TW202340455A - Improved virus-specific t cell - Google Patents

Abstract

The disclosure provides, at least in part, compositions and methods for generating antigen-specific T cells, optionally a virus-specific T cell. The disclosure further provides methods for generating an expanded population of said T cells. The disclosure further provides exemplary methods of administering a therapeutically effective amount of a composition comprising an antigen-specific T cell to a subject for the treatment of disease or disorder.

Description

Improved virus-specific T cells

The present invention generally relates to compositions and methods for generating antigen-specific T cells from monocytes. The monocytes may be peripheral blood monocytes (PBMC). Other aspects and advantages of the present invention will be apparent from the following embodiments of the present invention.

The following description includes information applicable to understanding the invention. There is no admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Disease epidemics remain a problem in today's society. Organisms are constantly exposed to and become victims of viral or pathogenic agents and/or malignancies that can lead to physiological consequences, physical damage or even death. For example, hepatitis B is a serious liver infection caused by hepatitis B virus (HBV) and is considered a major public health problem. For some people, hepatitis B infection becomes chronic (long-term) and is associated with an increased risk of liver failure, liver cancer, or cirrhosis (a condition that damages the liver over time). Individuals chronically infected with HBV have a deficiency of endogenous T cells, including dysfunctional and exhausted HBV-reactive cells. Human herpesvirus 8 (HHV8) (also known as Kaposi sarcoma-associated herpesvirus) is also an example of a public health problem associated with serious disease. HHV8 establishes latency after primary infection and can be reactivated in immunocompromised individuals. Reactivation of HHV8 infection can lead to Kaposi's sarcoma, primary effusion lymphoma (PEL), and multi-centric Castleman's disease (MCD).

There is a long-standing unmet need in the art for effective compositions that can treat disease and for adequate methods of producing such compositions. The present invention provides a solution to these unmet needs.

The following embodiments and aspects thereof are described in conjunction with systems, tools, and methods that are intended to be illustrative, non-limiting in scope.

In some aspects, the invention provides an ex vivo method for expanding T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; contacting the cells with one or more compositions, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; (ii) comprising IL-4 and IL -7 two or more interleukins; and (iii) IL-15; and culturing the cells with the components of (i), (ii) and (iii) sufficient to expand the T cells time period; thereby expanding the T cells.

In some aspects, the invention provides an ex vivo method for expanding T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; contacting the cells with one or more compositions, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; (ii) comprising IL-4 and IL -7 two or more interleukins; and (iii) IL-15; and culturing the cells with the components of (i), (ii) and (iii) to expand the T cells; These T cells are thereby expanded.

In some or any of the foregoing or related embodiments, the cells are contacted with the components of (i) and (ii) from about 0 hours to about 48 hours before being contacted with the component of (iii).

In some or any of the foregoing or related embodiments, the two or more interleukins do not comprise IL-2.

In some or any of the foregoing or related embodiments, the ingredients of (i) and (ii) are provided in a composition. In some or any of the foregoing or related embodiments, the ingredients of (i) and (ii) are provided in separate compositions. In some or any of the foregoing or related embodiments, the component of (iii) is added to a composition comprising the components of (i) and (ii). In some or any of the foregoing or related embodiments, the cells are contacted with components of (i) and (ii) simultaneously. In some or any of the foregoing or related embodiments, the cells are contacted with components of (i) and (ii) sequentially.

In some or any of the foregoing or related embodiments, the cells are about 0 hours, about 2 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours before being contacted with the component of (iii). , about 36 hours, or about 48 hours of contact with ingredients (i) and (ii).

In some or any of the foregoing or related embodiments, the cells are in contact with the component of (iii) from about 0 hours to about 48 hours, from about 1 hour to about 48 hours, from about 2 hours to about 48 hours. , about 12 hours to about 48 hours, about 24 hours to about 48 hours, about 36 hours to about 48 hours, about 0 hours to about 24 hours, about 2 hours to about 24 hours, about 6 hours to about 24 hours, about 12 hours to about 24 hours, or about 18 hours to about 24 hours in contact with ingredients (i) and (ii).

In some or any of the foregoing or related embodiments, the ingredients of (i), (ii) and (iii) are provided in separate compositions. In some or any of the foregoing or related embodiments, ingredients (i) and (ii) are provided in a composition. In some or any of the foregoing or related embodiments, the component of (iii) is added to a composition comprising the components of (i) and (ii). In some or any of the foregoing or related embodiments, the ingredients of (i), (ii) and (iii) are provided in a composition.

In some or any of the foregoing or related embodiments, about 0 days, about 1 day, about 2 days, about 3 days, about 4 days after the cells are contacted with the components of (i) and (ii) , about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, so that the cells are contacted with the component of (iii). In some or any of the foregoing or related embodiments, about 0 days to about 6 days, about 0 days to about 5 days, about 0 days after the cells are contacted with the components of (i) and (ii) The cells are contacted with the component of (iii) to about 4 days, from about 0 days to about 3 days, from about 0 days to about 2 days, from about 0 days to about 1 day. In some or any of the foregoing or related embodiments, the period of time sufficient to expand the T cells is about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days. In some or any of the foregoing or related embodiments, the cells are cultured for about 7 days to about 14 days, about 8 days to about 10 days, about 8 days to about 14 days, about 8 days to about 18 days. days, about 14 days to about 18 days, about 1 week to about 2 weeks, about 1 week to about 3 weeks, or about 1 week to about 4 weeks. In some or any of the foregoing or related embodiments, the cells are cultured for about 18 days or less, optionally about 14 days.

In some or any of the foregoing or related embodiments, the cells are cultured for an amount of time sufficient to produce cells that have completed log phase growth.

In some or any of the foregoing or related embodiments, the period during which the cells are in contact with component (i) is the priming period. In some or any of the foregoing or related embodiments, the activation period is day 0. In some or any of the foregoing or related embodiments, when ingredients (i) and (ii) are added on day 0, the start-up period includes adding ingredient (ii). In some or any of the foregoing or related embodiments, when ingredients (i), (ii) and (iii) are added on day 0, the start-up period includes adding ingredient (iii). In some or any of the foregoing or related embodiments, priming is sufficient to stimulate T cells.

In some or any of the foregoing or related embodiments, the expansion period begins after contacting the cells with components (i) and (ii). In some or any of the foregoing or related embodiments, the expansion period begins on day 1 or day 2 of culture. In some or any of the foregoing or related embodiments, the expansion phase includes contacting the cells with component (iii).

In some or any of the foregoing or related embodiments, the expansion period begins on day 1 or day 2 of culture. In some or any of the foregoing or related embodiments, when ingredient (ii) is added on day 1 or day 2, the expansion period includes the addition of ingredient (ii). In some or any of the foregoing or related embodiments, when ingredient (iii) is added on day 1 or day 2, the expansion period includes the addition of ingredient (iii).

In some or any of the foregoing or related embodiments, the expansion period continues until the cells have completed log phase growth.

In some or any of the foregoing or related embodiments, the expanded T cells comprise T cells specific for at least one target antigen. In some or any of the foregoing or related embodiments, the expanded T cells are enriched with T cells specific for at least one target antigen.

In some or any of the foregoing or related embodiments, the monocytes are peripheral blood monocytes (PBMC). In some or any of the foregoing or related embodiments, the PBMCs are human PBMCs, donor PBMCs, and/or allogeneic PBMCs. In some or any of the foregoing or related embodiments, the T cells are human T cells, donor T cells, and/or allogeneic T cells.

In some or any of the foregoing or related embodiments, the cells are cultured in the presence of VST expansion medium.

In some or any of the foregoing or related embodiments, the cells are contacted with a pepmix at a concentration of about 0.2 nanogram/peptide/ml to about 20 nanogram/peptide/ml. In some or any of the foregoing or related embodiments, the cells are contacted with the mixed peptide at a concentration of about 2 nanograms/peptide/ml.

In some or any of the foregoing or related embodiments, the cells are contacted with IL-4 at a concentration of about 400 U/mL to about 1200 U/mL. In some or any of the foregoing or related embodiments, the cells are contacted with IL-4 at a concentration of about 800 U/mL.

In some or any of the foregoing or related embodiments, the cells are contacted with IL-7 at a concentration of about 5 ng/mL to about 30 ng/mL. In some or any of the foregoing or related embodiments, the cells are contacted with IL-7 at a concentration of about 20 ng/mL.

In some or any of the foregoing or related embodiments, the cells are contacted with IL-15 at a concentration of about 1 ng/mL to about 20 ng/mL. In some or any of the foregoing or related embodiments, the cells are contacted with IL-15 at a concentration of about 5 ng/mL.

In some or any of the foregoing or related embodiments, the cells are mixed with mixed peptide at a concentration of about 2 nanograms/peptide/ml, IL-7 at a concentration of about 20 ng/mL, and a concentration of about 800 U /mL of IL-4 and IL-15 at a concentration of approximately 5 ng/mL.

In some or any of the foregoing or related embodiments, the cells are contacted with IL-15 on day 0, day 1 or day 2 of culture.

In some or any of the foregoing or related embodiments, the interleukins are recombinant interleukins. In some or any of the foregoing or related embodiments, the interleukins are human recombinant interleukins. In some or any of the foregoing or related embodiments, the interleukins are human interleukins.

In some or any of the foregoing or related embodiments, the cells are present at about 1.5 × 10 ⁶ cells/cm2 to about 4 × 10 ⁶ cells/cm2 or about 2 × 10 ⁶ cells/cm2 cm to about 4 × 10 ⁶ cells/cm2, and culture at a cell seeding density of about 3 × 10 ⁶ cells/cm2 depending on the situation.

In some or any of the foregoing or related embodiments, the number of expanded T cells or the number of cells in the expanded T cell population is at least 2-fold greater than the number of cells in the starting cell population. In some or any of the foregoing or related embodiments, the number of expanded T cells or the number of cells in the expanded T cell population is about 2-fold to about 7-fold greater than the number of cells in the starting cell population. .

In some or any of the foregoing or related embodiments, the expanded T cells or the expanded T cell population comprise antigen-specific T cells. In some or any of the foregoing or related embodiments, the antigen-specific T cells are pathogen-specific T cells (PST) or virus-specific T cells (VST). In some or any of the foregoing or related embodiments, the antigen-specific T cells are tumor-specific T cells (TST).

In some or any of the preceding or related embodiments, the expanded T cells or population of expanded T cells comprise VSTs that recognize at least one antigen from hepatitis B virus (HBV).

In some or any of the preceding or related embodiments, the expanded T cells or population of expanded T cells comprise VSTs that recognize at least one antigen from human herpesvirus 8 (HHV-8).

In some or any of the foregoing or related embodiments, the peptide mixture includes from about 2 to about 750 different peptides. In some or any of the foregoing or related embodiments, each peptide in the peptide mixture has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 with at least one other peptide. At least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 amino acids overlap. In some or any of the foregoing or related embodiments, the peptides are at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 , at least 15, at least 16, at least 17, at least 18 amino acids long.

In some or any of the foregoing or related embodiments, the peptides are about 15 amino acids long and overlap by about 11 amino acids. In some or any of the foregoing or related embodiments, the peptides are 15 amino acids long and overlap by 11 amino acids.

In some or any of the preceding or related embodiments, the peptide mixture comprises one or more target antigens from at least one pathogen, or a portion thereof. In some or any of the foregoing or related embodiments, the at least one pathogen is selected from cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, Varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), human herpes virus 8 (HHV -8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, bocavirus, coronavirus , Lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile West Nile virus (WNV), Zika virus, Ebola virus and human T-lymphotropic virus. In some or any of the foregoing or related embodiments, the at least one pathogen is hepatitis B virus (HBV). In some or any of the foregoing or related embodiments, the at least one pathogen is human herpesvirus 8 (HHV-8).

In some or any of the foregoing or related embodiments, the pathogen is HBV, and the one or more target antigens include E antigen (HBeAg), P antigen (HBP), and LE antigen (HBsAg). In some or any of the foregoing or related embodiments, the pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP) ), X antigen (HBX) or a combination thereof. In some or any of the foregoing or related embodiments, the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX ) or a combination thereof. In some or any of the foregoing or related embodiments, the HBeAg (E antigen) comprises pre-core (PreC) and core (HBcAg) antigens.

In some or any of the foregoing or related embodiments, the peptide mixture comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2) , AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and/or EYLVSFGVWIRTPPA (SEQ ID NO: 5).

In some or any of the foregoing or related embodiments, the one or more HBV antigens are selected from one or more HBV genotypes.

In some or any of the foregoing or related embodiments, at least two antigens are selected from the same HBV genotype. In some or any of the foregoing or related embodiments, at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first genotype and the second genotype Different types. In some or any of the foregoing or related embodiments, at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

In some or any of the foregoing or related embodiments, the amino acid sequence of each HBV antigen differs by at least one amino acid.

In some or any of the foregoing or related embodiments, the expanded T cells or population of expanded T cells are responsive upon stimulation.

In some or any of the foregoing or related embodiments, at least a portion of the T cell reactivity is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted.

In some or any of the preceding or related embodiments, the pathogen is: (a) EBV, and the at least one target antigen is EBNA1, LMP2, BZLF1 or a combination thereof; (b) CMV, and the at least one target antigen is IE1, pp65 or a combination thereof; (c) Adv, and the at least one target antigen is Hexon, Penton or a combination thereof; (d) BK virus, and the at least one target antigen is LT, VP-1 or a combination thereof; (e) HHV6, and the at least one target antigen is U14, U11, U71, U54, U90 or a combination thereof; (f) RSV, and the at least one target antigen is N, F or a combination thereof; (g) Influenza virus, and the at least one target antigen is MP1, NP1 or a combination thereof; (h) Parainfluenza virus type (PIV), and the at least one target antigen is F, N, M, M2-1, HN or a combination thereof; (i) hMPV, and the at least one target antigen is F, N, M2-1, M or a combination thereof; (j) HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( Kaposin) (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof; and/or (j) SARS-CoV2, and the at least one target antigen is spike protein (Spike) (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4 , nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some or any of the foregoing or related embodiments, the pathogen is HHV8, and the one or more target antigens include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72) , RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70). In some or any of the foregoing or related embodiments, the pathogen is HHV8, and the one or more target antigens comprise:

One or more latent antigens, including Kaposi protein (ORF12, K12), LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vFLIP (ORF71), vCYC (ORF72) and RTA (ORF50); and/or

One or more lytic antigens, including gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some or any of the foregoing or related embodiments, the pathogen is HHV8 and the one or more target antigens comprise LANA-1 (ORF3) and LANA-2 (vIRF3, K10.5). In some or any of the foregoing or related embodiments, the pathogen is HHV8 and the one or more target antigens comprise LANA-1 (ORF3) and gB (ORF8).

In some or any of the foregoing or related embodiments, the pathogen is SARS-CoV, and the one or more target antigens include spike protein (S), membrane protein (M), nucleocapsid protein (N) , accessory protein 7a (AP7a) and nonstructural protein 4 (nsp4).

In some or any of the foregoing or related embodiments, the peptide mixture comprises a peptide comprising or consisting of a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7) , TTDTHSPSALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

In some or any of the foregoing or related embodiments, the method includes obtaining expanded T cells or a population of expanded T cells. In some or any of the foregoing or related embodiments, the method includes the step of preparing a pharmaceutical composition, wherein to the expanded T cells or expanded T cell population a diluent, a stabilizer, a preservative, and a /or other pharmaceutically acceptable excipients.

In some aspects, the invention provides an expanded T cell population obtained by the method of some or any of the foregoing or related embodiments.

In some or any of the foregoing or related embodiments, the T cell population includes: (a) At least 70% CD3+ T cells; and/or (b) CD4+ and CD8+ T cells.

In some or any of the foregoing or related embodiments, the expanded T cell population comprises VST.

In some or any of the foregoing or related embodiments, (a) Approximately 30% or less of the cells in the expanded T cell population are natural killer (NK) cells; (b) More than approximately 80% of the T cell population includes CD4+ and/or CD8+ T cells; (c) The T cells are multi-strain; and/or (d) At least 1% of such CD3+ T cells can produce TNFα.

In some or any of the preceding or related embodiments, at least one T cell recognizes one or more target antigens from at least one pathogen, or a portion thereof.

In some or any of the foregoing or related embodiments, the at least one pathogen is selected from cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, Varicella zoster virus (VZV), Estén-Barr virus (EBV), cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human Herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, bocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumovirus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and Human T-lymphotropic virus.

In some or any of the foregoing or related embodiments, the at least one pathogen is hepatitis B virus (HBV). In some or any of the foregoing or related embodiments, the at least one pathogen is human herpesvirus 8 (HHV-8).

In some or any of the foregoing or related embodiments, (a) The pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX) or a combination thereof; (b) The pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX) or a combination thereof; and/or (c) The pathogen is HBV, and the at least one target antigen is HBcAg, HBsAg or a combination thereof.

In some or any of the foregoing or related embodiments, the HBeAg (E antigen) comprises pre-core (PreC) and core (HBcAg) antigens. In some or any of the foregoing or related embodiments, the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and EYLVSFGVWIRTPPA (SEQ ID NO: 5).

In some or any of the foregoing or related embodiments, at least two HBV antigens are selected from different HBV genotypes. In some or any of the foregoing or related embodiments, at least two antigens are selected from the same HBV genotype. In some or any of the foregoing or related embodiments, (a) at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first genotype and the The second genotype is different; and/or (b) at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

In some or any of the foregoing or related embodiments, the amino acid sequence of each HBV antigen differs from the amino acid sequence of each other HBV antigen by at least one amino acid.

In some or any of the foregoing or related embodiments, the expanded T cell population is reactive upon stimulation; optionally, wherein at least a portion of the T cell reactivity is affected by HLA-DR, HLA-DQ or HLA-DP restrictions.

In some or any of the foregoing or related embodiments, (a) The pathogen is EBV, and the at least one target antigen is EBNA1, LMP2, BZLF1 or a combination thereof; (b) The pathogen is CMV, and the at least one target antigen is IE1, pp65 or a combination thereof; (c) The pathogen is Adv, and the at least one target antigen is hexon, penton or a combination thereof; (d) The pathogen is BK virus, and the at least one target antigen is LT, VP-1 or a combination thereof; (e) The pathogen is HHV6, and the at least one target antigen is U14, U11, U71, U54, U90 or a combination thereof; (f) The pathogen is RSV, and the at least one target antigen is N, F or a combination thereof; (g) The pathogen is influenza virus, and the at least one target antigen is MP1, NP1 or a combination thereof; (h) The pathogen is a parainfluenza virus (PIV), and the at least one target antigen is F, N, M, M2-1, HN or a combination thereof; (i) The pathogen is hMPV, and the at least one target antigen is F, N, M2-1, M or a combination thereof; (j) The pathogen is HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kapos Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof; and/or (k) The pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4 , nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some or any of the foregoing or related aspects, the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: ( SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

In some aspects, the invention provides a plurality of ex vivo expanded T cell populations, each according to some or any of the foregoing or related embodiments, wherein each ex vivo expanded T cell population comprises a population obtained from T cells generated from donor monocytes from different donors; optionally, the HLA type of each donor is different from at least one HLA allele gene of at least one of the other donors.

In some aspects, the invention provides a universal antigen-specific T cell therapy product comprising an ex vivo expanded T cell population or a plurality of expanded T cells according to some or any of the foregoing or related embodiments. The T cell population includes T cells generated from donor monocytes obtained from different donors.

In some or any of the foregoing or related embodiments, (a) The HLA type of each donor is different from at least one HLA allele gene of at least one of the other donors; (b) The product exhibits reduced or negligible alloreactivity to partially HLA-matched and/or HLA-mismatched target cells; and/or (c) The product contains a VST that recognizes one or more target antigens from at least one pathogen, or a portion thereof.

In some aspects, the invention provides a pharmaceutical composition comprising an ex vivo expanded T cell population, a plurality of expanded T cell populations, or a universal population of some or any of the foregoing or related embodiments. Antigen-specific T cell therapy products.

In some or any of the preceding or related embodiments, the pharmaceutical composition is formulated for intravenous delivery.

In some or any of the foregoing or related embodiments, (a) The pharmaceutical composition is negative for bacteria and fungi during continuous culture for at least 7 days; (b) The pharmaceutical composition exhibits endotoxin levels below 5 EU/ml; and/or (c) The pharmaceutical composition is negative for Mycoplasma species.

In some aspects, the invention provides a population of ex vivo expanded VSTs, wherein the VSTs are specific for two or more HBV antigens selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX.

In some aspects, the invention provides a population of ex vivo expanded VSTs, wherein the VSTs are specific for HBV antigens including E antigen (HBeAg), P antigen (HBP), and LE antigen (HBsAg).

In some or any of the foregoing or related embodiments, the antigens E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg) are immunodominant antigens. In some or any of the foregoing or related embodiments, the VSTs are specific for two or more HBV antigens selected from HBsAg, HBeAg, HBP and HBX. In some or any of the foregoing or related embodiments, the VSTs are specific for the HBV antigens HBcAg and HBsAg. In some or any of the foregoing or related embodiments, the E antigen (HBeAg) includes pre-core (PreC) and core (HBcAg) antigens.

In some aspects, the invention provides a population of ex vivo expanded VST pairs, wherein the VST pairs are selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) are specific for two or more HHV8 antigens .

In some aspects, the invention provides a population of ex vivo expanded VST pairs, wherein the VST pairs include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA ( ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) are specific for HHV8 antigens.

In some aspects, the invention provides a population of ex vivo expanded VSTs, wherein the VSTs are specific for HHV8 antigens comprising: (a) One or more latent antigens, including Kaposi protein (ORF12, K12), LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vFLIP (ORF71), vCYC (ORF72) and RTA ( ORF50); and/or (b) One or more lytic antigens, including gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some aspects, the invention provides a population of ex vivo expanded VSTs specific for HHV8 antigens including LANA-1 (ORF3) and LANA-2 (vIRF3, K10.5).

In some aspects, the invention provides a population of ex vivo expanded VSTs specific for HHV8 antigens including LANA-1 (ORF3) and gB (ORF8).

In some aspects, the invention provides a population of ex vivo expanded VST pairs, wherein the VST pairs comprise spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a ( The SARS-CoV antigens of AP7a) and non-structural protein 4 (nsp4) are specific.

In some or any of the foregoing or related embodiments, the VST cells are human VST cells, donor VST cells, and/or allogeneic VST cells.

In some or any of the foregoing or related embodiments, the population includes (a) At least 70% CD3+ T cells; (b) approximately 30% or less CD56+ cells; and/or (c) CD4+ and CD8+ T cells.

In some or any of the foregoing or related embodiments, the VSTs have specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10 ⁵ cells.

In some or any of the foregoing or related embodiments, no more than 20% of the VSTs express a T cell exhaustion marker; optionally, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or Or TIGIT.

In some or any of the foregoing or related embodiments, the T cell exhaustion marker is a co-expression of PD1 and/or TIM3.

In some or any of the foregoing or related embodiments, the VSTs express one or more markers associated with T cell activation; optionally, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

In some or any of the foregoing or related embodiments, the VSTs exhibit one or more markers associated with central or effector cell memory; optionally, wherein the one or more markers associated with central or effector cell memory The species system is selected from CD45RO and CD62L.

In some or any of the foregoing or related embodiments, the VSTs are multifunctional.

In some or any of the foregoing or related embodiments, the VSTs produce two or more effector molecules when stimulated with two or more antigens; optionally, wherein the effector molecules are selected from IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b.

In some or any of the foregoing or related embodiments, the VSTs are enriched by at least 1000-fold, at least 3,000 times or at least 6,000 times. In some or any of the foregoing or related embodiments, the VSTs are enriched by at least 1000-fold, at least 3,000 times or at least 6,000 times.

In some or any of the foregoing or related embodiments, the VSTs have reduced alloreactivity or negligible alloreactivity. In some or any of the foregoing or related embodiments, the VSTs have reduced reactivity against the allogeneic target or negligible reactivity against the allogeneic target. In some or any of the foregoing or related embodiments, the VST has reduced or negligible autoreactivity.

In some aspects, the present invention provides a pharmaceutical composition comprising an ex vivo expanded multi-strain population of VST of some or any of the foregoing or related embodiments and a pharmaceutically acceptable carrier.

In some aspects, the invention provides a kit comprising a multi-strain population of ex vivo expanded VSTs of some or any of the foregoing or related embodiments.

In some aspects, the invention provides a donor minibank comprising a multi-strain population of amplified VSTs of some or any of the foregoing or related embodiments.

In some aspects, the invention provides a method of treating or preventing viral infection comprising administering to an individual a population of ex vivo expanded VSTs of some or any of the foregoing or related embodiments.

In some aspects, the invention provides a method of treating or preventing viral infection, comprising administering to an individual a pharmaceutical composition of some or any of the foregoing or related embodiments.

In some or any of the foregoing or related embodiments, the individual is immunocompetent or immunocompromised.

In some or any of the foregoing or related embodiments, the pharmaceutical composition is administered by intravenous injection or infusion. In some or any of the foregoing or related embodiments, the pharmaceutical composition is administered to the individual a plurality of times.

In some or any of the foregoing or related embodiments, about 5×10 ⁶ to about 5×10 ⁸ cells/cm2 are administered to the individual.

In some or any of the foregoing or related embodiments, the individual is infected with or at risk of infection with a virus. In some or any of the foregoing or related embodiments, administration of the pharmaceutical composition is effective to treat or prevent the viral infection in the individual.

In some or any of the foregoing or related embodiments, the individual a.Have received solid organ transplant; b.Have received chemotherapy; c. Suffering from HIV infection; d. Suffering from genetic immunodeficiency; e. Suffering from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation.

In some or any of the foregoing or related embodiments, the pharmaceutical composition comprises the universal antigen-specific T cell therapy product of some or any of the foregoing or related embodiments.

In some or any of the foregoing or related embodiments, the pharmaceutical composition is administered to the individual without knowledge of the individual's HLA type. In some or any of the preceding or related embodiments, the individual is a human.

In some aspects, the invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual an expanded VST of some or any of the foregoing or related embodiments. group.

In some aspects, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual an expanded VST of some or any of the foregoing or related embodiments. group.

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for two or more HBV antigens.

In some or any of the foregoing or related embodiments, the two or more HBV antigens are selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX. In some or any of the foregoing or related embodiments, the two or more HBV antigens are selected from the group consisting of HBsAg, HBeAg, HBP and HBX. In some or any of the foregoing or related embodiments, the two or more HBV antigens are HBcAg and HBsAg.

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST includes responses to HBV antigens including E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg). specificity.

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the antigens E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg) are immunodominant antigens.

In some or any of the foregoing or related embodiments, the E antigen (HBeAg) includes pre-core (PreC) and core (HBcAg) antigens.

In some or any of the foregoing or related embodiments, the VST cells are human VST cells, donor VST cells, and/or allogeneic VST cells.

In some or any of the foregoing or related embodiments, the population of VSTs produces two or more effector molecules. In some or any of the foregoing or related embodiments, the VSTs produce two or more effector molecules when stimulated with the two or more HBV antigens. In some or any of the foregoing or related embodiments, the effector molecules are selected from IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b.

In some or any of the foregoing or related embodiments, the composition is produced in vitro or ex vivo.

In some or any of the foregoing or related embodiments, the VSTs are multifunctional.

In some or any of the foregoing or related embodiments, the population of VSTs includes (a) At least 70% CD3+ T cells; (b) No more than 30% CD56+ cells; and/or (c) CD4+ and CD8+ T cells.

In some or any of the foregoing or related embodiments, the VSTs have specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10e5 cells .

In some or any of the foregoing or related embodiments, no more than 20% of the VSTs express a T cell exhaustion marker; optionally, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or Or TIGIT.

In some or any of the foregoing or related embodiments, the T cell exhaustion marker is PD1 and/or TIM3.

In some or any of the foregoing or related embodiments, the VSTs express one or more markers associated with T cell activation; optionally, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

In some or any of the foregoing or related embodiments, the VSTs exhibit one or more markers associated with central or effector cell memory; optionally, wherein the one or more markers associated with central or effector cell memory The species system is selected from CD45RO and CD62L.

In some or any of the foregoing or related embodiments, the VSTs are enriched at least 1000-fold after expansion in IL4, IL7 and IL15 in the presence of HBV antigen compared to the cell population before expansion, At least 3,000 times or at least 6,000 times.

In some or any of the foregoing or related embodiments, the multi-strain population of VST is an antigen-specific multi-strain population of VST.

In some or any of the foregoing or related embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HBV antigens.

In some or any of the foregoing or related embodiments, the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from HBsAg; optionally, wherein the antigen-specific multi-strain population of VST The strain population is specific for at least one peptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. In some or any of the foregoing or related embodiments, the antigen-specific polystrain population of VST is specific for one or more peptide sequences derived from HBeAg; optionally, wherein the antigen-specific polystrain population of VST is specific for one or more peptide sequences derived from HBeAg; The strain population is specific for at least one peptide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5. In some or any of the foregoing or related embodiments, the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from HBsAg and one or more peptide sequences derived from HBeAg; Optionally, wherein the antigen-specific multi-strain population of VST pairs at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO : At least one of the 5 peptide sequences is specific.

In some or any of the foregoing or related embodiments, the population of strains of VST comprises specificity for two or more HHV8 antigens. In some or any of the foregoing or related embodiments, the two or more HHV8 antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises a pair of pairs comprising LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC ( Specificity of HHV8 antigens of ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), and TS (ORF70).

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for an HHV8 antigen comprising: (a) One or more latent antigens, including Kaposi protein (ORF12, K12), LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vFLIP (ORF71), vCYC (ORF72) and RTA ( ORF50); and/or (b) One or more lytic antigens, including gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises response to HHV8 antigens including LANA-1 (ORF3) and LANA-2 (vIRF3, K10.5) specificity.

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for HHV8 antigens including LANA-1 (ORF3) and gB (ORF8).

In some or any of the foregoing or related embodiments, the VST cells are human VST cells, donor VST cells, and/or allogeneic VST cells.

In some or any of the foregoing or related embodiments, the population of VSTs produces two or more effector molecules. In some or any of the foregoing or related embodiments, the VSTs produce two or more effector molecules when stimulated with the two or more HHV8 antigens.

In some or any of the foregoing or related embodiments, the effector molecules are selected from IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b.

In some or any of the foregoing or related embodiments, the composition is produced in vitro or ex vivo.

In some or any of the foregoing or related embodiments, the VSTs are multifunctional.

In some or any of the foregoing or related embodiments, the VSTs include (a) CD4+ and CD8+ T cells; (b) At least 70% CD3+ T cells; and/or (c) About 30% or less CD56+ cells.

In some or any of the foregoing or related embodiments, the VSTs have specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10 ⁵ cells.

In some or any of the foregoing or related embodiments, no more than 20% of the VSTs express a T cell exhaustion marker; optionally, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or Or TIGIT.

In some or any of the foregoing or related embodiments, the T cell exhaustion marker is a co-expression of PD1 and/or TIM3.

In some or any of the foregoing or related embodiments, the VSTs express one or more markers associated with T cell activation; optionally, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

In some or any of the foregoing or related embodiments, the VSTs exhibit one or more markers associated with central or effector cell memory; optionally, wherein the one or more markers associated with central or effector cell memory The species system is selected from CD45RO and CD62L.

In some or any of the foregoing or related embodiments, the VSTs are enriched at least 1000-fold after expansion in IL4, IL7 and IL15 in the presence of HHV8 antigen compared to the cell population before expansion, At least 3,000 times or at least 6,000 times.

In some or any of the foregoing or related embodiments, the multi-strain population of VST is an antigen-specific population of VST.

In some or any of the foregoing or related embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HHV8 antigens.

In some or any of the foregoing or related embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1; optionally, wherein at least one peptide sequence is selected from SEQ ID NO: 6-12. In some or any of the foregoing or related embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from gB; optionally, wherein at least one peptide sequence is selected from SEQ ID NO: 13-15. In some or any of the foregoing or related embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1 and one or more peptide sequences derived from gB; Optionally, wherein at least one peptide sequence is selected from SEQ ID NO: 6-12 and at least one peptide sequence is selected from SEQ ID NO: 13-15.

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST includes pairs of proteins including spike protein (S), membrane protein (M), nucleocapsid protein (N) , accessory protein 7a (AP7a) and non-structural protein 4 (nsp4) specificity of SARS-CoV antigens.

