immunogen is a viral antigen derived from an oncogenic virus. Exemplary oncogenic viruses include, but are not limited to, EBV, HPV, HBV, HCV, HTLV, and KSHV. Exemplary viral antigens derived from oncogenic viruses that can be used in the present disclosure include, but are not limited to, EBV: EBNA-1, LMP-1, LMP-2A; HPV: E6, E7, E5; HBV: HBx; HCV: Core, NS3, Ns5A; HTLV: Tax, HBZ; KSHV: vFLIP, LANA, vGPCR, vIRF-1.

[0117] In some embodiments, the immunogen is an Epstein-Barr virus antigen (EBV antigen). The EBV antigen can be an EBV VLP. In a preferred embodiment, the EBV viral antigen is preferably EBV glycoprotein 350 / 220 (gp350 / 220). An exemplary EBV gp350 protein is shown in the UniProt database, entry P03200-1, version 1, July 21, 1986. An exemplary EBV gp220 is shown in the same database entry, but with positions 502-750 deleted. In some embodiments, the immunogen is an EBV VLP.

[0118] EBV VLPs and immunogenic compositions containing EBV VLPs In some embodiments, the immunogenic compositions of the present disclosure comprise VLPs of Epstein-Barr virus (EBV), i.e., EBV-VLPs.

[0119] In some embodiments of the present disclosure, the EBV-VLP comprises (i) at least one EBV structural polypeptide, (ii) at least one EBV lytic polypeptide, and (iii) membrane lipids, and preferably, the VLP has the following properties: (a) lacking EBV DNA; (b) one or more B-cell transforming abilities of EBV required for B-cell transformation are disabled or eliminated, while their immunogenicity is maintained; and / or (c) the EBV does not contain one or more EBV polypeptides required for B cell transformation selected from the group consisting of LMP1, EBNA2, EBNA3a, EBNA3b, and EBNA3c. The present invention has one or more of the following:

[0120] In some embodiments of the EBV VLPs of the present disclosure, the VLPs comprise at least one EBV polypeptide of gp350 and / or further comprise at least one EBV latency polypeptide.

[0121] The EBV polypeptide gp350 (glycoprotein 350) is a membrane-bound glycoprotein. Its specificity for B cells is due to its binding to CD21 on the cell surface of B cells. Additional auxiliary viral polypeptides may contribute to a fully efficient infection (Chesnokova et al., 2009; Omerovic et al., 2005; Silva et al., 2004; Sorem and Longnecker, 2009). Low-efficiency infection by recombinant EBV particles lacking gp350 has also been demonstrated (Janz et al., 2000). Recent studies also postulate the involvement of gp350 after internalization and possibly during the release of viral capsids from endosomal compartments (Busse et al., 2010). Although not critical for VLPs according to the present invention, it is preferred that gp350 be contained within the membrane of the VLP particle, as the immune response generated upon administration of the vaccine closely resembles the immune response elicited upon infection with wild-type EBV.

[0122] The term "EBV," as used herein, refers to any wild-type, i.e., naturally occurring, EBV strain and is not limited to one particular viral strain. Specifically, EBV type 1 and EBV type 2 strains are well known in the art and have been extensively characterized. These two EBV types differ significantly in the nuclear polypeptide genes encoding EBNA-LP, EBNA-2, EBNA-3A, EBNA-3B, and EBNA-3C. Other than differences related to genes encoding EBNA family polypeptides, the genomes of type 1 and type 2 differ little. Type 1 is predominantly prevalent in developed country populations, while type 2 is also prevalent in equatorial Africa and New Guinea (for a review, see Kieff and Rickinson, 2007). Furthermore, the term EBV polypeptide also includes polypeptides that are not identical in sequence to wild-type EBV strains, but include proteins that share at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, and at least 75% sequence identity (for each value) with wild-type EBV polypeptides. The degree of polypeptide sequence identity can be calculated by methods well known to those skilled in the art and can include automated implementation of algorithms that align sequence data and calculate sequence homology. The EBV polypeptides of the particles can be derived from different EBV strains; preferably, from one strain. As mentioned above, the EBV polypeptides required to be contained in the particles belong to the groups of EBV structural polypeptides and EBV lytic polypeptides. As will be understood by those skilled in the art, a particular EBV polypeptide can belong to more than one of the above groups of polypeptides. In other words, the EBV polypeptide can refer to a structural polypeptide as well as a lytic polypeptide, as will be apparent from the specific EBV polypeptides mentioned in the following paragraphs. In the latter case, the particles do not need to contain additional EBV polypeptides, either structural or lytic polypeptides. Preferably, the particles contain at least one distinct EBV polypeptide for each of the above-mentioned groups of EBV polypeptides, as this typically increases the antigenic potential of the vaccine.Preferably, at least (for each value) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or at least 12 distinct polypeptides are independently part of each of the polypeptides contained in the particles of the vaccine.

[0123] The term "latency polypeptide" relates to an EBV polypeptide that is involved in the induction and maintenance of the EBV latent cycle and / or is expressed as a result of the induction of the latent cycle. Preferably, at least one latency polypeptide is LMP-1 (also called BNLF1) and / or LP-2.

[0124] The term "structural polypeptide" of EBV refers to a polypeptide involved in the structural assembly of EBV. Said polypeptide is preferably selected from the group consisting of membrane polypeptides, tegument polypeptides, and capsid polypeptides. EBV membrane polypeptides include polypeptides selected from the group consisting of BALF4, BLLF1 (also called gp350), BDLF2, BDLF3, BKRF2, BLRF1, BNLF1 (also called LMP-1), TP (also called LMP-2a), BXLF2, BZLF2, and any combination thereof. EBV tegument polypeptides include polypeptides selected from the group consisting of BBRF2, BGLF2, BLLF1, BNRF1, BOLF1, BPLF1, BTRF1, BVRF1, and any combination thereof. EBV capsid polypeptides include polypeptides selected from the group consisting of BBRF1, BcLF1, BDLF1, BFRF3, and any combination thereof. Preferably, the at least one structural polypeptide is selected from the group consisting of BLLF1, BMLF1, BNRF1, or any combination thereof, such as BLLF1 and BMLF1, BLLF1 and BNRF1, or BMLF1 and BNRF1.

[0125] The term "lytic polypeptide" relates to an EBV polypeptide that is involved in the induction and maintenance of the EBV lytic cycle (also referred to herein as the productive phase) and / or is expressed as a result of the induction of the lytic cycle. The lytic polypeptide is preferably selected from the group comprising immediate early genes, early genes, and late lytic genes (Kieff and Rickinson, 2007). The lytic cycle is initiated by the expression of BZLF1 and BRLF1, both immediate early proteins, followed by the expression of early and late proteins. After induction, cells that have become permissive for viral replication undergo cytopathic changes typical of herpesviruses (Kieff and Rickinson, 2007). Exemplary lytic polypeptides of interest for use in accordance with the present invention are selected from the group comprising BZLF1, BRLF1, BMRF1, BMLF1, BALF2, BALF5, BGL2, BHRF1, BALF4, BDLF3, and any combination thereof. Preferably, the at least one lytic polypeptide is BLLF1 (also called gp350) or any combination thereof.

[0126] The term "membrane lipid," as used in accordance with the present invention, refers to a lipid capable of spontaneously arranging to form a lipid bilayer. Such membrane lipids are lipids comprising hydrophobic and hydrophilic regions, such that after self-assembly, the hydrophobic regions of the membrane lipid form the inner portion of the bilayer, while the hydrophilic regions form the outer surface of the membrane. Preferably, the membrane lipid is a lipid that naturally forms a cell membrane, such as an amphipathic phospholipid. It is also preferred that the membrane lipid is derived from a host cell in which wild-type EBV is capable of replicating. More preferably, the membrane lipid is derived from a cell according to the present invention. According to the present invention, the membrane contained in the particle is present in an amount sufficient to form a membrane constituting the outer shell of the particle. The particle must possess the membrane shell, preferably comprising at least one EBV structural polypeptide. As outlined below, preferred examples of membrane-associated EBV structural polypeptides are gp350 polypeptide and LP-1 polypeptide. As further detailed below, the B cell transforming ability of LMP-1 may be impaired. It is also preferred that the membrane of the particle comprises additional membrane components that are also naturally found in the EBV membrane, such as additional membrane polypeptides that can be found on the inner surface, on the outer surface or throughout the membrane.

[0127] In some embodiments of the immunogenic compositions of the invention, the EBV VLP comprises glycoprotein 350 / 220 (gp350) protein or a fragment thereof, and optionally, the VLP comprises one, two, three, or more polypeptide sequences that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 102-117.

[0128] In some embodiments, the EBV VLPs comprise:

[0129] [ka]

[0130] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0131] In some embodiments, the EBV VLPs comprise:

[0132] [ka]

[0133] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0134] In some embodiments, the EBV VLPs comprise:

[0135] [ka]

[0136] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0137] In some embodiments, the EBV VLPs comprise:

[0138] [ka]

[0139] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0140] In some embodiments, the EBV VLPs comprise:

[0141] [ka]

[0142] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0143] In some embodiments, the EBV VLPs comprise:

[0144] [ka]

[0145] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0146] In some embodiments, the EBV VLPs comprise:

[0147] [ka]

[0148] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0149] In some embodiments, the EBV VLPs comprise:

[0150] [ka]

[0151] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0152] In some embodiments, the EBV VLPs comprise:

[0153] [ka]

[0154] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0155] In some embodiments, the EBV VLPs comprise:

[0156] [ka]

[0157] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0158] In some embodiments, the EBV VLPs comprise:

[0159] [ka]

[0160] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0161] In some embodiments, the EBV VLPs comprise:

[0162] [ka]

[0163] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0164] In some embodiments, the EBV VLPs comprise:

[0165] [ka]

[0166] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0167] In some embodiments, the EBV VLPs comprise:

[0168] [ka]

[0169] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0170] In some embodiments, the EBV VLPs comprise:

[0171] [ka]

[0172] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0173] In some embodiments, the EBV VLPs comprise:

[0174] [ka]

[0175] or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

[0176] In some preferred embodiments of the immunogenic compositions of the invention, the EBV-VLPs are selected from the group consisting of gp350 (UniProt entry P03200), BKRF4 (UniProt entry P30117), BVRF1 (UniProt entry P03233), BDLF3 (UniProt entry P03224), BZLF2 (UniProt entry P03205), BXLF2 (UniProt entry P03231), BNRF1 (UniProt entry P03179), and the like. , BALF4 (UniProt entry P03188), and BZLF1 (UniProt entry P03206); or a functional variant thereof that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to the polypeptide sequences described herein.

[0177] In some preferred embodiments of the immunogenic compositions of the invention, the EBV-VLPs are selected from the group consisting of gp350 (UniProt entry P03200), BKRF4 (UniProt entry P30117), BVRF1 (UniProt entry P03233), BDLF3 (UniProt entry P03224), BZLF2 (UniProt entry P03205), BXLF2 (UniProt entry P03231), BNRF1 (UniProt entry P03179), BALF4 (UniProt entry P03179), and BALF5 (UniProt entry P03206). (UniProt entry P03188) and BZLF1 (UniProt entry P03206), or functional variants thereof that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein, the presence of two or more of said EBV proteins contributing to the enhanced immunogenicity of the VLP vaccine.

[0178] In some preferred embodiments of the immunogenic compositions of the invention, the EBV-VLPs do not contain LMP1 (UniProt entry P03230), EBNA2 (UniProt entry P12978), EBNA3a (UniProt entry P12977), EBNA3b (UniProt entry P03203), and EBNA3c (UniProt entry P03204), and the absence of these B-cell transforming EBV proteins contributes to the enhanced safety of the VLP vaccines.

[0179] [Table 1A]

[0180] [Table 1B]

[0181] The term "individual" is used interchangeably herein with "host" and "subject" and includes, but is not limited to, humans, non-human primates, all livestock and pets, wild mammals and poultry, including, but not limited to, cattle, horses, cows, pigs, sheep, goats, dogs, cats, rabbits, deer, mink, chickens, ducks, geese, turkeys, fighting hens, and the like.

[0182] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Various examples of cancer are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In some examples, the cancer is selected from the group consisting of B-cell lymphoma, T-cell lymphoma, multiple myeloma, chronic myelogenous leukemia (CML), acute myeloma leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloproliferative neoplasm (MPN), B-cell acute lymphoblastic leukemia (B-ALL), solid tumor, carcinoma, or sarcoma; and preferably, the cancer is a solid tumor. In another example, the cancer is a virus-specific cancer. In some examples, the cancer is a lymphoproliferative disorder (LPD) such as Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), B-cell lymphoma including diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma, NKT-cell lymphoma, NK-cell lymphoma, or post-transplant lymphoproliferative disorder (PTLD), or an EBV-associated cancer selected from epithelial carcinoma (nasopharyngeal, lung, breast), lymphoepithelioma, carcinoma with lymphocytic infiltration (GCLS, e.g., gastric cancer), or glioma.

[0183] DLBCL is a cancer of B cells. Typically, DLBCL arises from normal B cells, but it can also represent malignant transformation of other types of lymphoma or leukemia. Underlying immunodeficiency is a significant risk factor, and infection with Epstein-Barr virus has also been found to contribute to the development of DLBCL. In one embodiment, the herpesvirus-associated cancer is DLBCL.

[0184] Burkitt lymphoma is a cancer of B lymphocytes found in the lymphatic system, particularly in germinal centers. Burkitt lymphoma can be divided into three major clinical variants: endemic, sporadic, and immunodeficiency-associated. EBV infection is found in nearly all endemic variants. In one embodiment, the herpesvirus-associated cancer is Burkitt lymphoma.

[0185] Treatment or prevention of post-transplant lymphoproliferative disorder (PTLD), epithelial carcinoma (nasopharyngeal, lung, breast), lymphoepithelioma, cancer with lymphocytic infiltration (GCLS, e.g., gastric cancer), or glioma is also important within the context of the present disclosure. PTLD is the name given to B-cell proliferation resulting from therapeutic immunosuppression after organ transplantation. These patients may develop infectious mononucleosis-like lesions or polyclonal polymorphic B-cell hyperplasia. Thus, the present disclosure also relates to the treatment of immunodeficient or immunocompromised patients following chemotherapy, radiation, immunosuppression, or transplantation. In one embodiment, the herpesvirus-associated cancer is PTLD.

[0186] Nasopharyngeal carcinoma (NPC) is the most common cancer occurring in the nasopharynx (most commonly in the posterior nasopharynx or pharyngeal recesses, accounting for 50% of cases). NPC occurs in children and adults. An association between Epstein-Barr virus and nasopharyngeal carcinoma is evident in World Health Organization (WHO) type II and III tumors. In one embodiment, the herpesvirus-associated cancer is NPC.