In some or any of the foregoing or related embodiments, the VST cells are human VST cells, donor VST cells, and/or allogeneic VST cells. In some or any of the foregoing or related embodiments, the multi-strain population of VST has reduced alloreactivity or negligible alloreactivity. In some or any of the foregoing or related embodiments, the multi-strain population of VST has reduced reactivity against the allogeneic target or negligible reactivity against the allogeneic target. In some or any of the foregoing or related embodiments, the multi-strain population of VST has reduced autoreactivity or negligible autoreactivity.

In some or any of the foregoing or related embodiments, the composition includes a pharmaceutically acceptable diluent.

In some or any of the preceding or related embodiments, the composition is formulated for intravenous administration.

In some aspects, the present invention provides a pharmaceutical composition comprising the composition of some or any of the foregoing or related embodiments and a pharmaceutically acceptable carrier.

In some aspects, the invention provides a kit comprising a composition of some or any of the foregoing or related embodiments.

In some aspects, the invention provides a method of treating a viral infection, comprising administering to an individual a composition of some or any of the foregoing or related embodiments or some or any of the foregoing or related embodiments. of pharmaceutical compositions.

In some aspects, the present invention provides a method of preventing viral infection, comprising administering to an individual a composition of some or any of the foregoing or related embodiments or some or any of the foregoing or related embodiments. of pharmaceutical compositions.

In some or any of the foregoing or related embodiments, the individual is immunocompetent or immunocompromised.

In some or any of the foregoing or related embodiments, the pharmaceutical composition is administered by intravenous injection or infusion.

In some or any of the foregoing or related embodiments, the pharmaceutical composition is administered to the individual a plurality of times.

In some or any of the foregoing or related embodiments, about 5×10 ⁶ to about 5×10 ⁸ cells/cm2 are administered to the subject.

In some or any of the foregoing or related embodiments, the individual is infected with or at risk of infection with a virus.

In some or any of the foregoing or related embodiments, administration of the pharmaceutical composition is effective to treat or prevent the viral infection in the individual.

In some or any of the foregoing or related embodiments, the individual a.Have received solid organ transplant; b.Have received chemotherapy; c. Suffering from HIV infection; d. Suffering from genetic immunodeficiency; e. Suffering from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation.

In some or any of the preceding or related embodiments, the individual is a human.

In some aspects, the present invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual a composition of some or any of the preceding or related embodiments.

In some aspects, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a composition of some or any of the foregoing or related embodiments.

In some aspects, the invention provides a method for treating or preventing SARS-CoV infection in an individual in need thereof, comprising administering to the individual a composition of some or any of the foregoing or related embodiments.

In some or any of the preceding or related embodiments, the composition is a pharmaceutical composition.

The present invention provides, at least in part, methods for expanding T cells (eg, virus-specific T cells), compositions thereof, and methods of using the same to treat or prevent viral infections. In some aspects, the invention provides an in vitro method for expanding T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; adding one or more compositions to the cell culture , wherein the one or more compositions comprise: i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and ii) one or more components selected from IL -4. Exogenous interleukins other than IL-7 and IL-15; and culturing the cells for a period of time sufficient to expand the T cells; thereby amplifying the T cells.

In some aspects, the invention provides an in vitro method for expanding T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; adding one or more compositions to the cell culture , wherein the one or more compositions comprise: i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; or ii) one or more components selected from IL -4. Exogenous interleukins other than IL-7 and IL-15; and culturing the cells for a period of time sufficient to expand the T cells; thereby amplifying the T cells.

In some aspects, the invention provides an in vitro method for generating an expanded population of T cells, comprising: providing a starting population of mononuclear spheroids in a cell culture; adding to the cell culture a or Compositions, wherein the one or more compositions comprise: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more an exogenous interleukin selected from the group consisting of IL-4, IL-7, and IL-15; and culturing the cells for a period of time sufficient to expand the T cells, thereby producing a population of expanded T cells.

In some aspects, the invention provides an in vitro method for generating an expanded population of T cells, comprising: providing a starting population of mononuclear spheroids in a cell culture; adding to the cell culture a or Compositions, wherein the one or more compositions comprise: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; or ii) one or more an exogenous interleukin selected from the group consisting of IL-4, IL-7, and IL-15; and culturing the cells for a period of time sufficient to expand the T cells, thereby producing a population of expanded T cells.

In some or any of the foregoing or related aspects, the expanded T cells comprise T cells specific for at least one target antigen.

In some or any of the foregoing or related aspects, the expanded T cells are enriched with T cells specific for at least one target antigen.

In some or any of the foregoing or related aspects, the expanded T cell population includes T cells specific for at least one target antigen.

In some or any of the foregoing or related aspects, the expanded T cell population is enriched with T cells specific for at least one target antigen.

In some or any of the foregoing or related aspects, the monocytes are peripheral blood monocytes (PBMC).

In some or any of the foregoing or related aspects, the one or more exogenous interleukins comprise IL-4 and IL-7.

In some or any of the foregoing or related aspects, the one or more exogenous interleukins comprise IL-4, IL-7, and IL-15.

In some or any of the foregoing or related aspects, the method does not include adding exogenous IL-2. In some or any of the foregoing or related aspects, the method does not include adding IL-2.

In some or any of the foregoing or related aspects, there is added to the cell culture a composition comprising: (i) at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a a peptide or peptide mixture that is part of the target antigen; and (ii) one or more exogenous interleukins selected from the group consisting of IL-4, IL-7 and IL-15.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising: (i) at least one target antigen or a portion of a target antigen or corresponding to at least one target an antigen or a peptide or peptide mixture that is part of an antigen of interest; and a second composition comprising: (ii) one or more exogenous interleukins selected from the group consisting of IL-4, IL-7 and IL-15. In some aspects, the first composition and the second composition are added to the cell culture simultaneously. In some aspects, the second composition is added to the cell culture after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 1 minute to about 30 minutes after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 30 minutes to about 60 minutes after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 1 hour to about 2 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 2 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 6 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 12 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 18 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 24 hours after the first composition is added to the cell culture.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen; a second composition comprising IL-4 and IL-7; and a third composition comprising IL-15. In some aspects, the first composition and the second composition are added to the cell culture simultaneously. In some aspects, the second composition is added to the cell culture after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 1 minute to about 30 minutes after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 30 minutes to about 60 minutes after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 1 hour to about 2 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 2 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 6 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 12 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 18 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 24 hours after the first composition is added to the cell culture. In some aspects, the second composition and the third composition are added after the first composition. In some aspects, the third composition is added after the first composition and the second composition. In some aspects, the first composition, the second composition, and the third composition are added simultaneously. In some aspects, the first composition, the second composition, and the third composition are added separately. In some aspects, the second composition and the third composition are added simultaneously. In some aspects, the third composition is added to the cell culture after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture approximately 12 hours after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture approximately 24 hours after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture approximately 48 hours after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture approximately 72 hours after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture about 1 day after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture about 2 days after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture about 3 days after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture about 4 days after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture about 5 days after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture about 6 days after the second composition is added to the cell culture. In some aspects, the third composition is added to the cell culture about 7 days after the second composition is added to the cell culture.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target A peptide or peptide mixture that is part of an antigen, IL-4 and IL-7; and a second composition comprising IL-15. In some aspects, the first composition and the second composition are added simultaneously. In some aspects, the second composition is added to the cell culture after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 12 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 24 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 48 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture approximately 72 hours after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 1 day after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 2 days after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 3 days after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 4 days after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 5 days after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 6 days after the first composition is added to the cell culture. In some aspects, the second composition is added to the cell culture about 7 days after the first composition is added to the cell culture.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen; a second composition comprising IL-4; and a third composition comprising IL-7 and IL-15.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen; a second composition comprising IL-7; and a third composition comprising IL-4 and IL-15.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen; a second composition comprising IL-4; a third composition comprising IL-7; and a fourth composition comprising IL-15.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen, and IL-4; a second composition comprising IL-7; and a third composition comprising IL-15.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen, and IL-7; a second composition comprising IL-4; and a third composition comprising IL-15.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen, and IL-4; and a second composition comprising IL-7 and IL-15.

In some or any of the foregoing or related aspects, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target a peptide or peptide mixture that is part of an antigen, and IL-7; and a second composition comprising IL-4 and IL-15.

In some or any of the foregoing or related aspects, the cells are cultured for about 18 days or less, and optionally about 14 days.

In some or any of the foregoing or related aspects, the cells are cultured for about 8 days to about 10 days.

In some or any of the foregoing or related aspects, the cells are cultured for about 8 days to about 14 days.

In some or any of the foregoing or related aspects, the cells are cultured for about 8 days to about 18 days.

In some or any of the foregoing or related aspects, the cells are cultured for about 1 week to about 2 weeks.

In some or any of the foregoing or related aspects, the cells are cultured for about 1 week to about 3 weeks.

In some or any of the foregoing or related aspects, the cells are cultured for about 1 week to about 4 weeks.

In some or any of the foregoing or related aspects, the cells are cultured for an amount of time sufficient to produce cells that have completed logarithmic phase growth. In some aspects, the cells are cultured for an amount of time sufficient to produce cells that have completed logarithmic phase growth, wherein the cells are cultured for about 8 days to about 18 days.

In some or any of the foregoing or related aspects, the cells are cultured in the presence of T cell expansion medium.

In some or any of the foregoing or related aspects, the cells are cultured at a cell seeding density of about 2 × 10 ⁶ cells/cm2 to about 4 × 10 ⁶ cells/cm2. In some aspects, the cells are cultured at a cell seeding density of approximately 3 × 10 ⁶ cells/cm2. In some aspects, the cells are cultured at a cell seeding density of 3 × 10 ⁶ cells/cm2.

In some or any of the foregoing or related aspects, the number of expanded T cells is at least 2-fold greater than the number of cells in the starting cell population.

In some or any of the foregoing or related aspects, the expanded T cell population has a number of expanded T cells that is at least 2-fold greater than the number of cells in the starting cell population.

In some or any of the foregoing or related aspects, the expanded T cells comprise antigen-specific T cells. In some aspects, the antigen-specific T cells are pathogen-specific T cells (PST). In some aspects, the antigen-specific T cells are virus-specific T cells (VST). In some aspects, the antigen-specific T cells are tumor-specific T cells (TST).

In some or any of the foregoing or related aspects, the expanded T cell population includes antigen-specific T cells. In some aspects, the antigen-specific T cells are pathogen-specific T cells (PST). In some aspects, the antigen-specific T cells are virus-specific T cells (VST). In some aspects, the antigen-specific T cells are tumor-specific T cells (TST).

In some or any of the foregoing or related aspects, the expanded T cells comprise VSTs that recognize at least one antigen from hepatitis B virus (HBV).

In some or any of the foregoing or related aspects, the expanded T cell population comprises VSTs that recognize at least one antigen from hepatitis B virus (HBV).

In some or any of the foregoing or related aspects, the expanded T cells comprise a VST that recognizes at least one antigen from human herpesvirus 8 (HHV-8).

In some or any of the foregoing or related aspects, the expanded T cell population comprises VSTs that recognize at least one antigen from human herpesvirus 8 (HHV-8).

In some or any of the foregoing or related aspects, the peptide mixture includes between 2 and 750 peptides.

In some or any of the foregoing or related aspects, each peptide in the peptide mixture has at least 2 amino acid overlap with at least one other peptide. In some aspects, the peptides are at least 7 amino acids long.

In some or any of the foregoing or related aspects, the peptide mixture comprises one or more target antigens from at least one pathogen, or a portion thereof. In some aspects, the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), Hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 ( HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV) , mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T lymphocyte Cellular viruses. In some aspects, the at least one pathogen is hepatitis B virus (HBV). In some aspects, the at least one pathogen is human herpesvirus 8 (HHV-8).

In some or any of the foregoing or related aspects, the pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP) ), X antigen (HBX) or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX ) or a combination thereof.

In some or any of the foregoing or related aspects, the HBeAg (E antigen) includes pre-core (PreC) and core (HBcAg) antigens.

In some or any of the foregoing or related aspects, the peptide mixture comprises a peptide comprising a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and EYLVSFGVWIRTPPA (SEQ ID NO: 5).

In some or any of the foregoing or related aspects, the peptide mixture comprises a peptide consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and EYLVSFGVWIRTPPA (SEQ ID NO: 5).

In some or any of the foregoing or related aspects, the one or more HBV antigens are selected from one or more HBV genotypes.

In some or any of the foregoing or related aspects, at least two antigens are selected from the same HBV genotype.

In some or any of the foregoing or related aspects, at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first genotype and the second genotype Different types.

In some or any of the foregoing or related aspects, at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

In some or any of the foregoing or related aspects, the amino acid sequence of each HBV antigen differs by at least one amino acid.

In some or any of the foregoing or related aspects, the expanded T cells are responsive upon stimulation. In some aspects, at least a portion of the T cell reactivity is CD4+. In some aspects, at least a portion of the T cell reactivity is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted.

In some or any of the foregoing or related aspects, the expanded T cell population is responsive upon stimulation. In some aspects, at least a portion of the T cell reactivity is CD4+. In some aspects, at least a portion of the T cell reactivity is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted.

In some or any of the foregoing or related aspects, the pathogen is EBV, and the at least one target antigen is EBNAl, LMP2, BZLF1, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is CMV, and the at least one target antigen is IEl, pp65, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is Adv and the at least one target antigen is hexon, penton, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is BK virus, and the at least one target antigen is LT, VP-1, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is HHV6, and the at least one target antigen is U14, U11, U71, U54, U90, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is RSV and the at least one target antigen is N, F, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is an influenza virus, and the at least one target antigen is MP1, NP1, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is a parainfluenza virus type (PIV) and the at least one target antigen is F, N, M, M2-1, HN, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is hMPV, and the at least one target antigen is F, N, M2-1, M, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70), or combinations thereof.

In some or any of the foregoing or related aspects, the peptide mixture comprises a peptide comprising a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13) , DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

In some or any of the foregoing or related aspects, the peptide mixture comprises a peptide consisting of a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13) , DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

In some or any of the foregoing or related aspects, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), matrix protein (M), nuclear Capsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some or any of the foregoing or related aspects, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), membrane protein (M), nuclear Capsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some or any of the foregoing or related aspects, the method includes obtaining the expanded T cells.

In some or any of the foregoing or related aspects, the method includes obtaining the expanded T cell population.

In some or any of the foregoing or related aspects, the method further includes the step of preparing a pharmaceutical composition, wherein diluents, stabilizers, preservatives and/or other pharmaceuticals are added to the expanded T cells. Scientifically acceptable excipients.

In some or any of the foregoing or related aspects, the method further includes the step of preparing a pharmaceutical composition, wherein diluents, stabilizers, preservatives and/or other pharmaceuticals are added to the expanded T cell population. Scientifically acceptable excipients.

In some aspects, the invention provides a population of expanded T cells. In some aspects, the invention provides an expanded T cell population obtained by any of the methods of the invention.

In some or any of the foregoing or related aspects, the expanded T cell population comprises at least 70% CD3+ T cells.

In some or any of the foregoing or related aspects, the expanded T cell population includes VST.

In some or any of the foregoing or related aspects, no more than 30% of the cells in the expanded T cell population are natural killer (NK) cells.

In some or any of the foregoing or related aspects, the expanded T cell population includes CD4+ and CD8+ T cells.

In some or any of the foregoing or related aspects, more than 80% of the expanded T cell population includes CD4+ and CD8+ T cells.

In some or any of the foregoing or related aspects, more than 80% of the expanded T cell population includes CD4+ T cells.

In some or any of the foregoing or related aspects, more than 80% of the expanded T cell population includes CD8+ T cells.

In some or any of the foregoing or related aspects, the T cells of the expanded T cell population are polyclonal.

In some or any of the foregoing or related aspects, the expanded T cell population includes CD3+ T cells, and at least 1% of the CD3+ T cells can produce TNFα.

In some or any of the foregoing or related aspects, at least one T cell of the expanded T cell population recognizes one or more target antigens from at least one pathogen, or a portion thereof.

In some or any of the foregoing or related aspects, the at least one pathogen is selected from the group including: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV) , Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus (HDV), Herpes simplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV- 2), HIV, varicella-zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), human herpes virus 8 (HHV -8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, bocavirus, coronavirus, lymphocyte Choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonitis virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus , Ebola virus and human T-lymphotropic virus. In some aspects, the at least one pathogen is hepatitis B virus (HBV). In some aspects, the at least one pathogen is human herpesvirus 8 (HHV-8).

In some or any of the foregoing or related aspects, the pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP) ), X antigen (HBX) or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX ) or a combination thereof. In some aspects, the HBeAg (E antigen) includes precore (PreC) and core (HBcAg) antigens.

In some or any of the foregoing or related aspects, the pathogen is HBV, and the at least one target antigen is HBcAg, HBsAg, or a combination thereof.

In some aspects, the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and/or EYLVSFGVWIRTPPA (SEQ ID NO: 5).

In some or any of the foregoing or related aspects, at least two HBV antigens are selected from different HBV genotypes. In some aspects, at least two antigenic lines are selected from the same HBV genotype. In some aspects, at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first genotype is different from the second genotype. In some aspects, at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

In some or any of the foregoing or related aspects, the amino acid sequence of each HBV antigen differs from the amino acid sequence of each other HBV antigen by at least one amino acid.

In some or any of the foregoing or related aspects, the expanded T cell population is responsive upon stimulation. In some embodiments, at least a portion of the T cell reactivity is HLA-DR, HLA-DQ or HLA-DP restricted.

In some or any of the foregoing or related aspects, the pathogen is EBV, and the at least one target antigen is EBNAl, LMP2, BZLF1, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is CMV, and the at least one target antigen is IEl, pp65, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is Adv and the at least one target antigen is hexon, penton, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is BK virus, and the at least one target antigen is LT, VP-1, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is HHV6, and the at least one target antigen is U14, U11, U71, U54, U90, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is RSV and the at least one target antigen is N, F, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is an influenza virus, and the at least one target antigen is MP1, NP1, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is a parainfluenza virus type (PIV) and the at least one target antigen is F, N, M, M2-1, HN, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is hMPV, and the at least one target antigen is F, N, M2-1, M, or a combination thereof.

In some or any of the foregoing or related aspects, the pathogen is HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70), or combinations thereof.

In some aspects, the at least one target antigen comprises a peptide comprising a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

In some aspects, the at least one target antigen comprises a peptide consisting of a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

In some or any of the foregoing or related aspects, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), matrix protein (M), nuclear Capsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some or any of the foregoing or related aspects, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), membrane protein (M), nuclear Capsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some aspects, the invention provides a plurality of expanded T cell populations. In some aspects, each expanded T cell population is an expanded T cell population of the invention.

In some or any of the foregoing or related aspects, each expanded T cell population includes T cells generated from donor monocytes obtained from a different donor.

In some or any of the foregoing or related aspects, the HLA type of each donor differs from at least one of the other donors by at least one HLA allele.

In some aspects, the invention provides a universal antigen-specific T cell therapy product comprising an expanded T cell population of the invention.

In some aspects, the invention provides a universal antigen-specific T cell therapy product comprising a plurality of expanded T cell populations of the invention.

In some or any of the foregoing or related aspects, the universal antigen-specific T cell therapy product comprises T cells generated from donor monocytes obtained from different donors. In some aspects, each donor's HLA type differs from at least one of the other donors by at least one HLA allele.

In some or any of the foregoing or related aspects, the universal antigen-specific T cell therapy product exhibits reduced or negligible alloreactivity to partially HLA matched and/or to HLA mismatched target cells.

In some or any of the foregoing or related aspects, the universal antigen-specific T cell therapy product comprises a VST that recognizes one or more target antigens from at least one pathogen, or a portion thereof.

In some aspects, the invention provides a pharmaceutical composition comprising the expanded T cell population of the invention.

In some aspects, the invention provides a pharmaceutical composition comprising the VST of the invention.

In some aspects, the invention provides a pharmaceutical composition comprising the universal antigen-specific T cell therapy product of the invention.

In some or any of the foregoing or related aspects, the pharmaceutical composition is formulated for intravenous delivery.

In some or any of the foregoing or related aspects, the pharmaceutical composition is negative for bacteria and fungi during continuous culture for at least 7 days.

In some or any of the foregoing or related aspects, the pharmaceutical composition exhibits less than 5 EU/ml of endotoxin.

In some or any of the foregoing or related aspects, the pharmaceutical composition is negative for Mycoplasma species.

In some aspects, the invention provides a population of amplified VSTs, wherein the VSTs are specific for one or more viral antigens.

In some aspects, the invention provides a population of amplified VSTs, wherein the VSTs are specific for two or more HBV antigens selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX.

In some or any of the foregoing or related aspects, the VSTs are specific for two or more HBV antigens selected from HBsAg, HBeAg, HBP and HBX.

In some or any of the foregoing or related aspects, the VSTs are specific for the HBV antigens HBcAg and HBsAg.

In some aspects, the invention provides a population of amplified VSTs, wherein the VST pairs are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50 ), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) are specific for two or more HHV8 antigens.

In some or any of the foregoing or related aspects, the population of expanded VSTs comprises at least 70% CD3+ T cells.

In some or any of the foregoing or related aspects, the population of expanded VSTs comprises no more than 30% CD56+ cells.

In some or any of the foregoing or related aspects, the VST has a specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10 ⁵ cells.

In some or any of the foregoing or related aspects, the VSTs comprise CD4+ and CD8+ T cells.

In some or any of the foregoing or related aspects, no more than 20% of such VSTs exhibit markers of T cell exhaustion. In some aspects, the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT. In some aspects, the T cell exhaustion marker is a co-expression of PD1 and TIM3.

In some or any of the foregoing or related aspects, the VSTs exhibit one or more markers associated with T cell activation. In some aspects, the one or more markers associated with T cell activation are selected from CD25 and CD69.

In some or any of the foregoing or related aspects, the VSTs exhibit one or more markers associated with central or effector cell memory. In some aspects, the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

In some or any of the foregoing or related aspects, the VSTs are multifunctional.

In some or any of the foregoing or related aspects, the VSTs produce two or more effector molecules when stimulated with two or more antigens. In some aspects, the effector molecules are selected from the group consisting of IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a, and MIP-1b.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST Enriched at least 1000-fold.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST Enriched at least 3000-fold.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST Enriched at least 6000-fold.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen, the VST Enriched at least 1000-fold.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen, the VST Enriched at least 3000-fold.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen, the VST Enriched at least 6000-fold.

In some or any of the foregoing or related aspects, the VSTs have reduced alloreactivity.

In some or any of the foregoing or related aspects, the VST has negligible alloreactivity.

In some or any of the foregoing or related aspects, the VSTs have reduced reactivity against allogeneic targets.

In some or any of the foregoing or related aspects, the VSTs have negligible reactivity against allogeneic targets.

In some or any of the foregoing or related aspects, the VST has reduced autoreactivity.

In some or any of the foregoing or related aspects, the VST has negligible autoreactivity.

In some aspects, the invention provides a pharmaceutical composition comprising the expanded VST population of the invention and a pharmaceutically acceptable carrier.

In some aspects, the invention provides a kit comprising a population of expanded VSTs of the invention.

In some aspects, the invention provides a method of preventing or treating viral infection comprising administering to an individual a population of expanded VSTs of the invention.

In some aspects, the present invention provides a method of preventing or treating viral infection, comprising administering to an individual a pharmaceutical composition of the present invention. In some aspects, the pharmaceutical composition includes expanded T cells. In some aspects, the pharmaceutical composition includes an expanded population of T cells. In some aspects, the pharmaceutical composition includes multiple strains of VST. In some aspects, the pharmaceutical composition includes a population of multiple strains of VST. In some aspects, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

In some or any of the foregoing or related aspects, the individual is immunocompetent or immunocompromised.

In some or any of the foregoing or related aspects, the pharmaceutical composition is administered by intravenous injection or infusion.

In some or any of the foregoing or related aspects, the pharmaceutical composition is administered to the individual a plurality of times.

In some or any of the foregoing or related aspects, about 5×10 ⁶ to about 5×10 ⁸ cells/square meter are administered to the individual.

In some or any of the foregoing or related aspects, the individual is infected with or at risk of infection with the virus.

In some or any of the foregoing or related aspects, administration of the pharmaceutical composition is effective to treat or prevent the viral infection in the individual.

In some or any of the foregoing or related aspects, the individual has received a solid organ transplant; has received chemotherapy; has an HIV infection; has a genetic immunodeficiency; has a chronic viral infection; and/or has received Allogeneic or autologous stem cell transplantation.

In some or any of the foregoing or related aspects, the pharmaceutical composition comprises the universal antigen-specific T cell therapy product of the invention.

In some or any of the foregoing or related aspects, the pharmaceutical composition is administered to the individual without knowledge of the individual's HLA type.

In some or any of the foregoing or related aspects, the individual is a human being.

In some aspects, the invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual a population of expanded VSTs of the invention.

In some aspects, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a population of expanded VSTs of the invention.

In some aspects, the invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in a cell culture; (b) providing the cell culture with One or more compositions are added thereto, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen; and (ii) one or more an exogenous interleukin selected from the group consisting of IL-4, IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to stimulate expansion of VST.

In some aspects, the invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in a cell culture; (b) providing the cell culture with One or more compositions are added thereto, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen; or (ii) one or more an exogenous interleukin selected from the group consisting of IL-4, IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to stimulate expansion of VST.

In some aspects, the invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in cell culture; (b) performing a priming step, wherein The priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen ; and (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to stimulate expansion of VST.

In some aspects, the invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in cell culture; (b) performing a priming step, wherein The priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen ; or (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to stimulate expansion of VST.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is about 18 days or less, optionally about 14 days.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is from about 8 days to about 10 days.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is from about 8 days to about 14 days.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is from about 8 days to about 18 days.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is from about 1 week to about 2 weeks.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is from about 1 week to about 3 weeks.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is from about 1 week to about 4 weeks.

In some or any of the foregoing or related aspects, the period of time sufficient to stimulate expansion of the VST is an amount of time sufficient to produce cells that have completed log phase growth.

In some aspects, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in a cell culture; (b) adding to the cell culture One or more compositions, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen; and (ii) one or more compositions selected from IL -4. Exogenous interleukins other than IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to expand the VSTs.

In some aspects, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in a cell culture; (b) adding to the cell culture One or more compositions, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen; or (ii) one or more compositions selected from IL -4. Exogenous interleukins other than IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to expand the VSTs.

In some aspects, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) performing a priming step, wherein the priming The steps comprise adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen; and (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to expand the VSTs.

In some aspects, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) performing a priming step, wherein the priming The step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen; or (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to expand the VSTs.

In some or any of the foregoing or related aspects, the period of time sufficient to amplify the VST is about 18 days or less, and optionally about 14 days.

In some or any of the foregoing or related aspects, the period of time sufficient to amplify the VST is from about 8 days to about 10 days.

In some or any of the foregoing or related aspects, the period of time sufficient to amplify the VST is from about 8 days to about 14 days.

In some or any of the foregoing or related aspects, the period of time sufficient to amplify the VST is from about 8 days to about 18 days.

In some or any of the foregoing or related aspects, the period of time sufficient to amplify the VSTs is from about 1 week to about 2 weeks.

In some or any of the foregoing or related aspects, the period of time sufficient to amplify the VST is from about 1 week to about 3 weeks.

In some or any of the foregoing or related aspects, the period of time sufficient to amplify the VST is from about 1 week to about 4 weeks.

In some or any of the foregoing or related aspects, the period of time sufficient to expand the VSTs is an amount of time sufficient to produce cells that have completed log phase growth.

In some or any of the foregoing or related aspects, adding IL-15 to the cell culture (step c) occurs after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 12 hours after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 24 hours after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 48 hours after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 72 hours after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs about 1 day after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 2 days after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 3 days after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 4 days after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 5 days after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 6 days after the priming step. In some aspects, adding IL-15 to the cell culture (step c) occurs approximately 7 days after the priming step.

In some or any of the foregoing or related aspects, the in vitro method for amplifying VSTs results in expansion of the VSTs but not NK cells.

In some or any of the foregoing or related aspects, the in vitro method for stimulating expansion of VSTs results in expansion of the VSTs but not NK cells.

In some or any of the foregoing or related aspects, the presence of IL-15 causes accelerated expansion of the cells in culture.

In some or any of the foregoing or related aspects, the method generates an expanded population of VSTs that includes a broader repertoire of enriched VSTs than when the cells are contacted with: (i) IL-4 only; (ii) IL-7 only; (iii) IL-15 only; (iv) IL-7 and IL-15 without IL-4; or (v) IL-4 and IL-15 without IL-7.

In some or any of the foregoing or related aspects, the method generates an expanded population of VSTs comprising an increased number of VSTs compared to when the cells are contacted with: (i) IL-4 alone ; (ii) IL-7 only; or (iii) IL-4 and IL-7, without IL-15.

In some or any of the foregoing or related aspects, the duration of the initiation step is from about 1 to about 2 days. In some aspects, the activation step lasts for more than about 24 hours. In some aspects, the activation step lasts less than 24 hours. In some aspects, the duration of the activation step is from about 0 to about 12 hours. In some aspects, the duration of the activation step is from about 0 to about 24 hours. In some aspects, the duration of the activation step is from about 24 to about 48 hours. In some aspects, the duration of the activation step is from about 24 to about 72 hours. In some aspects, the activation step lasts for less than about 144 hours.

In some aspects, the duration of the priming step is sufficient to produce at least a 2-fold greater amplification of the number of VSTs.

In some or any of the foregoing or related aspects, the method does not induce higher levels of NK than would be expected if the mononuclear population were simultaneously exposed to IL-4, IL-7, and IL-15. Cell expansion.

In some or any of the foregoing or related aspects, the VST targets one or more antigens from a latent virus.

In some or any of the foregoing or related aspects, the method includes obtaining the amplified VSTs.

In some aspects, the invention provides a two-step method for selectively expanding VSTs in a heterogeneous cell population, comprising: first culturing the VSTs in the presence of a stimulating antigen and IL-4 and IL-7. step; and a second step of culturing the VST in the presence of IL-15.

In some aspects, the present invention provides a composition comprising VST expanded in a two-step process comprising: a first step of culturing the VST in the presence of IL-4 and IL-7; and The second step is to culture the VSTs in the presence of IL-15.

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for two or more HBV antigens.

In some aspects, the invention provides a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for two or more HHV8 antigens.

In some or any of the foregoing or related aspects, the population of VSTs includes CD4+ and CD8+ cells.

In some or any of the foregoing or related aspects, the population of VSTs produces two or more effector molecules.

In some or any of the foregoing or related aspects, the composition is produced in vitro.

In some or any of the foregoing or related aspects, the composition is produced ex vivo.

In some or any of the foregoing or related aspects, the VSTs produce two or more effector molecules when stimulated with the two or more HBV antigens.

In some or any of the foregoing or related aspects, the two or more HBV antigens are selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX.

In some or any of the foregoing or related aspects, the two or more HBV antigens are selected from the group consisting of HBsAg, HBeAg, HBP and HBX.

In some or any of the foregoing or related aspects, the two or more HBV antigens are HBcAg and HBsAg.

In some or any of the foregoing or related aspects, the VSTs produce two or more effector molecules when stimulated with the two or more HHV8 antigens.

In some or any of the foregoing or related aspects, the two or more HHV8 antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some or any of the foregoing or related aspects, the effector molecules are selected from the group including: IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP- 1b.

In some or any of the foregoing or related aspects, the VSTs are multifunctional.

In some or any of the foregoing or related aspects, the population of VSTs comprises at least 70% CD3+ T cells.

In some or any of the foregoing or related aspects, the composition comprises no more than 30% CD56+ cells.

In some or any of the foregoing or related aspects, the VST has a specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10e5 cells .

In some or any of the foregoing or related aspects, the VSTs comprise CD4+ and CD8+ T cells.

In some or any of the foregoing or related aspects, no more than 20% of such VSTs exhibit markers of T cell exhaustion. In some aspects, the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT. In some aspects, the T cell exhaustion markers are PD1 and TIM3.

In some or any of the foregoing or related aspects, the VSTs exhibit one or more markers associated with T cell activation. In some aspects, the one or more markers associated with T cell activation are selected from CD25 and CD69.

In some or any of the foregoing or related aspects, the VSTs exhibit one or more markers associated with central or effector cell memory. In some aspects, the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST Enriched at least 1000-fold. In some aspects, the VSTs are enriched at least 3000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. In some aspects, the VSTs are enriched at least 6000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen, the VST Enriched at least 1000-fold. In some aspects, the VSTs are enriched at least 3000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen compared to the cell population prior to expansion. In some aspects, the VSTs are enriched at least 6000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen compared to the cell population prior to expansion.

In some or any of the foregoing or related aspects, the multi-strain population of VST is an antigen-specific multi-strain population of VST.

In some or any of the foregoing or related aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HBV antigens. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg. In some aspects, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBeAg. In some aspects, the antigen-specific multi-strain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg and one or more peptide sequences derived from HBeAg. In some aspects, the antigen-specific multi-strain population of VST pairs at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 4. At least one peptide sequence of ID NO: 5 is specific.

In some or any of the foregoing or related aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HHV8 antigens. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1. In some aspects, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NOs: 6-12. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from gB. In some aspects, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NOs: 13-15. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1 and one or more peptide sequences derived from gB. In some aspects, the antigen-specific multi-strain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 6-12 and at least one peptide sequence selected from SEQ ID NO: 13-15.

In some or any of the foregoing or related aspects, the multi-strain population of VST has reduced alloreactivity.

In some or any of the foregoing or related aspects, the multi-strain population of VST has negligible alloreactivity.

In some or any of the foregoing or related aspects, the multi-strain population of VST has reduced reactivity against the allogeneic target.

In some or any of the foregoing or related aspects, the multi-strain population of VST has negligible reactivity against the allogeneic target.

In some or any of the foregoing or related aspects, the multi-strain population of VST has reduced autoreactivity.

In some or any of the foregoing or related aspects, the multi-strain population of VST has negligible autoreactivity.

In some or any of the foregoing or related aspects, the composition includes a pharmaceutically acceptable diluent. In some aspects, the compositions are formulated for intravenous administration.

In some aspects, the invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some aspects, the multi-strain population of VST includes specificity for two or more HBV antigens.

In some aspects, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of the VST of the invention. In some aspects, the multi-strain population of VST includes specificity for two or more HHV8 antigens.

In some or any of the foregoing or related aspects, the population of VSTs includes CD4+ and CD8+ cells.

In some or any of the foregoing or related aspects, the population of VSTs produces two or more effector molecules.

In some or any of the foregoing or related aspects, the composition is produced in vitro.

In some or any of the foregoing or related aspects, the composition is produced ex vivo.

In some or any of the foregoing or related aspects, the VSTs produce two or more effector molecules when stimulated with the two or more HBV antigens.

In some or any of the foregoing or related aspects, the two or more HBV antigens are selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX.

In some or any of the foregoing or related aspects, the two or more HBV antigens are selected from the group consisting of HBsAg, HBeAg, HBP and HBX.

In some or any of the foregoing or related aspects, the two or more HBV antigens are HBcAg and HBsAg.

In some or any of the foregoing or related aspects, the VSTs produce two or more effector molecules when stimulated with the two or more HHV8 antigens.

In some or any of the foregoing or related aspects, the two or more HHV8 antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some or any of the foregoing or related aspects, the effector molecules are selected from the group including: IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP- 1b.

In some or any of the foregoing or related aspects, the VSTs are multifunctional.

In some or any of the foregoing or related aspects, the population of VSTs comprises at least 70% CD3+ T cells.

In some or any of the foregoing or related aspects, the composition comprises no more than 30% CD56+ cells.

In some or any of the foregoing or related aspects, the VST has a specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10e5 cells .

In some or any of the foregoing or related aspects, the VSTs comprise CD4+ and CD8+ T cells.

In some or any of the foregoing or related aspects, no more than 20% of such VSTs exhibit markers of T cell exhaustion. In some aspects, the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT. In some aspects, the T cell exhaustion markers are PD1 and TIM3.

In some or any of the foregoing or related aspects, the VSTs exhibit one or more markers associated with T cell activation. In some aspects, the one or more markers associated with T cell activation are selected from CD25 and CD69.

In some or any of the foregoing or related aspects, the VSTs exhibit one or more markers associated with central or effector cell memory. In some aspects, the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST Enriched at least 1000-fold. In some aspects, the VSTs are enriched at least 3000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. In some aspects, the VSTs are enriched at least 6000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion.

In some or any of the foregoing or related aspects, after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, the VST Enriched at least 1000-fold. In some aspects, the VSTs are enriched at least 3000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen compared to the cell population prior to expansion. In some aspects, the VSTs are enriched at least 6000-fold after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen compared to the cell population prior to expansion.

In some or any of the foregoing or related aspects, the multi-strain population of VST is an antigen-specific multi-strain population of VST.

In some or any of the foregoing or related aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HBV antigens. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg. In some aspects, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBeAg. In some aspects, the antigen-specific multi-strain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg and one or more peptide sequences derived from HBeAg. In some aspects, the antigen-specific multi-strain population of VST pairs at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 4. At least one peptide sequence of ID NO: 5 is specific.

In some or any of the foregoing or related aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HHV8 antigens. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1. In some aspects, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NOs: 6-12. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from gB. In some aspects, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NOs: 13-15. In some aspects, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1 and one or more peptide sequences derived from gB. In some aspects, the antigen-specific multi-strain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 6-12 and at least one peptide sequence selected from SEQ ID NO: 13-15.

In some or any of the foregoing or related aspects, the multi-strain population of VST has reduced alloreactivity.

In some or any of the foregoing or related aspects, the multi-strain population of VST has negligible alloreactivity.

In some or any of the foregoing or related aspects, the multi-strain population of VST has reduced reactivity against the allogeneic target.

In some or any of the foregoing or related aspects, the multi-strain population of VST has negligible reactivity against the allogeneic target.

In some or any of the foregoing or related aspects, the multi-strain population of VST has reduced autoreactivity.

In some or any of the foregoing or related aspects, the multi-strain population of VST has negligible autoreactivity.

In some or any of the foregoing or related aspects, the composition is administered to the subject at a dose of about 5×10 ⁶ to about 5×10 ⁸ cells/square meter.

In some or any of the foregoing or related aspects, the composition is administered to the subject once a week, once every two weeks, once every three weeks, or once every four weeks.

In some or any of the foregoing or related aspects, the composition is administered to the individual a plurality of times.

In some or any of the foregoing or related aspects, the individual is immunocompromised.

In some or any of the foregoing or related aspects, the individual has received a solid organ transplant; has received chemotherapy; has an HIV infection; has a genetic immunodeficiency; has a chronic viral infection; and/or has received Allogeneic or autologous stem cell transplantation.

In some or any of the foregoing or related aspects, the individual is an allogeneic hematopoietic stem cell transplant (allo-HCT) recipient.

In some aspects, the invention provides a method for expanding T cells in vitro, comprising the steps of: a) contacting a starting population of mononuclear spheres with one or more compositions, the one or more compositions Comprising: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more compounds comprising at least IL-4, IL-7 and IL -15 interleukin, thereby generating an expanded T cell population.

In some aspects, the invention provides a method for expanding T cells in vitro, comprising the steps of: a) contacting a starting population of mononuclear spheres with one or more compositions, the one or more compositions Comprising: i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and ii) one or more cells comprising at least IL-4 and IL-7 interleukins, wherein the interleukins do not comprise IL-15; b) subsequently contacting the cell population with IL-15, thereby generating an expanded T cell population.

In some embodiments, the monocytes are peripheral blood monocytes (PBMC).

In some embodiments, the method does not include adding IL-2.

In some embodiments, the starting population of mononuclear spheres is contacted with a composition comprising (a)(i) and a separate composition comprising (a)(ii). In some embodiments, the starting population of mononuclear spheres is contacted with a composition comprising (a)(i) and (a)(ii). In some embodiments, (a)(i) is at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, (a)(ii) is one or more interleukins comprising at least IL-4 and IL-7, wherein the interleukins do not comprise IL-15. In some embodiments, (a)(ii) is one or more interleukins comprising at least IL-4 and IL-7. In some embodiments, (a)(ii) is one or more interleukins comprising at least IL-4 and IL-7, but not IL-2. In some embodiments, (a)(i) is at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen, and (a)(ii) is One or more interleukins comprising at least IL-4 and IL-7, wherein the interleukins do not comprise IL-15. In some embodiments, (a)(i) is at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen, and (a)(ii) is One or more interleukins comprising at least IL-4 and IL-7. In some embodiments, (a)(i) is at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen, and (a)(ii) is One or more interleukins comprising at least IL-4 and IL-7 but not IL-2.

In some embodiments, the starting population of mononuclear spheres is contacted with: a first composition comprising (a) (i) at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a a peptide or peptide mixture that is part of an antigen of interest; and at least a second separate composition comprising (a)(ii) one or more interleukins comprising at least IL-4 and IL-7, wherein the interleukins are not Contains IL-15.

In some embodiments, the starting population of monocytes is contacted with a first composition comprising: (a) (i) at least one target antigen or a portion of a target antigen or corresponding to at least one target an antigen or a peptide or mixture of peptides that is part of an antigen of interest; and (a)(ii) one or more interleukins comprising at least IL-4 and IL-7, wherein the interleukins do not comprise IL-15. In some embodiments, IL-4 and IL-7 are added simultaneously. In some embodiments, IL-4 and IL-7 are added in parallel.

In some embodiments, IL-4 and IL-7 are added separately.

In some embodiments, IL-4 and IL-7 are added within 24 hours of each other.

In some embodiments, step (b) is performed within about 10 days of step (a). In some embodiments, step (b) is performed within about 9 days of step (a). In some embodiments, step (b) is performed within about 8 days of step (a). In some embodiments, step (b) is performed within about 7 days of step (a). In some embodiments, step (b) is performed within about 6 days of step (a). In some embodiments, step (b) is performed within about 5 days of step (a). In some embodiments, step (b) is performed within about 4 days of step (a). In some embodiments, step (b) is performed within about 3 days of step (a). In some embodiments, step (b) is performed within about 2 days of step (a). In some embodiments, step (b) is performed within about 1 day of step (a). In some embodiments, step (b) is performed within about 18 hours of step (a). In some embodiments, step (b) is performed within about 12 hours of step (a). In some embodiments, step (b) is performed within about 6 hours of step (a). In some embodiments, step (b) is performed within about 1 hour of step (a).

In some embodiments, step (b) occurs about 10 days after step (a). In some embodiments, step (b) occurs about 9 days after step (a). In some embodiments, step (b) occurs about 8 days after step (a). In some embodiments, step (b) occurs about 7 days after step (a). In some embodiments, step (b) occurs about 6 days after step (a). In some embodiments, step (b) occurs about 5 days after step (a). In some embodiments, step (b) is performed about 4 days after step (a). In some embodiments, step (b) is performed about 3 days after step (a). In some embodiments, step (b) is performed about 2 days after step (a). In some embodiments, step (b) occurs about 48 hours after step (a).

In some embodiments, step (b) occurs approximately 24 hours after step (a).

In some embodiments, step (b) is performed on the same day as step (a), optionally within 18 hours of step (a). In some embodiments, step (b) is performed on the same day as step (a), and optionally within 12 hours of step (a). In some embodiments, step (b) is performed on the same day as step (a), optionally within 6 hours of step (a). In some embodiments, step (b) is performed on the same day as step (a), optionally within 4 hours of step (a). In some embodiments, step (b) is performed on the same day as step (a), and optionally within 2 hours of step (a). In some embodiments, step (b) is performed on the same day as step (a), and optionally within 1 hour of step (a).

In some embodiments, step (b) followed by contacting the population of cells with IL-15 is performed within about 7 days of step (a) of contacting the initial population of mononuclear spheres with one or Contacting a plurality of compositions, the one or more compositions comprising: i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and ii) one or more Cytokines including at least IL-4 and IL-7. In some embodiments, step (b) is performed within 7 days of step (a). In some embodiments, step (b) is performed within about 2 days of step (a). In some embodiments, step (b) is performed within 2 days of step (a). In some embodiments, step (b) is performed within about 48 hours after step (a). In some embodiments, step (b) is performed within about 24 hours after step (a). In some embodiments, step (b) is performed within 48 hours after step (a). In some embodiments, step (b) is performed within 24 hours after step (a). In some embodiments, step (b) occurs about 48 hours after step (a). In some embodiments, step (b) occurs approximately 24 hours after step (a). In some embodiments, step (b) is performed 48 hours after step (a). In some embodiments, step (b) is performed 24 hours after step (a). In some embodiments, step (b) is performed on the same day as step (a), and optionally within 12 hours of step (a). In some embodiments, step (b) is performed on the same day as step (a), and optionally within 2 hours of step (a).

In some embodiments, the amplification is performed for 18 days or less, optionally 14 days.

In some embodiments, the amplification is performed for an amount of time sufficient to produce cells that have completed logarithmic phase growth. In some embodiments, the amplification is performed for an amount of time sufficient to produce cells that have completed log phase growth, wherein the amplification is performed for about 9 days to about 18 days. In some embodiments, the amplification is performed for about 9 days. In some embodiments, the amplification is performed for about 10 days. In some embodiments, the amplification is performed for about 11 days. In some embodiments, the amplification is performed for about 12 days. In some embodiments, the amplification is performed for about 13 days. In some embodiments, the amplification is performed for about 14 days. In some embodiments, the amplification is performed for about 15 days. In some embodiments, the amplification is performed for about 16 days. In some embodiments, the amplification is performed for about 17 days. In some embodiments, the amplification is performed for about 18 days. In some embodiments, the amplification is performed for about 19 days. In some embodiments, the amplification is performed for about 20 days. In some embodiments, the amplification is performed for about 21 days.

In some embodiments, the expansion is performed in the presence of T cell expansion medium. In some embodiments, the T cell expansion medium is any suitable medium for T cell expansion. Those skilled in the art will be able to select appropriate T cell expansion media. In some embodiments, the T cell expansion medium is a commercially available T cell expansion medium. An illustrative example of a commercially available T cell expansion medium is TexMAC™ GMP medium (Miltenyi Biotec).

In some embodiments, the amplification is performed using a seeding density of between 2 × 10 ⁶ cells/cm2 and 4 × 10 ⁶ cells/cm2, and optionally 3 × 10 ⁶ cells/cm2. In some embodiments, the amplification is performed using a seeding density of about 2 × 10 ⁶ cells/cm2 to about 4 × 10 ⁶ cells/cm2. In some embodiments, the amplification is performed at a seeding density of about 3 × ¹⁰ cells/cm2.

In some embodiments, the expanded T cell population has a cell number that is at least about 3 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least about 2-fold greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least about 2.5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 3 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least about 3 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 4 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least about 4 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least about 5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 6 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least about 6 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 7 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least about 7 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 2-fold higher to about 7-fold higher than the starting cell population.

In some embodiments, the expanded T cell population includes antigen-specific T cells, wherein the antigen-specific T cells are pathogen-specific T cells (PST), virus-specific T cells (VST), or tumor-specific T cells (TST).

In some embodiments, the peptide mixture contains between 2 and 750 different peptides. In some embodiments, the peptide mixture includes about 2 to about 750 different peptides. In some embodiments, the peptide mixture contains 2 to 750 different peptides.

In some embodiments of the methods of the invention, the peptide mixture contains between 2 and 750 different peptides.

In some embodiments, each peptide in the peptide mixture has at least 2 amino acid overlap with at least one other peptide. In some embodiments, each peptide in the peptide mixture has at least 3 amino acid overlap with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 4 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture has at least 5 amino acid overlap with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 6 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 7 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture has at least 8 amino acid overlap with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 9 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 10 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 11 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 12 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 13 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 14 amino acids with at least one other peptide. In some embodiments, each peptide in the peptide mixture overlaps at least 15 amino acids with at least one other peptide.

In some embodiments, each peptide in the peptide mixture overlaps with at least one other peptide by about 6 amino acids to about 15 amino acids. In some embodiments, each peptide in the peptide mixture overlaps with at least one other peptide by about 8 amino acids to about 14 amino acids. In some embodiments, each peptide in the peptide mixture overlaps with at least one other peptide by about 9 amino acids to about 12 amino acids. In some embodiments, each peptide in the peptide mixture overlaps with at least one other peptide by about 10 amino acids to about 14 amino acids. In some embodiments, each peptide in the peptide mixture overlaps with at least one other peptide by about 11 amino acids to about 14 amino acids. In some embodiments, each peptide in the peptide mixture overlaps with at least one other peptide by about 8 amino acids to about 11 amino acids.

In some embodiments, the peptides are at least 7 amino acids long. In some embodiments, the peptides are at least 8 amino acids long. In some embodiments, the peptides are at least 9 amino acids long. In some embodiments, the peptides are at least 10 amino acids long. In some embodiments, the peptides are at least 11 amino acids long. In some embodiments, the peptides are at least 12 amino acids long. In some embodiments, the peptides are at least 13 amino acids long. In some embodiments, the peptides are at least 14 amino acids long. In some embodiments, the peptides are at least 15 amino acids long. In some embodiments, the peptides are at least 16 amino acids long. In some embodiments, the peptides are at least 17 amino acids long. In some embodiments, the peptides are at least 18 amino acids long. In some embodiments, the peptides are at least 19 amino acids long. In some embodiments, the peptides are at least 20 amino acids long. In some embodiments, the peptides are about 8 amino acids in length to about 18 amino acids in length. In some embodiments, the peptides are about 12 amino acids in length to about 18 amino acids in length. In some embodiments, the peptides are about 14 amino acids in length to about 16 amino acids in length. In some embodiments, the peptides are about 12 amino acids in length to about 15 amino acids in length. In some embodiments, the peptides are about 15 amino acids in length to about 18 amino acids in length.

In some embodiments, the peptide mixture includes one or more tumor-associated antigens or portions thereof.

In some embodiments, the one or more tumor-associated antigens are selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2 , TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu, MAGE A3, p53 non-mutant, NY -ESO-1, PSMA, GD2, Melan A (Melan A)/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, survivin, PSA, hTERT , EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GMl, mesothelin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydride Enzyme IX, PAX5, OY-TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fas-related antigen 1.

In some embodiments, the peptide mixture includes one or more target antigens from at least one pathogen, or a portion thereof. In some embodiments, the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), Hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 ( HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV) , mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T lymphocyte Cellular viruses.

In some embodiments, the at least one pathogen is selected from the group consisting of cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus ( HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), Estén-Barr virus (EBV), cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, Measles virus, human metapneumovirus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T-lymphotropic virus.

In some embodiments, the pathogen is HBV, and the at least one target antigen is selected from the group consisting of surface antigen (HBsAg), core antigen (HBcAg), pre-core antigen (HBeAg), DNA polymerase, and combinations thereof.

In some embodiments, the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), core antigen (HBcAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or its combination. In some embodiments, the target antigens include each of HBsAg, HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the peptide mixture includes a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3).

In some embodiments, a portion of the target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3). In some embodiments, PLPVLQAGFFLLTRI, FFLLTRILTIPQSLD, and AKYLWEWASVRFSWL are peptide sequences derived from HBsAg.

In some embodiments, one or more HBV antigens are selected from one or more HBV genotypes.

In some embodiments, one or more HBV antigens of interest (or portions of the target antigens) are derived from one HBV genotype. In some embodiments, one or more HBV antigens (or portions of the target antigens) are derived from sequences conserved in more than one HBV genotype.

In some embodiments, at least one antigenic line is selected from HBV genotype A. In some embodiments, at least one antigenic line is selected from HBV genotype C. In some embodiments, at least two antigenic lines are selected from the same HBV genotype. In some embodiments, at least one antigenic line is selected from HBV genotype A and at least one antigenic line is selected from HBV genotype C.

In some embodiments, the amino acid sequence of each HBV antigen differs from the amino acid sequence of each other HBV antigen by at least one amino acid.

In some embodiments, at least a portion of the T cell response is CD4+.

In some embodiments, at least a portion of the T cell response is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted.

In some embodiments, the pathogen is EBV, and the at least one target antigen is selected from the group consisting of EBNAl, LMP2, BZLF1, and combinations thereof.

In some embodiments, the pathogen is EBV and the at least one target antigen is EBNAl, LMP2, BZLF1, or a combination thereof.

In some embodiments, the pathogen is CMV and the at least one target antigen is selected from IEl, pp65, and combinations thereof.

In some embodiments, the pathogen is CMV and the at least one target antigen is IEl, pp65, or a combination thereof.

In some embodiments, the pathogen is Adv, and the at least one target antigen is selected from the group consisting of hexons, pentons, and combinations thereof.

In some embodiments, the pathogen is Adv and the at least one target antigen is hexon, penton, or a combination thereof.

In some embodiments, the pathogen is BK virus, and the at least one target antigen is selected from LT, VP-1, and combinations thereof.

In some embodiments, the pathogen is BK virus and the at least one target antigen is LT, VP-1, or a combination thereof.

In some embodiments, the pathogen is HHV6, and the at least one target antigen is selected from the group consisting of U14, U11, U71, U54, U90, and combinations thereof.

In some embodiments, the pathogen is HHV6 and the at least one target antigen is U14, U11, U71, U54, U90, or a combination thereof.

In some embodiments, the pathogen is RSV, and the at least one target antigen is selected from N, F, and combinations thereof.

In some embodiments, the pathogen is RSV and the at least one target antigen is N, F, or a combination thereof.

In some embodiments, the pathogen is an influenza virus, and the at least one target antigen is selected from MP1, NP1, and combinations thereof.

In some embodiments, the pathogen is influenza virus and the at least one target antigen is MP1, NP1, or a combination thereof.

In some embodiments, the pathogen is parainfluenza virus type 3 (PIV3), and the at least one target antigen is selected from the group consisting of F, N, M, M2-1, HN, and combinations thereof.

In some embodiments, the pathogen is a parainfluenza virus (PIV) and the at least one target antigen is F, N, M, M2-1, HN, or a combination thereof.

In some embodiments, the pathogen is hMPV, and the at least one target antigen is selected from the group consisting of F, N, M2-1, M, and combinations thereof.

In some embodiments, the pathogen is hMPV and the at least one target antigen is F, N, M2-1, M, or a combination thereof.

In some embodiments, the pathogen is HHV8, and the at least one target antigen is selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) and their combinations.

In some embodiments, the pathogen is HHV8 and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof. In some embodiments, the target antigens include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12 , K12), each of gB (ORF8), MIR1 (K3), SSB (ORF6), and TS (ORF70).

In some embodiments, the pathogen is SARS-CoV2, and the at least one target antigen is selected from the group consisting of spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14 and their combinations.

In some embodiments, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), matrix protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some embodiments, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some embodiments, the methods of the invention further comprise the step of preparing a pharmaceutical composition, wherein diluents, stabilizers, preservatives and/or other pharmaceutically acceptable excipients are added to the expanded T cell population. agent.

In some aspects, the invention provides an expanded T cell population obtained by the methods of the invention. In some embodiments, the T cell population includes at least 70% CD3+ T cells. In some embodiments, the expanded T cell population includes at least 70% CD3+ T cells. In some embodiments, the expanded T cell population includes about 70% CD3+ T cells. In some embodiments, the expanded T cell population includes at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% CD3+ T cells. In some embodiments, the expanded T cell population includes about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% CD3+ T cells.

In some embodiments, the expanded T cell population includes virus-specific T cells (VST).

In some embodiments, no more than 30% of the cells in the population are natural killer (NK) cells. In some embodiments of the expanded T cell population of the invention, no more than 10%, no more than 20%, no more than 30%, no more than 40%, or no more than 50% of the cells in the population are natural killers (NK) cells. In some embodiments, about 30% or less of the cells in the population are natural killer (NK) cells. In some embodiments of the expanded T cell population of the invention, about 10% or less, about 20% or less, about 30% or less, about 40% or less, or about 50% of the population % or less of the cells are natural killer (NK) cells.

In some embodiments of the expanded T cell population of the invention, about 10%, about 20%, about 30%, about 40%, or about 50% of the cells in the population are natural killer (NK) cells.

In some embodiments of the expanded T cell population of the invention, no more than 30% of the cells in the population are NK cells.

In some embodiments, the T cell population includes CD4+ and CD8+ T cells.

In some embodiments, more than 80% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments of the expanded T cell population of the invention, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 99% of the T cell population consists of CD4+ and/or CD8+ T cell composition. In some embodiments, more than about 80% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments of the expanded T cell population of the invention, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or more than about 99% of the T cell population. Composed of CD4+ and/or CD8+ T cells.

In some embodiments of the expanded T cell population of the invention, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the T cell population consists of CD4+ and/or CD8+ T cell composition.

In some embodiments of the expanded T cell population of the invention, more than 80% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, the T cells are polyline.

In some embodiments, at least 1% of the CD3+ T cells produce TNFα. In some embodiments, at least 0.5%, at least 1%, at least 1.5%, or at least 2% of the CD3+ T cells can produce TNFα.

In some embodiments, about 0.5%, about 1%, about 1.5%, or about 2% of the CD3+ T cells can produce TNFα.

In some embodiments, at least 1% of the CD3+ T cells produce TNFα.

In some embodiments, at least one T cell recognizes one or more tumor-associated antigens, or portions thereof. In some embodiments, the one or more tumor-associated antigens are selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2 , TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu, MAGE A3, p53 non-mutant, NY -ESO-1, PSMA, GD2, melanin A/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP , ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl, mesothelin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5 , OY-TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT- 2 and Fas-related antigen 1.

In some embodiments, at least one T cell recognizes one or more target antigens, or portions thereof, from at least one pathogen. In some embodiments, the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), Hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 ( HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV) , mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T lymphocyte Cellular viruses.

In some embodiments, the at least one pathogen is selected from the group consisting of cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus ( HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), Estén-Barr virus (EBV), cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, Measles virus, human metapneumovirus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T-lymphotropic virus.

In some embodiments, the pathogen is HBV, and the at least one target antigen is selected from the group consisting of surface antigen (HBsAg), core antigen (HBcAg), pre-core antigen (HBeAg), DNA polymerase, and combinations thereof. In some embodiments, the pathogen is HBV and the at least one target antigen is HBsAg, HBcAg, HBeAg, HBP, HBX, or a combination thereof. In some embodiments of the expanded T cell populations of the invention, the target antigens include each of HBsAg, HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3). In some embodiments, a portion of the target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3). In some embodiments, PLPVLQAGFFLLTRI, FFLLTRILTIPQSLD, and AKYLWEWASVRFSWL are peptide sequences derived from HBsAg.

In some embodiments, one or more HBV antigens of interest (or portions of the target antigens) are derived from one HBV genotype. In some embodiments, one or more HBV antigens (or portions of the target antigens) are derived from sequences conserved in more than one HBV genotype.

In some embodiments, at least one antigenic line is selected from HBV genotype A. In some embodiments, at least one antigenic line is selected from HBV genotype C. In some embodiments, at least two HBV antigen lines are selected from different HBV genotypes. In some embodiments, at least two antigenic lines are selected from the same HBV genotype. In some embodiments, at least one antigenic line is selected from HBV genotype A and at least one antigenic line is selected from HBV genotype C.

In some embodiments, the amino acid sequence of each HBV antigen differs from the amino acid sequence of each other HBV antigen by at least one amino acid.

In some embodiments, at least a portion of the T cell response is CD4+.

In some embodiments, at least a portion of the T cell response is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted. In some embodiments, at least a portion of the T cell response is HLA-DR, HLA-DQ or HLA-DP restricted.

In some embodiments, the pathogen is EBV, and the at least one target antigen is selected from the group consisting of EBNAl, LMP2, BZLF1, and combinations thereof.

In some embodiments, the pathogen is EBV and the at least one target antigen is EBNAl, LMP2, BZLF1, or a combination thereof.

In some embodiments, the pathogen is CMV and the at least one target antigen is selected from IEl, pp65, and combinations thereof.

In some embodiments, the pathogen is CMV and the at least one target antigen is IEl, pp65, or a combination thereof.

In some embodiments, the pathogen is Adv, and the at least one target antigen is selected from the group consisting of hexons, pentons, and combinations thereof.

In some embodiments, the pathogen is Adv and the at least one target antigen is hexon, penton, or a combination thereof.

In some embodiments, the pathogen is BK virus, and the at least one target antigen is selected from LT, VP-1, and combinations thereof.

In some embodiments, the pathogen is BK virus and the at least one target antigen is LT, VP-1, or a combination thereof.

In some embodiments, the pathogen is HHV6, and the at least one target antigen is selected from the group consisting of U14, U11, U71, U54, U90, and combinations thereof.

In some embodiments, the pathogen is HHV6 and the at least one target antigen is U14, U11, U71, U54, U90, or a combination thereof.

In some embodiments, the pathogen is RSV, and the at least one target antigen is selected from N, F, and combinations thereof.

In some embodiments, the pathogen is RSV and the at least one target antigen is N, F, or a combination thereof.

In some embodiments, the pathogen is an influenza virus, and the at least one target antigen is selected from MP1, NP1, and combinations thereof.

In some embodiments, the pathogen is influenza virus and the at least one target antigen is MP1, NP1, or a combination thereof.

In some embodiments, the pathogen is parainfluenza virus type 3 (PIV3), and the at least one target antigen is selected from the group consisting of F, N, M, M2-1, HN, and combinations thereof.

In some embodiments, the pathogen is a parainfluenza virus (PIV) and the at least one target antigen is F, N, M, M2-1, HN, or a combination thereof.

In some embodiments, the pathogen is hMPV, and the at least one target antigen is selected from the group consisting of F, N, M2-1, M, and combinations thereof.

In some embodiments, the pathogen is hMPV and the at least one target antigen is F, N, M2-1, M, or a combination thereof.

In some embodiments, the pathogen is HHV8, and the at least one target antigen is selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) and their combinations. In some embodiments, the pathogen is HHV8 and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof. In some embodiments, the target antigens include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12 , K12), each of gB (ORF8), MIR1 (K3), SSB (ORF6), and TS (ORF70).

In some embodiments, the pathogen is SARS-CoV2, and the at least one target antigen is selected from the group consisting of spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14 and their combinations.

In some embodiments, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), matrix protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some embodiments, the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof.

In some aspects, the invention provides a plurality of expanded T cell populations, each of which is an expanded T cell according to the invention, wherein each population includes T cells generated from donor monocytes obtained from a different donor. .

In some embodiments, each donor's HLA type differs from at least one of the other donors by at least one HLA allele.

In some aspects, the invention provides a universal antigen-specific T cell therapy product comprising an expanded T cell population or a plurality of expanded T cell populations according to the invention, wherein the product comprises a population obtained from a different supplier. T cells generated from donor monocytes.

In some embodiments, each donor's HLA type differs from at least one of the other donors by at least one HLA allele.

In some embodiments, the product is not alloreactive to partially HLA matched and/or to HLA mismatched target cells.

In some embodiments, the product includes virus-specific T cells (VST) that recognize one or more target antigens from at least one pathogen, or portions thereof.

In some aspects, the invention provides a pharmaceutical composition comprising an expanded T cell population, a plurality of expanded T cell populations, or a universal antigen-specific T cell therapy product of the invention.

In some embodiments, the pharmaceutical composition is formulated for intravenous delivery.

In some embodiments, the composition is negative for bacteria and fungi during continued culture for at least 7 days.

In some embodiments, the composition exhibits less than 5 EU/ml of endotoxin.

In some embodiments, the composition is negative for Mycoplasma species.

In some aspects, the present invention provides a method of preventing and/or treating viral infection, which includes administering a pharmaceutical composition of the present invention to an individual.

In some embodiments, the individual is immunocompetent or immunocompromised.

In some embodiments, the pharmaceutical composition is administered by injection or infusion.

In some embodiments, the pharmaceutical composition is administered to the individual once.

In some embodiments, the pharmaceutical composition is administered to the individual multiple times.

In some embodiments, between 5 × 10 ⁶ and 5 × 10 ⁷ cells/square meter are administered to the subject. In some embodiments, about 5 × 10 ⁶ to about 5 × 10 ⁷ cells/square meter are administered to the individual. In some embodiments, about 5 × 10 ⁶ to about 5 × 10 ⁸ cells/square meter are administered to the subject. In some embodiments, the individual is infected or at risk of becoming infected with a virus.

In some embodiments, administration of the pharmaceutical composition is effective to treat and/or prevent the viral infection in the individual.

In some embodiments, the individual: a. has received a solid organ transplant; b. has received chemotherapy; c. has an HIV infection; d. has a genetic immunodeficiency; e. has a chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation.

In some embodiments, the individual is immunocompetent.

In some embodiments, the pharmaceutical composition includes the universal antigen-specific T cell therapy product of the invention.

In some embodiments, the pharmaceutical composition is administered to the individual without knowledge of the individual's HLA type.

In some embodiments, the individual is a human.