[0187] Lymphoepithelioma is a type of poorly differentiated nasopharyngeal carcinoma characterized by significant lymphocyte infiltration in the tumor-involved area. Lymphoepithelioma is also known as "Class III nasopharyngeal carcinoma" in the WHO classification system. In one embodiment, the herpesvirus-associated cancer is lymphoepithelioma.

[0188] Gastric carcinoma with lymphocytic infiltration (GCLS) is a unique histological subtype of gastric cancer characterized by undifferentiated carcinoma mixed with significant lymphocytic infiltration. More than 80% of GCLS cases are associated with EBV infection, but it is unclear whether the virus influences disease progression. In one embodiment, the herpesvirus-associated cancer is GCLS.

[0189] Glioma is a type of tumor that originates in glial cells in the brain or spine. Glioma accounts for approximately 30% of all brain tumors and central nervous system tumors, and 80% of all malignant brain tumors. Studies have shown that EBV is present at an elevated frequency in glioma patients, indicating the potential targeting of EBV-associated gliomas using the present disclosure. In one embodiment, the herpesvirus-associated cancer is glioma.

[0190] The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid tumors or liquid tumors, e.g., diffuse tumors or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-cancerous conditions as well as malignant cancers and tumors.

[0191] The term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with the cancerous condition. An "anti-cancer effect" can also be manifested by the ability of the composition, peptide, and / or cell in preventing the development of cancer in the first place.

[0192] The term "specifically binds" refers to an antigen-binding domain (antibody) or ligand that recognizes and binds to a cognate binding partner (e.g., a stimulatory and / or costimulatory molecule present on a T cell) protein present in a sample, but that does not substantially recognize or bind to other molecules in the sample.

[0193] The term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular domain comprising an antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain" or "intracellular domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in a CAR polypeptide construct are in the same polypeptide chain, e.g., comprising a chimeric fusion protein.

[0194] In one aspect, the intracellular domain comprises at least one activation domain (e.g., the activation domain of CD3 zeta). In one aspect, the intracellular domain further comprises one or more costimulatory molecules derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing of the CAR and localization to the cell membrane.

[0195] A CAR comprising an antigen-binding domain (e.g., an scFv, a single-domain antibody, or a TCR (e.g., a TCR alpha-binding domain or a TCR beta-binding domain)) that targets a specific tumor marker X, where X can be a tumor marker as described herein, is also referred to as an XCAR. For example, a CAR comprising an antigen-binding domain that targets CD19 is referred to as a CD19CAR. A CAR can be expressed in any cell, for example, an immune effector cell (e.g., a T cell or an NK cell) as described herein.

[0196] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting a message within a cell to regulate cellular activity through a predetermined signaling pathway by generating second messengers or functioning as an effector by responding to such messengers. The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0197] The term "antibody fragment" refers to at least a portion of an intact antibody, or a recombinant variant thereof, and refers to an antigen-binding domain, e.g., the antigenically-determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (either VF or VH), camelid VHH domains, and multispecific molecules formed from antibody fragments such as two or more, e.g., two Fab fragments, linked by a disulfide bond at the hinge region, or a bivalent fragment comprising two or more linked antibodies, e.g., two isolated CDRs or other epitope-binding fragments. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0198] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked via a short, flexible polypeptide linker, capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv as used herein can have the VF and VH variable regions in either order; for example, relative to the N- and C-termini of the polypeptide, an scFv can comprise VF-linker-VH or VH-linker-VF.

[0199] The term "complementarity-determining region" or "CDR," as used herein, refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are typically three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (FCDR1, FCDR2, and FCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat (Rabat) et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat (Rabat)" numbering scheme), those described by Al-Fazikani et al. (1997) JMB 273, 927-948 (the "Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).

[0200] Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Under the combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.

[0201] The portion of the CAR composition of the present disclosure that comprises an antibody or antibody fragment thereof can exist in various forms, e.g., the antigen-binding domain is expressed as part of a polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), or a humanized antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present disclosure comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0202] As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "anti-target binding domain") refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In embodiments, the antibody molecule is a multispecific antibody molecule, e.g., comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the antigen-binding domain is an scFv domain.

[0203] The term "autologous" refers to any material derived from the same individual that is to be subsequently reintroduced into the individual.

[0204] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another if their genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may not be sufficiently genetically similar to interact as an antigen.

[0205] "Derived from," as the term is used herein, indicates a relationship between a first molecule and a second molecule. The term generally refers to a structural similarity between the first and second molecules and does not imply or include a limitation on the process or source from which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure so that it has the required function, i.e., the ability to generate a signal under appropriate conditions. The term does not imply or include a limitation on the particular process by which the intracellular signaling domain is generated, e.g., it does not mean that one must start with a CD3 zeta sequence and delete or mutate undesired sequences to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0206] The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex in a stimulatory manner with respect to at least some aspect of the T cell signaling pathway. In some embodiments, an ITAM-containing domain within a CAR recapitulates primary TCR signaling independent of the endogenous TCR complex. In one aspect, the primary signal is initiated, for example, by binding of the TCR / CD3 complex with an MF1C molecule attached to a peptide, and leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. A primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAMs comprising primary cytoplasmic signaling sequences of particular use in the present disclosure include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FceRI and CD66d, DAP10, and DAP12. In specific CARs of the present disclosure, the intracellular signaling domain in any one or more CARs of the present disclosure comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3 zeta. The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0207] "Intracellular signaling domain" or "intracellular domain," as the term is used herein, refers to the intracellular portion of a molecule. In embodiments, the intracellular signaling domain transduces an effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be utilized, in many cases it is not necessary to use the entire chain. To the extent a truncated portion of an intracellular signaling domain is used, such truncated portion can be used in place of the intact chain, so long as it transduces the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0208] The intracellular signaling domain generates a signal that promotes immune effector function of the CAR-containing cell, e.g., a CART cell, such as cytolytic and helper activity, including cytokine secretion.

[0209] In embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0210] The primary intracellular signaling domain can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAMs comprising primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FceRI, CD66d, DAP10, and DAP12. The term "zeta" or alternatively "zeta chain," "CD3 zeta," or "TCR zeta" refers to CD247. Swiss-Prot Accession Number P20963 provides an exemplary human CD3 zeta amino acid sequence. "Zeta stimulatory domain" or alternatively "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" refers to the stimulatory domain of CD3 zeta or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, fragment, insertion, or deletion). In one embodiment, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1, or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In one embodiment, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as SEQ ID NO: 641 or 643, or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion).

[0211] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1 BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, I TGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB 2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile) , CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0212] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

[0213] The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment thereof. The term "4-1BB" refers to CD137 or tumor necrosis factor receptor superfamily member 9. Swiss-Prot Accession Number P20963 provides an exemplary human 4-1BB amino acid sequence. "4-1BB costimulatory domain" refers to the costimulatory domain of 4-1BB, or a variant thereof (e.g., a molecule having mutations, such as point mutations, fragments, insertions, or deletions).

[0214] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0215] The term "endogenous" means any material that originates from or is produced within an organism, cell, tissue, or system.

[0216] The term "exogenous" means any material introduced from or produced outside an organism, cell, tissue or system.

[0217] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence. In some embodiments, expression includes the translation of mRNA introduced into a cell.

[0218] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

[0219] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions, e.g., conservative substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions, e.g., conservative substitutions, can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0220] The terms "cancer-associated antigen" or "tumor antigen" refer interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either in whole or as a fragment (e.g., MHC / peptide), and that is useful for preferential targeting of drugs to cancer cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed on cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more, compared to normal cells. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains a deletion, addition, or mutation compared to the molecule expressed on normal cells. In some embodiments, a tumor antigen, either in whole or as a fragment (e.g., MHC / peptide), will be expressed exclusively on the cell surface of cancer cells and will not be synthesized or expressed on the surface of normal cells. In some embodiments, CARs of the present disclosure include CARs comprising an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the pocket of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified from screening libraries such as human scFv phage display libraries.

[0221] The term "subject" is intended to include living organisms in which an immune response can be mounted (eg, mammals, humans).

[0222] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0223] Compositions and Medical Uses In some aspects, the present disclosure provides a composition for improving immune cell therapy, the composition comprising: (a) polyinosinic-polycytidylic acid (PIC); (b) a stabilizing agent; and (c) at least one cation. In some embodiments, the composition is useful for generating, inducing, strengthening, and / or enhancing an immune response, which may be an innate and / or adaptive immune response mediated by immune cell therapy. In some embodiments, the immune cell is an immune effector cell (e.g., a T cell or an NK cell) that expresses a chimeric antigen receptor (CAR) molecule, e.g., a CAR molecule that binds to a tumor antigen, e.g., an antigen expressed on the surface of a solid tumor or a hematological tumor.

[0224] In some embodiments, the present disclosure provides immunogenic compositions comprising: (a) polyinosinic-polycytidylic acid (PIC); (b) a stabilizer that is an aminoglycoside antibiotic or a non-aminoglycoside amine; and (c) at least one cation, preferably calcium. In some embodiments, the compositions further comprise at least one immunogen or antigen. In some embodiments, the immunogen or antigen is a recombinant protein, virus-like particle (VLP), peptide, mRNA, or vaccine.

[0225] In some embodiments, the present disclosure relates to an immunogenic composition comprising an Epstein-Barr virus (EBV) VLP. The immunogenic composition can be used to induce and / or enhance activation of an immune response in an individual, such as the immune response of immune cell therapy (preferably CAR-T cell therapy) for cancer treatment. The immunogenic composition can be used in combination with engineered immune cells (e.g., CAR-T cells or engineered TCR-T cells) in the treatment of cancer. In some examples, the immunogenic composition is administered in combination (concurrently or sequentially) with engineered immune cells, preferably CAR-T cells, more preferably CAR-T cells targeting EBV-associated cancer.

[0226] In some embodiments, the immunogenic composition may be for use in inducing and / or enhancing activation of an immune response in an individual, such as the immune response of immune cell therapy (preferably CAR-T cell therapy) for the treatment of cancer. In some further embodiments, the immunogenic composition may also be useful for use in combination with engineered immune cells (preferably CAR-T cells or engineered TCR-T cells) in the treatment of cancer. In one embodiment, the immunogenic composition enhances the anti-cancer effect of cell therapy.

[0227] PICs are typically double-stranded polymers comprising one strand of an inosinic acid polymer (polyinosinic acid; polyI) and one strand of a cytidylic acid polymer (polycytidylic acid; polyC). The polymer backbone can be a deoxyribonucleic acid backbone or a ribonucleic acid backbone. PICs can be oligonucleotide analogs. In a preferred embodiment, polyI is preferably polyriboinosinic acid. In a preferred embodiment, PolyC is preferably polyribocytidylic acid. In a preferred embodiment, the PIC is polyriboinosinic acid:polyribocytidylic acid, i.e., a double-stranded RNA (dsRNA)-like polymer.

[0228] In some embodiments, the concentration of the PIC is between 0.5 mg / ml and 10 mg / ml.

[0229] In some embodiments, the PIC is heterogeneous with respect to molecular weight, with an average molecular weight of 66,000 daltons or greater. The value of 66,000 daltons corresponds to a molecular size of 6.4 sedimentation units (Svedberg). In some embodiments, the PIC has an average molecular weight of 150,000 daltons or greater or an average molecular size of 9.3 Svedbergs or greater. In some embodiments, the PIC has an average molecular weight of 250,000 daltons or greater or an average molecular size of 11.8 Svedbergs or greater. In some embodiments, the PIC has an average molecular weight of 350,000 daltons or greater or an average molecular size of 15.3 Svedbergs or greater.

[0230] In some embodiments, the molecular weight of the PIC is 66,000-2,000,000 daltons. In some embodiments, the molecular weight of the PIC is 66,000-1,200,000 daltons (equivalent to 6.4-24.0 sedimentation units). In some embodiments, the molecular weight of the PIC is 66,000-660,000 daltons, or the molecular size range is about 6.4-18.3 Svedberg. In some embodiments, the PIC has a molecular weight range of about 300,000-1,200,000 daltons or a size range of about 6.4-24.0 Svedberg. In some embodiments, the PIC has a molecular weight range of about 300,000-660,000 daltons or a molecular size range of about 12.8-18.3 Svedberg. In some other embodiments, the molecular weight of the PIC is between 100,000 and 200,000 daltons, or between 300,000 and 4,000,000 daltons, or between 500,000 and 1,000,000 daltons, or between 1,000,000 and 1,500,000 daltons, or between 1,500,000 and 2,000,000 daltons, or between 2,000,000 and 3,000,000 daltons. 00 to 2,500,000 daltons, or 2,500,000 to 3,000,000 daltons, or 3,000,000 to 3,500,000 daltons, or 3,500,000 to 4,000,000 daltons, or 4,000,000 to 4,500,000 daltons, or 4,500,000 to 5,000,000 daltons.

[0231] In some embodiments, the PIC composition is obtained by mixing polyinosinic acid and polycytidylic acid in a specific ratio. Preferably, the ratio can be 0.5:1.0, 0.6:1.0, 0.7:1.0, 0.8:1.0, 0.9:1.0, 1.0:1.0, 1.0:1.1, 1.0:1.2, 1.0:1.3, 1.0:1.4, 1.0:1.5, 1.5:1.0, 1.4:1.0, 1.3:1.0, 1.2:1.0, 1.1:1.0, 1.0:0.9, 1.0:0.8, 1.0:0.7, 1.0:0.6, or 1.0:0.5. In a preferred embodiment, the ratio is 1:1. Furthermore, the mixture is further mixed with 200-2000 IU of kanamycin and 0.02-10 mM CaCl2.

[0232] In certain embodiments, the stabilizer is selected from the group consisting of tacrolamycin, anthracycline, butyrin sulfate, gentamicin, hygromycin, amikacin, dideoxykanamycin, nebramycin, β-lactam, neomycin, puromycin, streptomycin, streptozocin, and any combination thereof. The polyamine compound is selected from the group consisting of arginine salt, spermidine, N-(3-aminopropyl), N-(3-aminopropyl)-1,4-butanediamine, spermine, OS-dimethylaminothiophosphate, polylysine, aminoglycosides, and any combination thereof. In certain embodiments, the stabilizer is kanamycin. In other specific embodiments, the stabilizer is ε-polylysine, hexylglucosamine, polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, and galactose or acetylglucosamine. In some embodiments, the concentration of the stabilizer in the composition is from 10 units / ml to 100,000 units / ml, preferably from 100 units / ml to 10,000 units / ml, and more preferably from 500 units / ml to 5,000 units / ml.

[0233] In some embodiments, the cation is a cation and is selected from the group consisting of calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, zinc, and any combination thereof. In certain embodiments, the cation is calcium. The cation can be in the form of any suitable salt or organic complex, including, but not limited to, chloride, fluoride, hydroxide, phosphate, or sulfate. For example, when the cation is calcium, the calcium ion can be in the form of calcium carbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphate, or calcium sulfate. In some embodiments, the concentration of the cation in the composition is 0.01 pmol to 10 mmol / ml, preferably 0.02 pmol to 5 mmol / ml, more preferably 0.1 pmol to 1 mmol / ml, and most preferably 0.1 pmol to 100 pmol / ml.

[0234] In some embodiments, the immunogen is a recombinant protein. In some embodiments, the immunogen is a virus-like particle (VLP). In some embodiments, the immunogen is a peptide vaccine. In some embodiments, the immunogen is an mRNA vaccine. In some other embodiments, the immunogen can be one or more polypeptides / peptides derived from cancer cells or antigenic fragments or variants thereof. It will be understood that the immunogens described herein can further comprise additional components. For example, one or more immunogens can be contained in a lipid or liposome. In some embodiments, peptides or polypeptides corresponding to cancer antigens can generally be 10-20 amino acid residues in length and can include two or more peptide determinants, or up to about 30-50 residues, etc. In some embodiments, polypeptides are 10 to about 150 residues in length or longer. In some embodiments, longer peptides or polypeptides can also be prepared, e.g., by recombinant means. In certain embodiments, nucleic acids encoding the antigenic compositions and / or components described herein can be used, e.g., to generate antigenic compositions in vitro or in vivo for various compositions and methods of the present disclosure. For example, in certain embodiments, the nucleic acid encoding the antigen is contained in a vector, e.g., in a recombinant cell. The nucleic acid can be expressed to produce a peptide or polypeptide comprising the antigenic sequence. The peptide or polypeptide can be secreted from the cell or contained as part of or within the cell. In some embodiments, the immunogen is the same as or derived from the same antigen recognized by the CAR.

[0235] In some embodiments, the immunogen is a "tumor-associated antigen" or "cancer antigen" and is selected from the group consisting of CTA, NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7, CT10, GAGE, PRAME; BAGE; RAGE, SAGE, HAGE, MPHOSPH1, DEPDC1, IMP3, and MAGE-A, and T antigens BK, p53, Ras, c-Myc, A-Raf, B-Raf, C-Raf, cyclin-dependent kinase, MAGE-A2, MAGE-A6, MAGE-A10, MAGE-A12, MART -1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK, PRAME, P15, RU1, RU2, SART-1, SART-3 receptor, Wilms tumor antigen (WT1), AFP, β-catenin / ш, caspase-8 / m, CE A, CDK-4 / m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCRABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RARa, tumor-associated calcium signaling factor 1 (TACSTD1), TACSTD2 receptor tyrosine kinase, epidermal growth factor receptor (EGFR) , EGFRvIII, platelet growth factor receptor (PDGFR), vascular endothelial growth factor receptor ia (VEGFR), cytoplasmic tyrosine kinase, src family, syk-ZAP70, integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STAT5 and STAT6, hypoxia-induced factors, HIF-1a and HIF-2a, nuclear factor kappa in (NF-kB), Notch, Notch1-4, c-Met receptor, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), PMSA,PR-3, MDM2, mesothelin, carcinoma kidney cells-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation control point, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, zclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesotheliana, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NYBR-1, RG The antigens may be selected from SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, and fos-related antigen 1.

[0236] In another embodiment, the immunogen is a viral antigen derived from an oncogenic virus. Exemplary oncogenic viruses include, but are not limited to, EBV, HPV, HBV, HCV, HTLV, and KSHV. Exemplary viral antigens derived from oncogenic viruses that can be used in the present disclosure include, but are not limited to, EBV: EBNA-1, LMP-1, LMP-2A; HPV: E6, E7, E5; HBV: HBx; HCV: Core, NS3, Ns5A; HTLV: Tax, HBZ; KSHV: vFLIP, LANA, vGPCR, vIRF-1.

[0237] In some embodiments, the immunogen is a virus-like particle (VLP). In other embodiments, the immunogen is an Epstein-Barr virus (EBV) VLP, preferably comprising glycoprotein 350 / 220 (gp350) protein or a fragment thereof.

[0238] In some embodiments, the composition is administered parenterally, intramuscularly, intraperitoneally, intrathoracically, intravenously, subcutaneously, intrapericardially, by inhalation, rectally, by suppository, intranasally, ophthalmically, transdermally, or orally.

[0239] In some embodiments, the PIC is preferably synthetic and preferably obtained by de novo chemical synthesis. Synthetic PIC molecules can be obtained from commercial sources (e.g., Sigma or Midland Certified). The PIC molecule can be synthesized with or chemically modified to include a 2'-position modification, such as 2'-O-methyl, 2'-fluoro, or 2'-NH.

[0240] In some embodiments, the immune cells described herein can be selected from the group consisting of T lymphocytes, NK cells, macrophages, and dendritic cells, and the T lymphocytes are preferably cytotoxic T lymphocytes or T helper cells, more preferably cytotoxic T lymphocytes. The immune cells can be T lymphocytes. The T lymphocytes can be cytotoxic T lymphocytes. The T lymphocytes can be T helper cells. The immune cells can be NK cells. The immune cells can be macrophages. The immune cells can be dendritic cells. These immune cells are known in the art to exhibit cytotoxic activity and / or other beneficial activity in response to unwanted agents, cells, or pathogens, such as cells infected with herpesviruses. By directing the activity of these cells to specific immunogenic targets, i.e., herpesvirus antigens described herein, the infected pathogenic cells can be eliminated by the corresponding activity of the immune cells described herein.

[0241] Chimeric antigen receptor (CAR) In one aspect, disclosed herein are methods of using cells (e.g., populations of cells) that express a CAR molecule. In one aspect, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an antigen-binding domain (e.g., an antigen-binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one aspect, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain (e.g., an antigen-binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and / or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).

[0242] CAR antigen-binding domain In one aspect, the portion of the CAR that comprises an antigen-binding domain comprises an antigen-binding domain that targets a tumor antigen, e.g., a tumor antigen described herein. In some embodiments, the antigen-binding domain targets CD19; CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule 1, CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family members; B-cell maturation antigen (BCMA); Tn antigen (Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Fike tyrosine kinase 3 (FFT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2; mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine kinase protein kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1; tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5);High molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); glob oH glycoceramide (GloboH) hexasaccharide moiety; mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1, recognized by T cells melanoma antigen 1; rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC);Tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-Fike, squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (FCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mutated) hsp70-2; CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican 3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1);

[0243] In some embodiments, the antigen recognized by the CAR is a viral antigen. In some embodiments, the antigen recognized by the CAR is an Epstein-Barr virus antigen (EBV antigen). In some embodiments, the EBV antigen may be present on the surface of an EBV-infected cell, preferably an EBV-infected cancer cell, an EBV-infected B cell, or an EBV-infected epithelial cell. The EBV antigen may be an EBV virion envelope protein or a protein of the EBV envelope complex (such as gB, gL, or gH). The EBV viral antigen is preferably EBV glycoprotein 350 / 220 (gp350 / 220). In preferred embodiments, the present disclosure focuses on targeting EBV antigens and treating EBV-associated medical conditions. An exemplary EBV gp350 protein is shown in the UniProt database, entry P03200-1, version 1, dated July 21, 1986. An exemplary EBV gp220 is shown in the same database entry, but with positions 502-750 missing.

[0244] In some embodiments, a CAR of the present disclosure comprises an antigen-binding domain comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) as provided in Table 1 and Table 2, or a specific VH and VL combination as provided in Table 1 and Table 2. In another embodiment, a CAR comprises an antigen-binding domain comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) having at least 60%, 70%, 80%, 85%, 90%, 95%, 99% identity to the VH and / or VL amino acid sequences as provided in Table 1 and Table 2. In another embodiment, the CAR comprises antigen-binding domains that bind to the same epitope on EBV gp350 or competitively bind to EBV gp350 and include antigen-binding domains comprising specific combinations of VH and VL as provided in Table 1 and Table 2.

[0245] In some embodiments, a CAR of the present disclosure comprises an antigen-binding domain that binds to EBV glycoprotein 350 / 220 (gp350 / 220), wherein the antigen-binding domain is (1) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 1 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 6 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (2) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 2 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 7 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (3) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 3 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 8 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (4) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 4 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 9 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (5) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 5 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 10 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; (6) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (7) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (8) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (9) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (10) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (12) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (13) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof with one, two, or three amino acid substitutions relative to the sequence; (14) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence; or (15) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof in which one, two, or three amino acids are substituted relative to the sequence. Includes.

[0246] In some embodiments, a CAR of the present disclosure comprises an antigen-binding domain comprising one, two, three or more HCDRs and / or one, two, three or more LCDRs as provided in Table 3 and Table 4, or specific HCDR1-3 and LCDR1-3 combinations as provided in Table 3 and Table 4.

[0247] In some embodiments, a CAR of the present disclosure comprises an antigen-binding domain that binds to EBV glycoprotein 350 / 220 (gp350 / 220), wherein the antigen-binding domain is (1) SEQ ID NOs: 11, 12, 13, 26, 27, and 28, respectively; (2) SEQ ID NOs: 14, 15, 16, 29, 30, and 31, respectively; (3) SEQ ID NOs: 17, 18, 19, 32, 33, and 34, respectively; (4) SEQ ID NOs: 20, 21, 22, 35, 36, and 37, respectively; or (5) SEQ ID NOs: 23, 24, 25, 38, 39 and 40, respectively or an EBV glycoprotein 350 / 220 antigen-binding domain that competes therewith or binds to the same epitope in EBV glycoprotein 350 / 220 as bound by any one of the antigen-binding domains (1) to (5) of the CAR as described herein.

[0248] In some embodiments, a CAR of the disclosure comprises an amino acid sequence as provided in Table 5, preferably, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144 to 161. In other embodiments, the CAR comprises at least 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a CAR sequence as provided in Table 5, preferably, the CAR is selected from the group consisting of SEQ ID NOs: 144 to 161.

[0249] In yet another embodiment, the CAR of the present disclosure comprises an antigen-binding domain comprising: Heavy chain complementarity determining regions: H-CDR1 (GLSLTSN) according to SEQ ID NO: 118, H-CDR2 (WSNGG) according to SEQ ID NO: 119, and H-CDR3 (PRYNSGYFFDY) according to SEQ ID NO: 120, or one or more corresponding CDR sequences having at least 80% sequence identity to SEQ ID NOs: 118 to 120 a variable heavy chain (VH) comprising: and Light chain complementarity determining regions: L-CDR1 (KASESVSTRMH) according to SEQ ID NO: 121, L-CDR2 (KTSNLAS) according to SEQ ID NO: 122, and L-CDR3 (QQSWNGPLT) according to SEQ ID NO: 123, or one or more corresponding CDR sequences with at least 80% sequence identity to SEQ ID NOs: 121 to 123 variable light chain (VL) containing The present invention is characterized by comprising:

[0250] In yet another embodiment, the CAR of the present disclosure comprises an antigen-binding domain comprising: Heavy chain complementarity determining regions: H-CDR1 (GFSLTSY) according to SEQ ID NO: 124, H-CDR2 (WSDGD) according to SEQ ID NO: 125, and H-CDR3 (LQSEDTATYYCARLQVFGYPGIRDYVMDA) according to SEQ ID NO: 126, or one or more corresponding CDR sequences having at least 80% sequence identity to SEQ ID NOs: 124 to 126 a variable heavy chain (VH) comprising: and Light chain complementarity determining region: L-CDR1 (KSSQSLLSSRHQKNFLA) according to SEQ ID NO: 127, L-CDR2 (HASTRQS) according to SEQ ID NO: 128, and L-CDR3 (LQHYTSPYT) according to SEQ ID NO: 129, or a sequence having at least 80% sequence identity to SEQ ID NOs: 127 to 129 variable light chain (VL) containing The present invention is characterized by comprising:

[0251] In yet another embodiment, a CAR of the present disclosure comprises a VH domain according to SEQ ID NO: 130:

[0252] [ka]

[0253] and (7A1) a VL domain according to SEQ ID NO: 131: (DTVLTQSPALAVSPGERVTISCKASESVSTRMHWYRQKPGQQPKLLIYKTSNLASGVPARFSGSGSGTDFTLTIDPVEADDTATYFCQQSWNGPLTFGSGTKLEIKR), or (6G4) VH domain according to SEQ ID NO: 132:

[0254] [ka]

[0255] and (6G4) a VL domain according to SEQ ID NO: 133:

[0256] [ka]

[0257] Includes.

[0258] In yet another embodiment, the CAR of the disclosure has the sequence of SEQ ID NO: 134:

[0259] [ka]

[0260] or SEQ ID NO:135:

[0261] [ka]

[0262] or SEQ ID NO:136:

[0263] [ka]

[0264] or SEQ ID NO:137:

[0265] [ka]

[0266] It comprises or consists of a sequence according to

[0267] The antigen-binding domain can be any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and single-domain antibodies, such as, but not limited to, heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-derived nanobodies (VHH), as well as functional fragments thereof, including alternative scaffolds known in the art that function as antigen-binding domains, such as recombinant fibronectin domains, T cell receptors (TCR), or fragments thereof, e.g., single-chain TCRs, etc. In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to include human or humanized residues relative to the antigen-binding domain of an antibody or antibody fragment.

[0268] CAR transmembrane domain With regard to the transmembrane domain, in various embodiments, a CAR can be designed to include a transmembrane domain linked to the extracellular domain of the CAR. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is associated with one of the other domains of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In a different embodiment, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with the binding domain of a native binding partner present in the same CART.

[0269] The transmembrane domain can be derived from either natural or recombinant sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever the CAR binds to the target. Transmembrane domains of particular use in the present disclosure can include at least the transmembrane regions of, for example, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is selected from the group consisting of, for example, KIR2DS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49 a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c , ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It can include at least the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and NKG2C.

[0270] In some cases, the transmembrane domain can be linked to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a human protein hinge. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge.

[0271] In one embodiment, the hinge or spacer comprises an IgG4 hinge. In one embodiment, the hinge or spacer comprises an IgD hinge. In one embodiment, the transmembrane domain may be recombinant, in which case it will comprise primarily hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine residues can be found at each end of the recombinant transmembrane domain. Optionally, a short oligo- or polypeptide linker, 2-10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine double residue provides a particularly suitable linker. In one embodiment, the hinge or spacer comprises a KIR2DS2 hinge.

[0272] Cytoplasmic domain The cytoplasmic domain or region of the CAR comprises an intracellular signaling domain, which is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced.