In some embodiments, the invention provides a population of expanded virus-specific T cells specific for at least one HBV antigen selected from the group consisting of HBsAg, HBcAg, HBeAg, HBP, and HBX. In some embodiments, the invention provides a population of expanded virus-specific T cells specific for two or more HBV antigens selected from the group consisting of HBsAg, HBcAg, HBeAg, HBP and HBX. In some embodiments, the invention provides a population of expanded virus-specific T cells specific for three or more HBV antigens selected from the group consisting of HBsAg, HBcAg, HBeAg, HBP, and HBX. In some embodiments, the expanded population of virus-specific T cells is specific for each of the HBV antigens HBsAg, HBcAg, HBeAg, HBP, and HBX. In some embodiments, the invention provides a population of expanded virus-specific T cells with pairs selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA ( ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), one of the HHV8 antigens is specific. In some embodiments, the invention provides a population of expanded virus-specific T cells with pairs selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA ( At least one HHV8 antigen of ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) is specific. In some embodiments, the invention provides a population of expanded virus-specific T cells with pairs selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA ( Two or more HHV8 antigens including ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) are specific. In some embodiments, the expanded population of virus-specific T cells is responsive to the HHV8 antigens LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), 3, 4, 5, 6, 7, 8 or 9 are specific sex. In some embodiments, the expanded population of virus-specific T cells is responsive to the HHV8 antigens LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( Each of ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) is specific.

In some embodiments, the population of antigen-specific T cells comprises at least 70%, at least 75%, or at least 80% CD3+ T cells. In some embodiments, the population of antigen-specific T cells comprises at least 70%, at least 75%, at least 80%, at least 90%, or at least 99% CD3+ T cells. In some embodiments, the population of antigen-specific T cells comprises at least 70% CD3+ T cells.

In some embodiments, the population of antigen-specific T cells includes CD4+ and CD8+ T cells.

In some embodiments, the population of antigen-specific T cells comprises no more than 50%, no more than 40%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10% NK cells (e.g. CD56+ cells). In some embodiments, the population of antigen-specific T cells includes no more than 30% NK cells.

In some embodiments, the antigen-specific T cells are virus-specific T cells.

In some embodiments, the specificity of the virus-specific T cells for the target antigen is measured by spot-forming cells (SFC). SFC can be measured using any suitable assay in this technology, including but not limited to ELISpot (enzyme-linked immunosorbent spot) analysis.

In some embodiments, the specificity of the virus-specific T cells for the target antigen is measured by ELISpot analysis. In some embodiments, the specificity of the virus-specific T cells for the target antigen is measured by IFNγ ELISpot analysis. In some embodiments, the specificity of the VSTs for two or more antigens is measured by IFNγ ELISpot analysis. In some embodiments, the specificity of the VSTs for the three or more antigens is measured by IFNγ ELISpot analysis. In some embodiments, the specificity of the VSTs for the four or more antigens is measured by IFNγ ELISpot analysis. In some embodiments, the specificity of the VSTs for the five or more antigens is measured by IFNγ ELISpot analysis. In some embodiments, the virus-specific T cells have a specificity for the target antigen of at least 10 SFC/2 × ¹⁰ cells, at least 20 SFC/2 × ¹⁰ cells, at least 30 SFC/2 × 10 ⁵ cells, at least 40 SFC/2 × 10 ⁵ cells, at least 50 SFC/2 × 10 ⁵ cells, at least 60 SFC/2 × 10 ⁵ cells, at least 70 SFC/2 × 10 ⁵ cells, at least 80 SFC/2 × 10 ⁵ cells, at least 90 SFC/2 × 10 ⁵ cells, or at least 100 SFC/2 × 10 ⁵ cells. In some embodiments, the virus-specific T cells are specific for the target antigen from about 10 SFC/2 × 10 ⁵ cells to about 1000 SFC/2 × 10 ⁵ cells. In some embodiments, the virus-specific T cells are specific for the target antigen from about 10 SFC/2 × 10 ⁵ cells to about 500 SFC/2 × 10 ⁵ cells. In some embodiments, the virus-specific T cells are specific for the target antigen from about 10 SFC/2 × 10 ⁵ cells to about 100 SFC/2 × 10 ⁵ cells. In some embodiments, the virus-specific T cells have specificity for the two or more antigens, as measured by an IFNγ ELISpot assay, of at least about 1,000 SFC/2 × ¹⁰ cells, or at least about 1,500 SFC/2 × 10 ⁵ cells, or at least about 2,000 SFC/2 × 10 ⁵ cells, or at least about 2,500 SFC/2 × 10 ⁵ cells, or at least about 3,000 SFC/2 × 10 ⁵ cells.

In some embodiments, the virus-specific T cells comprise no more than 25%, no more than 20%, no more than 15%, or no more than 10% of cells expressing T cell exhaustion markers. In some embodiments, the virus-specific T cells comprise no more than 20% of cells expressing markers of T cell exhaustion. For example, in some embodiments, no more than 20% of the virus-specific T cells express a T cell exhaustion marker selected from Tim3, PD1, Lag3, and TIGIT.

In some embodiments of the expanded population of virus-specific T cells of the invention, the virus-specific T cells are multifunctional. For example, in some embodiments, a portion of the virus-specific T cells produce two or more effector molecules when stimulated with one of the HBV or HHV8 antigens. In some embodiments, the effector molecules are selected from the group consisting of: IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a, and MIP-1b.

In some aspects, the invention provides methods for treating HBV infection in an individual in need thereof, comprising administering to the individual an amplified virus provided herein that is specific for two or more HBV antigens. A population of specific T cells. In some aspects, the invention provides methods for treating HHV8 infection in an individual in need thereof, comprising administering to the individual an amplified virus provided herein that is specific for two or more HHV8 antigens. A population of specific T cells. In some embodiments, the HBV or HHV8 infection is a reactivated infection in an immunocompromised individual.

In some aspects, the present invention provides an in vitro method for stimulating the selective expansion of VST cells, comprising contacting a starting population of mononuclear spheres in culture with or corresponding to at least one target antigen. The peptide or peptide mixture is contacted first in the presence of IL-4 and IL-7 and then in the presence of IL-15.

In some aspects, the present invention provides an in vitro method for stimulating the selective expansion of VST cells, comprising contacting a starting population of mononuclear spheres in culture with or corresponding to at least one target antigen. The peptide or peptide mixture is contacted in the presence of IL-4, IL-7 and IL-15.

In some embodiments, the cells are contacted with IL-4 and IL-7 in culture for a first period that ranges between about 1 day and about 2 days.

In some embodiments, contact with IL-4 and IL-7 results in selective expansion of these VSTs but not NK cells.

In some embodiments, contact with IL-15 results in accelerated expansion of the cells in culture.

In some embodiments, contacting IL-4 and IL-7 prior to subsequent contacting with IL-15 results in an expanded population of VSTs that comprise an expanded population of VSTs obtainable by contacting the cells with Enriched populations compared to a broader spectrum of enriched VST for: (i) IL-4 and IL-7, without IL-15; (ii) IL-7 and IL-15, without IL-4; or (iii) both IL-4, IL-7 and IL-15.

In some aspects, the invention provides an in vitro method for selectively amplifying virus-specific T (VST) cells, comprising: a) contacting a starting population of mononuclear spheres with one or more compositions; For a period of time, the one or more compositions comprise: (i) at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen; and (ii) IL-4 and IL-7; and b) from (a) ) cells are exposed to IL-15 for the second period.

In some embodiments, the first period exceeds about 24 hours.

In some embodiments, the first period of time is between about 24 and about 48 hours.

In some embodiments, the first period of time is less than about 144 hours.

In some embodiments, the first period of time is sufficient to produce at least a 2-fold higher amplification of the number of VSTs. In some embodiments, the first period of time is sufficient to increase the number of VSTs by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least twice, at least three times. times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times.

In some embodiments, the first period does not result in higher levels of NK cell expansion than would be expected if the mononuclear population were simultaneously exposed to IL-4, IL-7, and IL-15.

In some embodiments, the VSTs target one or more antigens from latent viruses.

In some aspects, the invention provides a two-step method for selectively expanding VSTs in a heterogeneous cell population, comprising: first culturing the VSTs in the presence of a stimulating antigen and IL-4 and IL-7. step; and a second step of culturing the VST in the presence of IL-15.

In some aspects, the present invention provides a composition comprising VST expanded in a two-step process comprising: a first step of culturing the VST in the presence of IL-4 and IL-7; and The second step is to culture the VSTs in the presence of IL-15.

Cross-references to related applications

This application proposes U.S. Provisional Application No. 62/282,670 filed on November 23, 2021, U.S. Provisional Application No. 63/349,910 filed on June 7, 2022, and U.S. Provisional Application No. 63/349,910 filed on June 7, 2022. Priority and rights of application No. 63/349,934. The contents of the aforementioned application are incorporated into this application by reference.

Some viruses can cause chronic viral diseases and/or virus-related malignancies. This example includes hepatitis B virus (HBV), which causes short-term illness in many people. But for others, hepatitis B virus can become a long-term, chronic infection that can lead to serious and even life-threatening health problems, such as cirrhosis or liver cancer. Similarly, primary infection with the latent virus human herpesvirus 8 (HHV8) is generally asymptomatic in immunocompetent individuals. However, in immunocompromised patients, initial infection can present with lymphadenopathy, acute pancytopenia, and rapid progression to disseminated disease. In fact, HHV8 is the causative agent of all types of Kaposi's sarcoma (KS). Endogenous T cells reactive against these viruses often circulate at low frequencies and exhibit features of dysfunction/exhaustion. The present invention provides, at least in part, improved methods for enriching and expanding such T cells that support maintenance of virus specificity and increase functional efficacy.

The present invention provides, at least in part, improved methods for making antigen-specific T cells, such as virus-specific T cells (VST), compositions comprising such cells, and methods of using the cells and compositions in therapy. In some embodiments, the present invention provides stabilization for selectively expanding a population of T cells specific for one or more targeted antigens without substantially expanding undesirable non-specific cells, such as, for example, NK cells. method. Both IL-15 and IL-2 are used in cell culture methods to support T cell proliferation. (Rochman et al. Nat. Rev. Immunol. 9(7):480; July 2009). The present invention is based, at least in part, on the inventors' unexpected discovery that including IL-15 but not IL-2 in a VST manufacturing protocol resulted in a significant increase in specific VST expansion without significant expansion of non-specific cell populations. . In some embodiments, including IL-15 within a critical window of the amplification process results in maximal specific VST amplification. In some embodiments, including IL-15 during the amplification process results in maximal specific VST amplification. Without being bound by theory, it is believed that the methods of the present invention provide a solution to the need in the art for stable and specific amplification protocols capable of generating antibodies against antigens with different immunogenicity, including those from latent and/or VST products of chronic viruses, such as antigens of HBV and HHV-8). In some embodiments, VSTs produced using the methods of the present invention are specific, mobile, Th1 polarized, and multifunctional. Without being bound by theory, it is believed that VSTs produced using the method of the present invention provide superior treatment results compared to other methods.

In some embodiments, the invention provides a method for expanding T cells (e.g., VST) from mononuclear cells (optionally peripheral blood mononuclear cells (PBMC)) using methods including: (1) Contacting a population of mononuclear spheres with at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; (2) exposing a population of mononuclear spheres to one or more interleukins that bias expansion of the VST, wherein the interleukins do not include IL-15; and (3) Contacting a population of monocytes with IL-15, thereby generating an expanded T cell population containing cells with antigen specificity. In some embodiments, the above steps (1) and (2) in this paragraph are performed simultaneously. In some embodiments, the above steps (1) and (2) in this paragraph are performed sequentially. When proceeding in sequence, step (1) may be performed first or step (2) may be performed first, depending on the situation. In some embodiments, step (3) is performed after steps (1) and (2). In some embodiments, step (3) and step (1) are performed simultaneously. In some embodiments, step (3) and step (2) are performed simultaneously. In some embodiments, steps (1), (2) and (3) are performed simultaneously. Any interleukin suitable for selective amplification of VST may be utilized in step (2), including those disclosed herein. In some embodiments, the interleukin included in step (2) includes or consists of IL-4 and IL-7.

In some embodiments, the terms "antigen" and "target antigen" are used interchangeably.

In some embodiments, the invention provides a method for expanding T cells (e.g., VST) from mononuclear cells (optionally peripheral blood mononuclear cells (PBMC)) using methods including: (1) Contacting a population of mononuclear spheres with at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; (2) Contacting a population of mononuclear spheres with one or more interleukins that bias expansion of the VST, thereby generating a population of expanded T cells that includes cells with antigen specificity. In specific embodiments, the interleukin included in step (2) includes or consists of IL-4, IL-7 and IL-15.

Other features, objects, and advantages of the invention will be apparent from the detailed description and claims. definition

Before the present invention is explained in more detail, it may be helpful to provide definitions of certain terms that will be used herein to assist in understanding the invention.

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 invention belongs. Any materials and methods similar or equivalent to those described herein can be used in the practice of the present invention. The practice of the present invention may employ techniques commonly known in the art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology. Such techniques are fully explained in literature such as: Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (ed. MJ. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E. Cellis, 1998) Academic Press; Animal Cell Culture (edited by R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. Griffiths, and D.G. Newell, 1993- 1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by D.M. Weir and CC. Blackwell) ); Gene Transfer Vectors for Mammalian Cells (edited by J.M. Miller and M.P. Calos, 1987); Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., 1987); PCR: The Polymerase Chain Reaction, (edited by Mullis et al., 1994); Current Protocols in Immunology (J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies : a practical approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by V. T. DeVita et al., J.B. Lippincott) Company, 1993). Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, illustrative methods, devices, and materials are described herein. For the purposes of this invention, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of this invention, the following terms are defined below.

As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or this specification may mean "a" , but it is also consistent with the meaning of "one or more (species)", "at least one (species)" and "one (species) or more than one (species)". By way of example, "an element" means one element or more than one element. Some embodiments of the invention may consist of or consist essentially of one or more elements, one or more method steps and/or one or more methods of the invention. It is contemplated that any method or composition described herein may be practiced in accordance with any other method or composition described herein.

When immediately preceding a numerical value, the term "about" means ± 0% to 10%, ± 0% to 10%, ± 0% to 9%, ± 0% to 8%, ± 0% to 7 %, ± 0% to 6%, ± 0% to 5%, ± 0% to 4%, ± 0% to 3%, ± 0% to 2%, ± 0% to 1%, ± 0% to less than 1 %, or any other value or range of values therein. For example, "about 40" means ± 0% to 10% of 40 (i.e., 36 to 44).

Unless otherwise indicated, the term "and/or" is used in this disclosure to mean "and" or "or".

Unless the context otherwise requires, throughout this specification the words "comprise", "comprises" and "comprising" shall be understood to mean the inclusion of a stated step or element or group of steps or element, but not to the exclusion of any other step or element or other group of steps or elements. "Consisting of" means including and being limited to anything in the middle of the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or required and no other elements may be present. "Consisting essentially of" is meant to include any of the elements listed in the middle of the phrase, and is limited to other elements that do not interfere with or promote the activity or role of the listed elements as specified in the invention. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or required, but that other elements are optional and may be present depending on whether they materially affect the activity or effect of the listed elements. Or it may not exist.

Unless otherwise indicated, the term "disorder" is used herein to mean the term disease, condition or illness and it may be used interchangeably with these terms.

When used in conjunction with a therapeutic agent, such as an antigen-specific T cell product or cell line disclosed herein, an "effective amount" is an amount effective to treat or prevent a disease or disorder in an individual as described herein.

As used herein, the terms "priming phase", "priming period" and "priming step" are used interchangeably and refer to a cell and at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen in the method of the invention or a period of contact with a peptide or mixture of peptides that is part of a target antigen. Two or more interleukins (eg, IL-4 and IL-7) can be added in parallel. In some embodiments, the priming phase includes adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen, IL-4, and IL-7. In some embodiments, the priming phase includes adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen, IL-4, IL-7, and IL-15 . In some embodiments, the start-up period includes day 0 of the culture period.

In some embodiments, the priming period is sufficient to stimulate T cells. In some embodiments, the priming phase is sufficient to stimulate T cells by exposure to at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, stimulation of T cells results in upregulation of cell surface markers. In some embodiments, cell surface markers include CD25, CD40L, 41BB, and/or CD69. In some embodiments, the T cells are memory T cells.

As used herein, the terms "amplification phase," "amplification period," and "amplification step" are used interchangeably and refer to cells with at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or The period of time following contact between a peptide or peptide mixture that is part of a target antigen and two or more interleukins (eg, IL-4 and IL-7). In some embodiments, the expansion phase includes adding IL-15 on day 1 or 2 of the culture period.

In some embodiments, the culture time includes a start-up period and an expansion period.

When used in conjunction with the time in culture to generate a population of expanded T cells (eg, antigen-specific T cells), a "sufficient amount of time" is an amount of time sufficient to allow the cells to complete logarithmic phase growth. In some embodiments, log phase growth is complete when cell numbers plateau. In some embodiments, log phase growth is monitored using methods known in the art. In some embodiments, log phase growth is measured by serial cell counting of cell cultures. In some embodiments, log phase growth is measured by consumption of nutrients by cells. In some embodiments, the nutrient includes glucose. In some embodiments, log phase growth is measured by consumption of glucose by cells. In some embodiments, log-phase growth is measured by glucose monitoring, wherein stabilization of glucose levels indicates completion of log-phase growth. In some embodiments, log phase growth is measured by the cell's metabolite production. In some embodiments, the metabolite includes lactate. In some embodiments, log phase growth is measured by lactate production by cells. In some embodiments, log-phase growth is measured by lactate monitoring, wherein a stable increase in lactate content indicates completion of log-phase growth.

The term " eg " is used herein to mean "for example" and should be understood to mean the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or Other group steps or elements.

The terms "ex vivo" and "ex vivo" are used interchangeably herein to refer to the methods described herein for expanding, generating, and/or stimulating T cells.

The terms "contacting" and "adding" are used interchangeably herein to refer to contacting one or more compositions (e.g., interleukins and/or At least one target antigen or a portion of a target antigen or a peptide or a mixture of peptides (eg, mixed peptides)) or one or more compositions is added to the cell culture.

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes circumstances in which the event or circumstance occurs as well as circumstances in which it does not occur.

The term "tumor-associated antigen" as used herein refers to an antigenic substance produced/expressed on tumor cells that triggers an immune response in the host.

Exemplary tumor antigens include at least the following: carcinoembryonic antigen (CEA) for intestinal cancer; CA-125 for ovarian cancer; MUC-1 or epithelial tumor antigen (ETA) or CA15-3 for breast cancer; tyrosinase for malignant melanoma Or melanoma-associated antigen (MAGE); and abnormal products of ras and p53 in various types of tumors; α-fetoprotein in hepatocellular carcinoma, ovarian cancer or testicular cancer; β-subunit of hCG in men with testicular cancer; prostate-specific in prostate cancer Sexual antigen; β2 microglobulin in multiple myeloma and some lymphomas; CA19-9 in colorectal cancer, cholangiocarcinoma, and pancreatic cancer; staining granulin A in lung and prostate cancer; melanoma, soft tissue sarcoma, and breast cancer. TA90 for colorectal and lung cancer. Examples of tumor antigens are known in the art, for example Cheever et al., 2009, which is incorporated herein by reference in its entirety.

For example, specific examples of tumor antigens include at least CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4 -1BB, CT26, GITR, OX40, TGF-α, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, melanin A/MART1, Ras mutants, gp 100, p53 mutants, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, PSCA, MAGE A1 , sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK , HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2 and Fos-related antigen 1.

The term "viral antigen" as used herein refers to an antigen that is proteinaceous in nature and closely associated with viral particles. In specific embodiments, the viral antigen is a coat protein.

Specific non-limiting examples of viral antigens include at least one antigen derived from a virus selected from: EBV, CMV, adenovirus, BK virus, JC virus, HHV6, RSV, influenza virus, parainfluenza virus, Boca virus, coronavirus Viruses, LCMV, mumps virus, measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, herpes simplex virus, HPV, HBV, HIV, HTLV1, HHV8 and West Nile virus, hereby Kavirus, Ebola virus. In some embodiments, the viral antigen is derived from a latent virus. In some embodiments, the viral antigen is from a lytic virus.

The terms "virus-specific T cells" or "VST" or "virus-specific T cell strains" or "VST cell strains" are used interchangeably herein to refer to cells that have been expanded outside an individual and/or, for example, as described herein. Create a T cell strain that is specific and potent against one or more viruses of interest. The term "VST" as used herein means virus-specific T cells.

In some embodiments, a VST may be monoclonal or oligoclonal. In certain embodiments, the VST is multi-strain. As described herein, in some embodiments, a viral antigen or several viral antigens are presented to naive T cells or memory T cells in peripheral blood mononuclear spheres, and in response, a viral antigen(s) specific for the viral antigen(s) is Initial CD4+ and/or CD8+ T cell population expansion. For example, virus-specific T cells directed against EBV in a PBMC sample obtained from a suitable donor may recognize (bind to) an EBV antigen (e.g., a peptide epitope from the EBV antigen, optionally presented by the MHC), and this can Triggers the expansion of T cells specific for EBV. In another example, virus-specific T cells against BK virus in a PBMC sample obtained from a suitable donor and virus-specific T cells against adenovirus in the PBMC sample can recognize and bind to BK virus antigens and adenovirus, respectively. Antigens (such as peptide epitopes from BK virus antigens and adenovirus antigens respectively, as appropriate presented by the MHC), and this can trigger the expansion of T cells specific for BK virus and T cells specific for adenovirus increase.

In some embodiments, the T cells are human T cells. In some embodiments, the T cells are donor T cells. In some embodiments, the T cells are allogeneic T cells.

In some embodiments, the antigen-specific T cells are human antigen-specific T cells. In some embodiments, the antigen-specific T cells are donor antigen-specific T cells. In some embodiments the antigen-specific T cells are alloantigen-specific T cells.

In some embodiments, the VST is a human VST. In some embodiments, the VST is a donor VST. In some embodiments the VST is an allogeneic VST.

In some embodiments, the monocytes are human monocytes. In some embodiments, the monospheres are donor monospheres. In some embodiments the mononuclear spheres are allogeneic mononuclear spheres.

In some embodiments, the PBMC are human PBMC. In some embodiments, the PBMC are donor PBMC. In some embodiments, the PBMC are allogeneic PBMC.

As used herein, the term "exogenous" refers to a substance introduced into a composition, cell, cell culture, container, suspension, and the like, which substance is present in the composition, cell, cell culture, Generated externally to the container or suspension. The term "endogenous" refers to any substance produced internally from a composition, cell, cell culture, container or suspension.

As used herein, the term "cell therapy product" refers to a cell line that is expanded and/or produced outside an individual, for example, as described herein. For example, the term "cell therapy product" encompasses cell lines produced in culture. A cell line may comprise or consist essentially of effector cells. The cell line may comprise or consist essentially of NK cells. The cell line may comprise or consist essentially of T cells. For example, the term "cell therapy product" encompasses antigen-specific T cell lines produced in culture. In some cases, such antigen-specific T cell lines include expanded populations of memory T cells, expanded populations of T cells generated by stimulating naive T cells, and engineered T cells (e.g., CAR-T cells and expanded populations of T cells expressing exogenous proteins such as chimeric or recombinant T cell receptors, costimulatory receptors, and the like. In particular, in some embodiments, the term "cell therapy product" includes virus-specific T cell lines or tumor-specific T cell lines (eg, TAA-specific T cell lines). Cell lines can be monoculture or oligoculture. In certain embodiments, the cell lines are polystrain. In some embodiments, such multiple cell lines comprise expanded populations of multiple cells with different antigen specificities (eg, antigen-specific T cells). For example, one non-limiting example of a cell line encompassed by the term "cell therapy product" includes a multi-strain population of virus-specific T cells, which includes an expanded homogeneous population of a plurality of T cells, at least two of which They are specific for different viral antigens. Such multi-strain virus-specific T cells are known in the art and are disclosed in various patent applications filed by the present inventors, including WO2011028531, WO2013119947, WO2017049291 and PCT/US2020/024726, each of which It is incorporated herein by reference in its entirety.

The term "donor minibank" as used herein refers to a cell bank that contains a plurality of cell therapy products (e.g., antigen-specific T cell lines) that are collectively derived from a diverse pool of donors, such that the donor minibank contains At least one cell therapy product (eg, an antigen-specific T cell strain) that is a sufficient match for a defined percentage of patients in the target patient population. For example, in certain embodiments, the donor minibanks described herein include at least one donor that is a good match for 95% of a target patient population, such as, for example, allogeneic hematopoietic stem cell transplant recipients or immunocompromised individuals. Cell therapy products (e.g., antigen-specific T cell lines). The term "donor bank" as used herein refers to a plurality of donor minibanks. In various embodiments, it may be beneficial to create several non-duplicate mini-libraries for inclusion in a "donor library", thus ensuring that two or more well-matched cell therapy products are available for each potential patient. Cell banks can be stored frozen. Cryopreservation methods are known in the art and may include, for example, storage of cell therapy products (eg, antigen-specific T cell lines) at -70°C in a controlled access area, such as in vapor phase liquid nitrogen. Individual aliquots of cell therapy products can be prepared and stored in multiple containers (eg, vials) in calibrated liquid nitrogen dewars. Containers (eg vials) can be marked with a unique identification number that enables retrieval.

As used herein, the terms "patient" or "individual" are used interchangeably to refer to any mammal, including humans, domestic and farm animals, and zoo, sporting and pet animals, such as dogs, horses, cats, cattle, sheep, pigs , goats, rats, guinea pigs or non-human primates such as monkeys, chimpanzees, baboons or rhesus monkeys. A preferred mammal is a human being, including adults, children and the elderly.

As used herein, the term "population" refers to one or more cells in this specification. In some embodiments, the methods described herein are used to generate a population of cells; for example, a population of expanded T cells. In some embodiments, the expanded T cells described herein are a population of cells. In some embodiments, the antigen-specific T cells described herein are a population of cells. In some embodiments, the virus-specific T cells described herein are a population of cells. In some embodiments, the cell population is a starting cell population. In some embodiments, the cell population is a mononuclear spheroid population; for example, a PBMC population.

Unless otherwise indicated, the terms "treat", "treating", "treatment" and similar terms as used herein refer to the reversal, alleviation, inhibition of the disease, condition to which such terms apply. or condition or the course of one or more symptoms of such disease, condition or condition, or the prevention of a disease, condition or condition or one or more symptoms of such disease, condition or condition to which such terms apply, and includes Any of the compositions, pharmaceutical compositions or dosage forms described herein is administered to prevent the onset of a symptom or complication, or to alleviate a symptom or complication, or to eliminate a disease, symptom or condition. In some cases, treatment is curative or ameliorative.

In some embodiments, references herein to the term "third party" mean an individual (eg, a patient) that is different from the donor. Thus, for example, a reference to treating an individual with a "third party antigen-specific T cell product" (e.g., a third party VST product) means that the product is derived from donor tissue (e.g., PBMC isolated from donor blood) and that the individual (e.g. the patient) and the donor are not the same individual. In various embodiments, allogeneic cell therapy (eg, alloantigen-specific T cell therapy) is a "third party" cell therapy.

In relation to an individual, the term "prevent" or "preventing" means preventing the individual from contracting a disease or condition. Prevention includes preventive treatment. For example, prevention can include administering to an individual a compound disclosed herein before the individual develops the disease, and such administration will prevent the individual from developing the disease.

As used herein, the terms "administering," "administer," "administration" and similar terms refer to the transfer, delivery, introduction or delivery of a therapeutic agent to a patient in need of such treatment. Any pattern of an individual being treated with a pharmaceutical. Such modes include, but are not limited to, intraocular, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal and subcutaneous administration.

As used herein, the term "potential donor" refers to an individual (eg, a healthy individual) who is seropositive for one or more antigens to be targeted by the cell therapy products (eg, antigen-specific T cells) disclosed herein. In some embodiments, all potential donors eligible for inclusion in the donor pool are pre-screened and/or deemed seropositive for the target antigen.

In some embodiments, the term "target patient population" is used to describe a plurality of patients (or, interchangeably, "individuals") who are in need of a cell therapy product (eg, an antigen-specific T cell product) described herein. In some embodiments, this term encompasses the entire worldwide allogeneic HSCT population. In some embodiments, this term encompasses the entire United States-wide allogeneic HSCT population. In some embodiments, this term encompasses all patients included in the National Marrow Donor Program (NMDP) database, which is available at bioinformatics.bethematchclinical.org. In some embodiments, this term encompasses all patients included in the European Society for Blood and Marrow Transplantation (EBMT) database, which is available on the global information network at: ebmt.org/ebmt- patient-registry obtained. In some embodiments, this term encompasses the entire worldwide population of children aged ≤ 16 years with allogeneic HSCT. In some embodiments, this term encompasses the entire United States population of children aged ≤ 16 years with allogeneic HSCT. In some embodiments, this term encompasses the entire worldwide population of children aged ≤ 5 years with allogeneic HSCT. In some embodiments, this term encompasses the entire United States population of children aged ≤ 5 years with allogeneic HSCT. In some embodiments, this term encompasses the entire worldwide population of allogeneic HSCT individuals aged ≥ 65 years. In some embodiments, this term encompasses the entire United States population of allogeneic HSCT individuals aged ≥ 65 years.

In various embodiments, the term "well-matched" is used herein with reference to a given patient and a given cell therapy product (eg, an antigen-specific T cell line) to describe that the patient and the cell therapy product share at least two HLA alleles (That is, there are at least two HLA allele matching situations).

It is to be understood, however, that the embodiments and specific examples are intended to indicate specific embodiments of the invention, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from these embodiments. Given by means of illustration only.

The following discussion is directed to various embodiments of the invention. The term "present invention" is not intended to refer to any specific embodiment or to otherwise limit the scope of the invention. Although one or more of these embodiments may be preferred, the disclosed embodiments should not be construed or otherwise used to limit the scope of the invention, including technical solutions. Additionally, those skilled in the art will understand that the following description has broad application and that discussion of any embodiment is merely meant to be illustrative of that embodiment and is not intended to imply that the scope of the invention (including the scope of the claims) is limited to such embodiment. Example.

All publications and patents cited herein are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of conflict, the present application, including any definitions contained herein, will control. However, the mention of any references, articles, publications, patents, patent publications and patent applications cited herein is not and shall not be regarded as an admission or in any way an indication that they constitute valid prior art or form the basis of prior art in any country in the world. part of public knowledge.

However, the mention of any references, articles, publications, patents, patent publications and patent applications cited herein is not and shall not be regarded as an admission or in any way an indication that they constitute valid prior art or form the basis of prior art in any country in the world. part of public knowledge.

The section headings used in this article are for organizational purposes only and should not be construed as limiting the subject matter described. Overview

In particular, the present invention provides compositions and improved methods for making antigen-specific T cells, such as virus-specific T cells (VST), and methods of using such T cells in therapy. In some embodiments, the present invention provides stabilization for selectively expanding a population of T cells specific for one or more targeted antigens without substantially expanding undesirable non-specific cells, such as, for example, NK cells. method. The present invention is based in part on the inventors' unexpected discovery that including IL-15 but not IL-2 in a VST manufacturing protocol resulted in a significant increase in specific VST expansion without significant expansion of non-specific cell populations. In some embodiments, including IL-15 within a critical window of the amplification process results in maximal specific VST amplification. The methods described herein provide a solution to the need in this technology for stable and specific amplification protocols capable of generating VST products against antigens with different immunogenicity, including antigens from chronic viruses. . Importantly, VSTs produced using the methods disclosed herein are specific, mobile, Th1 polarizing and multifunctional. This indicates that VST produced via the methods disclosed herein will provide superior treatment results compared to prior art methods. Methods to expand T cells

In some aspects, the invention includes methods for producing cell therapy products, such as antigen-specific T cell lines, expanded antigen-specific T cell populations, selectively expanded antigen-specific T cells Cell populations and a plurality of expanded T cell populations. The invention further provides one or more T cell populations that are combined to produce a universal antigen-specific T cell product (eg, a universal virus-specific T cell product). In some embodiments, the antigen-specific T cells of the invention are virus specific. In some embodiments, virus-specific T cells of the invention are antigen-specific. In some embodiments, the antigen-specific T cells of the invention are virus-specific T cells. In some embodiments, T cells of the invention or generated using the methods of the invention are antigen-specific. In some embodiments, T cells of the invention or generated using the methods of the invention are virus specific. In some embodiments, T cells of the invention or generated using the methods of the invention are antigen-specific and virus-specific. In some embodiments, T cells of the invention or T cells produced using the methods of the invention are antigen-specific. In some embodiments, the T cells of the invention or T cells generated using the methods of the invention are virus specific. In some embodiments, T cells of the invention or T cells generated using the methods of the invention are antigen-specific and virus-specific.