[0273] Examples of intracellular signaling domains for use in the CARs described herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as derivatives or variants of any of these sequences and any recombinant sequences that have the same functional capability.

[0274] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary and / or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0275] The primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0276] Examples of ITAMs comprising primary intracellular signaling domains of particular use in the present disclosure include those of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FceRI, DAP10, DAP12, and CD66d. In one embodiment, a CAR of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain, of CD3 zeta, e.g., a CD3 zeta sequence described herein.

[0277] In one embodiment, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain that has altered (e.g., increased or decreased) activity when compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In embodiments, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0278] Costimulatory signaling domain The intracellular signaling domain of the CAR can comprise a CD3 zeta signaling domain by itself, or can be combined with any other desired intracellular signaling domain useful in the context of the CAR of the present disclosure. For example, the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain. A costimulatory signaling domain refers to a portion of the CAR that comprises the intracellular domain of a costimulatory molecule. In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of CD28. In one aspect, the intracellular domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of ICOS.

[0279] A costimulatory molecule can be a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Examples of such molecules include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death 1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen 1 (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, and MHC class I molecules. Proteins, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta , IL-2R Gamma, IL-7R Alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD 84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.For example, CD27 costimulation has been demonstrated to enhance the expansion, effector function, and survival of human CART cells in vitro, and enhance human T cell persistence and anti-tumor activity in vivo (Song et al., Blood. 2012;H9(3):696-706). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, F1VEM (LIGF1TR), SLAMF7, NKp80 (KLRF1), NKp30, NKp44, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, and CD29 , ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, NKG2D, NKG2C, and PAG / Cbp. The intracellular signaling sequences within the cytoplasmic portion of the CAR can be linked to each other randomly or in a specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, can form the link between the intracellular signaling sequences. In one embodiment, a glycine-serine double residue can be used as a suitable linker. In one embodiment, a single amino acid, e.g., alanine, glycine, can be used as a suitable linker.

[0280] In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., two, three, four, five, or more, costimulatory signaling domains. In embodiments, the two or more, e.g., two, three, four, five, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0281] In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of CD28. In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of 4-1BB.

[0282] In one embodiment, the intracellular signaling domain is designed to include the signaling domain of CD3 zeta and the signaling domain of CD27.

[0283] In one aspect, a CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR comprising a different antigen-binding domain, e.g., to the same target or a different target (e.g., a target other than a cancer-associated antigen described herein or a different cancer-associated antigen described herein, e.g., CD19, CD33, CLL-1, CD34, FLT3, or folate receptor beta). In one embodiment, the second CAR comprises an antigen-binding domain to a target expressed in the same cancer cell type as the cancer-associated antigen. In one embodiment, the CAR-expressing cell comprises a first CAR that targets a first antigen and comprises an intracellular signaling domain with a costimulatory signaling domain but without a primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain with a primary signaling domain but without a costimulatory signaling domain. Without wishing to be bound by theory, the placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, ICOS, CD27, or OX-40, on a first CAR and a primary signaling domain, e.g., CD3 zeta, on a second CAR may limit CAR activity to cells in which both targets are expressed. In one embodiment, a CAR-expressing cell comprises a first cancer-associated antigen CAR comprising an antigen-binding domain that binds to a target antigen described herein, a transmembrane domain, and a costimulatory domain, and a second CAR that targets a different target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, a CAR-expressing cell comprises a first CAR comprising an antigen-binding domain that binds to a target antigen described herein, a transmembrane domain, and a primary signaling domain, and a second CAR that targets an antigen other than the first target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and comprises an antigen-binding domain, a transmembrane domain, and a costimulatory signaling domain for the antigen.

[0284] In another aspect, the disclosure features a population of CAR-expressing cells, e.g., CART cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CART cells can comprise a first cell expressing a CAR having an antigen-binding domain for a cancer-associated antigen described herein, and a second cell expressing a CAR having a different antigen-binding domain, e.g., an antigen-binding domain for a different cancer-associated antigen described herein, e.g., an antigen-binding domain for a cancer-associated antigen described herein that is different from the cancer-associated antigen to which the antigen-binding domain of the CAR expressed by the first cell binds. As another example, the population of CAR-expressing cells can comprise a first cell expressing a CAR comprising an antigen-binding domain for a cancer-associated antigen described herein, and a second cell expressing a CAR comprising an antigen-binding domain for a target other than the cancer-associated antigen as described herein. In one embodiment, the population of CAR-expressing cells comprises, for example, a first cell expressing a CAR comprising a primary intracellular signaling domain and a second cell expressing a CAR comprising a secondary signaling domain.

[0285] In another aspect, the present disclosure features a population of cells, wherein at least one cell in the population expresses a CAR having an antigen-binding domain for a cancer-associated antigen described herein, and a second cell expresses another substance, e.g., a substance that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the substance can be a substance that inhibits an inhibitory molecule. In some embodiments, an inhibitory molecule, such as PD-1, can reduce the ability of the CAR-expressing cell to initiate an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF (e.g., TGF beta). In one embodiment, the substance that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to a cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide of an inhibitory molecule, such as, for example, PD-1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGF-beta, or a fragment of any thereof, and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, OX40, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., a CD3-zeta signaling domain described herein)). In one embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof, and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3-zeta signaling domain described herein).

[0286] Nucleic acid construct encoding a CAR The present disclosure also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

[0287] In some embodiments, the present disclosure relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, e.g., a costimulatory signaling domain, and / or a primary signaling domain, e.g., a zeta chain.

[0288] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain it, or by isolating it directly from cells and tissues containing it, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0289] The present disclosure also provides a vector into which the DNA of the present disclosure has been inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. Lentiviral vectors also have the additional advantage of low immunogenicity. Retroviral vectors can also be, for example, gammaretroviral vectors. Gammaretroviral vectors can include, for example, a promoter, a packaging signal (y), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, such as a gene encoding a CAR. Gammaretroviral vectors may lack viral structural genes, such as gag, pol, and env. Exemplary gammaretroviral vectors include murine leukemia virus (MLV), spleen-limited focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application," Viruses. 2011 June;3(6):677-713.

[0290] In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the present disclosure is an adenoviral vector (A5 / 35). In another embodiment, expression of the nucleic acid encoding the CAR can be achieved using transposons such as sleeping beauty, CRISPR, CAS9, and zinc finger nucleases. See June et al., 2009 Nature Reviews Immunology 9.10:704-716, incorporated herein by reference. In brief summary, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0291] The expression constructs of the present disclosure can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present disclosure provides a gene therapy vector.

[0292] Nucleic acids can be cloned into numerous types of vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0293] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vol. 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0294] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, a selected gene can be inserted into a vector and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to the subject's cells either in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, adenoviral vectors are used.

[0295] A number of adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0296] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located in the region 30–110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, preserving promoter function when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp until activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription.

[0297] An example of a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor 1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into lentiviral vectors. See, e.g., Milone et al., Mol. Ther. 17(8):1453-1464 (2009).

[0298] Another example of a promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, elongation factor 1a promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the present disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of an operably linked polynucleotide sequence when such expression is desired and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Another example of a promoter is the phosphoglycerate kinase (PGK) promoter. In embodiments, a truncated PGK promoter (e.g., a PGK promoter having one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotide deletions compared to the wild-type promoter sequence) may be desirable.

[0299] Vectors can also include, for example, a signal sequence to facilitate secretion, a polyadenylation signal and a transcription terminator (e.g., from the bovine growth hormone (BGH) gene), elements allowing episomal replication and replication in prokaryotes (e.g., an SV40 origin or others known in the art), and / or elements allowing selection (e.g., an ampicillin resistance gene and / or a Zeocin marker).

[0300] The subject expression vector introduced into cells to assess expression of a CAR polypeptide or a portion thereof can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0301] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes encoding polypeptides that are not present or expressed in the recipient organism or tissue and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimal 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to alter promoter-driven transcription.

[0302] In one embodiment, the vector can further comprise a nucleic acid encoding a second CAR. In one embodiment, the second CAR comprises an antigen-binding domain for a target expressed on acute myeloid leukemia cells, such as CD123, CD34, CLL-1, folate receptor beta, or FLT3; or a target expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, or CD79a. In one embodiment, the vector comprises a nucleic acid sequence encoding a first CAR that specifically binds to a first antigen and comprises an intracellular signaling domain with a costimulatory signaling domain but no primary signaling domain, and a nucleic acid encoding a second CAR that specifically binds to a second, different antigen and comprises an intracellular signaling domain with a primary signaling domain but no costimulatory signaling domain.

[0303] In one embodiment, the vector comprises a nucleic acid encoding a CAR and a nucleic acid encoding an inhibitory CAR described herein. In one embodiment, the inhibitory CAR comprises an antigen-binding domain that binds to an antigen found on normal cells but not on cancer cells. In one embodiment, the inhibitory CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can be the intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.

[0304] In embodiments, the vector can include two or more nucleic acid sequences encoding a CAR, e.g., a CAR described herein, and a second CAR, e.g., an inhibitory CAR or a CAR that specifically binds to a different antigen. In such embodiments, the two or more nucleic acid sequences encoding the CARs are encoded by a single nucleic acid molecule in the same frame and as a single polypeptide chain. In this aspect, the two or more CARs can be separated, for example, by one or more peptide cleavage sites (e.g., autocleavage sites or substrates for intracellular proteases).

[0305] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0306] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-4, Cold Spring Harbor Press, NY. A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection. Biological methods for introducing a polynucleotide of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0307] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods for targeted delivery of modern nucleic acids are available, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems. When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is considered for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be associated with a lipid. Lipid-associated nucleic acids can be encapsulated in the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, linked to liposomes via linker molecules associated with both the liposomes and the oligonucleotides, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained or complexed in micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, micelles, or "folded" structures. They can also simply be dispersed in solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include lipid droplets naturally occurring in cytoplasm and classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0308] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories, Inc. (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of closed lipid bilayers or aggregates.

[0309] Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0310] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose the cells to the inhibitors of the present disclosure, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify substances within the scope of the present disclosure.

[0311] The present disclosure further provides a vector comprising a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly transduced into a cell, for example, a T cell or an NK cell.

[0312] In one aspect, the vector is a cloning or expression vector, such as, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR construct in mammalian T cells or NK cells. In one aspect, the mammalian T cells are human T cells. In one aspect, the mammalian NK cells are human NK cells. In one embodiment, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0313] Also disclosed are vectors comprising a nucleic acid molecule encoding an RNA molecule disclosed herein, e.g., an immunostimulatory RNA molecule disclosed herein. In one embodiment, the vector is capable of directly transducing a cell, e.g., a T cell or an NK cell. In one aspect, the vector is a cloning or expression vector, such as, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral, and lentiviral vector constructs. In one aspect, the vector is capable of expressing the RNA molecule in a mammalian T cell or an NK cell. In one aspect, the mammalian T cell is a human T cell. In one aspect, the mammalian NK cell is a human NK cell. In one embodiment, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule encoding a CAR and the RNA molecule, e.g., the nucleic acid molecule encoding an immunostimulatory RNA molecule, are located on a single vector. In some embodiments, the nucleic acid molecule encoding the CAR and the nucleic acid molecule encoding the RNA molecule, e.g., the immunostimulatory RNA molecule, are located on separate vectors.

[0314] In some embodiments, the present disclosure provides nucleic acid molecules (or vectors) encoding a polypeptide comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) as provided in Table 1 and Table 2, or a specific VH and VL combination as provided in Table 1 and Table 2. In other embodiments, the present disclosure provides nucleic acid molecules encoding a polypeptide comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) having at least 60%, 70%, 80%, 85%, 90%, 95%, 99% identity to the VH and / or VL amino acid sequences as provided in Table 1 and Table 2.

[0315] In some embodiments, the present disclosure provides nucleic acid molecules (or vectors) encoding polypeptides comprising one, two, three or more HCDRs and / or one, two, three or more LCDRs as provided in Table 3 and Table 4, or specific HCDR1-3 and LCDR1-3 combinations as provided in Table 3 and Table 4.

[0316] In some embodiments, the disclosure provides a nucleic acid molecule (or vector) encoding a polypeptide comprising an amino acid sequence as provided in Table 5. In another embodiment, the polypeptide comprises at least 60%, 70%, 80%, 85%, 90%, 95%, 99% identity to a CAR sequence as provided in Table 5. In another embodiment, the polypeptide is a CAR.

[0317] In some embodiments, the disclosure provides a nucleic acid molecule (or vector) comprising a nucleic acid sequence as provided in Table 6. In other embodiments, the disclosure provides a nucleic acid molecule (or vector) comprising a nucleic acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 99% identity to a nucleic acid sequence as provided in Table 6.

[0318] Combination therapy A composition or immunogenic composition comprising a PIC as described herein can be used in combination with CAR-expressing cells (CAR therapy). In some embodiments, in addition to combination with CAR-expressing cells, the composition or immunogenic composition can be used in combination with other known drugs and therapies. "Administered in combination," as used herein, means that two (or more) different therapies are administered to a subject during the course of the subject's suffering from a disorder, e.g., two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or resolved or the treatments are stopped for other reasons. In some embodiments, the delivery of one treatment is still ongoing when the delivery of a second treatment begins, thereby resulting in an overlap in administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective due to the combined administration. For example, the second treatment may be more effective, e.g., a comparable effect may be seen with less of the second treatment, or the second treatment may reduce symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation may be seen with the first treatment. In some embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be observed with one treatment delivered in the absence of the other. The effects of the two treatments can be partially additive, fully additive, or greater than additive. Delivery can be such that the effect of the first treatment delivered is still detectable at the time the second treatment is delivered.

[0319] The compositions or immunogenic compositions in combination with CAR-expressing cells as described herein and at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the compositions or immunogenic compositions in combination with CAR-expressing cells as described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

[0320] CAR therapy and / or other therapeutic agents, procedures, or modalities can be administered during periods of active disorder or during periods of remission or less active disease. CAR therapy can be administered before, concurrently with, or after other treatments, or during remission of a disorder.

[0321] When administered in combination, the composition or immunogenic composition, CAR therapy, and additional agent (e.g., a second or third agent), or all of them, can be administered in amounts or doses that are higher, lower, or the same as the amount or dosage of each agent used individually, e.g., as monotherapy. In certain embodiments, the administered amount or dosage of the composition or immunogenic composition, CAR therapy, additional agent (e.g., a second or third agent), or all of them, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as monotherapy. In other embodiments, the amount or dosage of the composition or immunogenic composition, CAR therapy, additional agent (e.g., a second or third agent), or all of them, that produces the desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent used individually, e.g., as monotherapy, required to achieve the same therapeutic effect.

[0322] In some embodiments, the present disclosure discloses a combination therapy comprising a composition or immunogenic composition described herein, a CAR-expressing cell therapy described herein, and an additional therapeutic agent.

[0323] PD-1 inhibitors In some embodiments, the additional therapeutic agent is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck & Co.), pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune). In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule as described in U.S. Patent Application Publication No. 2015 / 0210769, published on July 30, 2015, entitled "Antibody Molecules to PD-1 and Uses Thereof," which is incorporated by reference in its entirety. In one embodiment, the anti-PD-1 antibody molecule comprises the CDRs, variable regions, heavy chain and / or light chain of BAP049-Clone-E or BAP049-Clone-B disclosed in U.S. Patent Application Publication No. 2015 / 0210769. The antibody molecules described herein can be made by the vectors, host cells, and methods described in U.S. Patent Application Publication No. 2015 / 0210769, which is incorporated by reference in its entirety.

[0324] In one embodiment, the anti-PD-1 antibody molecule is nivolumab (Bristol-Myers Squibb), also known as MDX-1106, MDX-1106-04, ONO-4538, BMS-936558, or OPDIVO®. Nivolumab (clone 5C4) and other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-1 antibody molecule is pembrolizumab (Merck & Co.), also known as lambrolizumab, MK-3475, MK03475, SCH-900475, or KEYTRUDA®. Pembrolizumab and other anti-PD-1 antibodies are disclosed in Hamid, O. et al., (2013) New England Journal of Medicine 369(2):134-44, U.S. Pat. No. 8,354,509, and WO2009 / 114335, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-1 antibody molecule is pidilizumab (CureTech), also known as CT-011. Pidilizumab and other anti-PD-1 antibodies are disclosed in Rosenblatt, J. et al., (2011) J Immunotherapy 34(5):409-18, U.S. Pat. No. 7,695,715, U.S. Pat. No. 7,332,582, and U.S. Pat. No. 8,686,119, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDI0680 and other anti-PD-1 antibodies are disclosed in U.S. Patent No. 9,205,148 and WO 2012 / 145493, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (Regeneron). In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer). In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 or BGB-108 (Beigene). In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Incyte), also known as INCSHR01210 or SHR-1210.In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tesaro), also known as ANB011. Additional known anti-PD-1 antibody molecules include those described in WO2015 / 112800, WO2016 / 092419, WO2015 / 085847, WO2014 / 179664, WO2014 / 194302, WO2014 / 209804, WO2015 / 200119, U.S. Patent No. 8,735,553, U.S. Patent No. 7,488,802, U.S. Patent No. 8,927,697, U.S. Patent No. 8,993,731, and U.S. Patent No. 9,102,727, which are incorporated by reference in their entireties.

[0325] In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, e.g., as described in U.S. Patent No. 8,907,053, which is incorporated by reference in its entirety. In one embodiment, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In one embodiment, the PD-1 inhibitor is AMP-224 (B7-DCIg (Amplimmune), e.g., as disclosed in WO2010 / 027827 and WO2011 / 066342, which are incorporated by reference in their entireties).

[0326] PD-L1 inhibitors In some embodiments, the additional therapeutic agent is a PD-L1 inhibitor, hi some embodiments, the PD-L1 inhibitor is selected from FAZ053 (Novartis), atezolizumab (Genentech / Roche), avelumab (Merck Serono and Pfizer), durvalumab (MedImmune / AstraZeneca), or BMS-936559 (Bristol-Myers Squibb).

[0327] In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule as disclosed in U.S. Patent Application Publication No. 2016 / 0108123, published on April 21, 2016, entitled "Antibody Molecules to PD-L1 and Uses Thereof," which is incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises the CDRs, variable regions, heavy chain and / or light chain of BAP058-Clone O or BAP058-Clone N disclosed in U.S. Patent Application Publication No. 2016 / 0108123.

[0328] In one embodiment, the anti-PD-L1 antibody molecule is atezolizumab (Genentech / Roche), also known as MPDL3280A, RG7446, R05541267, YW243.55.S70, or TECENTRIQ™. Atezolizumab and other anti-PD-L1 antibodies are disclosed in U.S. Patent No. 8,217,149, which is incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule is avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab and other anti-PD-L1 antibodies are disclosed in WO2013 / 079174, which is incorporated by reference in its entirety. In one MEDI4736 embodiment, the anti-PD-L1 antibody molecule is durvalumab (MedImmune / AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in U.S. Patent No. 8,779,108, which is incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), also known as MDX-1105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in U.S. Patent No. 7,943,743 and WO2015 / 081158, which are incorporated by reference in their entireties. Additional known anti-PD-L1 antibodies include those described in, for example, WO2015 / 181342, WO2014 / 100079, WO2016 / 000619, WO2014 / 022758, WO2014 / 055897, WO2015 / 061668, WO2013 / 079174, WO2012 / 145493, WO2015 / 112805, WO2015 / 109124, WO2015 / 195163, U.S. Patent No. 8,168,179, U.S. Patent No. 8,552,154, U.S. Patent No. 8,460,927, and U.S. Patent No. 9,175,082, which are incorporated by reference in their entireties.

[0329] LAG-3 inhibitors In some embodiments, the additional therapeutic agent is a LAG-3 inhibitor, hi some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), or TSR-033 (Tesaro).

[0330] In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule as disclosed in U.S. Patent Application Publication No. 2015 / 0259420, published September 17, 2015, entitled "Antibody Molecules to LAG-3 and Uses Thereof," which is incorporated by reference in its entirety. In one embodiment, the anti-LAG-3 antibody molecule comprises the CDRs, variable regions, heavy chain and / or light chain of BAP050-Clone I or BAP050-Clone J disclosed in U.S. Patent Application Publication No. 2015 / 0259420.

[0331] In one embodiment, the anti-LAG-3 antibody molecule is BMS-986016 (Bristol-Myers Squibb), also known as BMS986016. BMS-986016 and other anti-LAG-3 antibodies are disclosed in WO 2015 / 116539 and U.S. Patent No. 9,505,839, which are incorporated by reference in their entireties. In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Tesaro). In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and Prima BioMed). IMP731 and other anti-LAG-3 antibodies are disclosed in WO 2008 / 132601 and U.S. Patent No. 9,244,059, which are incorporated by reference in their entireties. In one embodiment, the anti-LAG-3 antibody molecule is IMP761 (Prima BioMed). Additional known anti-LAG-3 antibodies include those described in, e.g., WO2008 / 132601, WO2010 / 019570, WO2014 / 140180, WO2015 / 116539, WO2015 / 200119, WO2016 / 028672, U.S. Patent No. 9,244,059, and U.S. Patent No. 9,505,839, which are incorporated by reference in their entireties. In one embodiment, the anti-LAG-3 inhibitor is a soluble LAG-3 protein, e.g., IMP321 (Prima BioMed), as disclosed in WO2009 / 044273, which is incorporated by reference in its entirety.

[0332] TIM-3 inhibitors In some embodiments, the additional therapeutic agent is a TIM-3 inhibitor, hi some embodiments, the TIM-3 inhibitor is MGB453 (Novartis) or TSR-022 (Tesaro).

[0333] In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule. In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule as disclosed in U.S. Patent Application Publication No. 2015 / 0218274, published August 6, 2015, entitled "Antibody Molecules to TIM-3 and Uses Thereof," which is incorporated by reference in its entirety. In one embodiment, the anti-TIM-3 antibody molecule comprises the CDRs, variable regions, heavy chain and / or light chain of ABTIM3-hum11 or ABTIM3-hum03 disclosed in U.S. Patent Application Publication No. 2015 / 0218274.

[0334] In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio / Tesaro). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or all of the CDR sequences collectively), heavy or light chain variable region sequences, or heavy or light chain sequences of APE5137 or APE5121. APE5137, APE5121, and other anti-TIM-3 antibodies are disclosed in WO2016 / 161270, which is incorporated by reference in its entirety. In one embodiment, the anti-TIM-3 antibody molecule is antibody clone F38-2E2. Additional known anti-TIM-3 antibodies include those described in, for example, WO2016 / 111947, WO2016 / 071448, WO2016 / 144803, U.S. Pat. No. 8,552,156, U.S. Pat. No. 8,841,418, and U.S. Pat. No. 9,163,087, which are incorporated by reference in their entireties.

[0335] Inhibitors of pro-M2 macrophage molecules In some embodiments, the additional therapeutic agent is an inhibitor of a pro-M2 macrophage molecule. Macrophages with an M2 phenotype are known to function in inhibiting T cell function, including cytotoxic function. Some cytokines, such as IL-13, IL-4, IL-10, CSF-1, TGF-beta, and GM-CSF, are known to polarize macrophages, for example, toward the M2 phenotype (in the case of IL-13 and / or IL-4), by interacting with the IL-13Ra1 and / or IL-4Ra chains expressed on macrophages. Molecules that inhibit such molecules are useful in the methods and compositions described herein. Exemplary inhibitors of pro-M2 macrophage molecules include, for example, inhibitors of IL-13, IL-4, IL-13Ra1, and / or IL-4Ra, as described herein.

[0336] Inhibitors of pro-M2 macrophage molecules include, for example, small molecules. An example of a small molecule inhibitor that can be administered with the CAR-expressing cells disclosed herein and the RNA molecules disclosed herein is pterostilbene (see, e.g., Huang et al., Oncotarget. 2016 Jun. 28;7(26):39363-39375, which is incorporated herein by reference in its entirety).

[0337] Inhibitors of pro-M2 macrophage molecules include, for example, antibody molecules, polypeptides, such as fusion proteins, or inhibitory nucleic acids, such as siRNA or shRNA, or CAR-expressing cells that bind to one or more surface antigens on MDSCs or TAMs.

[0338] In one embodiment, the inhibitor of pro-M2 macrophage molecules is an anti-IL-13 antibody. The production of such an antibody can be accomplished by methods known in the art. An example of an anti-IL-13 antibody is, for example, lebrikizumab (see CAS No. 953400-68-5). Another example of an anti-IL-13 antibody is tralokinumab (CAS No. 1044515-88-9). Another example of an anti-IL-13 antibody is or includes the anti-IL-13 binding domain of GSK2434735. Another example of an anti-IL-13 antibody is QAX576 (see, for example, Rothenberg et al., J. Allergy Clin. Immunol., 2015, 135(2), pp. 500-507, which is incorporated herein by reference in its entirety).

[0339] In another embodiment, the inhibitor of pro-M2 macrophage molecules is an anti-IL-4 antibody or an anti-IL-4Ra antibody. Such antibodies can be produced by methods known in the art. An example of an anti-IL-4 antibody is, for example, the anti-IL-4 binding domain of GSK2434735. Another example of an anti-IL-4 antibody is, for example, dupilumab (see CAS No. 1190264-60-8).

[0340] In another embodiment, the inhibitor of pro-M2 macrophages is an inhibitor of IL-13 and / or IL-4. An example of an IL-13 and IL-4 inhibitor that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is the vitamin A derivative fenretinide ((e.g., 4-HPR) see, e.g., Dong et al., Cancer Letters. 2017 Mar. 1. Vol. 388, pp. 43-53, which is incorporated by reference in its entirety).

[0341] In another embodiment, the inhibitor of a pro-M2 macrophage molecule is an anti-CSF-1 antibody or a small molecule inhibitor of CSF-1. The generation of such antibodies can be accomplished by methods known in the art. An example of an anti-CSF-1 antibody is emactuzumab. Another example of a CSF-1 inhibitor is BLZ945 (see, e.g., Strachan, DC et al., Oncoimmunology, December 1, 2013; 2(12):e26968, incorporated herein by reference in its entirety). Another example of a CSF-1 inhibitor that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is nintedanib (see, e.g., Tandon et al., American Journal of Respiratory and Critical Care Medicine 2017;195:A2397, incorporated herein by reference in its entirety). BLZ945 is a small molecule inhibitor of colony-stimulating factor 1 receptor (CSF1R). See, e.g., Pyonteck et al., Nat. Med. 19(2013):1264-72. The structure of BLZ945 is shown below.

[0342] [ka]

[0343] In another aspect, the inhibitor of a pro-M2 macrophage molecule is a CAR-expressing cell that binds to an antigen expressed on the surface of MDSCs or TAMs (i.e., a TAM antigen), for example, an antigen that is upregulated on the surface of MDSCs or TAMs compared to other macrophages. In embodiments, CAR-expressing cells that bind to MDSC or TAM antigens bind to CD123. In embodiments, CAR-expressing cells that bind to MDSC or TAM antigens bind to CSF1R. In embodiments, CAR-expressing cells that bind to MDSC or TAM antigens bind to CD68. In embodiments, CAR-expressing cells that bind to MDSC or TAM antigens bind to CD206.

[0344] In another embodiment, the inhibitor of pro-M2 macrophages is a JAK2 inhibitor. An example of a JAK2 inhibitor that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is ruxolitinib (see, e.g., Chen et al., Clinical Lymphoma, Myeloma and Leukemia, Vol. 17, No. 1, p. e93, 2017, incorporated herein by reference in its entirety). In another embodiment, the inhibitor of pro-M2 macrophage molecules is a cell surface molecule. An example of a cell surface molecule that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is dipeptidyl peptidase 4 (DPP-4) or CD26 (see, e.g., Zhuge et al., Diabetes 2016 Oct;65(10):2966-2979, incorporated herein by reference in its entirety).

[0345] In another embodiment, the inhibitor of pro-M2 macrophage molecules is an HDAC inhibitor. An example of an HDAC inhibitor that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is suberanilohydroxamic acid (SAHA). In another embodiment, the inhibitor of pro-M2 macrophage molecules is an inhibitor of the glycolytic pathway. An example of an inhibitor of the glycolytic pathway that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is 2-deoxy-d-glucose ((2-DG), see, e.g., Zanganeh, Nat Nanotechnol. 2016 Nov;11(11):986-994, which is incorporated by reference in its entirety.

[0346] In another embodiment, the inhibitor of pro-M2 macrophage molecules is a mitochondrial-targeted antioxidant. An example of a mitochondrial-targeted antioxidant that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is MitoQ (Formentini et al., Cell Reports, Vol. 19, No. 6, May 9, 2017, pp. 1202-1213, incorporated herein by reference in its entirety). In another embodiment, the inhibitor of pro-M2 macrophage molecules is an iron oxide. An example of an iron oxide that can be administered with the CAR-expressing cells disclosed herein and the compositions or immunogenic compositions disclosed herein is ferumoxytol (see, e.g., Zanganeh, Nat Nanotechnol. 2016 Nov;11(11):986-994, incorporated herein by reference in its entirety).