Methods of the present invention for generating and expanding antigen-specific T cells generally comprise: obtaining a sample of cells from a donor, wherein the cells are mononuclear spheroids; contacting a starting population of mononuclear spheroids with one or more compositions, The one or more compositions comprise: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more interleukins; whereby This generates a population of expanded T cells. Generation of antigen-specific T cells

In some embodiments, methods for generating antigen-specific T cells as described herein include harvesting blood from one or more donors. In some embodiments, the sample is used independently to generate antigen-specific T cell lines or is included in a donor library. The term "donor bank" as used herein refers to a plurality of donor minibanks. As used herein, the term "donor minibank" refers to a cell bank that contains a plurality of cell therapy products (e.g., antigen-specific or virus-specific T cell lines) that are collectively derived from a diverse donor pool such that the donor The in vivo mini-library contains at least one cell therapy product (eg, antigen-specific or virus-specific T cell line) that is a good match for a defined percentage of patients in the target patient population. In some embodiments, methods of the present invention include harvesting blood from each donor included in a donor bank or donor minibank.

In some embodiments, methods for generating antigen-specific T cells as described herein include harvesting blood from one or more donors. In some embodiments, the samples are independently used to generate antigen-specific T cell lines. In some embodiments, methods of the present invention include harvesting blood from a donor. In some embodiments, methods of the present invention include harvesting blood from one or more donors of a selected donor group. In some embodiments, methods of the present invention include harvesting blood from each donor of the selected donor group.

In some embodiments, methods of the invention comprise harvesting blood from thirty or fewer different donors. In some embodiments, methods of the invention comprise harvesting blood from four to thirty different donors. In some embodiments, methods of the invention comprise harvesting blood from 4, 5, 6, 7, 8, 9 or 10 different donors.

In some embodiments, methods of the invention comprise harvesting mononuclear pellets (MNCs) from a donor. In some embodiments, methods of the invention comprise harvesting mononuclear pellets (MNCs) from one or more donors. In some embodiments, methods of the invention comprise harvesting MNCs from each donor of the selected donor group. In some embodiments, methods of the invention comprise harvesting mononuclear pellets (MNCs) from one or more donors included in a donor bank or donor mini-bank. In some embodiments, methods of the invention comprise harvesting MNCs from each donor included in a donor library or donor mini-bank. In some embodiments, harvesting MNCs from each donor includes isolating MNCs or causing MNC isolation. In some embodiments, MNCs comprise peripheral blood mononuclear cells (eg, PBMCs). In some embodiments, the MNCs comprise blood hemocytocytes. In some embodiments, harvesting MNCs from each donor includes isolating PBMCs or allowing PBMCs to be isolated. In some embodiments, harvesting MNCs from each donor includes isolating PBMCs using methods known to those skilled in the art. In some embodiments, MNC separation is performed by ficoll gradient. In some embodiments, MNCs are separated by density gradient. In some embodiments, harvesting MNCs as disclosed herein includes culturing the cells. In some embodiments, harvesting MNCs as disclosed herein includes cryopreserved cells.

In some embodiments, methods for generating antigen-specific T cell lines include culturing MNC in the presence of one or more antigens or functional variants thereof and one or more interleukins. In some embodiments, the MNC are peripheral blood mononuclear cells (PBMC). In some embodiments, MNCs are cultured in a container containing a gas-permeable culture surface. In some embodiments, the container is an infusion bag with a breathable portion. In some embodiments, the container is a rigid container. In some embodiments, the vessel is a GRex bioreactor.

In some embodiments, culturing MNCs includes contacting the cells in culture with one or more target antigens or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, culturing MNCs comprises contacting the cells in culture with one or more target antigens or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen to stimulate antigen specificity. T cells.

In some embodiments, culturing MNCs includes contacting cells in culture with one or more target antigens or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and one or more cell mediators. elemental contact. In some embodiments, culturing MNCs includes contacting cells in culture with one or more target antigens or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and one or more cell mediators. exposure to stimulate and expand antigen-specific T cells. In some embodiments, the one or more interleukins include IL-4 and IL-7. In some embodiments, the one or more interleukins include IL-4, IL-7, and IL-15. In some embodiments, the one or more interleukins do not include IL-2.

In some embodiments, culturing MNCs comprises coordinating cells in culture with one or more target antigens or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and two or more interleukin exposure. In some embodiments, culturing MNCs comprises coordinating cells in culture with one or more target antigens or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and two or more exposure to interleukins to stimulate and expand antigen-specific T cells. In some embodiments, the two or more interleukins include IL-4 and IL-7. In some embodiments, the two or more interleukins include IL-4, IL-7, and IL-15. In some embodiments, the two or more interleukins do not include IL-2.

In some embodiments, culturing MNCs includes contacting the cells in culture with one or more interleukins under suitable culture conditions to stimulate and expand antigen-specific T cells. In some embodiments, culturing MNCs includes contacting the cells in culture with one or more interleukins under suitable culture conditions to expand antigen-specific T cells. In some embodiments, the interleukin includes IL-4. In some embodiments, the interleukin includes IL-7. In some embodiments, interleukins include IL-4 and IL-7. In some embodiments, the interleukins include IL-4 and IL-7, but not IL-2. In some embodiments, the interleukins include IL-4 and IL-7, but not IL-15. In some embodiments, when the interleukins include IL-4 and IL-7 but not IL-15, IL-15 is subsequently added to the culture at a later time during the culture. In some embodiments, interleukins include IL-4, IL-7, and IL-15. In some embodiments, interleukins include IL-4 and IL-7.

In some embodiments, MNCs are contacted with medium containing IL-4 in culture. In some embodiments, MNCs are contacted with medium containing IL-7 in culture. In some embodiments, MNCs are contacted with culture medium containing IL-4 and IL-7 in culture. In some embodiments, MNCs are contacted in culture with medium that includes IL-4 and IL-7 but does not include IL-2. In some embodiments, MNCs are contacted in culture with medium containing IL-4 and IL-7 but not IL-15, and IL-15 is subsequently added to the culture. In some embodiments, MNCs are contacted in culture with medium containing IL-4, IL-7, and IL-15.

In some embodiments, MNCs are contacted with culture medium containing antigen (eg, mixed peptides) and IL-4 in culture. In some embodiments, MNCs are contacted with culture medium containing antigen (eg, mixed peptides) and IL-7 in culture. In some embodiments, MNCs are contacted in culture with culture medium containing antigen (eg, mixed peptides), IL-4, and IL-7. In some embodiments, MNCs are contacted in culture with culture medium that includes antigen (eg, mixed peptides), IL-4, and IL-7, but not IL-2. In some embodiments, MNCs are contacted in culture with medium containing an antigen (eg, mixed peptides), IL-4, and IL-7, but not IL-15, and IL-15 is subsequently added to the culture. In some embodiments, MNCs are contacted in culture with culture medium that includes antigens (eg, mixed peptides), IL-4, IL-7, and IL-15.

In some embodiments, cultured MNCs can include contacting cells in culture with one or more antigens under suitable culture conditions to stimulate and expand antigen-specific T cells. In some embodiments, the method includes culturing cells (eg, PBMCs) in culture medium comprising an antigen (eg, mixed peptides) and an interleukin as described herein. In some embodiments, the method includes culturing cells (eg, PBMC) in culture medium comprising an antigen (eg, mixed peptides) and IL-4, IL-7, and IL-15. In some embodiments, the method includes culturing cells (eg, PBMC) in culture medium comprising an antigen (eg, mixed peptides) and IL-4 and IL-7. In some embodiments, the interleukin does not comprise IL-2. i.Antigen

In other embodiments, the one or more antigens contacted with the cell may comprise one or more pathogen antigens. In other embodiments, the one or more antigens contacted with the cell may comprise one or more viral antigens. In other embodiments, the one or more antigens contacted with the cell may include one or more tumor-associated antigens. In some embodiments, contacting the cell with one or more antigens may include a combination of one or more viral antigens and one or more tumor-associated antigens.

In some embodiments, one or more antigens may be in the form of whole proteins. In some embodiments, one or more antigens may be in the form of a mixed peptide comprising a series of overlapping peptides covering a portion or the entire sequence of each antigen. In some embodiments, one or more antigens may be in a combination of a whole protein form and a mixed peptide form that includes a series of overlapping peptides covering part or all of the sequence of each antigen. In some embodiments, one or more antigens may be present as a plurality of mixed peptides. In some embodiments, each of the plurality of mixed peptides may comprise a series of overlapping peptides covering a portion or the entire sequence of one or more antigens. In some embodiments, mixed peptides as described herein may comprise 10 to 20-mer peptides. In one embodiment, the sequences of the peptides covering the antigens in the mixed peptide may overlap by 5 to 15 amino acids. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide may overlap by 5 to 15 amino acids. In some embodiments, mixed peptides as described herein may comprise less than 10-mer peptides. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide may overlap by 1 to 10 amino acids. In some embodiments, mixed peptides as described herein may comprise more than 20-mer peptides. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptides may overlap by more than 10 amino acids. In some embodiments, a mixed peptide as described herein may comprise a 15-mer peptide. In one embodiment, the sequences of the peptides covering the antigen in the mixed peptide may overlap by 11 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 11 amino acids.

In some embodiments, MNCs can be PBMCs. In some embodiments, each mixed peptide may comprise a series of overlapping peptides covering a portion of the antigen. In some embodiments, each mixed peptide can comprise a series of overlapping peptides covering the entire sequence of the antigen.

In some embodiments, generating an antigen-specific T cell strain includes culturing MNC from a donor in the presence of at least 1 mixed peptide. In some embodiments, generating an antigen-specific T cell strain may comprise culturing MNC from a donor in the presence of at least 2 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 3 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 4 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 5 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 6 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 7 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 8 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 9 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 10 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 11 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 12 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 13 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 14 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 15 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 16 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 17 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 18 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 19 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least 20 different mixed peptides. In some embodiments, methods as described herein can comprise culturing MNC in the presence of at least more than 20 different mixed peptides.

In some embodiments, generating an antigen-specific T cell strain includes culturing MNC from a donor in the presence of at least 1 mixed peptide. In some embodiments, generating an antigen-specific T cell strain comprises culturing MNC from a donor in the presence of at least 2 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 3 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 4 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 5 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 6 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 7 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 8 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 9 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 10 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 11 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 12 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 13 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 14 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 15 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 16 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 17 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 18 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 19 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least 20 different mixed peptides. In some embodiments, methods as described herein comprise culturing MNC in the presence of at least more than 20 different mixed peptides.

In some embodiments, methods for generating antigen-specific T cell lines can include culturing donor-derived MNCs in the presence of a plurality of mixed peptides. In some embodiments, each mixed peptide can encompass at least one antigen that is different from the antigens encompassed by each of the other mixed peptides in the plurality of mixed peptides. In some embodiments, the plurality of mixed peptides may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 different antigens. In some embodiments, the plurality of mixed peptides may encompass at least more than 20 different antigens. In some embodiments, a plurality of mixed peptides may encompass at least one antigen from at least 2 different viruses.

In some embodiments, methods for generating antigen-specific T cell lines comprise culturing donor-derived MNCs in the presence of a plurality of mixed peptides. In some embodiments, each mixed peptide encompasses at least one antigen that is different from the antigens encompassed by each of the other mixed peptides in the plurality of mixed peptides. In some embodiments, the plurality of mixed peptides encompass at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 different antigens. In some embodiments, the plurality of mixed peptides encompass at least more than 20 different antigens. In some embodiments, the plurality of mixed peptides encompass at least one antigen from at least 2 different viruses.

In some embodiments, the antigens used in the methods described herein are from latent viruses. In some embodiments, the antigens used in the methods described herein are from lytic viruses. In some embodiments, the antigens used in the methods for generating antigen-specific T cell lines as described herein can be from EBV (Estembar virus). In some embodiments, the antigens used in the methods described herein are from EBV. In some embodiments, antigens used in methods as described herein can be from CMV (cytomegalovirus). In some embodiments, antigens used in methods as described herein can be from adenovirus. In some embodiments, antigens used in methods as described herein can be from BK virus. In some embodiments, antigens used in methods as described herein may be from JC virus (John Cunningham virus). In some embodiments, the antigen used in the methods as described herein can be from HHV6 (Herpesvirus 6). In some embodiments, the antigen used in the methods as described herein can be from HHV8 (Herpesvirus 8). In some embodiments, antigens used in methods as described herein can be from HBV (hepatitis B virus). In some embodiments, the antigens used in the methods as described herein can be from RSV (Human Respiratory Synthetic Virus). In some embodiments, antigens used in methods as described herein can be from influenza viruses. In some embodiments, antigens used in methods as described herein can be from parainfluenza viruses. In some embodiments, the antigen used in the methods as described herein can be from Bocavirus. In some embodiments, antigens used in methods as described herein can be from coronaviruses. In some embodiments, antigens used in methods as described herein can be from LCMV (lymphocytic choriomeningitis virus). In some embodiments, the antigen used in the methods as described herein can be from the mumps virus. In some embodiments, the antigen used in the methods as described herein can be from measles virus. In some embodiments, antigens used in methods as described herein can be from human metapneumovirus. In some embodiments, the antigen used in the methods as described herein can be from parvovirus B. In some embodiments, antigens used in methods as described herein can be from rotavirus. In some embodiments, the antigen used in the methods as described herein can be from Merkel cell virus. In some embodiments, antigens used in methods as described herein can be from herpes simplex virus. In some embodiments, the antigens used in the methods as described herein can be from HPV (human papillomavirus). In some embodiments, antigens used in methods as described herein can be from HIV (human immunodeficiency virus). In some embodiments, the antigen used in the methods as described herein can be from HTLV1 (human T-cell leukemia virus type 1). In some embodiments, antigens used in methods as described herein can be from West Nile virus. In some embodiments, the antigen used in the methods as described herein can be from Zika virus. In some embodiments, the antigen used in the methods as described herein can be from Ebola virus.

In some embodiments, the antigen used in the methods for generating antigen-specific T cell lines as described herein is from EBV (Estembar virus). In some embodiments, the antigens used in the methods described herein are from EBV. In some embodiments, the antigens used in the methods as described herein are from CMV (cytomegalovirus). In some embodiments, the antigens used in the methods as described herein are from adenovirus. In some embodiments, the antigens used in the methods as described herein are from BK virus. In some embodiments, the antigen used in the methods as described herein is from JC virus (John Cunningham virus). In some embodiments, the antigen used in the methods as described herein is from HHV6 (Herpesvirus 6). In some embodiments, the antigen used in the methods as described herein is from HHV8 (Herpesvirus 8). In some embodiments, the antigens used in the methods as described herein are from HBV (hepatitis B virus). In some embodiments, the antigens used in the methods as described herein are from RSV (Human Respiratory Synthetic Virus). In some embodiments, the antigens used in the methods as described herein are from influenza viruses. In some embodiments, the antigens used in the methods as described herein are from parainfluenza viruses. In some embodiments, the antigen used in the methods as described herein is from Bocavirus. In some embodiments, the antigens used in the methods as described herein are from coronaviruses. In some embodiments, the antigen used in the methods as described herein is from LCMV (lymphocytic choriomeningitis virus). In some embodiments, the antigen used in the methods as described herein is from the mumps virus. In some embodiments, the antigen used in the methods as described herein is from measles virus. In some embodiments, the antigen used in the methods as described herein is from human metapneumovirus. In some embodiments, the antigen used in the methods as described herein is from parvovirus B. In some embodiments, the antigens used in the methods as described herein are from rotavirus. In some embodiments, the antigen used in the methods as described herein is from Merkel cell virus. In some embodiments, the antigen used in the methods as described herein is from herpes simplex virus. In some embodiments, the antigens used in the methods as described herein are from HPV (human papillomavirus). In some embodiments, the antigens used in the methods as described herein are from HIV (human immunodeficiency virus). In some embodiments, the antigen used in the methods as described herein is from HTLV1 (human T-cell leukemia virus type 1). In some embodiments, the antigen used in the methods as described herein is from West Nile virus. In some embodiments, the antigen used in the methods as described herein is from Zika virus. In some embodiments, the antigen used in the methods as described herein is from Ebola virus.

In some embodiments, at least one mixed peptide can encompass antigens from each of RSV, influenza, parainfluenza, and hMPV (human metapneumovirus). In some embodiments, the influenza virus antigen used in the mixed peptides as described herein can be the influenza A virus antigen NP1. In some embodiments, the influenza virus antigen used in the mixed peptides as described herein can be the influenza A virus antigen MP1. In some embodiments, the influenza virus antigens used in the mixed peptides as described herein can be influenza A virus antigens NP1 and MP1. In some embodiments, the RSV antigen used in the mixed peptides as described herein can be RSV N protein. In some embodiments, the RSV antigen used in the mixed peptides as described herein can be the RSV F protein. In some embodiments, the RSV antigens used in mixed peptides as described herein can be RSV N protein and RSV F protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein can be hMPV F protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein can be hMPV N protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein can be the hMPV M2-1 protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein can be hMPV M protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein can be a combination of hMPV F protein, hMPV N protein, hMPV M2-1, and hMPV M protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein can be the PIV M protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein can be the PIV HN protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein can be the PIV N protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein can be the PIV F protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein can be a combination of PIV M protein, PIV HN protein, PIV N protein, and PIV F protein.

In some embodiments, at least one mixed peptide encompasses antigens from each of RSV, influenza, parainfluenza, and hMPV (human metapneumovirus). In some embodiments, the influenza virus antigen used in the mixed peptides as described herein is influenza A virus antigen NP1. In some embodiments, the influenza virus antigen used in the mixed peptides as described herein is the influenza A virus antigen MP1. In some embodiments, the influenza virus antigens used in the mixed peptides as described herein are influenza A virus antigens NP1 and MP1. In some embodiments, the RSV antigen used in the mixed peptides as described herein is RSV N protein. In some embodiments, the RSV antigen used in the mixed peptides as described herein is RSV F protein. In some embodiments, the RSV antigens used in the mixed peptides as described herein are RSV N protein and RSV F protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein is hMPV F protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein is hMPV N protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein is hMPV M2-1 protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein is hMPV M protein. In some embodiments, the hMPV antigen used in the mixed peptides as described herein is a combination of hMPV F protein, hMPV N protein, hMPV M2-1 and hMPV M protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein is the PIV M protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein is the PIV HN protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein is the PIV N protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein is the PIV F protein. In some embodiments, the PIV antigen used in the mixed peptides as described herein is a combination of PIV M protein, PIV HN protein, PIV N protein, and PIV F protein.

In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise subjecting PBMC from a donor in the presence of mixed peptides encompassing influenza A virus antigen NP1 and influenza A virus antigen MP1 Cultivate. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing RSV Antigen N and RSV Antigen F. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing hMPV Antigen F. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing hMPV Antigen N. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing hMPV antigen M2-1. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing hMPV Antigen M. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing PIV Antigen M. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing the PIV antigen HN. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing PIV Antigen N. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing PIV Antigen F.

In some embodiments, methods for generating antigen-specific T cell lines as described herein comprise culturing PBMC from a donor in the presence of mixed peptides encompassing influenza A virus antigen NP1 and influenza A virus antigen MP1 . In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing RSV Antigen N and RSV Antigen F. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing hMPV Antigen F. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing hMPV Antigen N. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing hMPV antigen M2-1. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing hMPV Antigen M. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing PIV Antigen M. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the PIV antigen HN. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing PIV Antigen N. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing PIV Antigen F.

In some embodiments, methods for generating antigen-specific T cell lines as described herein can include combining PBMC from a donor with antigens encompassing each of EBV, CMV, adenovirus, BK virus, and HHV6 Cultured in the presence of mixed peptides. In some embodiments, methods for generating antigen-specific T cell lines as described herein can include converting PBMC from a donor into at least one molecule from each of EBV, CMV, adenovirus, BK virus, and HHV6. Culture is performed in the presence of a mixture of peptides from an antigen. In some embodiments, at least one mixed peptide can cover an antigen from EBV, at least one mixed peptide can cover an antigen from CMV, at least one mixed peptide can cover an antigen from adenovirus, and at least one mixed peptide can cover an antigen from BK virus. Antigens, and at least one mixed peptide may encompass antigens from HHV6. In some embodiments, the EBV antigen can be LMP2. In some embodiments, the EBV antigen can be EBNAl. In some embodiments, the EBV antigen can be BZLF1. In some embodiments, the EBV antigen can be a combination of CMV antigens. In some embodiments, the CMV antigen can be from IE1. In some embodiments, the CMV antigen can be from pp65. In some embodiments, the CMV antigen may be derived from a combination of 1El and pp65. In some embodiments, adenovirus antigens can be derived from hexons. In some embodiments, the adenovirus antigen can be derived from pentons. In some embodiments, adenovirus antigens can be derived from a combination of hexons and pentons. In some embodiments, the BK virus antigen can be from VP1. In some embodiments, the BK virus antigen may be derived from large T. In some embodiments, the BK virus antigen may be derived from a combination of VP1 and large T. In some embodiments, the HHV6 antigen can be from U90. In some embodiments, the HHV6 antigen can be from Ul 1. In some embodiments, the HHV6 antigen can be from U14. In some embodiments, the HHV6 antigen can be from a combination of U90, U11 and U14.

In some embodiments, methods for generating antigen-specific T cell lines as described herein comprise placing PBMC from a donor in a cell containing antigens from each of EBV, CMV, adenovirus, BK virus, and HHV6. Culture was performed in the presence of mixed peptides. In some embodiments, methods for generating antigen-specific T cell lines as described herein comprise converting PBMC from a donor into at least one enzyme encompassing each of EBV, CMV, adenovirus, BK virus, and HHV6 Culture is carried out in the presence of mixed peptides of the antigen. In some embodiments, at least one mixed peptide encompasses an antigen from EBV, at least one mixed peptide encompasses an antigen from CMV, at least one mixed peptide encompasses an antigen from adenovirus, at least one mixed peptide encompasses an antigen from BK virus, and at least A mixed peptide covers antigens from HHV6. In some embodiments, the EBV antigen is LMP2. In some embodiments, the EBV antigen is EBNAl. In some embodiments, the EBV antigen is BZLF1. In some embodiments, the EBV antigen is a combination of CMV antigens. In some embodiments, the CMV antigen is from IE1. In some embodiments, the CMV antigen is derived from pp65. In some embodiments, the CMV antigen is derived from a combination of 1El and pp65. In some embodiments, the adenovirus antigen is derived from hexons. In some embodiments, the adenovirus antigen is derived from penton. In some embodiments, the adenovirus antigen is derived from a combination of hexons and pentons. In some embodiments, the BK virus antigen is derived from VP1. In some embodiments, the BK virus antigen is derived from large T. In some embodiments, the BK virus antigen is derived from a combination of VP1 and large T. In some embodiments, the HHV6 antigen is derived from U90. In some embodiments, the HHV6 antigen is from Ul 1. In some embodiments, the HHV6 antigen is from U14. In some embodiments, the HHV6 antigen is derived from a combination of U90, U11 and U14.

In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise culturing PBMC from a donor in the presence of mixed peptides encompassing the EBV antigen LMP2. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing the EBV antigen EBNAl. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing the EBV antigen BZLF1. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing CMV antigen IE 1. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing the CMV antigen pp65. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing the adenoviral antigen hexon. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing pentons. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing the BK virus antigen VP1. In some embodiments, methods as described herein may comprise culturing PBMC in the presence of mixed peptides encompassing the BK virus antigen large T. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing HHV6 antigen U90. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing the HHV6 antigen U11. In some embodiments, methods as described herein can comprise culturing PBMC in the presence of mixed peptides encompassing the HHV6 antigen U14.

In some embodiments, methods for generating antigen-specific T cell lines as described herein comprise culturing PBMC from a donor in the presence of mixed peptides encompassing the EBV antigen LMP2. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the EBV antigen EBNAl. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the EBV antigen BZLF1. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing CMV antigen IE 1. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the CMV antigen pp65. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the adenoviral antigen hexon. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing pentons. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the BK virus antigen VP1. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the BK virus antigen large T. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing HHV6 antigen U90. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the HHV6 antigen U11. In some embodiments, methods as described herein comprise culturing PBMC in the presence of mixed peptides encompassing the HHV6 antigen U14.

In some embodiments, methods for generating antigen-specific T cell lines as described herein comprise dividing PBMCs from a donor into cells encompassing BKV antigens large T and VP1, AdV antigens hexon and penton, CMV antigen IE1 and cultured in the presence of mixed peptides of pp65, HHV-6 antigens U11, U14 and U90 and EBV antigens LMP2, EBNA1 and BZLF1.

In some embodiments, methods for generating antigen-specific T cell lines as described herein comprise combining PBMC from a donor in a group encompassing RSV antigens F and N, influenza virus antigens NP1 and MP1, PIV antigens M, N, HN and F and mixed peptides of hMPV antigens F, N, M2-1 and M were cultured.

In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise culturing PBMC from a donor in the presence of mixed peptides encompassing the EBV antigen LMP2. In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of mixed peptides encompassing the EBV antigen EBNAl. In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of mixed peptides encompassing the EBV antigen BZLF1. In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of mixed peptides covering CMV antigen IE 1. In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise culturing PBMC from a donor in the presence of mixed peptides covering the CMV antigen pp65. In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise culturing PBMC from a donor in the presence of mixed peptides encompassing the adenoviral antigen hexon. In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise culturing PBMC from a donor in the presence of a penton-containing mixed peptide. In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of mixed peptides covering the BK virus antigen VP1. In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise culturing PBMC from a donor in the presence of a mixed peptide covering the BK virus antigen large T. In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of mixed peptides covering the HHV6 antigen U90. In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of mixed peptides covering the HHV6 antigen U11. In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of mixed peptides covering the HHV6 antigen U14.

In some embodiments, virus-specific T cells (VST) target peptides, peptide fragments, antigens, or fragments of the antigens described herein. In some embodiments, virus-specific T cells (VST) target antigens of latent viruses. In some embodiments, virus-specific T cells (VST) target lytic viral antigens. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of hMPV, wherein the antigen is selected from F, N, M2-1, M, or a combination thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen or fragment of an antigen of SARS-CoV2, wherein the antigen is selected from the group consisting of spike protein (S), envelope protein (E) , membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B , Y14 or their combination. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen or fragment of an antigen of SARS-CoV2, wherein the antigen is selected from the group consisting of spike protein (S), membrane protein (M), Nucleocapsid protein (N), accessory protein 7a (AP7a) and non-structural protein 4 (NSP4) or combinations thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of EBV, wherein the antigen is selected from EBNAl, LMP2, BZLF1, or a combination thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of CMV, wherein the antigen is selected from IEl, pp65, or a combination thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of Adv, wherein the antigen is selected from hexon, penton, or a combination thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of BK virus, wherein the antigen is selected from LT, VP-1, or a combination thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of HHV6, wherein the antigen is selected from U14, U11, U71, U54, U90, or a combination thereof. In some embodiments, virus-specific T cells (VST) target an antigen of HHV6, wherein the antigen is selected from U14, U11, U90, or a combination thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of RSV, wherein the antigen is selected from N, F, or a combination thereof. In some embodiments, virus-specific T cells (VST) target a peptide, peptide fragment, antigen, or fragment of an antigen of influenza virus, wherein the antigen is selected from MP1, NP1, or a combination thereof. In some embodiments, the virus-specific T cells (VST) target a peptide, peptide fragment, antigen or fragment of an antigen of parainfluenza virus type 3 (PIV3), wherein the antigen is selected from the group consisting of F, N, M, M2- 1. HN or its combination. a.Coronavirus antigen

In some embodiments, methods for generating antigen-specific T cell lines as described herein can comprise culturing PBMC from a donor in the presence of a mixed peptide encompassing antigens from coronaviruses. In some embodiments, the coronavirus is a beta-coronavirus (β-CoV). In some embodiments, the coronavirus is an alpha-coronavirus (alpha-CoV). In some embodiments, the β-CoV is selected from the group consisting of SARS-CoV, MERS-CoV, HCoVHKU1, and HCoV-OC43. In some embodiments, the alpha-CoV is selected from HCoV-E229 and HCoV-NL63. In some embodiments, methods for constructing donor minilibraries of antigen-specific T cell lines as described herein may include culturing PBMC with a plurality of mixed peptide libraries, each mixed peptide library containing a SARS-CoV2 antigen or Multiple overlapping peptides from all or part of the antigens of one or more additional viruses. In some embodiments, VST is generated by contacting T cells with APCs (such as DCs) primed with a plurality of mixed peptide libraries, each mixed peptide library containing a plurality of overlapping peptides covering all or a portion of the viral antigen, wherein At least one of the plurality of mixed peptide libraries covers a first antigen from SARS-CoV2, and wherein at least one additional mixed peptide library (or a portion thereof) of the plurality of mixed peptide libraries covers each second antigen. In some embodiments, VST is performed by nucleofection of T cells with at least one DNA plasmid encoding at least one SARS-CoV2 antigen or a portion thereof and at least one DNA plasmid encoding each second antigen or a portion thereof. APC (such as DC) contact is generated. In some embodiments, the plasmid encodes at least one SARS-CoV2 antigen, or a portion thereof, and at least one of, or a portion thereof, additional antigens. In some embodiments, VST includes CD4+ T lymphocytes and CD8+ T lymphocytes. In some embodiments, VST expresses T cell receptors. In some embodiments, the VST is MHC restricted. In some embodiments, the SARS-CoV2 antigen comprises one or more antigens selected from the group consisting of: nsp 1; nsp3; nsp4; nsp5; nsp6; nsp7a, nsp8, nsp10; nsp12; nsp13; nsp14; nsp15; and nsp16 . In some embodiments, the SARS-CoV2 antigen comprises one or more antigens selected from the group consisting of: spike protein (S); envelope protein (E); matrix protein (M); and nucleocapsid protein (N ). In some embodiments, the SARS-CoV2 antigen comprises one or more antigens selected from the group consisting of: nsp 1; nsp3; nsp4; nsp5; nsp6; nsp7a, nsp8, nsp10; nsp12; nsp13; nsp14; nsp15; and nsp16 . In some embodiments, the SARS-CoV2 antigen comprises one or more antigens selected from the group consisting of: spike protein (S); envelope protein (E); membrane protein (M); and nucleocapsid protein (N ). In some embodiments, the SARS-CoV2 antigen comprises one or more antigens selected from the group consisting of: spike protein (S); membrane protein (M); nucleocapsid protein (N); accessory protein 7a (AP7a) ; and nonstructural protein 4 (NSP4). The term "matrix (M) protein" and its analogues (eg, M protein or M antigen) may be used interchangeably herein with the term "membrane (M) protein" and its analogues (eg, membrane (M) matrix protein). In some embodiments, the SARS-CoV2 antigen comprises one or more antigens selected from the group consisting of: SARS-CoV-2 (AP3A); SARS-CoV-2 (NSS); SARS-CoV-2 (ORFIO); SARS-CoV-2 (ORF9B); and SARS-CoV-2 (Yl4). In some embodiments, methods for constructing donor minibanks of antigen-specific T cell lines as described herein may include combining PBMCs from selected donors in groups encompassing one or more SARS-CoV2 antigens and one or more additional Culture is performed in the presence of a mixed peptide of antigens, the one or more additional antigens being selected from the group consisting of: PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza virus antigen NP1, influenza virus antigen MP1, RSV Antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-l, hMPV antigen F, hMPV antigen N, AdV antigen hexon and AdV antigen penton. In some embodiments, the one or more additional antigens include PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza virus antigen NP1, influenza virus antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M , hMPV antigen M2-1, hMPV antigen F, hMPV antigen N, AdV antigen hexon, AdV antigen penton or combinations thereof. b. HBV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines may comprise culturing PBMC from a selected donor in the presence of mixed peptides encompassing antigens from hepatitis B virus (HBV). In some embodiments, the HBV antigen is selected from the group consisting of HBV core antigen (HBcAg or C), HBV surface antigen (HBsAg or LE), E antigen (HBeAg or E), DNA polymerase (HBP or P), and HBV X protein ( X antigen, HBX or X). In some embodiments, the HBV antigen is selected from the group consisting of HBV surface antigen (HBsAg or LE), E antigen (HBeAg or E), DNA polymerase (HBP or P), and HBV X protein (X antigen, HBX or X). In some embodiments, HBeAg includes core (HBcAg) and pre-core (PreC) antigens.

In some embodiments, the HBV antigen is HBcAg. In some embodiments, the HBV antigen is HBsAg. In some embodiments, the HBV antigen is HBeAg. In some embodiments, the HBV antigen is HBP. In some embodiments, the HBV antigen is HBX.