[0347] In embodiments, the present disclosure includes a composition comprising an inhibitor of a pro-M2 macrophage molecule and a pharmaceutically acceptable carrier.

[0348] Flt3 ligand polypeptide In some embodiments, the additional therapeutic agent is an Fms-like tyrosine kinase 3 ligand (Flt3 ligand) polypeptide. Flt3 ligand is a cytokine that affects the growth, survival, and / or differentiation of cells in the hematopoietic lineage. In combination with other growth factors, Flt3 ligand can stimulate the proliferation and development of various cell types, including stem cells, myeloid and lymphoid progenitor cells, dendritic cells, and NK cells. Exemplary Flt3 ligand polypeptides are disclosed in U.S. Patent Nos. 5,554,512, 6,291,661, 7,294,331, 7,361,330, and 9,486,519, which are incorporated by reference in their entireties.

[0349] chemotherapy drugs In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab). , antimetabolites (including, for example, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide). Common chemotherapy agents considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Cyclohexanone®), cyclosporine ... Platin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,Docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitabine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Flydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6 -mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Mylotarg, paclitaxel (Taxol®), Phoenix (yttrium 90 / MX-DTPA), pentostatin, polipheprosan 20 carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0350] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard, etc.) Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipob These include romane (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (also known as actinomycin-D, Cosmegen®); melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cisplatin (also known as cisplatin, Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); Clofosfamide (Cytoxan® and Neosar®); dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); ifosfamide (Ifex®); prednumustine; procarbazine (Matulane®); mechlorethamine (nitrogen mustard, mucosal) These include Mustargen®, also known as benzodiazepine and mechlorethamine hydrochloride; streptozocin (Zanosar®); thiotepa (thiophosphoamide, also known as TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine HCl (Treanda®).

[0351] Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formerly known as deferolimus, (1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0] 4,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication WO 03 / 064383; everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51 -3; temsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (2-Amino-8-| s-4-(2-hydroxybenzoyl)cyclohexyl]-6-(6-mcthoxy-3-pyridinyl)-4-mcthyl-pyrido|2,3-r / ]pyrimidin-7(8 / / )-onc) (PF04691502, CAS1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine ( / V2-| 1,4-dioxo-4-| |4-(4-oxo-8-phenyl-4 / / - 1 -bcnzopyran-2-yl)morpholinium-4-yl ]mcthoxy]butyl ]-L-arginylglycyl-La-aspartylL-serine-) (SEQ ID NO: 846), inner salt (SF1126, CAS936487-67-1), and XL765.

[0352] Exemplary immunomodulatory agents include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (a mixture of human cytokines including interleukin-1, interleukin-2, and interferon-g, CAS951209-71-5, available from IRX Therapeutics).

[0353] Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin.

[0354] Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

[0355] Exemplary proteosome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, (S)-4-methyl-N-[(S)-1-[[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]-2-[(S)-2-(2-morpholineacetamido)-4-phenylbutanamido]-pentanamide ((S)-4-Methyl- / V-((S)-1 -(((S)-4-methyl- 1 -((R)-2-methyloxiran-2-yl)-1 -oxopentan-2-yl)amino)- 1 -oxo-3- phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (O-Methyl- / V-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl- / V-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (phenylmethyl)ethyl]- L-serinamide) (ONX-0912).

[0356] Pharmaceutical Compositions and Treatments The pharmaceutical compositions of the present disclosure can include CAR-expressing cells, e.g., multiple CAR-expressing cells, combined with a composition or immunogenic composition as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer such as neutral buffered saline, phosphate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, or the like; a protein; an amino acid such as a polypeptide or glycine; an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide); and a preservative. In one embodiment, the compositions of the present disclosure are formulated for intravenous administration. The pharmaceutical compositions of the present disclosure can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but appropriate dosages can be determined through clinical trials.

[0357] In one embodiment, the pharmaceutical composition is substantially free, e.g., has no detectable levels of contaminants selected from the group consisting of endotoxin, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, bacteria, and fungi. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Group A hemolytic streptococcus.

[0358] Where an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present disclosure to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). Pharmaceutical compositions comprising T cells described herein are intended to be administered in amounts up to 10, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 1 4 ~10 9 cells / kg body weight, in some cases 10 5 ~10 6 It can be generally stated that T cell compositions can be administered in doses of 1000 cells / kg body weight. The T cell compositions can also be administered multiple times at these doses. The cells can be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0359] In certain embodiments, it may be desirable to administer activated T cells to a subject, followed by subsequent blood collection (or apheresis), activating T cells derived therefrom according to the present disclosure, and re-infusing these activated and expanded T cells back into the patient. This process can be performed multiple times, every few weeks. In certain embodiments, T cells can be activated from a blood collection of 10 cc to 400 cc. In certain embodiments, T cells are activated from a blood collection of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

[0360] Administration of the subject compositions can be by any convenient mode, including aerosol inhalation, injection, oral ingestion, infusion, implant, or transplant. The compositions described herein can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intralymph node, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In one embodiment, a T cell composition of the present disclosure is administered to a patient by intradermal or subcutaneous injection. In one embodiment, a CAR-expressing cell (e.g., T cell or NK cell) composition of the present disclosure is administered by iv infusion. A CAR-expressing cell (e.g., T cell or NK cell) composition can be injected directly into a tumor, lymph node, or site of infection.

[0361] In certain exemplary embodiments, a subject can undergo leukapheresis, in which leukocytes are collected, enriched, or ex vivo depleted to select and / or isolate cells of interest, e.g., immune effector cells (e.g., T cells or NK cells). These immune effector cell (e.g., T cell or NK cell) isolates can be expanded by methods known in the art and treated to allow for the introduction of one or more CAR constructs of the present disclosure, thereby generating CAR-expressing cells (e.g., CAR T cells or CAR-expressing NK cells) of the present disclosure. Subjects in need thereof can then undergo standard treatments, including high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after or concurrently with transplantation, the subject receives an infusion of the expanded CAR-expressing cells (e.g., CAR T cells or NK cells) in combination with a composition or immunogenic composition of the present disclosure. In additional aspects, the expanded cells combined with the RNA molecules described herein are administered before or after surgery. In embodiments, prior to administration of one or more cells expressing a CAR in combination with a composition or immunogenic composition as described herein, for example, lymphocyte depletion therapy is administered to the subject. In embodiments, the lymphocyte depletion therapy comprises administering one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0362] The dosage of the above-described treatments administered to a patient will vary depending on the condition being treated and the exact nature of the recipient of the treatment. Adjustments to dosages for human administration can be made according to art-accepted practices. For example, dosages for CAMPATH will generally be in the range of 1 to about 100 mg for an adult patient, usually administered daily for 1 to 30 days. A preferred daily dose is 1 to 10 mg / day, although higher doses of up to 40 mg / day can be used in some cases (as described in U.S. Pat. No. 6,120,766).

[0363] In one embodiment, the CAR is introduced into immune effector cells (e.g., T cells or NK cells), e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of the CAR immune effector cells (e.g., T cells or NK cells) of the present disclosure and one or more subsequent administrations of the CAR immune effector cells (e.g., T cells or NK cells) of the present disclosure, where the one or more subsequent administrations are administered less than 15 days after the previous administration, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In one embodiment, two or more administrations of the CAR immune effector cells (e.g., T cells or NK cells) of the present disclosure are administered to the subject (e.g., human) per week, e.g., two, three, or four administrations of the CAR immune effector cells (e.g., T cells or NK cells) of the present disclosure are administered per week. In one embodiment, a subject (e.g., a human subject) receives two or more administrations of CAR immune effector cells (e.g., T cells or NK cells) per week (e.g., two, three, or four administrations per week) (also referred to herein as cycles), followed by a week without CAR immune effector cells (e.g., T cells or NK cells), after which the subject is administered one or more additional administrations of CAR immune effector cells (e.g., T cells or NK cells) (e.g., two or more administrations of CAR immune effector cells (e.g., T cells or NK cells) per week). In another embodiment, a subject (e.g., a human subject) receives two or more cycles of CAR immune effector cells (e.g., T cells or NK cells), with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells (e.g., T cells or NK cells) are administered every other day for three doses per week. In one embodiment, the CAR immune effector cells (e.g., T cells or NK cells) of the disclosure are administered for at least 2, 3, 4, 5, 6, 7, 8 or more weeks.

[0364] In one aspect, the CAR-expressing cells (e.g., CART- or CAR-expressing NK cells) are generated using a lentiviral viral vector, such as a lentivirus, and the CAR-expressing cells (e.g., CART- or CAR-expressing NK cells) generated in that manner will have stable CAR expression. In one aspect, the disclosure features a cell that expresses a stimulatory RNA molecule, e.g., an immunostimulatory RNA molecule, disclosed herein, wherein the cell is generated using a lentiviral viral vector, such as a lentivirus.

[0365] In one embodiment, CAR-expressing cells, for example, CARTs, are produced using viral vectors, such as gammaretroviral vectors, for example, gammaretroviral vectors, as described herein.CARTs produced using these vectors can have stable CAR expression.In one embodiment, cells expressing stimulatory RNA molecules, for example, immunostimulatory RNA molecules, as disclosed herein, are produced using viral vectors, such as gammaretroviral vectors, for example, gammaretroviral vectors, as described herein.

[0366] In one embodiment, the CAR-expressing cells (e.g., CART- or CAR-expressing NK cells) transiently express the CAR vector for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of the CAR can be achieved by RNA CAR vector delivery. In one embodiment, the CAR RNA is transduced into cells, e.g., T cells or NK cells, by electroporation. In one embodiment, cells expressing a stimulatory RNA molecule, e.g., an immunostimulatory RNA molecule, disclosed herein transiently express the RNA molecule. In one embodiment, the stimulatory RNA molecule is delivered to the cell by electroporation. A potential problem that can arise in patients treated with transiently expressing CAR-expressing cells (e.g., CART- or CAR-expressing NK cells), particularly with murine scFv-bearing CAR-expressing cells (e.g., CART- or CAR-expressing NK cells), is anaphylaxis after multiple treatments. Without wishing to be bound by theory, it is believed that such anaphylactic responses can be triggered in patients who have developed a humoral anti-CAR response, i.e., anti-CAR antibodies with the anti-IgE isotype. The patient's antibody-producing cells are thought to undergo class switching from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype when there is a 10-14 day hiatus from exposure to the antigen.

[0367] If a patient is at high risk of developing an anti-CAR antibody response during the course of transient CAR therapy (such as that generated by RNA transduction), interruption of CAR-expressing cell (e.g., CART or CAR-expressing NK cell) infusion should not last more than 10-14 days.

[0368] The disclosures of each and every patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. While the present disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the present disclosure. It is intended that the appended claims be construed to include all such embodiments and equivalent variations. [Example]

[0369] The present disclosure will be further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise detailed. Therefore, the present disclosure should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0370] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compositions of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0371] Example 1 Preparation of immunogenic compositions containing polyinosinic-polycytidylic acid (PIC) kanamycin and calcium chloride or ZNP (a polyinosinic-polycytidylic acid-based adjuvant) An immunogenic composition containing polyinosinic-polycytidylic acid-based adjuvant (ZNP), polyinosinic-polycytidylic acid (PIC), kanamycin, and calcium chloride was prepared as previously reported (Cell Mol Immunol. 2007 April; 4(2):113-20). Under sterile conditions, the ZNP composition will be formulated in a physiologically acceptable buffer. The concentration and volume of the formulated composition can be adjusted according to factors such as the subject (including, but not limited to, age, sex, weight, and health condition), cancer status (including, but not limited to, cancer type and severity), administration route, and administration frequency. For the same therapeutically effective amount, a higher concentration of the composition requires a smaller administration volume; a lower concentration of the composition requires a larger administration volume.

[0372] PIC is unstable in the human body and can be rapidly degraded by nucleases, which limits its effective use in the human body. In this disclosure, the inventors envisioned that the presence of antibiotics (or polyamine compounds) and cations (calcium) in the ZNP composition can form a stable three-dimensional structure containing PIC, thereby increasing the PIC stability of the ZNP composition and enabling effective therapeutic use in the human body.

[0373] In the illustrative examples below, representative ZNP compositions are prepared at concentrations of 0.5 mg / ml to 10 mg / ml, and the PIC has a molecular weight ranging from 66,000 to 2,000,000 daltons.

[0374] Example 2A Preparation of immunogenic compositions containing EBV VLPs Epstein-Barr virus (EBV), a widespread human gamma-herpesvirus, causes persistent infection in over 95% of the world's population. Primary EBV infection is usually asymptomatic and often occurs during childhood. EBV is the causative agent of infectious mononucleosis and numerous malignancies, including several types of lymphomas, such as Hodgkin's lymphoma, Burkitt's lymphoma, and malignant B-cell lymphoma; age-related EBV-positive B-lymphoproliferative disorders (LPDs); T-cell and natural killer (NK)-cell LPDs; NK / T-cell lymphomas; leiomyosarcoma; and cancers such as nasopharyngeal carcinoma (NPC), EBV-associated gastric cancer (EBV-GC), and breast, lung, colon, and rectal cancer. EBV encodes multiple envelope glycoproteins. The most abundant glycoprotein on the virion surface, gp350, has been one of the most studied targets for the development of prophylactic subunit vaccines to neutralize infection of B cells.

[0375] An immunogenic composition containing EBV VLPs was prepared. Epstein-Barr virus virus-like particles (EB-VLPs) were prepared as described in WO 2022 / 084373. Briefly, EB-VLP producer cells 87H7 were plated in RPMI 1640 cell culture medium supplemented with 10% FBS, penicillin (100 U / ml), streptomycin (100 mg / ml), and puromycin (0.5 pg / ml) for 24 hours. The cells were then switched to additive-free RPMI 1640 cell culture medium containing 1 pM 4-hydroxytamoxifen to induce EB-VLP production for 4 days. The supernatant containing the secreted EB-VLPs was then collected and centrifuged at 300 g for 10 minutes and at 2,000 g for 20 minutes, respectively, to remove any cells and debris. After filtration through a 0.45 μm low protein-binding filter, EB-VLPs in the conditioned medium were purified by ultracentrifugation at 100,000 g for 2 h and 160,000 g for 1.5 h, respectively, and subsequently resuspended in filtered PBS for storage.

[0376] In an exemplary embodiment, the EB-VLP particles produced contain most of the EBV-encoded proteins, including gp350, BKRF4, BVRF1, BDLF3, BZLF2, BXLF2, BNRF1, BALF4, and BZLF1, but excluding LMP1, EBNA2, EBNA3a, EBNA3b, and EBNA3c. In another example, full-length or truncated proteins were expressed as fusions with the NDV M, NP, F, and HN proteins.

[0377] Example 2B Preparation of an immunogenic composition comprising polyinosinic-polycytidylic acid (PIC), kanamycin, and calcium chloride (i.e., ZNP) and EBV VLPs to form a ZNP-EBV VLP vaccine The ZNP composition (ZNP) described in Example 1 will be mixed with Epstein-Barr virus (EBV) virus-like particles (VLPs) described in Example 2A to form an EBV vaccine composition (referred to as "ZNP-EB-VLP vaccine") comprising polyinosinic-polycytidylic acid (PIC), kanamycin, and calcium chloride (i.e., ZNP) and EB-VLPs.

[0378] Example 3 Construction of a chimeric CAR construct containing anti-EBV Gp350 scFv Chimeric CAR constructs containing anti-EBV Gp350 scFvs that specifically bind to gp350 protein were designed and constructed (Tables 5 and 6). The amino acid sequences of the heavy chain variable region (VH), light chain variable region (VL), and their respective CDRs of anti-EBV Gp350 scFvs with the overall structure of VH-linker-VL or VL-linker-VH are shown in Tables 1 to 4. The linker used is S(G4S)3 (SEQ ID NO: 101). V01, V02, and V04 are sequences that contain / encode the hinge, transmembrane, and cytoplasmic regions of the CAR, respectively (the underlined sequences in Tables 5 and 6). The hinge region of the V01 sequence contains TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, the hinge region of V02 contains ESKYGPPCPPCP, and the hinge region of the V04 sequence contains ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0379] [Table 2]

[0380] [Table 3]

[0381] [Table 4]

[0382] [Table 5]

[0383] Table 6A

[0384] Table 6B

[0385] Table 6C

[0386] Table 6D

[0387] Table 6E

[0388]

Table 6F

[0389]

Table 6G

[0390] Table 6H

[0391] Table 6I

[0392]

Table 6J

[0393]

Table 6K

[0394]

Table 6L

[0395]

Table 6M

[0396]

Table 6N

[0397] Table 6O

[0398] [Table 6P]

[0399]

Table 6Q

[0400] Table 7A

[0401] Table 7B

[0402] Table 7C

[0403] Table 7D

[0404] Table 7E

[0405] Table 7F

[0406]

Table 7G

[0407]

Table 7H

[0408] Table 7I

[0409]

Table 7J

[0410]

Table 7K

[0411]

Table 7L

[0412]

Table 7M

[0413]

Table 7N

[0414] Table 7O

[0415] [Table 7P]

[0416]

Table 7Q

[0417]

Table 7R

[0418]

Table 7S

[0419]

Table 7T

[0420]

Table 7U

[0421]

Table 7V

[0422]

Table 7W

[0423]

Table 7X

[0424]

Table 7Y

[0425]

Table 7Z

[0426]

Table 7AA

[0427]

Table 7BB

[0428]

Table 7CC

[0429]

Table 7DD

[0430] Table 7EE

[0431]

Table 7FF

[0432]

Table 7GG

[0433]

Table 7HH

[0434] Table 7II

[0435]

Table 7JJ

[0436]

Table 7KK

[0437]

Table 7LL

[0438]

Table 7MM

[0439]

Table 7NN

[0440]

Table 700

[0441]

Table 7PP

[0442]

Table 7 QQ

[0443]

Table 7 RR

[0444]

Table 7SS

[0445]

Table 7TT

[0446]

Table 7UU

[0447]

Table 7VV

[0448]

Table 7WW

[0449]

Table 7XX

[0450]

Table 7YY

[0451]

Table 7ZZ

[0452]

Table 7AAA

[0453]

Table 7BBB

[0454]

Table 7 CCC

[0455] [Table 7DDD]

[0456] [Table 7EEE]

[0457] [Table 7FFF]

[0458] Example 4 Generation of CAR T cells containing anti-EBV Gp350 scfv To generate anti-EBV Gp350 CAR-T cells, PBMCs were first purified from buffy coat samples using Ficoll gradient density media. T cells were purified from PBMCs using a commercially available T cell isolation kit. Briefly, a third-generation recombinant lentiviral packaging system was utilized to prepare CAR-encoding lentiviral vectors. Seventy-two hours after transfection of the transfer plasmid and three helper plasmids into HEK293T cells, viral particles in the culture supernatant were concentrated using a Takara Lenti-X™ Concentrator according to the product manual. Subsequently, for CAR cell generation, the lentivirus was applied to infect enriched T cells preactivated with TransACT (Miltenyi Biotec) for 2 days in TexMACS medium supplemented with 10 ng / ml or 5 ng / ml of IL-7 and IL-15, respectively. Immunophenotypic characterization of CAR T cells during the process was analyzed using the LIVE / DEAD™ Violet Viability Kit for viability cell grouping and antibodies from BioLengend and Jackson Immunoresearch Laboratories to examine effector / memory and exhaustion states (CD3, CD4, CD8, CD45RA, CD62L, CCR7, Lags, Tim3, PD-1) (Chimeric Antigen Receptor T Cells, Development and Production, Springer (2020)). FACS studies were performed to confirm that T cells were successfully transduced with the anti-EBV Gp350 CAR, which is expressed on the surface of primary T cells and can recognize recombinant EBV Gp350 (data not shown).

[0459] The cytotoxic activity of an exemplary anti-EBV Gp350 CAR-T cell (CAR_EBV-Gp350-003) was evaluated. Specifically, anti-EBV Gp350 CAR-T cells were incubated with the gp350-expressing PCI-gp350 cell line (or PCI, a cell line without gp350 expression) at effector-to-target (E:T) ratios ranging from 5:1 to 10:1 in T cell expansion medium. Cell viability was measured, and cytotoxic activity was calculated in terms of % cytotoxicity.

[0460] As shown in Figure 1, an exemplary anti-EBV Gp350 CAR-T cell (CAR_EBV-Gp350-003) demonstrated potent killing of PCI-gp350 cells but no detectable activity in PCI-g cells, which lack gp350 expression.

[0461] Example 5 ZNP compositions (and ZNP-EBV-VLP compositions) promote T cell proliferation, cytokine release, and / or cytotoxicity in vitro T cells (or CAR-T cells) purified from human peripheral blood mononuclear cells (PBMCs) were pre-stained with CellTrace CFSE (carboxyfluorescein succinimidyl ester) staining solution according to the manufacturer's instructions for in vitro labeling of T cells to track T cell proliferation (multiple cell generation) by flow cytometry using dye dilution. T cells were co-incubated with PBMCs and the ZNP composition described in Example 1 at a predetermined working concentration (50, 100, or 200 μg / ml) or with ZNP-EBV-VLPs for 4 days. T cell proliferation was analyzed using a flow cytometer equipped with appropriate 488 nm excitation and emission filters for fluorescein.

[0462] As shown in Figure 2, co-incubation of the ZNP composition prepared in Example 1 with T cells and PBMCs promoted T cell activation and proliferation in a ZNP concentration-dependent manner, suggesting that the ZNP composition (or ZNP-EBV-VLP) may be useful in enhancing the efficacy of T cell therapy, including CAR-T cells.

[0463] Co-treatment with ZNP compositions (or ZNP-EBV-VLPs) will promote cytokine release from activated T cells (or CAR-T cells) and in vitro cytotoxicity. Activated T cells (or CAR-T cells) and target cancer cells will be co-cultured for 24 hours before supernatant analysis using an enzyme-linked immunosorbent assay (ELISA) to quantify released cytokines using an ELISA MAX™ standard set. The xCELLigence system will be used to evaluate T cell-mediated cytotoxicity at different effector-target (E:T) ratios. Cell-mediated killing will be quantified using electrical impedance readings every 30 minutes for the next 48 hours. % specific lysis values ​​will be calculated using GraphPad Prism software v6 for each replicate at each time point.

[0464] Example 6 Cytokine release and cell killing assays to assess the activity of anti-EBV Gp350 CAR T cells alone and in combination with ZNP or ZNP-EBV-VLP compositions Real-time cell analysis for the xCELLigence cytotoxicity assay: The xCELLigence system is utilized for the assessment of T cell-mediated cytotoxicity. 4Target-expressing cells will be placed into each well of the E-Plate and grown overnight, and electrical impedance will be quantified using an RTCA SP analyzer system. Approximately 24 hours later, 50 μL of anti-EBV Gp350 CAR-T cells will be added at different effector-to-target (E:T) ratios, or 50 μL of medium or 10% Triton-X100 will be added as negative and positive controls, respectively. Cell-mediated killing will be quantified based on electrical impedance readings every 30 minutes for the next 48 hours. Percent specific lysis values ​​will be calculated for each replicate at each time point using GraphPad Prism software v6.

[0465] Cytokine release assay: CAR-T cells and target cancer cells will be co-cultured in 96-well plates at a predetermined E:T ratio for 24 hours. The supernatant will then be analyzed using an enzyme-linked immunosorbent assay (ELISA) to quantify released cytokines according to the ELISA MAX™ standard set manufacturer's instructions. If necessary, the supernatant will be centrifuged to remove debris prior to analysis.

[0466] Example 7 Epstein-Barr virus (EBV) VLPs enhance the cytotoxicity of anti-EBV Gp350 CAR-T cells The cytotoxic activity of exemplary anti-EBV Gp350 CAR-T cells (CAR_EBV-Gp350-003) was evaluated in combination with or without EBV VLPs (EBV-VLP particles containing gp350, BKRF4, BVRF1, BDLF3, BZLF2, BXLF2, BNRF1, BALF4, and BZLF1) prepared in Example 2. The xCELLigence system was utilized for evaluation of T cell-mediated cytotoxicity.

[0467] Specifically, target cells (PCI-gp350 cell line expressing gp350 or negative control PCI cells) were cultured at 1 × 10 in 130 μL of DMEM medium + 10% FBS. 6Cells / well were initially seeded one day prior in xCELLigence E-Plates 96. Separately, 1 x 10 4 Raji cells (human B lymphoblastoid cell line as VLP target cells) were cultured at 1.25 × 10 cells per well in 50 μL RPMI medium + 10% FBS at 37 °C for 1 hour before loading onto xCELLigence E plates. 6 ;1.25×10 7 , or 1.25 × 10 8 After incubation, the cells were incubated with EBV VLPs. After incubation, the cells were subsequently incubated with anti-EBV Gp350 CAR-T cells and Raji cells (1 × 10 4 Cells) were seeded onto xCELLigence E plates for co-culture with target cells at an effector-target (E:T) ratio of 5:1. Cell-mediated killing was quantified using electrical impedance readouts every 30 minutes for the next 68 hours. Specific cell lysis (%) was calculated for each replicate at each time point using GraphPad Prism software v6.

[0468] As shown in Figure 3, co-incubation of EBV VLPs with Raji cells increased the number of VLPs in a manner correlated with the amount of VLPs (PCI-g+1.25 x 10 compared to PCI-g+0 VLPs). 7 VLP, PCI-g + 1.25 × 10 8 VLP) significantly enhances the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against the target cell PCI-gp350 cell line. In contrast, EBV VLP does not increase the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against control PCI cells.

[0469] Figure 4 further shows that the enhanced cytotoxicity of anti-EBV Gp350 CAR-T cells against target PCI-gp350 cells in the presence of EBV VLPs is dependent on co-incubation with VLP-targeted Raji cells. 1.25 x 10 8 VLP sample (Raji + 1.25 × 10 8VLPs) without co-incubation with Raji cells. 8 VLP sample (without Raji + 1.25 × 10 8 It exhibits much higher cytotoxicity than VLPs.

[0470] Figure 5 shows that anti-EBV Gp350 CAR-T cells exhibit little or low cytotoxicity against PCI cell lines without the gp350 antigen in the absence or presence of EBV VLPs.

[0471] Figure 6 shows the results of another set of experiments in which co-incubation of EBV VLPs with Raji cells significantly enhanced the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against the target cell PCI-gp350 cell line (PCI-g) in the VLP concentration regime (CAR+VLPe7 or CAR+VLPe8 compared to CAR alone). In contrast, EBV VLPs do not increase the cytotoxicity (% cytolysis) of anti-EBV Gp350 CAR-T cells against control PCI cells. EBV VLPs alone do not increase the cytotoxicity (% cytolysis) of mock T cells or PCI cells without gp350 expression.

[0472] Example 8 In vivo evaluation of anti-EBV Gp350 scfv-containing CAR T cells in tumor mouse models 4 × 10 6-week-old NOD-scid IL2Rγ null mice (NOG) (Charles River) 6 A xenograft model of nasopharyngeal carcinoma was established by subcutaneous (sc) injection of 100 μL of nasopharyngeal carcinoma cells (C666-1, genetically engineered to express gp350 protein and a luciferase tracker). The following day, mice were examined by IVIS imaging and then divided into groups for intravenous administration of T cells.

[0473] For imaging studies, mice were anesthetized with inhaled isoflurane and maintained at 1.5–2% isoflurane throughout the imaging procedure. Luciferase-based bioluminescence imaging was performed using an IVIS Lumina Series III imaging system equipped with a camera box and heated stage. After intraperitoneal injection of 150 mg / kg D-luciferin dissolved in phosphate-buffered saline (PBS) for 15 min, mouse images were acquired, and bioluminescence intensity was quantified and analyzed. The same region of interest (ROI) was selected across each mouse for total flux determination, presented as photons (p) / second (sec).

[0474] Gp350-targeting CAR-T cells were prepared using PBMCs from healthy donors according to an in-house protocol. At the time of T cell administration, 4 × 10 6 / 100μL or 2×10 6 CAR-T cells, mock T cells, or PBS were injected intravenously into C666-1 mouse models at 100 μL / 100 μL. Body weight and tumor growth were monitored 2-3 times weekly. Bioluminescence intensity levels were examined on days 7, 14, 18, and 21 to monitor changes in tumor cells. Necropsies were performed at 18 or 24 days to collect blood for FACS analysis; tumors and major organs were collected and weighed.

[0475] Figure 7 shows that Gp350 CAR-T cells inhibited the growth of nasopharyngeal carcinoma cell C666-1 and suppressed tumor formation in vivo. C666-1 (genetically engineered for gp350 and luciferase expression) cells were subcutaneously injected into mice, and 4 x 10 6 (4E6) or 2×10 6 This was followed by administration of (4E6)gp350 CAR-T or mock T cells. Figure 7A shows the results of the 4E6 gp350 CAR-T or mock T-cell administration on days 0, 7, 14, and 18 (4 × 10 6 group) or day 21 (2 × 10 6 The results of luminescence imaging of tumor cell growth at the time points (groups 4×10 and PBS) were recorded and compared, demonstrating specific tumor formation suppression by gp350 CAR-T cells. Figure 7B shows that 4×106 Figure 7C shows the mean tumor cell luminescence intensity change at days 0, 7, 14, 18, or 21 after CAR-T injection for the (4E6) CAR-T cell or mock T cell treatment groups. 6 Figure 7D shows the mean tumor cell luminescence intensity change after CAR-T injection at days 0, 7, 14, 18, or 21 for (2E6) CAR-T cells, mock T cells, or PBS treatment groups. Figure 7D shows the mean tumor volume change pattern after CAR-T injection at days 0, 7, 14, 18, or 21. Figure 7E shows dissection and tumor tissue mass assessment in mice across different experimental groups, demonstrating reduced tumor growth in the CAR-T treatment groups.