In some embodiments, methods of the present invention for generating antigen-specific T cell lines may comprise placing PBMC from a selected donor in the presence of mixed peptides covering one or more target antigens from hepatitis B virus (HBV). culture below. In some embodiments, the target antigen includes HBV core antigen (HBcAg or C), HBV surface antigen (HBsAg or LE), E antigen (HBeAg or E), DNA polymerase (HBP or P), HBV X protein (X antigen , HBX or X) or combinations thereof. In some embodiments, the target antigen includes HBcAg. In some embodiments, the target antigen includes HBsAg. In some embodiments, the target antigen includes HBeAg. In some embodiments, the target antigen includes HBP. In some embodiments, the target antigen includes HBX.

In some embodiments, target antigens include HBsAg and HBcAg. In some embodiments, target antigens include HBsAg and HBeAg. In some embodiments, target antigens include HBsAg and HBP. In some embodiments, target antigens include HBsAg and HBX. In some embodiments, target antigens include HBcAg and HBeAg. In some embodiments, target antigens include HBcAg and HBP. In some embodiments, target antigens include HBcAg and HBX. In some embodiments, target antigens include HBeAg and HBP. In some embodiments, target antigens include HBeAg and HBX. In some embodiments, target antigens include HBP and HBX.

In some embodiments, target antigens include HBsAg, HBcAg, and HBeAg. In some embodiments, target antigens include HBsAg, HBcAg, and HBP. In some embodiments, target antigens include HBsAg, HBcAg, and HBX. In some embodiments, target antigens include HBsAg, HBeAg, and HBP. In some embodiments, target antigens include HBsAg, HBeAg, and HBX. In some embodiments, target antigens include HBsAg, HBP, and HBX. In some embodiments, target antigens include HBcAg, HBeAg, and HBP. In some embodiments, target antigens include HBcAg, HBeAg, and HBX. In some embodiments, target antigens include HBcAg, HBP, and HBX. In some embodiments, target antigens include HBeAg, HBP, and HBX.

In some embodiments, target antigens include HBsAg, HBcAg, HBeAg, and HBP. In some embodiments, target antigens include HBsAg, HBcAg, HBeAg, and HBX. In some embodiments, target antigens include HBsAg, HBcAg, HBP, and HBX. In some embodiments, target antigens include HBsAg, HBeAg, HBP, and HBX. In some embodiments, target antigens include HBcAg, HBeAg, HBP, and HBX.

In some embodiments, HBV target antigens include E antigen (HBeAg), P antigen (HBP), and LE antigen (HBsAg). In some embodiments, HBeAg (E antigen) includes pre-core (PreC) and core (HBcAg) antigens.

In some embodiments, the antigen is an immunodominant antigen. In some embodiments, the immunodominant antigen includes one or more of LE, P, and E, or a combination thereof. In some embodiments, the immunodominant antigen is LE. In some embodiments, the immunodominant antigen is P. In some embodiments, the immunodominant antigen is E. c. HHV-8 antigen

In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise subjecting PBMC from a donor in the presence of mixed peptides encompassing antigens from human herpesvirus-8 (HHV-8) Cultivate. In some embodiments, the HHV-8 antigen comprises a latent antigen. In some embodiments, the HHV-8 antigen comprises a soluble antigen. In some embodiments, HHV-8 antigens comprise one or more latent and lytic antigens. In some embodiments, the HHV-8 antigen is selected from the group consisting of LANA-1 (ORF3); LANA-2 (vIRF3, K10.5); vCYC (ORF72); RTA (ORF50); vFLIP (ORF71); Kaposi protein (ORF12, K12); gB (ORF8); MIR1 (K3); SSB (ORF6); and TS (ORF70).

In some embodiments, methods for generating antigen-specific T cell lines as described herein may comprise mixing PBMC from a donor in a mixture encompassing one or more target antigens from human herpesvirus-8 (HHV-8). Cultivate in the presence of peptides. In some embodiments, at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12 , K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70), or combinations thereof. In some embodiments, a target antigen is selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the two target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the two target antigens are LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the two target antigens are LANA1 (ORF3) and gB (ORF8). In some embodiments, the two target antigens are LANA2 (ORF3) and gB (ORF8). In some embodiments, the at least two target antigens are LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the at least two target antigens are LANA1 (ORF3) and gB (ORF8). In some embodiments, the at least two target antigens are LANA2 (ORF3) and gB (ORF8).

In some embodiments, the pathogen is HHV8, and the three target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least three target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the four target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least four target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the five target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least five target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the six target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least six target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the seven target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least seven target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the eight target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least eight target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the nine target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least nine target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the ten target antigens are LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), card Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least ten target antigens are LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, HHV8 target antigens include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the target antigen includes LANA-1 (ORF3). In some embodiments, the target antigen includes LANA-2 (vIRF3, K10.5). In some embodiments, the target antigen includes vCYC (ORF72). In some embodiments, the target antigen includes RTA (ORF50). In some embodiments, the target antigen includes vFLIP (ORF71). In some embodiments, the target antigen includes Kaposi proteins (ORF12, K12). In some embodiments, the target antigen includes gB (ORF8). In some embodiments, the target antigen includes MIR1 (K3). In some embodiments, the target antigen includes SSB (ORF6). In some embodiments, the target antigen includes TS (ORF70).

In some embodiments, the target antigen is LANA-1 (ORF3). In some embodiments, the target antigen is LANA-2 (vIRF3, K10.5). In some embodiments, the target antigen is vCYC (ORF72). In some embodiments, the target antigen is RTA (ORF50). In some embodiments, the target antigen is vFLIP (ORF71). In some embodiments, the target antigen is Kaposi protein (ORF12, K12). In some embodiments, the target antigen is gB (ORF8). In some embodiments, the target antigen is MIR1 (K3). In some embodiments, the target antigen is SSB (ORF6). In some embodiments, the target antigen is TS (ORF70).

In some embodiments, VST targets LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12 ), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the peptide comprises a polypeptide sequence from LANA-1. In some embodiments, the peptide comprises the polypeptide sequence from SEQ ID NO: 16. In some embodiments, the peptide comprises a polypeptide of amino acids 49-63 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 65-83 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 125-139 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 325-339 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 901-915 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 905-919 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 1093-1115 of LANA-1. In some embodiments, the peptide comprises a polypeptide sequence selected from the group consisting of: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO : 4), EYLVSFGVWIRTPPA (SEQ ID NO: 5), ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 13) NO: 15) or its functional variant. In some embodiments, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are peptide sequences derived from HBsAg. In some embodiments, SEQ ID NO: 4 and SEQ ID NO: 5 are peptide sequences derived from HBeAg. In some embodiments, SEQ ID NOs: 6-12 are peptide sequences derived from HHV8 LANA1. In some embodiments, SEQ ID NOs: 13-15 are peptide sequences derived from HHV8 gB.

In some embodiments, the peptide comprises a polypeptide sequence from gB. In some embodiments, the peptide comprises a polypeptide of amino acids 105-123 of gB. In some embodiments, the peptide comprises a polypeptide of amino acids 588-602 of gB. In some embodiments, the peptide comprises a polypeptide of amino acids 748-770. d. BKV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from BKV. In some embodiments, the BKV antigen is selected from BKV large T and VP1. In some embodiments, the BKV antigen is large T. In some embodiments, the BKV antigen is VP1. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering the BKV antigens large T and VP1. e. AdV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from AdV. In some embodiments, the AdV antigen is selected from Adv hexon and penton. In some embodiments, the AdV antigen is hexon. In some embodiments, the AdV antigen is penton. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering the hexon and penton of the AdV antigen. f. CMV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from CMV. In some embodiments, the CMV antigen is selected from CMV IE1 and pp65. In some embodiments, the CMV antigen is pp65. In some embodiments, the CMV antigen is IEl. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering CMV antigens IE1 and pp65. g. HHV-6 antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from HHV-6. In some embodiments, the HHV-6 antigen is selected from HHV-6 U11, U14, and U90. In some embodiments, the HHV-6 antigen is selected from HHV-6 U11, U14, U54, U71, and U90. In some embodiments, the HHV-6 antigen is U11. In some embodiments, the HHV-6 antigen is U14. In some embodiments, the HHV-6 antigen is U54. In some embodiments, the HHV-6 antigen is U71. In some embodiments, the HHV-6 antigen is U90. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering HHV-6 antigens U11, U14, and U90. In some embodiments, the HHV-6 antigen is U90. In some embodiments, methods of the invention for generating antigen-specific T cell lines comprise subjecting PBMC from a selected donor in the presence of mixed peptides covering HHV-6 antigens U11, U14, U54, U71 and U90 Cultivate. h. EBV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from EBV. In some embodiments, the EBV antigen is selected from LAMP2, EBNAl, and BZLF1. In some embodiments, the EBV antigen is EBNAl. In some embodiments, the EBV antigen is LAMP2. In some embodiments, the EBV antigen is BZLF1. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering the EBV antigens LAMP2, EBNAl, and BZLF1. i. RSV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from RSV. In some embodiments, the RSV antigen is selected from RSV F and N. In some embodiments, the RSV antigen is F. In some embodiments, the RSV antigen is N. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering RSV antigens F and N. j. IFV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from IFV. In some embodiments, the IFV antigen is selected from IFV MP1 and NP1. In some embodiments, the IFV antigen is MP1. In some embodiments, the IFV antigen is NP1. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering the IFV antigens NP1 and MP1. k. PIV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides encompassing antigens from PIV. In some embodiments, the PIV antigen is selected from PIV M, HN, N, and F. In some embodiments, the PIV antigen is M. In some embodiments, the PIV antigen is HN. In some embodiments, the PIV antigen is N. In some embodiments, the PIV antigen is F. In some embodiments, methods of the invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of a mixed peptide covering PIV antigens M, HN, N, and F. l. hMPV antigen

In some embodiments, methods of the present invention for generating antigen-specific T cell lines comprise culturing PBMC from a selected donor in the presence of mixed peptides covering antigens from hMPV. In some embodiments, the hMPV antigen is selected from hMPV F, N, M2-1, and M. In some embodiments, the hMPV antigen is F. In some embodiments, the hMPV antigen is N. In some embodiments, the hMPV antigen is M2-1. In some embodiments, the hMPV antigen is M. In some embodiments, methods of the present invention for generating antigen-specific T cell lines include culturing PBMC from a selected donor in the presence of mixed peptides covering hMPV antigens F, N, M2-1, and M. ii. Generation of mixed peptide library

In some embodiments of the invention, a peptide library is provided for peripheral blood mononuclear cells (PBMC) to ultimately generate antigen-specific T cells. In some embodiments, a peptide library is provided for the generation of virus-specific T cells (VST) from peripheral blood mononuclear cells (PBMC). In some embodiments, a peptide library is provided for monocytes to generate antigen-specific T cells. In some embodiments, a peptide library is provided for the generation of virus-specific T cells (VST) from monocytes. In some embodiments, the antigen-specific T cells comprise VST. In some embodiments, the library includes a mixture of peptides covering part or all of the same antigen ("mixed peptides"). In some aspects, the mixed peptides used in the present invention can be obtained from commercially available peptide libraries and are composed of peptides that are 15 amino acids long and overlap each other by 11 amino acids. In some embodiments, the mixed peptides comprise a series of overlapping peptides covering part or the entire sequence of each antigen. In some embodiments, one or more antigens are in a combination of a complete protein form and a mixed peptide form that includes a series of overlapping peptides covering part or the entire sequence of each antigen. In some embodiments, one or more antigens are present as a plurality of mixed peptides. In some embodiments, each of the plurality of mixed peptides includes a series of overlapping peptides covering a portion or the entire sequence of one or more antigens. In some embodiments, the mixed peptides may comprise 10 to 20-mer peptides. In some embodiments, the mixed peptides comprise 10-mer peptides. In some embodiments, the mixed peptides comprise 11-mer peptides. In some embodiments, the mixed peptides comprise 12-mer peptides. In some embodiments, the mixed peptides comprise 13-mer peptides. In some embodiments, the mixed peptides comprise 14-mer peptides. In some embodiments, the mixed peptides comprise 15-mer peptides. In some embodiments, the mixed peptides comprise 16-mer peptides. In some embodiments, the mixed peptides comprise 17-mer peptides. In some embodiments, the mixed peptides comprise 18-mer peptides. In some embodiments, the mixed peptides comprise 19-mer peptides. In some embodiments, the mixed peptides comprise 20-mer peptides. In some embodiments, the mixed peptides comprise less than 10-mer peptides. In some embodiments, the mixed peptides comprise more than 20-mer peptides.

In some embodiments, the sequences of the peptides encompassing the antigens in the mixed peptide overlap by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. . In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide overlap by 5 to 15 amino acids. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide overlap by 1 to 10 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 1 amino acid. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 2 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 3 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 4 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 5 amino acids. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide overlap by 6 amino acids. In some embodiments, the sequences of the peptides encompassing the antigens in the mixed peptide overlap by 7 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 8 amino acids. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide overlap by 9 amino acids. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide overlap by more than 10 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 11 amino acids. In some embodiments, the sequences of the peptides encompassing the antigens in the mixed peptide overlap by 12 amino acids. In some embodiments, the sequences of the peptides covering the antigens in the mixed peptide overlap by 13 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 14 amino acids. In some embodiments, the sequences of the peptides covering the antigen in the mixed peptide overlap by 15 amino acids.

In some embodiments, the mixed peptides include between 2 and 750 different peptides. In some embodiments, the mixed peptides include between 2 and 20 different peptides. In some embodiments, the mixed peptides include between 2 and 30 different peptides. In some embodiments, the mixed peptides include between 2 and 40 different peptides. In some embodiments, the mixed peptides include between 2 and 50 different peptides. In some embodiments, the mixed peptides include between 2 and 100 different peptides. In some embodiments, the mixed peptides include between 50 and 100 different peptides. In some embodiments, the mixed peptides include between 50 and 200 different peptides. In some embodiments, the mixed peptides include between 100 and 200 different peptides. In some embodiments, the mixed peptides include between 150 and 300 different peptides. In some embodiments, the mixed peptides include between 200 and 500 different peptides. In some embodiments, the mixed peptides include between 300 and 600 different peptides. In some embodiments, the mixed peptides include between 400 and 800 different peptides. In some embodiments, the mixture of peptides contains between 500 and 1000 different peptides. In some embodiments, the mixed peptide includes 2 different peptides. In some embodiments, the mixed peptides include 5 peptides. In some embodiments, the mixed peptides include 10 different peptides. In some embodiments, the mixed peptides include 20 different peptides. In some embodiments, the peptide mixture contains 30 peptides. In some embodiments, the mixed peptides include 40 different peptides. In some embodiments, the mixed peptides include 50 different peptides. In some embodiments, the mixed peptides include 75 different peptides. In some embodiments, the mixture of peptides contains 100 different peptides. In some embodiments, the peptide mixture contains 150 different peptides. In some embodiments, the peptide mixture contains 200 different peptides. In some embodiments, the peptide mixture contains 300 different peptides. In some embodiments, the peptide mixture contains 400 different peptides. In some embodiments, the peptide mixture contains 500 different peptides. In some embodiments, the peptide mixture contains 600 different peptides. In some embodiments, the peptide mixture contains 700 different peptides. In some embodiments, the peptide mixture contains 750 different peptides. In some embodiments, the peptide mixture contains 800 different peptides. In some embodiments, the peptide mixture contains 850 different peptides. In some embodiments, the peptide mixture contains 900 different peptides. In some embodiments, the mixture of peptides contains 950 different peptides. In some embodiments, the peptide mixture contains 1000 different peptides.

In some embodiments, the compositions comprise from about 0.2 nanogram/peptide/ml to about 20 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 0.5 nanogram/peptide/ml to about 10 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 0.5 nanogram/peptide/ml to about 5 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 0.5 nanogram/peptide/ml to about 3 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 1 nanogram/peptide/ml to about 3 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 1 nanogram/peptide/ml to about 5 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 1 nanogram/peptide/ml to about 10 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 1 nanogram/peptide/ml to about 20 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 1.5 nanogram/peptide/ml to about 2.5 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 1 nanogram/peptide/ml to about 2 nanogram/peptide/ml of mixed peptides. In some embodiments, the compositions comprise from about 2 nanogram/peptide/ml to about 3 nanogram/peptide/ml of mixed peptides. In some embodiments, the composition includes 0.2 nanogram/peptide/ml of mixed peptides. In some embodiments, the composition includes 0.5 nanogram/peptide/ml of mixed peptides. In some embodiments, the composition includes 1 nanogram/peptide/ml of mixed peptides. In some embodiments, the composition includes 1.5 nanogram/peptide/ml of mixed peptides. In some embodiments, the composition includes 2 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 2.5 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 3 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 4 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 5 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 6 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 7 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 8 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 9 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 10 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 11 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 12 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 13 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 14 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 15 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 16 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 17 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 18 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 19 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes 20 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 0.2 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 0.5 nanogram/peptide/ml of mixed peptides. In some embodiments, the composition includes about 1 nanogram/peptide/ml of mixed peptides. In some embodiments, the composition includes about 1.5 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 2 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 2.5 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 3 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 4 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 5 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 6 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 7 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 8 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 9 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 10 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 11 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 12 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 13 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 14 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 15 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 16 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 17 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 18 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 19 nanograms/peptide/ml of mixed peptides. In some embodiments, the composition includes about 20 nanograms/peptide/ml of mixed peptides.

In some embodiments, the peptides can be produced synthetically. Examples include those from JPT Technologies (Springfield, VA) or Miltenyi Biotec (Auburn, CA). In some embodiments, the peptides are, for example, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or more amino acids long, and in particular embodiments, there are, for example, at least 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33 or 34 amino acid length overlap. In some embodiments, the peptides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 or more amino acids long. In some embodiments, the peptides are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acid length overlap.

In some embodiments, the amino acids used in the hybrid peptide have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% pure, including all ranges and subranges therebetween. In some embodiments, purity refers to the amount of synthetic peptide that corresponds to the full length of the peptide intended to be synthesized. In some embodiments, the amino acids used in the mixed peptides as herein are at least 95% pure. In some embodiments, the amino acids used in the mixed peptides as herein are at least 90% pure. In some embodiments, the amino acids used in the mixed peptides as herein are at least 85% pure. In some embodiments, the amino acids used in the mixed peptides as herein are at least 80% pure. In some embodiments, the amino acids used in the mixed peptides as herein are at least 75% pure. In some embodiments, the amino acids used in the mixed peptides as herein are at least 70% pure. A mixture of different peptides may include any ratio of different peptides, but in some embodiments, each particular peptide is present in the mixture in substantially the same amount as another particular peptide. Methods for preparing and generating mixed peptides for multi-viral antigen-specific T cells with broad specificity are described in US2018/0187152, which is incorporated by reference in its entirety.

In some embodiments, the mixed peptides used to construct a population of antigen-specific T cells are chemically synthesized. In some embodiments, the mixed peptides are optionally >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90% pure, including any therebetween. All ranges and subranges. In some embodiments, the mixed peptides are optionally >90% pure. In some embodiments, the mixed peptides used to construct a multi-strain population of antigen-specific T cells are chemically synthesized. In some embodiments, the mixed peptides are optionally >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90% pure, including any therebetween. All ranges and subranges. In some embodiments, the mixed peptides are optionally >90% pure.

In some embodiments, the hybrid peptides of the invention encompass at least one antigen that is different from the antigens encompassed by each of the other hybrid peptides in the plurality of hybrid peptides. In some embodiments, the plurality of mixed peptides encompass at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 different antigens. In some embodiments, the plurality of mixed peptides encompasses at least 20 different antigens. In some embodiments, a plurality of mixed peptides may encompass at least one antigen from at least 2 different viruses. i. Antigen of mixed peptide

In some embodiments, hybrid peptides of the invention comprise viral antigens. In some embodiments, hybrid peptides of the invention comprise viral antigens from latent viruses. In some embodiments, hybrid peptides of the invention comprise viral antigens from lytic viruses.

In some embodiments, the mixed peptide of the present invention includes or consists of the following: selected from the group consisting of HBV core antigen (HBcAg or C), HBV surface antigen (HBsAg or LE), E antigen (HBeAg or E), DNA polymerase ( All or part of the HBV antigen of HBP or P) and HBV X protein (X antigen, HBX or X). In some embodiments, the mixed peptide includes or consists of all or part of HBV antigen E antigen (HBeAg), P antigen (HBP), and LE antigen (HBsAg). In some embodiments, HBeAg includes core (HBcAg) and pre-core (PreC) antigens.

In some embodiments, the mixed peptides of the invention comprise or consist of: all or a portion of HBcAg. In some embodiments, the mixed peptides of the invention comprise or consist of: all or a portion of HBsAg. In some embodiments, the mixed peptides of the invention comprise or consist of: all or a portion of HBeAg. In some embodiments, the hybrid peptides of the invention comprise or consist of: all or a portion of HBP. In some embodiments, the hybrid peptides of the invention comprise or consist of: all or a portion of HBX. In some embodiments, HBeAg includes core (HBcAg) and pre-core (PreC) antigens.

In some embodiments, the mixed peptide of the present invention includes or consists of all or part of HBsAg and HBcAg. In some embodiments, the mixed peptide of the invention includes or consists of all or part of HBsAg and HBeAg. In some embodiments, the mixed peptide of the invention includes or consists of all or part of HBsAg and HBP. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBsAg and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBcAg and HBeAg. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBcAg and HBP. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBcAg and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBeAg and HBP. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBeAg and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBP and HBX.

In some embodiments, the mixed peptides of the present invention include or consist of all or part of HBsAg, HBcAg and HBeAg. In some embodiments, the mixed peptides of the present invention include or consist of all or part of HBsAg, HBcAg and HBP. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBsAg, HBcAg and HBX. In some embodiments, the mixed peptides of the present invention include or consist of all or part of HBsAg, HBeAg and HBP. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBsAg, HBeAg and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBsAg, HBP and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBcAg, HBeAg and HBP. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBcAg, HBeAg and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBcAg, HBP and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBeAg, HBP and HBX.

In some embodiments, the mixed peptides of the present invention include or consist of all or part of HBsAg, HBcAg, HBeAg and HBP. In some embodiments, the mixed peptides of the present invention include or consist of all or part of HBsAg, HBcAg, HBeAg and HBX. In some embodiments, the mixed peptides of the present invention include or consist of all or part of HBsAg, HBcAg, HBP and HBX. In some embodiments, the mixed peptides of the present invention include or consist of all or part of HBsAg, HBeAg, HBP and HBX. In some embodiments, the mixed peptides of the invention comprise or consist of all or part of HBcAg, HBeAg, HBP and HBX.

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or a combination thereof. In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of all or part of LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the mixed peptides of the invention comprise or consist of all or part of LANA1 (ORF3) and gB (ORF8). In some embodiments, the mixed peptides of the invention comprise or consist of all or part of LANA2 (ORF3) and gB (ORF8). In some embodiments, the mixed peptides of the invention comprise or consist of all or part of LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the mixed peptides of the invention comprise or consist of all or part of LANA1 (ORF3) and gB (ORF8). In some embodiments, the mixed peptides of the invention comprise or consist of all or part of LANA2 (ORF3) and gB (ORF8).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of at least three target antigens of ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of the four target antigens of ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of at least four target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of the five target antigens of ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of at least five target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of the six target antigens: ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of at least six target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of the seven target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of at least seven target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of eight target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of at least eight target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of the nine target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the mixed peptides of the invention comprise or consist of the following: selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP ( All or part of at least nine target antigens including ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the mixed peptides of the invention comprise or consist of the following: LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) in whole or in part. In some embodiments, the mixed peptides of the invention comprise or consist of the following: at least LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) in whole or in part.

In some embodiments, the hybrid peptides of the invention comprise or consist of: all or a portion of LANA-1 (ORF3). In some embodiments, the mixed peptides of the invention comprise or consist of: all or a portion of LANA-2 (vIRF3, K10.5). In some embodiments, hybrid peptides of the invention comprise or consist of: all or a portion of vCYC (ORF72). In some embodiments, hybrid peptides of the invention comprise or consist of all or a portion of RTA (ORF50). In some embodiments, the hybrid peptides of the invention comprise or consist of: all or a portion of vFLIP (ORF71). In some embodiments, the mixed peptides of the invention comprise or consist of: all or part of kaposi proteins (ORF12, K12). In some embodiments, hybrid peptides of the invention comprise or consist of: all or a portion of gB (ORF8). In some embodiments, the hybrid peptides of the invention comprise or consist of: all or a portion of MIR1 (K3). In some embodiments, hybrid peptides of the invention comprise or consist of all or a portion of SSB (ORF6). In some embodiments, the hybrid peptides of the invention comprise or consist of: all or a portion of TS (ORF70).

In some embodiments, the peptide comprises a polypeptide sequence from LANA-1. In some embodiments, the peptide comprises the polypeptide sequence from SEQ ID NO: 16. In some embodiments, the peptide comprises a polypeptide of amino acids 49-63 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 65-83 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 125-139 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 325-339 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 901-915 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 905-919 of LANA-1. In some embodiments, the peptide comprises a polypeptide of amino acids 1093-1115 of LANA-1.

In some embodiments, the mixed peptides of the invention comprise or consist of one or more polypeptide sequences selected from the following sequences: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4), EYLVSFGVWIRTPPA (SEQ ID NO: 5), ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO : 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15) or its functional variant. In some embodiments, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are peptide sequences derived from HBsAg. In some embodiments, SEQ ID NO: 4 and SEQ ID NO: 5 are peptide sequences derived from HBeAg. In some embodiments, SEQ ID NOs: 6-12 are peptide sequences derived from HHV8 LANA1. In some embodiments, SEQ ID NOs: 13-15 are peptide sequences derived from HHV8 gB.

In some embodiments, the mixed peptides of the invention comprise or consist of: all or part of one or more antigens derived from one or more viruses selected from the following: EBV, CMV, adenovirus, BK virus, JC virus, HHV6, RSV, influenza virus, parainfluenza virus, pocavirus, coronavirus, LCMV, mumps virus, measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, herpes simplex virus, HPV, HBV, HIV, HTLV1, HHV8, West Nile virus, Zika virus and Ebola virus.

In some embodiments, the mixed peptides of the invention comprise at least one target antigen from EBV, wherein the antigen is selected from the group consisting of EBNAl, LMP2 and BZLF1. In some embodiments, the mixed peptides of the invention comprise at least one target antigen from CMV, wherein the antigen is selected from IE1 and pp65. In some embodiments, the hybrid peptides of the invention comprise at least one target antigen from AdV, wherein the antigen is selected from the group consisting of hexon and penton. In some embodiments, the mixed peptides of the invention comprise at least one target antigen from BKV, wherein the antigen is selected from LT and VP-1. In some embodiments, the mixed peptides of the invention comprise at least one target antigen from HHV-6, wherein the antigen is selected from the group consisting of U14, U11, U71, U54 and U90. In some embodiments, mixed peptides of the invention comprise at least one target antigen from RSV, wherein the antigen is selected from N and F. In some embodiments, the mixed peptides of the invention comprise at least one target antigen from influenza virus, wherein the antigen is selected from MP1 and NP1. In some embodiments, mixed peptides of the invention comprise at least one target antigen from PIV, wherein the antigen is selected from the group consisting of F, N, M, M2-1 and HN. In some embodiments, the mixed peptides of the invention comprise at least one target antigen from hMPV, wherein the antigen is selected from the group consisting of F, N, M2-1 and M.

In some embodiments, the mixed peptides of the invention comprise at least one target antigen from SARS-CoV, wherein the antigen is selected from the group consisting of spike protein (S), membrane protein (M), nucleocapsid protein (N), auxiliary protein protein 7a (AP7a) and nonstructural protein 4 (nsp4). In some embodiments, the mixed peptides of the invention comprise or consist of the following: spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a (AP7a) and non-structural protein 4 (nsp4) in whole or in part. iii. Cytokines

Interleukins serve as hormones of the immune system and have important roles related to T cell proliferation, differentiation, and survival. Type I interleukins have a common structure containing four alpha-helical bundles, and these interleukins include various interleukins as well as some growth and hematopoietic factors. An important family of type I interleukins is the common interleukin receptor gamma chain (γc) family, which includes interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL- 15 and IL-21, and are so named because the receptors for these interleukins share a common gamma chain (γc, also known as IL-2Rγ and CD132).

γc family interleukins transmit signals through the JAK-STAT (signal transducer and activator of transcription) pathway. Interestingly, IL-2, IL-7, IL-9, and IL-15 activate STAT5A and STAT5B (together referred to herein as STAT5), while IL-4 activates STAT6 and IL-21 activates STAT3. Without being bound by theory, it is believed that activation of different STAT proteins may explain the different effects of these interleukins (Rochman et al., 2009. Nature Reviews Immunology , Vol. 9, pp. 480-490).

In some embodiments, the present invention provides a method for expanding T cells (e.g., VST) from mononuclear cells (optionally peripheral blood mononuclear cells (PBMC)), using methods including: (1) using Contacting a population of mononuclear spheres with at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; (2) contacting a population of mononuclear spheres with one or more biased Exposure of VST to expanded interleukins, wherein the interleukins do not contain IL-15; and (3) contacting a population of mononuclear spheres with IL-15, thereby generating a process containing cells with antigen specificity. Expand T cell populations. In some embodiments, the method does not utilize IL-2. In some embodiments, the above steps (1) and (2) in this paragraph are performed simultaneously. In some embodiments, the above steps (1) and (2) in this paragraph are performed in parallel. In some embodiments, the above steps (1) and (2) in this paragraph are performed sequentially. When proceeding in sequence, step (1) may be performed first or step (2) may be performed first, depending on the situation. In some embodiments, step (1) is performed first. In some embodiments, step (2) is performed first. In some embodiments, the above steps (2) and (3) in this paragraph are performed simultaneously. In some embodiments, the above steps (2) and (3) in this paragraph are performed in parallel. In some embodiments, the above steps (2) and (3) in this paragraph are performed sequentially. When proceeding sequentially, step (2) may be performed first or step (3) may be performed first, depending on the situation. In some embodiments, step (2) is performed first. In some embodiments, step (3) is performed first. In some embodiments, step (3) is performed after steps (1) and (2). In some embodiments, step (3) and step (1) are performed simultaneously. In some embodiments, step (3) is performed in parallel with step (1). In some embodiments, step (1), step (2) and step (3) are performed simultaneously. In some embodiments, step (1), step (2) and step (3) are performed in parallel. In some embodiments, step (1), step (2) and step (3) are performed sequentially. In some embodiments, step (1) is performed before step (2) and step (3). Any interleukin suitable for selective amplification of VST may be utilized in step (2), including those disclosed herein. In specific embodiments, the interleukin included in step (2) includes or consists of IL-4 and IL-7. In certain embodiments, the interleukin included in step (2) does not include IL-2.

The present invention provides, at least in part, in vitro methods for expanding T cells and in vitro methods for expanding populations of T cells. In some embodiments, methods of the present invention include culturing monocytes with: at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and One or more exogenous interleukins. In some embodiments, methods of the invention comprise culturing monocytes with: at least one target antigen or fragment thereof corresponding to at least one antigen; and one or more exogenous interleukins. In some embodiments, methods of the invention comprise culturing monocytes with: at least one peptide or peptide mixture corresponding to at least one antigen; and one or more exogenous interleukins. In some embodiments, the exogenous interleukin is selected from IL-4, IL-7, and IL-15. In some embodiments, the interleukin is other than IL-2.

In some embodiments, methods of the present invention include culturing monocytes with: at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and One or more interleukins. In some embodiments, methods of the invention comprise culturing monocytes with: at least one target antigen or fragment thereof corresponding to at least one antigen; and one or more interleukins. In some embodiments, methods of the invention comprise culturing monocytes with: at least one peptide or peptide mixture corresponding to at least one antigen; and one or more interleukins. In some embodiments, the interleukin is selected from IL-4, IL-7, and IL-15. In some embodiments, the interleukin is other than IL-2.

In some embodiments, methods of the invention include culturing monocytes with: at least one peptide mixture (eg, mixed peptides) corresponding to at least one target antigen or a portion of a target antigen; and one or more exogenous Cytokinins. In some embodiments, methods of the present invention comprise culturing monocytes with at least one peptide mixture (eg, mixed peptides) corresponding to at least one antigen; and one or more exogenous interleukins. In some embodiments, the interleukin is selected from IL-4, IL-7, and IL-15. In some embodiments, the interleukin is other than IL-2.

In some embodiments, methods of the invention include culturing monocytes with: at least one peptide mixture (eg, mixed peptides) corresponding to at least one target antigen or a portion of a target antigen; and one or more interleukins . In some embodiments, methods of the invention comprise culturing monocytes with: a mixture of at least one peptide thereof (eg, a mixed peptide) corresponding to at least one antigen; and one or more interleukins. In some embodiments, the interleukin is selected from IL-4, IL-7, and IL-15. In some embodiments, the interleukin is other than IL-2.

In some embodiments, the present invention provides a method for expanding T cells (e.g., VST) from mononuclear cells (optionally peripheral blood mononuclear cells (PBMC)), using methods including: (1) using Contacting a population of mononuclear spheres with at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; (2) contacting a population of mononuclear spheres with one or more biased Expanded interleukin exposure of the VST thereby generates an expanded T cell population containing cells with antigen specificity. In specific embodiments, the interleukin included in step (2) includes or consists of IL-4, IL-7 and IL-15.

In some embodiments, the interleukin is IL-4. In some embodiments, the interleukin is IL-7. In some embodiments, the interleukin is IL-15. In some embodiments, the interleukins are IL-4 and IL-7. In some embodiments, the interleukins are IL-4 and IL-15. In some embodiments, the interleukins are IL-7 and IL-15. In some embodiments, the interleukins are IL-4, IL-7, and IL-15.

In some embodiments, one or more interleukins are added to the culture simultaneously. In some embodiments, one or more interleukins are added to the culture simultaneously with the addition of at least one target antigen. In some embodiments, one or more interleukins are added to the culture simultaneously with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. . In some embodiments, one or more interleukins are added to the culture in parallel. In some embodiments, one or more interleukins are added to the culture concurrently with the addition of at least one target antigen. In some embodiments, one or more interleukins are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. .

In some embodiments, one or more interleukins are added to the cell culture during the priming step. In some embodiments, the one or more interleukins are added to the in culture. In some embodiments, one or more interleukins are added during the priming step simultaneously with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. into the culture. In some embodiments, one or more interleukins are added during the priming step in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. into the culture. In some embodiments, one or more additional interleukins are added to the cell culture after the priming step. In some embodiments, the priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen Peptides or peptide mixtures; and (ii) IL-4 and IL-7. In some embodiments, the priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen Peptides or peptide mixtures; and (ii) IL-4, IL-7 and IL-15. In some embodiments, IL-15 is added to the cell culture during the priming step.

In some embodiments, IL-15 is added to the cell culture after the priming step. In some embodiments, IL-15 is added to the cell culture during the expansion step.

In some embodiments of the invention, IL-4, IL-7, and IL-4, IL-7, and IL-15 was added to the culture. In some embodiments, IL-4, IL-7, and IL-15 are added in parallel with the addition of at least one target antigen or a portion of a target antigen, or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. Add to culture. In some embodiments, IL-4, IL-7, and IL-15 are added to the culture concurrently with the addition of at least one target antigen or a portion of a target antigen. In some embodiments, IL-4, IL-7, and IL-15 are added to the culture concurrently with the addition of at least one target antigen or fragment thereof. In some embodiments, IL-4, IL-7, and IL-15 are added to the culture in parallel with the addition of at least one peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen.

In some embodiments of the invention, IL-4, IL-7 and IL-4, IL-7 and IL-4 are combined after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments of the invention, IL-4, IL-7, and IL-15 was added to the culture at the same time. In some embodiments of the invention, IL-4, IL-7, and IL-15 was added to the cultures in parallel. In some embodiments of the invention, IL-4, IL-7, and IL-15 are added to the culture in parallel after the addition of at least one target antigen or a portion of a target antigen. In some embodiments of the invention, IL-4, IL-7 and IL-15 are added to the culture in parallel after the addition of at least one target antigen or fragment thereof. In some embodiments of the invention, IL-4, IL-7 and IL-15 are added to the culture in parallel after adding at least one peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. .

In some embodiments, from about 0 to about 1 hour, about 2 hours, about 3 hours after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, IL-4 was added to the culture at approximately 36 hours, or approximately 48 hours. In some embodiments, from about 0 to about 1 hour, about 2 hours, about 3 hours after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, IL-7 was added to the culture at approximately 36 hours, or approximately 48 hours. In some embodiments, from about 0 to about 1 hour, about 2 hours, about 3 hours after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, IL-15 was added to the culture at approximately 36 hours, or approximately 48 hours. In some embodiments, from about 0 to about 1 hour, about 2 hours, about 3 hours after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, IL-4 and IL-7 were added to the culture at approximately 36 hours, or approximately 48 hours. In some embodiments, from about 0 to about 1 hour, about 2 hours, about 3 hours after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, IL-4 and IL-15 were added to the culture at approximately 36 hours, or approximately 48 hours. In some embodiments, from about 0 to about 1 hour, about 2 hours, about 3 hours after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, IL-7 and IL-15 were added to the culture at approximately 36 hours, or approximately 48 hours.

In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. middle.

In some embodiments, IL-15 is added to the culture about 1 day after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture about 2 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture about 3 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture about 4 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture about 5 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture approximately 6 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture approximately 7 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture about 8 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture about 9 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. In some embodiments, IL-15 is added to the culture about 10 days after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen.

In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 is added to the culture after about 0 to about 1 hour. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 is added to the culture after about 0 to about 12 hours. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 is added to the culture after about 0 to about 24 hours. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 is added to the culture after about 0 to about 48 hours. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 is added to the culture after about 1 to about 6 hours. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 is added to the culture after about 6 to about 12 hours. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture about 12 to about 18 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture about 18 to about 24 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture about 24 to about 36 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture about 36 to about 48 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 1 hour later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 2 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 6 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 12 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 18 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 24 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 36 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 48 hours later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 1 day later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 2 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 3 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 4 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 5 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 6 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 7 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 8 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 9 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture approximately 10 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture about 1 day to about 2 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture after about 1 day to about 3 days. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture after about 1 day to about 4 days. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture after about 1 day to about 5 days. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture after about 1 day to about 6 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture after about 1 day to about 7 days. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture after about 1 day to about 8 days later. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture after about 1 day to about 9 days. In some embodiments, IL-4 and IL-7 are added to the culture in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. , and IL-15 was added to the culture about 1 day later to about 10 days later.

In some embodiments, the IL-4, IL- 7 and IL-15 were added to the culture. In some embodiments, the IL-4, IL- 7 and IL-15 were added to the culture. In some embodiments, the IL-4, IL- 7 and IL-15 were added to the culture. In some embodiments, the IL-4, IL- 7 and IL-15 were added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 2 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 3 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added about 4 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 5 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 6 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 8 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 12 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 16 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 20 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 24 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 36 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture. In some embodiments, IL-4, IL-7 and IL-4, IL-7 and IL-4 are added approximately 48 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -15 was added to the culture.

In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen is added from about 0 hours to about 1 hour after adding IL-4, IL-7, and IL-15 to the culture. Or a peptide or peptide mixture that is part of a target antigen is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 1 hour after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 2 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 3 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 4 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 5 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 6 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 8 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 12 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added approximately 16 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 20 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 24 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture. In some embodiments, at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a target antigen is added about 48 hours after IL-4, IL-7, and IL-15 are added to the culture. A portion of the peptide or peptide mixture is added to the culture.

In some embodiments of the invention, IL-4 and IL-7 are added simultaneously with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. to the culture, and IL-15 was added to the culture at a later time point. In some embodiments of the invention, IL-4 and IL-7 are added in parallel with the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. to the culture, and IL-15 was added to the culture at a later time point. In some embodiments of the invention, IL-4 and IL-7 are added to the cultures, and IL-15 was added to the cultures at later time points. In some embodiments, IL-15 is added to the culture from about 0 hours to about 1 hour after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 1 hour after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 2 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 3 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 4 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 5 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 6 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 8 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 12 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 24 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 36 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 48 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 60 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 72 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 1 day after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 2 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 3 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 4 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 5 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 6 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 7 days after the addition of IL-4 and IL-7.

In some embodiments, IL-15 is added to the culture from about 0 hours to about 48 hours after the addition of IL-4 and IL7. In some embodiments, IL-15 is added to the culture from about 0 hours to about 24 hours after the addition of IL-4 and IL7. In some embodiments, IL-15 is added to the culture about 0 hours to about 12 hours after the addition of IL-4 and IL7. In some embodiments, IL-15 is added to the culture about 0 hours to about 6 hours after the addition of IL-4 and IL7. In some embodiments, IL-15 is added to the culture about 0 hours to about 2 hours after the addition of IL-4 and IL7. In some embodiments, IL-15 is added to the culture about 0 hours to about 1 hour after the addition of IL-4 and IL7. In some embodiments, IL-15 is added to the culture about 0 hours to about 30 minutes after the addition of IL-4 and IL7. In some embodiments, IL-15 is added to the culture concurrently with the addition of IL-4 and IL7.

In some embodiments of the invention, IL-4 and IL-7 are added to the cultures; and IL-15 was added to the cultures at later time points. In some embodiments, IL-15 is added to the culture from about 0 hours to about 1 hour after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 1 hour after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 2 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 3 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 4 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 5 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 6 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 8 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 12 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 24 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 36 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 48 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 60 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 72 hours after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 1 day after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 2 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 3 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 4 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 5 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 6 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 7 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 8 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 9 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture approximately 10 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture from about 0 days to about 1 day after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 0 days to about 2 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 0 days to about 3 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 0 days to about 4 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 0 days to about 10 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 1 day to about 2 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 1 day to about 3 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 1 day to about 4 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 1 day to about 5 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 1 day to about 6 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 2 days to about 4 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 2 days to about 6 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 4 days to about 8 days after the addition of IL-4 and IL-7. In some embodiments, IL-15 is added to the culture about 6 days to about 10 days after the addition of IL-4 and IL-7.

In some embodiments of the invention, IL-4 and IL-7 are added to the in culture. In some embodiments, the IL-4 and IL-4 are added from about 0 hours to about 1 hour after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 0 hours to about 2 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 0 hours to about 6 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 1 hour to about 2 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 1 hour to about 12 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 2 hours to about 6 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added about 6 hours to about 12 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 8 hours to about 18 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added about 12 hours to about 24 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 18 hours to about 36 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added about 24 hours to about 48 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, the IL-4 and IL-4 are added from about 36 hours to about 48 hours after the addition of at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. -7 added to cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added to in cell culture. In some embodiments, IL-4 and IL-7 are added about 48 hours after adding at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen. in cell culture.

In some embodiments, IL-4 is added to the cells at about 400 U/mL to about 1200 U/mL. In some embodiments, IL-4 is added to the cells at about 600 U/mL to about 1000 U/mL. In some embodiments, IL-4 is added to the cells at about 700 U/mL to about 900 U/mL. In some embodiments, IL-4 is added to the cells at about 800 U/mL to about 1000 U/mL. In some embodiments, IL-4 is added to the cells at about 600 U/mL to about 800 U/mL. In some embodiments, IL-4 is added to the cells at about 400 U/mL. In some embodiments, IL-4 is added to the cells at about 500 U/mL. In some embodiments, IL-4 is added to the cells at about 600 U/mL. In some embodiments, IL-4 is added to the cells at about 700 U/mL. In some embodiments, IL-4 is added to the cells at about 800 U/mL. In some embodiments, IL-4 is added to the cells at about 900 U/mL. In some embodiments, IL-4 is added to the cells at about 1000 U/mL. In some embodiments, IL-4 is added to the cells at 400 U/mL. In some embodiments, IL-4 is added to the cells at 500 U/mL. In some embodiments, IL-4 is added to the cells at 600 U/mL. In some embodiments, IL-4 is added to the cells at 700 U/mL. In some embodiments, IL-4 is added to the cells at 800 U/mL. In some embodiments, IL-4 is added to the cells at 900 U/mL. In some embodiments, IL-4 is added to the cells at 1000 U/mL. In some embodiments, IL-4 is added to the cells at 1100 U/mL. In some embodiments, IL-4 is added to the cells at 1200 U/mL. In some embodiments, 1 μg of IL-4 contains 2.9×10 ⁴ units (U) (R&D Systems).

In some embodiments, IL-7 is added to the cells at about 5 ng/mL to about 30 ng/mL. In some embodiments, IL-7 is added to the cells at about 10 ng/mL to about 25 ng/mL. In some embodiments, IL-7 is added to the cells at about 15 ng/mL to about 25 ng/mL. In some embodiments, IL-7 is added to the cells at about 15 ng/mL to about 20 ng/mL. In some embodiments, IL-7 is added to the cells at about 20 ng/mL to about 25 ng/mL. In some embodiments, IL-7 is added to the cells at about 5 ng/mL. In some embodiments, IL-7 is added to the cells at about 10 ng/mL. In some embodiments, IL-7 is added to the cells at about 15 ng/mL. In some embodiments, IL-7 is added to the cells at about 20 ng/mL. In some embodiments, IL-7 is added to the cells at about 25 ng/mL. In some embodiments, IL-7 is added to the cells at about 30 ng/mL. In some embodiments, IL-7 is added to the cells at 5 ng/mL. In some embodiments, IL-7 is added to the cells at 10 ng/mL. In some embodiments, IL-7 is added to the cells at 15 ng/mL. In some embodiments, IL-7 is added to the cells at 20 ng/mL. In some embodiments, IL-7 is added to the cells at 25 ng/mL. In some embodiments, IL-7 is added to the cells at 30 ng/mL.

In some embodiments, IL-15 is added to the cells at about 1 ng/mL to about 20 ng/mL. In some embodiments, IL-15 is added to the cells at about 1 ng/mL to about 15 ng/mL. In some embodiments, IL-15 is added to the cells at about 1 ng/mL to about 10 ng/mL. In some embodiments, IL-15 is added to the cells at about 2 ng/mL to about 8 ng/mL. In some embodiments, IL-15 is added to the cells at about 4 ng/mL to about 6 ng/mL. In some embodiments, IL-15 is added to the cells at about 4 ng/mL to about 5 ng/mL. In some embodiments, IL-15 is added to the cells at about 5 ng/mL to about 6 ng/mL. In some embodiments, IL-15 is added to the cells at about 1 ng/mL. In some embodiments, IL-15 is added to the cells at about 2 ng/mL. In some embodiments, IL-15 is added to the cells at about 3 ng/mL. In some embodiments, IL-15 is added to the cells at about 4 ng/mL. In some embodiments, IL-15 is added to the cells at about 5 ng/mL. In some embodiments, IL-15 is added to the cells at about 6 ng/mL. In some embodiments, IL-15 is added to the cells at about 7 ng/mL. In some embodiments, IL-15 is added to the cells at about 8 ng/mL. In some embodiments, IL-15 is added to the cells at about 9 ng/mL. In some embodiments, IL-15 is added to the cells at about 10 ng/mL. In some embodiments, IL-15 is added to the cells at 1 ng/mL. In some embodiments, IL-15 is added to the cells at 2 ng/mL. In some embodiments, IL-15 is added to the cells at 3 ng/mL. In some embodiments, IL-15 is added to the cells at 4 ng/mL. In some embodiments, IL-15 is added to the cells at 5 ng/mL. In some embodiments, IL-15 is added to the cells at 6 ng/mL. In some embodiments, IL-15 is added to the cells at 7 ng/mL. In some embodiments, IL-15 is added to the cells at 8 ng/mL. In some embodiments, IL-15 is added to the cells at 9 ng/mL. In some embodiments, IL-15 is added to the cells at 10 ng/mL. In some embodiments, IL-15 is added to the cells at 11 ng/mL. In some embodiments, IL-15 is added to the cells at 12 ng/mL. In some embodiments, IL-15 is added to the cells at 13 ng/mL. In some embodiments, IL-15 is added to the cells at 14 ng/mL. In some embodiments, IL-15 is added to the cells at 15 ng/mL. In some embodiments, IL-15 is added to the cells at 16 ng/mL. In some embodiments, IL-15 is added to the cells at 17 ng/mL. In some embodiments, IL-15 is added to the cells at 18 ng/mL. In some embodiments, IL-15 is added to the cells at 19 ng/mL. In some embodiments, IL-15 is added to the cells at 20 ng/mL.

In some embodiments, IL-4 is added to the cells at about 400 U/mL to about 1200 U/mL, and IL-7 is added to the cells at about 5 ng/mL to about 30 ng/mL. In some embodiments, IL-15 is added to the cells at about 1 ng/mL to about 20 ng/mL. In some embodiments, IL-4 is added to the cells at 800 U/mL and IL-7 is added to the cells at 20 ng/mL. In some embodiments, cells are contacted with a composition comprising 800 U/mL of IL-4 and 20 ng/mL of IL-7. In some embodiments, the cells are contacted with a composition comprising 5 ng/mL of IL-15. In some embodiments, the cells are contacted with a first composition comprising 800 U/mL of IL-4 and 20 ng/mL of IL-7, and the cells are contacted with a second composition comprising 5 ng/mL of IL-15 get in touch with. In some embodiments, cells are contacted with a composition comprising 800 U/mL of IL-4, 20 ng/mL of IL-7, and 5 ng/mL of IL-15. In some embodiments, the cells are contacted with a composition comprising 800 U/mL of IL-4 and 20 ng/mL of IL-7, and after 0 to 48 hours, the cells are contacted with 5 ng/mL of IL-15 . In some embodiments, cells are contacted with a composition comprising 800 U/mL of IL-4 and 20 ng/mL of IL-7, and after 0 to 48 hours, 5 ng/mL of IL-15 is added to In a composition containing 800 U/mL of IL-4 and 20 ng/mL of IL-7. In some embodiments, the composition includes mixed peptides (2 nanograms/peptide/ml). In some embodiments, the cells are combined with a composition comprising 800 U/mL of IL-4, 20 ng/mL of IL-7, 5 ng/mL of IL-15, and mixed peptides (2 nanograms/peptide/ml) get in touch with.

In some embodiments, cells are contacted with a composition comprising 800 U/mL of IL-4, 20 ng/mL of IL-7, and mixed peptides (2 nanograms/peptide/ml), and after 0 to 48 hours , cells were exposed to 5 ng/mL IL-15. In some embodiments, cells are contacted with a composition comprising 800 U/mL of IL-4, 20 ng/mL of IL-7, and mixed peptides (2 nanograms/peptide/ml), and after 0 to 48 hours , 5 ng/mL of IL-15 was added to a composition containing 800 U/mL of IL-4, 20 ng/mL of IL-7, and mixed peptides (2 nanograms/peptide/ml).

In some embodiments, the interleukin is utilized by the cells during the culture period. In some embodiments, the interleukin is utilized by the monocytes during the culture period. In some embodiments, the interleukin is utilized by the PBMC during the culture period. In some embodiments, the interleukin is utilized by antigen-specific T cells during the culture period. In some embodiments, interleukins are utilized by the VST during the culture period. In some embodiments, the interleukin is partially utilized by the cells during the culture period. In some embodiments, the interleukin is partially utilized by the monocytes during the culture period. In some embodiments, the interleukin is utilized partially by the PBMC during the culture period. In some embodiments, the interleukin is utilized in part by the antigen-specific T cells during the culture period. In some embodiments, the interleukin is utilized in part by the VST during the culture period.

In some embodiments, the interleukin is a human interleukin. In some embodiments, the interleukin is a recombinant interleukin. In some embodiments, the interleukin is a recombinant human interleukin.

In some embodiments, the IL-4 is human IL-4. In some embodiments, the IL-4 is recombinant IL-4. In some embodiments, the IL-4 is recombinant human IL-4.

In some embodiments, the IL-7 is human IL-7. In some embodiments, IL-7 is recombinant IL-7. In some embodiments, the IL-7 is recombinant human IL-7.

In some embodiments, the IL-15 is human IL-15. In some embodiments, IL-15 is recombinant IL-15. In some embodiments, IL-15 is recombinant human IL-15. iv.Cultivation time

The cells can be cultured for a sufficient duration to obtain a population of antigen-specific T cells. In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) culturing the cells for a first period of time; Culture for a second period in the presence of interleukins. In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) culturing the cells for a first period of time; Culture for the second period in the presence of IL-4. In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) culturing the cells for a first period of time; Culture for the second period in the presence of IL-7. In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) culturing the cells for a first period of time; Culture for the second period in the presence of IL-15. In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) culturing the cells for a first period of time; Culture for the second period in the presence of IL-4 and IL-7. In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) culturing the cells for a first period of time; Culture for the second period in the presence of IL-4, IL-7 and IL-15. In some embodiments, culturing MNC to generate antigen-specific T cells comprises: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; ii) culturing the cells in IL -4. Culturing the cells in the presence of IL-7 for a second period; and culturing the cells in the presence of IL-15 for a third period. In some embodiments, culturing MNC to generate antigen-specific T cells comprises: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen and IL-4 and IL-7; period; and ii) culturing the cells in the presence of IL-15 for a second period.

In some embodiments, the first period of time is less than about 24 hours. In some embodiments, the first period exceeds about 24 hours. In some embodiments, the first period of time is between about 24 and about 36 hours. In some embodiments, the first period of time is between about 24 and about 48 hours. In some embodiments, the first period of time is between about 0 and about 48 hours. In some embodiments, the first period of time is between about 24 and about 60 hours. In some embodiments, the first period is between about 24 and about 72 hours. In some embodiments, the first period of time is less than about 144 hours. In some embodiments, the first period is less than 24 hours. In some embodiments, the first period exceeds 24 hours. In some embodiments, the first period of time is from about 24 hours to about 36 hours. In some embodiments, the first period of time is from about 24 hours to about 48 hours. In some embodiments, the first period of time is from about 24 hours to about 60 hours. In some embodiments, the first period of time is from about 24 hours to about 72 hours. In some embodiments, the first period is less than 144 hours. In some embodiments, the first period exceeds about 144 hours. In some embodiments, the first period exceeds 144 hours. In some embodiments, the first period is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In some embodiments, the second period of time is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In some embodiments, the second period of time is about 1 to about 2 days. In some embodiments, the second period of time is from about 1 to about 5 days. In some embodiments, the second period of time is from about 1 to about 10 days. In some embodiments, the second period of time is from about 1 to about 14 days. In some embodiments, the second period of time is from about 1 to about 18 days. In some embodiments, the second period of time is from about 1 to about 20 days. In some embodiments, the second period of time is from about 2 to about 14 days. In some embodiments, the second period of time is from about 2 to about 18 days. In some embodiments, the second period of time is from about 14 to about 18 days. In some embodiments, the second period exceeds about 10 days. In some embodiments, the second period exceeds about 14 days. In some embodiments, the second period exceeds about 18 days. In some embodiments, the second period of time is about 1 day. In some embodiments, the second period of time is about 2 days. In some embodiments, the second period is about 3 days. In some embodiments, the second period is about 4 days. In some embodiments, the second period is about 5 days. In some embodiments, the second period is about 6 days. In some embodiments, the second period is about 7 days. In some embodiments, the second period is about 8 days. In some embodiments, the second period is about 9 days. In some embodiments, the second period is about 10 days. In some embodiments, the second period is about 11 days. In some embodiments, the second period is about 12 days. In some embodiments, the second period is about 13 days. In some embodiments, the second period is about 14 days. In some embodiments, the second period is about 15 days. In some embodiments, the second period is about 16 days. In some embodiments, the second period is about 17 days. In some embodiments, the second period is about 18 days. In some embodiments, the second period is about 19 days. In some embodiments, the second period is about 20 days. In some embodiments, the second period is about 21 days.

In some embodiments, the third period of time is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In some embodiments, the third period of time is about 1 to about 2 days. In some embodiments, the third period of time is from about 1 to about 5 days. In some embodiments, the third period of time is from about 1 to about 10 days. In some embodiments, the third period of time is from about 1 to about 14 days. In some embodiments, the third period of time is from about 1 to about 18 days. In some embodiments, the third period of time is from about 1 to about 20 days. In some embodiments, the third period of time is from about 2 to about 14 days. In some embodiments, the third period of time is from about 2 to about 18 days. In some embodiments, the third period of time is from about 14 to about 18 days. In some embodiments, the third period exceeds about 10 days. In some embodiments, the third period exceeds about 14 days. In some embodiments, the third period exceeds about 18 days. In some embodiments, the third period of time is about 1 day. In some embodiments, the third period is about 2 days. In some embodiments, the third period of time is about 3 days. In some embodiments, the third period is about 4 days. In some embodiments, the third period is about 5 days. In some embodiments, the third period is about 6 days. In some embodiments, the third period is about 7 days. In some embodiments, the third period is about 8 days. In some embodiments, the third period of time is about 9 days. In some embodiments, the third period is about 10 days. In some embodiments, the third period is approximately 11 days. In some embodiments, the third period is approximately 12 days. In some embodiments, the third period is about 13 days. In some embodiments, the third period is about 14 days. In some embodiments, the third period is about 15 days. In some embodiments, the third period is about 16 days. In some embodiments, the third period is about 17 days. In some embodiments, the third period is about 18 days. In some embodiments, the third period is approximately 19 days. In some embodiments, the third period is about 20 days. In some embodiments, the third period is approximately 21 days.

In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) incubating the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) IL-4, IL-7 and IL-15 are added to the cell culture from about 0 days to about 2 days after the first period. In some embodiments, cells are cultured for 7 to 14 days. In some embodiments, cells are cultured for 10 to 18 days. In some embodiments, cells are cultured for about 14 days. In some embodiments, cells are cultured for about 18 days.

In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells for a first period of time with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen, IL-4 and IL-7; and ii) IL-15 is added to the cell culture from about 0 days to about 2 days after the first period. In some embodiments, cells are cultured for 7 to 14 days. In some embodiments, cells are cultured for 10 to 18 days. In some embodiments, cells are cultured for about 14 days. In some embodiments, cells are cultured for about 18 days.

In some embodiments, contact of cells with IL-15 results in accelerated expansion of cells in culture. In some embodiments, contacting cells with IL-4 and IL-7 prior to subsequent contact with IL-15 generates an expanded population of antigen-specific T cells comprising an antigen obtainable by contacting the cells with Expanded populations of specific T cells compared to enriched antigen-specific T cells of a broader lineage: (i) IL-4 and IL-7, without IL-15; (ii) IL-7 and IL-15 , without IL-4; or (iii) simultaneously IL-4, IL-7 and IL-15.

In some embodiments, contact of cells with IL-15 results in accelerated expansion of cells in culture. In some embodiments, contacting cells with IL-4 and IL-7 prior to subsequent contact with IL-15 generates an expanded population of antigen-specific T cells comprising an antigen obtainable by contacting the cells with Expanded populations of specific T cells compared to enriched antigen-specific T cells of a broader repertoire of: (i) IL-4 and IL-7, without IL-15; or (ii) IL-7 and IL- 15, no IL-4.

In some embodiments, the cells are contacted with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen and IL-4, IL-7, and IL-15 such that a population of antigen-specific T cells has antigen specificity. Greater expansion than a population obtainable by contacting cells with: IL-4 and IL-7 without IL-15; or (ii) IL-4 and IL-7 with IL-2.

In some embodiments, the cells are contacted with at least one target antigen or a peptide or mixture of peptides corresponding to at least one target antigen and IL-4 and IL-7 prior to subsequent contact with IL-15 such that the cells have antigen specificity. A population of T cells with greater expansion than that obtainable by contacting the cells with: (i) IL-4 and IL-7, without IL-15; or (ii) IL-4 and IL- 7. Contains IL-2.

In some embodiments, the antigen-specific T cells have completed log phase growth. In some embodiments, the antigen-specific T cells are sufficiently expanded within 9 to 18 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded within 8 to 20 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded within 6 to 22 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded within 4 to 24 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded after 24 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 9 days to about 18 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 8 days to about 20 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 6 days to about 22 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 4 days to about 24 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 4 days to about 20 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 6 days to about 20 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 4 days to about 18 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded by about 6 days to about 18 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells are sufficiently expanded after about 24 days in culture such that they are ready for administration to the patient.

In some embodiments, whether a culture is ready for administration to a patient is determined by the physician. In some embodiments, a culture is considered ready for administration to a patient when it reaches a certain number of cells, displays a certain degree of specificity, reaches a certain level of multifunctionality, or a combination thereof.

In some embodiments, the antigen-specific T cells have expanded within 9 to 18 days of culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded within 8 to 20 days of culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded within 6 to 22 days of culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded within 4 to 24 days of culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded after 24 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 9 days to about 18 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 8 days to about 20 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 6 days to about 22 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 4 days to about 24 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 4 days to about 20 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 6 days to about 20 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 4 days to about 18 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 6 days to about 18 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 12 days to about 18 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 14 days to about 18 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded by about 14 days to about 21 days in culture such that they have completed log phase growth. In some embodiments, the antigen-specific T cells have expanded after about 24 days in culture such that they have completed log phase growth.

In some embodiments, methods for generating antigen-specific T cells as provided herein can result in about 7- to 9-fold expansion of cells compared to the starting cell population cultured. In some embodiments, methods of generating antigen-specific T cells as provided herein can result in about 5- to 10-fold expansion of cells compared to the starting cell population cultured. In some embodiments, methods of generating antigen-specific T cells as provided herein can result in about 2- to 20-fold expansion of cells compared to the starting population of cells cultured. In some embodiments, methods for generating antigen-specific T cells as provided herein can result in about 7-fold to about 9-fold expansion of cells compared to the starting population of cells cultured. In some embodiments, methods of generating antigen-specific T cells as provided herein can result in about 5-fold to about 10-fold expansion of cells compared to the starting cell population in culture. In some embodiments, methods of generating antigen-specific T cells as provided herein can result in about 2-fold to about 20-fold expansion of cells compared to the starting cell population in culture.

In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes a seeding density in the range of 0.5 × ¹⁰ cells per square centimeter to 6 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes a seeding density in the range of 2 × 10 ⁶ cells per square centimeter to 4 × 10 ⁶ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 0.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 1 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 1.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 2 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 2.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 3 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 3.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 4 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 4.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes 5.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for generating antigen-specific T cells as provided herein includes ⁶ × 10 cells per square centimeter.

In some embodiments, a seeding density of 3 million cells per square centimeter is selected as the optimal seeding density.

In some embodiments, the seeding density of the method for generating antigen-specific T cells of the present invention includes a seeding density of about 2 × 10 ⁶ cells per square centimeter to about 4 × 10 ⁶ cells per square centimeter. In some embodiments, the seeding density of the method for generating antigen-specific T cells of the present invention includes about 2 × 10 ⁶ cells per square centimeter. In some embodiments, the seeding density of the methods of the present invention for generating antigen-specific T cells includes about 2.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of the method for generating antigen-specific T cells of the present invention includes about 3 × 10 ⁶ cells per square centimeter. In some embodiments, the seeding density of the method for generating antigen-specific T cells of the present invention includes about 3.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of the method for generating antigen-specific T cells of the present invention includes about 4 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of the method for generating antigen-specific T cells of the present invention includes about 1 × 10 ⁶ cells per square centimeter. In some embodiments, the seeding density of the methods of the present invention for generating antigen-specific T cells includes about 5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of the method for generating antigen-specific T cells of the present invention includes about ⁶ × 10 cells per square centimeter.

In some embodiments, the present invention provides that such antigen-specific T cell lines can be prospectively generated and subsequently cryopreserved such that they are directly available as immunologically active "off-the-shelf" products. iii.Cultivation steps

In some embodiments, methods of generating antigen-specific T cells include at least one composition. In some embodiments, methods of generating antigen-specific T cells comprise at least two compositions. In some embodiments, methods of generating antigen-specific T cells comprise at least three compositions. In some embodiments, methods of generating antigen-specific T cells comprise at least four compositions.

In some embodiments, a method of generating antigen-specific T cells includes a first composition and a second composition. In some embodiments, a method of generating antigen-specific T cells includes a first composition, a second composition, and a third composition. In some embodiments, a method of generating antigen-specific T cells includes a first composition, a second composition, a third composition, and a fourth composition.

In some embodiments, the first composition comprises an antigen as described herein, or a fragment thereof. In some embodiments, the first composition includes a peptide or mixture of peptides described herein. In some embodiments, the first composition includes: (i) an antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) At least one interleukin.

In some embodiments, the first composition includes: (i) An antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-4.

In some embodiments, the first composition includes: (i) An antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-7.

In some embodiments, the first composition includes: (i) an antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-4 and IL-7.

In some embodiments, the first composition includes: (i) An antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-4, IL-7 and IL-15.

In some embodiments, the first composition includes: (i) an antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) At least one interleukin, but not IL-2.

In some embodiments, the first composition includes: (i) An antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-4, but not IL-2.

In some embodiments, the first composition includes: (i) An antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-7, but not IL-2.