[0476] Figure 8 shows the 4×10 6 (4E6) CAR T and Mock T treatment groups (Figure 8A), and 2 x 10 at day 24 6 Further shown are cell percentage and count comparisons of human CD45+, CD8+, CAR+, and CAR+ / CD8+ cells in blood or spleen samples from (2E6) CAR T cells and mock T cell-treated groups (Figure 8B). The results show that CAR-T cells were found to be present in the blood and spleen while suppressing tumor formation.

[0477] For in vivo studies in T-cell lymphoma models, 2 × 10 6 / 100μL Jurkat-gp350-luc cells were injected for 5 or 7 days for tumor formation in mice, followed by gp350-targeted CAR-T cell treatment. In the former group, mice were examined by IVIS imaging and 1 × 10 6 (1E6) / 100 μL CAR-T cells or mock T cells were administered; in the latter group, mice were examined by IVIS imaging and 2 × 10 6(2E6) / 100 μL CAR-T or mock T were administered, respectively. Mice were weighed and observed 2-3 times a week. On days 7, 14, and 21, bioluminescence intensity levels were examined to monitor changes in tumor cells. The results show that Gp350 CAR-T cells inhibited T-cell lymphoma cell growth and suppressed tumor formation in vivo (Figure 9). Figure 9A shows the tumor profile of a T-cell lymphoma mouse model injected with Jurkat-gp350-luc cells for 5 or 7 days for tumor formation. Mice were examined by IVIS imaging, and 2 × 10 6 (2E6) or 1 x 10 6 (1E6) CAR-T cells or mock T cells were administered. Luminescence imaging of tumor cell growth was recorded and compared on days 0, 7, 14, 21, 28, and 32, demonstrating specific tumor formation suppression by gp350 CAR-T cells. Figures 9B and 9C show the results of 2×10 CAR-T cell-mediated tumor suppression after CAR-T injection. 6 (2E6) or 1 x 10 6 The change in mean tumor cell luminescence intensity over time is shown in (1E6) CAR-T or mock T groups.

[0478] Example 9 Tumor shrinkage induced by anti-EBV Gp350-containing CAR T cells in patients diagnosed with B-cell acute lymphoblastic leukemia with central nervous system involvement A clinical trial was conducted to test the safety and efficacy of EBV Gp350-specific CAR-T cells for the treatment of human subjects. ZYH, a 36-year-old man diagnosed with B-cell acute lymphoblastic leukemia with central nervous system involvement, received approximately 2.87 x 10 6 EBV Gp350-specific CAR-T cells were administered. Figure 10(A) shows MRI imaging results demonstrating lesions in the brain before (upper panel) and after (lower panel) treatment, which largely disappeared after treatment; Figure 10(B) shows that the percentage of abnormal blasts in the CSF decreased from day 1 to day 129 after treatment. After treatment, the subject was discharged without CRS, ICANS, or significant abnormalities in cardiac, liver, and kidney function tests.

Claims

1. (a) Polyinosinic-polycytidylic acid (PIC); (b) a stabilizer that is an aminoglycoside antibiotic or a non-aminoglycoside amine; (c) at least one cation; and (d) optionally, an immunogen that is a recombinant protein, virus-like particle (VLP), peptide, mRNA, or vaccine. An immunogenic composition comprising:

2. 1. An immunogenic composition comprising a VLP of Epstein-Barr virus (EBV), preferably wherein the VLP comprises glycoprotein 350 / 220 (gp350) protein or a fragment thereof, and preferably wherein the VLP comprises one, two, three or more polypeptides at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 102-117. and more preferably, the EBV-VLPs comprise the EBV proteins gp350, BKRF4, BVRF1, BDLF3, BZLF2, BXLF2, BNRF1, BALF4 and BZLF1, or functional variants thereof that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the polypeptide sequences described herein.

3. 3. The composition of claim 1 or 2 for use in cancer treatment.

4. 10. Use of a composition according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of cancer.

5. 5. The composition or use of claim 3 or 4, wherein the cancer treatment is immune cell therapy (preferably CAR-T cell therapy), and optionally the composition enhances the anti-cancer effect of the cell therapy.

6. 6. The composition or use according to any one of claims 3 to 5, for use in combination with engineered immune cells (preferably CAR-T cells or engineered TCR-T cells) in the treatment of cancer, optionally wherein the immunogenic composition is administered to the subject before, after or simultaneously with the immune cells.

7. 7. The composition or use according to any one of claims 1 to 6, wherein the immunogen is a tumor-associated antigen or an antigen associated with a virus, preferably the immunogen is a VLP of Epstein-Barr virus (EBV) comprising glycoprotein 350 / 220 (gp350) protein or a fragment thereof.

8. 8. The composition or use of any one of claims 1 to 7, wherein the stabilizer is an aminoglycoside antibiotic selected from kanamycin, streptomycin, dihydrostreptomycin, mannoside streptomycin, amikacin, amikacin, dibekacin, vietomycin, gentamicin, and any combination thereof; and preferably, the stabilizer is kanamycin.

9. 8. The composition or use according to any one of claims 1 to 7, wherein the stabilizer is a non-aminoglycoside amine selected from the group consisting of polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, galactose, polylysine, protamine, shell oligosaccharide, chitosan, spermine, glucosamine, and any combination thereof; preferably, the stabilizer is polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, galactose, polylysine, chitin, chitosan, or glucosamine; more preferably, the stabilizer is polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, ε-polylysine, hexylglucosamine, or acetylglucosamine.

10. 10. The composition or use according to any one of claims 1 to 9, wherein the cation is selected from the group consisting of calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, zinc, and any combination thereof; and preferably, the cation is calcium.

11. 11. The composition or use of any one of claims 5 to 10, wherein the immune cell is a T cell expressing a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding domain that specifically binds to an antigen expressed on the surface of cancer, and preferably the antigen-binding domain is an scFv domain.

12. 12. The composition or use of claim 11, wherein the intracellular domain comprises at least one costimulatory domain.

13. The costimulatory domains of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death 1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen 1 (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor tyrosine kinase (TNF-κB), and IgG4 receptor tyrosine kinase (IGK) are also expressed. Proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7 R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CE The composition or use of claim 12, which is a signaling region of ACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

14. The composition or use of claim 13, wherein the costimulatory domain comprises the signal transduction region of 4-1BB / CD137.

15. 14. The composition or use of claim 13, wherein the costimulatory domain comprises the signal transduction region of CD28.

16. 16. The composition or use of any one of claims 12 to 15, wherein the CAR comprises two or more costimulatory domains.

17. 17. The composition or use of claim 16, wherein the CAR comprises two costimulatory domains, one of the two costimulatory domains being CD28 and the other costimulatory domain being selected from 4-1BB / CD137 or OX40.

18. 18. The composition or use of any one of claims 11 to 17, wherein the intracellular domain of the CAR comprises at least one activation domain.

19. 19. The composition or use of claim 18, wherein the activation domain comprises CD3, preferably said CD3 is CD3 zeta.

20. 11. The composition or use of any one of claims 5 to 10, wherein the immune cell is a T cell, a natural killer (NK) cell, a TCR-expressing cell, a dendritic cell, a gamma delta T cell, or an NK-T cell, preferably the immune cell is a CAR-T cell.

21. 21. A method of increasing an anti-cancer response of immune cell therapy in an individual, comprising administering to the individual an effective amount of the immunogenic composition of any one of claims 1 to 20.

22. (a) administering to an individual an effective amount of the immunogenic composition of any one of claims 1 to 10; and (b) administering to the individual an effective amount of the cells of any one of claims 11 to 20.

1. A method of treating an individual with cancer, comprising:

23. 23. The composition, use, or method of any one of claims 3 to 22, wherein the cancer is a lymphoproliferative disorder (LPD) such as Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), B-cell lymphoma including diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma, NK / T-cell lymphoma, or post-transplant lymphoproliferative disorder (PTLD), or an EBV-associated cancer selected from epithelial carcinoma (nasopharyngeal, lung, breast), lymphoepithelioma, carcinoma with lymphocytic infiltration (GCLS, e.g., gastric cancer) or glioma.

24. 24. The composition, use, or method of any one of claims 3 to 23, wherein the antigen recognized by the CAR is an Epstein-Barr virus antigen (EBV antigen), preferably wherein the EBV antigen is EBV glycoprotein 350 / 220 (gp350 / 220).

25. The cells comprise a CAR comprising an antigen-binding domain comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) provided in Table 1 and Table 2, or a specific VH and VL combination provided in Table 1 and Table 2; preferably, the antigen-binding domain is (1) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 1 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 6 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (2) a heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 2 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 7 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (3) a heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 3 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 8 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (4) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 4 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 9 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (5) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 5 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 10 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (6) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (7) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (8) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (9) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (10) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (12) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (13) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (14) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; or (15) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof in which one, two, or three amino acids are substituted relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof in which one, two, or three amino acids are substituted relative to said sequence.

25. The composition, use or method of any one of claims 5 to 24, comprising:

26. The cells comprise a CAR comprising an antigen-binding domain comprising one, two, three or more HCDRs and / or one, two, three or more LCDRs provided in Table 3 and Table 4, or a combination of specific HCDRs 1-3 and LCDRs 1-3 provided in Table 3 and Table 4; preferably, the antigen-binding domain that binds to EBV gp350 / 220 is (1) SEQ ID NOs: 11, 12, 13, 26, 27, and 28, respectively; (2) SEQ ID NOs: 14, 15, 16, 29, 30, and 31, respectively; (3) SEQ ID NOs: 17, 18, 19, 32, 33, and 34, respectively; (4) SEQ ID NOs: 20, 21, 22, 35, 36, and 37, respectively; or (5) SEQ ID NOs: 23, 24, 25, 38, 39 and 40, respectively a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequence of An EBV glycoprotein 350 / 220 antigen-binding domain that competes with or binds to the same epitope in EBV glycoprotein 350 / 220 as that bound by any one of the antigen-binding domains (1) to (5) of the CAR described herein.

25. The composition, use or method of any one of claims 5 to 24, comprising:

27. 25. The composition, use, or method of any one of claims 5 to 24, wherein the cell comprises a CAR comprising any one of the CAR amino acid sequences provided in Table 5.

28. 25. The composition, use or method of any one of claims 3 to 24, wherein the composition and cells are (to be) administered simultaneously or sequentially, for example, the composition is (to be) administered prior to or subsequent to administration of the cells.

29. 1. A CAR, or an immune cell (preferably a CAR-T cell) comprising a CAR, wherein the CAR comprises an EBV glycoprotein 350 / 220 antigen-binding domain, and the antigen-binding domain comprises a heavy chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 138, 139, or 140, and / or a light chain variable region having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 141, 142, or 143.

30. The antigen-binding domain that binds to EBV gp350 / 220 is (1) SEQ ID NOs: 11, 12, 13, 26, 27, and 28, respectively; (2) SEQ ID NOs: 14, 15, 16, 29, 30, and 31, respectively; (3) SEQ ID NOs: 17, 18, 19, 32, 33, and 34, respectively; (4) SEQ ID NOs: 20, 21, 22, 35, 36, and 37, respectively; or (5) SEQ ID NOs: 23, 24, 25, 38, 39 and 40, respectively a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequence of An EBV glycoprotein 350 / 220 antigen-binding domain that competes with or binds to the same epitope in EBV glycoprotein 350 / 220 as that bound by any one of the antigen-binding domains (1) to (5) of the CAR described herein.

30. The CAR or immune cell (preferably a CAR-T cell) of claim 29, comprising:

31. EBV glycoprotein 350 / 220 antigen-binding domain, (1) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 1 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 6 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (2) a heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 2 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 7 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (3) a heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 3 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 8 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (4) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 4 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 9 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (5) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 5 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 10 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; or (6) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (7) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (8) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 138 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (9) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (10) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (12) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 139 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (13) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 141 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; (14) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 142 or a variant thereof having one, two, or three amino acid substitutions relative to said sequence; or (15) A heavy chain variable region (VH) having the polypeptide sequence of SEQ ID NO: 140 or a variant thereof in which one, two, or three amino acids are substituted relative to said sequence, and a light chain variable region (VL) having the polypeptide sequence of SEQ ID NO: 143 or a variant thereof in which one, two, or three amino acids are substituted relative to said sequence.

31. A CAR or immune cell (preferably a CAR-T cell) according to claim 29 or 30, comprising any one of:

32. 32. The CAR or immune cell (preferably a CAR-T cell) of any one of claims 29 to 31, wherein the EBV glycoprotein 350 / 220 antigen-binding domain comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) provided in Table 1 and Table 2, or a specific VH and VL combination provided in Table 1 and Table 2.

33. 33. The CAR or immune cell (preferably a CAR-T cell) of any one of claims 29 to 32, wherein the EBV glycoprotein 350 / 220 antigen-binding domain comprises one, two, three or more HCDRs and / or one, two, three or more LCDRs provided in Table 3 and Table 4, or a combination of specific HCDRs 1-3 and LCDRs 1-3 provided in Table 3 and Table 4.

34. 34. The CAR or immune cell (preferably a CAR-T cell) of any one of claims 29 to 33, wherein the EBV glycoprotein 350 / 220 antigen-binding domain comprises an amino acid sequence of at least 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identity to any one of the CAR sequences provided in Table 5, and preferably the CAR is selected from the group consisting of SEQ ID NOs: 144 to 161.

35. 35. The immune cell (preferably a CAR-T cell) according to any one of claims 29 to 34, for use in cancer treatment, wherein preferably said cancer is a lymphoproliferative disorder (LPD) such as Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), B-cell lymphoma including diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma, NK / T-cell lymphoma, or post-transplant lymphoproliferative disorder (PTLD), or an EBV-associated cancer, for example selected from epithelial carcinoma (nasopharyngeal, lung, breast), lymphoepithelioma, carcinoma with lymphocytic infiltration (GCLS, e.g. gastric cancer) or glioma.

36. 36. An isolated nucleic acid molecule, preferably in the form of an isolated vector, such as an isolated viral vector, comprising a nucleotide sequence encoding a CAR polypeptide as defined in any one of claims 1 to 35.

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