In some embodiments, the first composition includes: (i) an antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-4 and IL-7, but not IL-2.

In some embodiments, the first composition includes: (i) An antigen as described herein or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and (ii) IL-4, IL-7 and IL-15, but not IL-2.

In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes at least one interleukin. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes IL-4. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes IL-4 and IL-7. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes IL-4, IL-7, and IL-15.

In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes at least one interleukin but not IL-2. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes IL-4 but not IL-2. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes IL-4 and IL-7 but not IL-2. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition includes IL-4, IL-7, and IL-15 but not IL- 2.

In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes at least one interleukin, the first composition and the second combination substances are administered into cells in parallel. In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4, the first composition and the second composition are in parallel. administered into cells. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4 and IL-7, the first composition and the second composition include IL-4 and IL-7. The two compositions are administered to the cells in parallel. In some embodiments, when the first composition includes an antigen or fragment thereof, or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4, IL-7, and IL-15, the first composition The composition and the second composition are administered to the cells in parallel.

In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition comprises IL-4 but not IL-2, the first composition and the second composition are administered to the cells in parallel. In some embodiments, when the first composition includes an antigen or fragment thereof, or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4 and IL-7 but not IL-2, The first composition and the second composition are administered to the cells in parallel. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4, IL-7, and IL-15 but does not include IL At -2, the first composition and the second composition are administered to the cells in parallel.

In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes at least one interleukin, the first composition during the first period administered within and the second composition is administered during a second period of time. In some embodiments, when the first composition comprises an antigen or fragment thereof, or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition comprises IL-4, the first composition is administered during the first period of time. and the second composition is administered during a second period of time. In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4 and IL-7, the first composition is is administered during one period of time and the second composition is administered during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof, or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4, IL-7, and IL-15, the first composition The composition is administered during a first period of time and the second composition is administered during a second period of time.

In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, and the second composition comprises IL-4 but not IL-2, the first composition The second composition is administered during a first period of time and the second composition is administered during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof, or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4 and IL-7 but not IL-2, The first composition is administered during a first period of time and the second composition is administered during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, and the second composition includes IL-4, IL-7, and IL-15 but does not include IL -2, the first composition is administered during the first period and the second composition is administered during the second period.

In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin, and the third composition includes IL-15. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least IL-4, and the third composition includes IL-15. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes IL-4 and IL-7, and the third composition includes IL-15 .

In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but not IL-2, and the third composition Contains IL-15 but not IL-2. In some embodiments, the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least IL-4 but not IL-2, and the third composition comprises IL-15 but not IL-2. In some embodiments, the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes IL-4 and IL-7 but not IL-2, and the third composition The composition contains IL-15 but not IL-2.

In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least one interleukin, and the third composition comprises IL-15 , the first composition, the second composition and the third composition are administered to the cells in parallel. In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least IL-4, and the third composition comprises IL-15, The first composition, the second composition and the third composition are administered to the cells in parallel. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7, and the third composition includes IL- At 15 o'clock, the first composition, the second composition and the third composition are administered to the cells in parallel.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but not IL-2, and the third combination When the composition contains IL-15 but does not contain IL-2, the first composition, the second composition and the third composition are administered to the cells in parallel. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least IL-4 but not IL-2, and the third composition When IL-15 is included but IL-2 is not included, the first composition, the second composition and the third composition are administered to the cells in parallel. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7 but not IL-2, and the second composition includes IL-4 and IL-7 but does not include IL-2. When the three compositions include IL-15 but do not include IL-2, the first composition, the second composition and the third composition are administered to the cells in parallel.

In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least one interleukin, and the third composition comprises IL-15 , the first composition is administered during a first period of time, the second composition is administered during a second period of time, and the third composition is administered to the cell during a third period of time. In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least IL-4, and the third composition comprises IL-15, The first composition is administered during a first period of time, the second composition is administered during a second period of time, and the third composition is administered into the cell during a third period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7, and the third composition includes IL- At 15 hours, the first composition is administered during a first time period, the second composition is administered during a second time period, and the third composition is administered into the cells during a third time period.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but not IL-2, and the third combination When the composition includes IL-15 but does not include IL-2, the first composition is administered during a first period of time, the second composition is administered during a second period of time, and the third composition is administered during a third period of time. in cells. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least IL-4 but not IL-2, and the third composition When IL-15 is included but IL-2 is not included, the first composition is administered during a first period of time, the second composition is administered during a second period of time, and the third composition is administered to the cell during a third period of time middle. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7 but not IL-2, and the second composition includes IL-4 and IL-7 but does not include IL-2. When three compositions include IL-15 but not IL-2, the first composition is administered during a first time period, the second composition is administered during a second time period, and the third composition is administered during a third time period into the cells.

In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least one interleukin, and the third composition comprises IL-15 , the first composition and the second composition are administered during a first period of time, and the third composition is administered to the cells during a second period of time. In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least IL-4, and the third composition comprises IL-15, The first composition and the second composition are administered during a first period of time, and the third composition is administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7, and the third composition includes IL- At 15 o'clock, the first composition and the second composition are administered during a first period of time, and the third composition is administered to the cells during a second period of time.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but not IL-2, and the third combination When the composition includes IL-15 but does not include IL-2, the first composition and the second composition are administered during a first period of time, and the third composition is administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least IL-4 but not IL-2, and the third composition When IL-15 is included but IL-2 is not included, the first composition and the second composition are administered during a first period of time and the third composition is administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7 but not IL-2, and the second composition includes IL-4 and IL-7 but not IL-2. When the three compositions include IL-15 but not IL-2, the first composition and the second composition are administered during a first period of time, and the third composition is administered to the cells during a second period of time.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but not IL-2, and the third combination When the object includes IL-15 but does not include IL-2, the first composition is administered during a first period of time, and the second and third compositions are administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least IL-4 but not IL-2, and the third composition When IL-15 is included but IL-2 is not included, the first composition is administered during a first period of time and the second and third compositions are administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7 but not IL-2, and the second composition includes IL-4 and IL-7 but does not include IL-2. When the three compositions include IL-15 but not IL-2, the first composition is administered during a first period of time, and the second and third compositions are administered to the cells during a second period of time.

In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least one interleukin, and the third composition comprises IL-15 , the first composition is administered during a first period of time, and the second and third compositions are administered to the cells during a second period of time. In some embodiments, when the first composition comprises an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition comprises at least IL-4, and the third composition comprises IL-15, The first composition is administered during a first period of time, and the second and third compositions are administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7, and the third composition includes IL- At 15 hours, the first composition is administered during a first period of time, and the second and third compositions are administered to the cells during a second period of time.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but not IL-2, and the third combination When the object includes IL-15 but does not include IL-2, the first composition is administered during a first period of time, and the second and third compositions are administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least IL-4 but not IL-2, and the third composition When IL-15 is included but IL-2 is not included, the first composition is administered during a first period of time and the second and third compositions are administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 and IL-7 but not IL-2, and the second composition includes IL-4 and IL-7 but does not include IL-2. When the three compositions include IL-15 but not IL-2, the first composition is administered during a first period of time, and the second and third compositions are administered to the cells during a second period of time.

In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least one interleukin, and the third composition includes at least one interleukin. , and when the fourth composition includes IL-15, the first composition is administered during the first period, the second composition is administered during the second period, the third composition is administered during the third period, and the Four compositions are administered to the cells during a fourth period. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4, the third composition includes IL-7, and the fourth When the composition includes IL-15, the first composition is administered during a first period of time, the second composition is administered during a second period of time, the third composition is administered during a third period of time, and the fourth composition is administered during administered into the cells during the fourth period. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-7, the third composition includes IL-4, and the fourth When the composition includes IL-15, the first composition is administered during a first period of time, the second composition is administered during a second period of time, the third composition is administered during a third period of time, and the fourth composition is administered during administered into the cells during the fourth period.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but does not include IL-2, and the third composition When at least one interleukin is included but does not include IL-2, and the fourth composition includes IL-15 but does not include IL-2, the first composition is administered during a first period of time and the second composition is administered during a second period of time. For internal administration, the third composition is administered during a third period of time, and the fourth composition is administered into the cell during a fourth period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 but not IL-2, and the third composition includes IL -7 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first composition is administered during the first time period and the second composition is administered during the second time period, The third composition is administered during a third period of time, and the fourth composition is administered to the cells during a fourth period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes IL-7 but not IL-2, and the third composition includes IL -4 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first composition is administered during the first time period and the second composition is administered during the second time period, The third composition is administered during a third period of time, and the fourth composition is administered to the cells during a fourth period of time.

In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least one interleukin, and the third composition includes at least one interleukin. , and when the fourth composition includes IL-15, the first composition is administered during the first period, and the second, third and fourth compositions are administered into the cells during the second period. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4, the third composition includes IL-7, and the fourth When the composition includes IL-15, the first composition is administered during a first period of time and the second, third and fourth compositions are administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-7, the third composition includes IL-4, and the fourth When the composition includes IL-15, the first composition is administered during a first period of time and the second, third and fourth compositions are administered to the cells during a second period of time.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but does not include IL-2, and the third composition When the first composition includes at least one interleukin but does not include IL-2, and the fourth composition includes IL-15 but does not include IL-2, the first composition is administered during the first period, the second, third and fourth The composition is administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 but not IL-2, and the third composition includes IL -7 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first composition is administered during the first period, and the second, third and fourth compositions are administered during the first period. administered to cells within two time periods. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-7 but not IL-2, and the third composition includes IL -4 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first composition is administered during the first period, and the second, third and fourth compositions are administered during the first period. administered to cells within two time periods.

In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least one interleukin, and the third composition includes at least one interleukin. , and when the fourth composition includes IL-15, the first, second and third compositions are administered within a first period of time, and the fourth composition is administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4, the third composition includes IL-7, and the fourth When the composition includes IL-15, the first, second and third compositions are administered during a first period of time and the fourth composition is administered to the cells during a second period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-7, the third composition includes IL-4, and the fourth When the composition includes IL-15, the first, second and third compositions are administered during a first period of time and the fourth composition is administered to the cells during a second period of time.

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but does not include IL-2, and the third composition When the first, second and third compositions comprise at least one interleukin but not IL-2, and the fourth composition comprises IL-15 but not IL-2, the first, second and third compositions are administered during the first period, and the Four compositions are administered to the cells during the second period. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 but not IL-2, and the third composition includes IL -7 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first, second, and third compositions are administered during the first period, and the fourth composition is administered during administered into the cells during the second period. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-7 but not IL-2, and the third composition includes IL -4 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first, second and third compositions are administered during the first period, and the fourth composition is administered during administered into the cells during the second period.

In some embodiments, when the first composition includes an antigen or a fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes at least one interleukin, and the third composition includes at least one interleukin. , and when the fourth composition includes IL-15, the first composition is administered during the first period, the second and third compositions are administered during the second period, and the fourth composition is administered during the third period. into the cells. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4, the third composition includes IL-7, and the fourth When the composition includes IL-15, the first composition is administered during a first period of time, the second and third compositions are administered during a second period of time, and the fourth composition is administered to the cell during a third period of time. . In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-7, the third composition includes IL-4, and the fourth When the composition includes IL-15, the first composition is administered during a first period of time, the second and third compositions are administered during a second period of time, and the fourth composition is administered to the cell during a third period of time. .

In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one antigen of interest, the second composition includes at least one interleukin but does not include IL-2, and the third composition When the fourth composition includes at least one interleukin but does not include IL-2, and the fourth composition includes IL-15 but does not include IL-2, the first composition is administered during the first period, and the second and third compositions are administered during is administered during a second period of time, and the fourth composition is administered to the cells during a third period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-4 but not IL-2, and the third composition includes IL -7 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first composition is administered during the first period and the second and third compositions are administered during the second period is administered, and the fourth composition is administered to the cell during a third period of time. In some embodiments, when the first composition includes an antigen or fragment thereof or a peptide or mixed peptide corresponding to at least one target antigen, the second composition includes IL-7 but not IL-2, and the third composition includes IL -4 but not IL-2, and the fourth composition includes IL-15 but not IL-2, the first composition is administered during the first period and the second and third compositions are administered during the second period is administered, and the fourth composition is administered to the cell during a third period of time.

In some embodiments, the first time period, the second time period, and the third time period are any of the time periods described herein. In some embodiments, the time between the first period and the second period is the same. In some embodiments, the time between the first period and the second period is different. In some embodiments, the time between the first period, the second period and the third period is the same. In some embodiments, the times between the first period, the second period and the third period are different. In some embodiments, the first period of time is about 1 day and the second period of time is about 1 day. In some embodiments, the first period is about 1 day and the second period is about 2 days. In some embodiments, the first period is about 1 day and the second period is about 3 days. In some embodiments, the first period is about 1 day and the second period is about 4 days. In some embodiments, the first period is about 1 day and the second period is about 5 days. In some embodiments, the first period is about 1 day and the second period is about 6 days. In some embodiments, the first period is about 1 day and the second period is about 7 days. In some embodiments, the first period is about 1 day and the second period is about 8 days. In some embodiments, the first period is about 1 day and the second period is about 9 days. In some embodiments, the first period is about 1 day and the second period is about 10 days.

In some embodiments, the first period of time is from about 0 days to about 2 days, and the second period of time is from about 8 days to about 12 days. In some embodiments, the first period of time is from about 0 days to about 1 day, and the second period of time is from about 7 days to about 13 days. In some embodiments, the first period of time is about 0 days and the second period of time is from about 8 days to about 14 days. In some embodiments, the first period of time is from about 0 hours to about 48 hours, and the second period of time is from about 8 days to about 12 days. In some embodiments, the first period of time is from about 0 hours to about 24 hours, and the second period of time is from about 7 days to about 13 days. In some embodiments, the first period of time is from about 0 minutes to about 30 minutes, and the second period of time is from about 8 days to about 14 days.

In some embodiments, the first period is about 2 days and the second period is about 1 day. In some embodiments, the first period is about 2 days and the second period is about 2 days. In some embodiments, the first period is about 2 days and the second period is about 3 days. In some embodiments, the first period is about 2 days and the second period is about 4 days. In some embodiments, the first period is about 2 days and the second period is about 5 days. In some embodiments, the first period is about 2 days and the second period is about 6 days. In some embodiments, the first period is about 2 days and the second period is about 7 days. In some embodiments, the first period is about 2 days and the second period is about 8 days. In some embodiments, the first period is about 2 days and the second period is about 9 days. In some embodiments, the first period is about 2 days and the second period is about 10 days.

In some embodiments, the first period is about 3 days and the second period is about 1 day. In some embodiments, the first period is about 3 days and the second period is about 2 days. In some embodiments, the first period is about 3 days and the second period is about 3 days. In some embodiments, the first period is about 3 days and the second period is about 4 days. In some embodiments, the first period is about 3 days and the second period is about 5 days. In some embodiments, the first period is about 3 days and the second period is about 6 days. In some embodiments, the first period is about 3 days and the second period is about 7 days. In some embodiments, the first period is about 3 days and the second period is about 8 days. In some embodiments, the first period is about 3 days and the second period is about 9 days. In some embodiments, the first period is about 3 days and the second period is about 10 days.

In some embodiments, the first period is about 4 days and the second period is about 1 day. In some embodiments, the first period is about 4 days and the second period is about 2 days. In some embodiments, the first period is about 4 days and the second period is about 3 days. In some embodiments, the first period is about 4 days and the second period is about 4 days. In some embodiments, the first period is about 4 days and the second period is about 5 days. In some embodiments, the first period is about 4 days and the second period is about 6 days. In some embodiments, the first period is about 4 days and the second period is about 7 days. In some embodiments, the first period is about 4 days and the second period is about 8 days. In some embodiments, the first period is about 4 days and the second period is about 9 days. In some embodiments, the first period is about 4 days and the second period is about 10 days.

In some embodiments, the first period is about 5 days and the second period is about 1 day. In some embodiments, the first period is about 5 days and the second period is about 2 days. In some embodiments, the first period is about 5 days and the second period is about 3 days. In some embodiments, the first period is about 5 days and the second period is about 4 days. In some embodiments, the first period is about 5 days and the second period is about 5 days. In some embodiments, the first period is about 5 days and the second period is about 6 days. In some embodiments, the first period is about 5 days and the second period is about 7 days. In some embodiments, the first period is about 5 days and the second period is about 8 days. In some embodiments, the first period is about 5 days and the second period is about 9 days. In some embodiments, the first period is about 5 days and the second period is about 10 days.

In some embodiments, the first period is about 6 days and the second period is about 1 day. In some embodiments, the first period is about 6 days and the second period is about 2 days. In some embodiments, the first period is about 6 days and the second period is about 3 days. In some embodiments, the first period is about 6 days and the second period is about 4 days. In some embodiments, the first period is about 6 days and the second period is about 5 days. In some embodiments, the first period is about 6 days and the second period is about 6 days. In some embodiments, the first period is about 6 days and the second period is about 7 days. In some embodiments, the first period is about 6 days and the second period is about 8 days. In some embodiments, the first period is about 6 days and the second period is about 9 days. In some embodiments, the first period is about 6 days and the second period is about 10 days.

In some embodiments, the first period is about 7 days and the second period is about 1 day. In some embodiments, the first period is about 7 days and the second period is about 2 days. In some embodiments, the first period is about 7 days and the second period is about 3 days. In some embodiments, the first period is about 7 days and the second period is about 4 days. In some embodiments, the first period is about 7 days and the second period is about 5 days. In some embodiments, the first period is about 7 days and the second period is about 6 days. In some embodiments, the first period is about 7 days and the second period is about 7 days. In some embodiments, the first period is about 7 days and the second period is about 8 days. In some embodiments, the first period is about 7 days and the second period is about 9 days. In some embodiments, the first period is about 7 days and the second period is about 10 days.

In some embodiments, the first period is about 8 days and the second period is about 1 day. In some embodiments, the first period is about 8 days and the second period is about 2 days. In some embodiments, the first period is about 8 days and the second period is about 3 days. In some embodiments, the first period is about 8 days and the second period is about 4 days. In some embodiments, the first period is about 8 days and the second period is about 5 days. In some embodiments, the first period is about 8 days and the second period is about 6 days. In some embodiments, the first period is about 8 days and the second period is about 7 days. In some embodiments, the first period is about 8 days and the second period is about 8 days. In some embodiments, the first period is about 8 days and the second period is about 9 days. In some embodiments, the first period is about 8 days and the second period is about 10 days.

In some embodiments, the first composition is added to the cells about 0 to 2 days after initiating cell culture. In some embodiments, cells are cultured in the first composition for 1 day. In some embodiments, cells are cultured in the first composition for 2 days. In some embodiments, cells are cultured in the first composition for 3 days. In some embodiments, cells are cultured in the first composition for 4 days. In some embodiments, cells are cultured in the first composition for 5 days. In some embodiments, cells are cultured in the first composition for 6 days. In some embodiments, cells are cultured in the first composition for 7 days. In some embodiments, cells are cultured in the first composition for 8 days. In some embodiments, cells are cultured in the first composition for 9 days.

In some embodiments, the first composition is added to the cells about 0 to 2 days after initiating cell culture. In some embodiments, the first composition is added to the cells about 0 to 2 days after initiating cell culture, and the second composition is added 2 days after initiating cell culture. In some embodiments, the first composition is added to the cells approximately 0 to 2 days after initiating cell culture, and the second composition is added 3 days after initiating cell culture. In some embodiments, the first composition is added to the cells about 0 to 2 days after initiating cell culture, and the second composition is added 4 days after initiating cell culture. In some embodiments, the first composition is added to the cells approximately 0 to 2 days after initiating cell culture, and the second composition is added 5 days after initiating cell culture. In some embodiments, the first composition is added to the cells about 0 to 2 days after initiating cell culture, and the second composition is added 6 days after initiating cell culture. In some embodiments, the first composition is added to the cells approximately 0 to 2 days after initiating cell culture, and the second composition is added 7 days after initiating cell culture. In some embodiments, the first composition is added to the cells approximately 0 to 2 days after initiating cell culture, and the second composition is added 8 days after initiating cell culture. In some embodiments, the first composition is added to the cells approximately 0 to 2 days after initiating cell culture, and the second composition is added 9 days after initiating cell culture.

In some embodiments, the first and second compositions are added to the cells about 0 to 2 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the third composition is added 2 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the third composition is added 3 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the third composition is added 4 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the third composition is added 5 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the third composition is added 6 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the third composition is added 7 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells about 0 to 2 days after initiating cell culture, and the third composition is added 8 days after initiating cell culture. In some embodiments, the first and second compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the third composition is added 9 days after initiating cell culture.

In some embodiments, the first, second, and third compositions are added to the cells about 0 to 2 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells about 0 to 2 days after initiating cell culture, and the fourth composition is added 2 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the fourth composition is added 3 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells about 0 to 2 days after initiating cell culture, and the fourth composition is added 4 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the fourth composition is added 5 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the fourth composition is added 6 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the fourth composition is added 7 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the fourth composition is added 8 days after initiating cell culture. In some embodiments, the first, second, and third compositions are added to the cells approximately 0 to 2 days after initiating cell culture, and the fourth composition is added 9 days after initiating cell culture. Donor bank and selection

In some aspects, the invention provides methods for producing cell therapy products. In some aspects, the invention provides methods for generating antigen-specific T cell lines. In some aspects, the invention provides methods for generating virus-specific T cell lines. In some aspects, the present invention provides methods for generating universal antigen-specific T cell products. In some aspects, the invention includes methods for generating a plurality of cell lines comprising cell therapy products. In some embodiments, the cell therapy product includes an antigen-specific T cell line. In some embodiments, the cell therapy product comprises a virus-specific T cell product. In some embodiments, the cell therapy product comprises a universal antigen-specific T cell product. In some embodiments, the cell therapy product includes an antigen-specific T cell strain, a virus-specific T cell product, a universal antigen-specific T cell product, or a combination thereof.

In some embodiments, cell lines of the invention are generated from a donor. In some embodiments, the cell therapy products of the invention are produced from a donor. In some embodiments, cell lines of the invention are generated from more than one donor. In some embodiments, cell therapy products of the invention are produced from more than one donor. In some embodiments, multiple cell lines of the invention are generated from a single donor. In some embodiments, multiple cell therapy products of the invention are produced from a single donor. In some embodiments, a plurality of cell lines of the invention are generated from more than one donor. In some embodiments, a plurality of cell therapy products of the invention are produced from more than one donor.

In some aspects, the invention includes methods for generating donor minibanks comprising cell therapy products, such as the antigen-specific T cell lines and universal antigen-specific T cell products provided herein. In some embodiments, the invention includes methods for identifying one or more suitable donors from at least one donor pool having various HLA (human leukocyte antigen) allele types that are compatible with a majority of potential patients. As used herein, the term "donor" refers to the organism from which the biological sample is generated. In some embodiments, the donor is human. In some embodiments, the donor is healthy. In some embodiments, the potential patient has undergone allogeneic hematopoietic stem cell transplantation (HSCT). In some embodiments, potential patients have suppressed immunity or are immunocompromised. In various embodiments, methods of the invention involve restoring T cell immunity in immunocompromised patients.

The term "donor minibank" as used herein refers to a cell bank that contains a plurality of cell therapy products (e.g., antigen-specific T cell lines) that are collectively derived from a diverse pool of donors, such that the donor minibank contains At least one cell therapy product (eg, an antigen-specific T cell strain) that is a sufficient match for a defined percentage of patients in the target patient population. For example, in some embodiments, the donor minibanks described herein include at least one donor that is a good match for at least 95% of a target patient population, such as, for example, allogeneic hematopoietic stem cell transplant recipients or immunocompromised individuals. Cell therapy products (e.g., antigen-specific T cell lines). The term "donor bank" as used herein refers to a plurality of donor minibanks. In various embodiments, it may be beneficial to create several non-duplicate mini-libraries for inclusion in a "donor library", thereby ensuring that two or more well-matched cell therapy products are available for each potential patient. Cell banks can be stored frozen. Cryopreservation methods are known in the art and may include, for example, storage of cell therapy products (eg, antigen-specific T cell lines) at -70°C in a controlled access area, such as in vapor phase liquid nitrogen. Individual aliquots of cell therapy products can be prepared and stored in multiple containers (eg, vials) in calibrated liquid nitrogen Dewars. Containers (eg vials) can be marked with a unique identification number that enables retrieval.

Embodiments of the invention include donor minibanks containing a plurality of cell therapy products (e.g., antigen-specific T cell lines) and donor libraries composed of a plurality of such donor minibanks, as well as the manufacture and use of such donor minibanks. Methods of using microbanks, donor libraries, and cell therapy products (eg, antigen-specific T cell lines) contained therein (alone or in combination as universal cell therapy products) for adoptive immunotherapy to treat diseases or conditions.

In some embodiments, the present invention includes methods and computer-implemented algorithms for identifying and selecting appropriately diverse sets of donors (with respect to their HLA typing) for use in constructing donors. The cell therapy products (e.g., antigen-specific T cell lines) contained in the donor mini-library are designed to ensure that each donor mini-library contains at least one cell therapy product (e.g., antigen-specific T cell lines) that is a sufficient match for the desired percentage of the target population. cell lines). As discussed further herein, in some embodiments, the percentage of the target population that will adequately match at least one cell therapy product (e.g., an antigen-specific T cell line) in a given minilibrary can be predetermined when constructing the minilibrary. and parameters based on the HLA type of the target population and the number of cell therapy products included in the donor mini-library. In some embodiments, each donor minilibrary contains at least one donor that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of the target population. , cell therapy products (such as antigen-specific T cell lines) that are fully matched to at least 99% and at least 99.9% of potential patients (including all ranges and sub-ranges therein). Accordingly, in some embodiments, the methods disclosed herein allow the construction of such donor mini-libraries with appropriate donor diversity (with respect to their HLA typing) to ensure at least one cell therapy in the donor mini-library. The product (eg, antigen-specific T cell line) will have at least 2 HLA allele matches to 95% or higher of a given target population.

Donors used to construct the donor minibanks disclosed herein may be pre-screened for seropositivity and/or the donors may be healthy. In some embodiments, the donor is a healthy donor. In some embodiments, donors used to prepare such cell therapy products (e.g., antigen-specific T cell lines) contained in such donor minibanks are carefully selected using the donor selection methods disclosed herein to Ensure sufficient HLA diversity among donors such that at least 95% of the target patient population has two or more HLA alleles matched to at least one cell therapy product (e.g., antigen-specific T cell line) in the mini-library. In some embodiments, the present invention is based at least in part on the unexpected discovery that partially HLA-matched cell therapies, such as antigen-specific T cell lines (eg, VST cell lines), are safe and effective in third parties.

In some embodiments, the invention includes donor mini-libraries (and donor libraries comprising a plurality of such donor mini-libraries) that include donors derived from individuals identified via the donor selection methods disclosed herein. such cell therapy products suitable for blood samples collected from third-party blood donors; and the manufacture, administration and use of such cell therapy products (including, for example, antigen-specific T cell line products, such as VST products) to treat or A method of preventing disease or illness. Accordingly, in some embodiments, such donor mini-libraries include a plurality of cell therapy products derived from samples (e.g., mononuclear spheroids, such as PBMCs) obtained from donors carefully selected using the donor selection methods disclosed herein. (e.g., antigen-specific T cell lines), and their corresponding cell therapy products contain sufficient HLA diversity between each other such that at least 95% of the target patient population is associated with at least one cell therapy product (e.g., antigen-specific T cell lines) with two or more HLA allele genes matching. Methods for identifying suitable donors and for constructing donor libraries and donor mini-libraries are discussed in PCT/US2020/044080, the entire contents of which are incorporated herein by reference.

In some embodiments, methods of the invention for generating antigen-specific T cell lines from donor minibanks comprise culturing MNCs in the presence of one or more antigens or functional variants thereof as provided herein and one or more interleukins. . Accordingly, in some embodiments, the invention provides donor minibanks and universal T cell products produced by methods for producing antigen-specific T cells provided herein. Accordingly, in some embodiments, the present invention provides donor minibanks and universal T cell products.

In some embodiments, methods of the invention for constructing donor minibanks of antigen-specific T cell lines comprise culturing the cells for a duration sufficient to obtain a population of antigen-specific T cells. In some embodiments, culturing MNC to generate antigen-specific T cells comprises: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) for a second period IL-4 and IL-7 were added to the cells during the period. In some embodiments, culturing MNC to generate antigen-specific T cells comprises: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) for a second period IL-4 and IL-7 are added to the cells during the third period; and iii) IL-15 is added to the cells during the third period. In some embodiments, culturing MNC to generate antigen-specific T cells includes: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) culturing the cells for a first period of time; Culture for the second period in the presence of IL-15. In some embodiments, culturing MNC to generate antigen-specific T cells comprises: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen for a first period of time; and ii) for a second period IL-4, IL-7 and IL-15 were added to the cells during the session. In some embodiments, culturing MNC to generate antigen-specific T cells comprises: i) culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen, IL-4 and IL-7; period; and ii) adding IL-15 to the cells during the second period. In some embodiments, culturing MNC to generate antigen-specific T cells includes culturing the cells with at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen, IL-4, IL-7, and IL-15.

In some embodiments, the first period of time is less than about 24 hours. In some embodiments, the first period exceeds about 24 hours. In some embodiments, the first period of time is between about 24 and about 36 hours. In some embodiments, the first period of time is between about 24 and about 48 hours. In some embodiments, the first period of time is between about 24 and about 60 hours. In some embodiments, the first period is between about 24 and about 72 hours. In some embodiments, the first period of time is from about 24 hours to about 36 hours. In some embodiments, the first period of time is from about 24 hours to about 48 hours. In some embodiments, the first period of time is from about 24 hours to about 60 hours. In some embodiments, the first period of time is from about 24 hours to about 72 hours. In some embodiments, the first period of time is less than about 144 hours. In some embodiments, the first period exceeds about 144 hours. In some embodiments, the first period is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In some embodiments, the second period of time is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In some embodiments, the second period of time is about 1 to about 2 days. In some embodiments, the second period of time is from about 1 to about 5 days. In some embodiments, the second period of time is from about 1 to about 10 days. In some embodiments, the second period exceeds about 10 days.

In some embodiments, contact of cells with IL-15 results in accelerated expansion of cells in culture. In some embodiments, contacting cells with IL-4 and IL-7 prior to subsequent contact with IL-15 generates an expanded population of antigen-specific T cells comprising an antigen obtainable by contacting the cells with Expanded populations of specific T cells compared to enriched antigen-specific T cells of a broader lineage: (i) IL-4 and IL-7, without IL-15; (ii) IL-7 and IL-15 , without IL-4; or (iii) simultaneously IL-4, IL-7 and IL-15.

In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded within 9 to 18 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded within 8 to 20 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded within 6 to 22 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded within 4 to 24 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded by about 9 to about 18 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded by about 8 to about 20 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded by about 6 to about 22 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded by about 4 to about 24 days in culture such that they are ready for administration to the patient. In some embodiments, the antigen-specific T cells of the donor minibank are sufficiently expanded after about 24 days in culture such that they are ready for administration to the patient. In some embodiments, the donor minibank's antigen-specific T cells are sufficiently expanded after 24 days in culture such that they are ready for administration to the patient.

In some embodiments, minibanks of antigen-specific T cells can be evaluated by testing the antigen-specific cytotoxicity of the antigen-specific T cells. In some embodiments, a mini-library of antigen-specific T cell lines can be generated by constructing a first donor mini-library of antigen-specific T cell lines as disclosed herein. In some embodiments, a mini-repertoire of antigen-specific T cell lines can be derived from a plurality of donors selected via methods as described herein. In some embodiments, a repertoire of antigen-specific T cell lines may comprise a plurality of mini-repertoires derived from a plurality of donors selected via methods as described herein. Exemplary ex vivo expansion of T cells

In some aspects, the invention provides an in vitro method for expanding T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; adding one or more compositions to the cell culture, wherein the one or more compositions comprise: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more substances selected from IL- 4. Exogenous interleukins such as IL-7 and IL-15; and culturing the cells for a period of time sufficient to expand T cells; thereby amplifying T cells.

In some aspects, the invention provides an in vitro method for expanding T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; adding one or more compositions to the cell culture, wherein the one or more compositions comprise: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; or ii) one or more substances selected from the group consisting of IL- 4. Exogenous interleukins such as IL-7 and IL-15; and culturing the cells for a period of time sufficient to expand T cells; thereby amplifying T cells.

In some aspects, the invention provides an in vitro method for generating an expanded T cell population, comprising: providing a starting population of mononuclear spheroids in a cell culture; adding to the cell culture one or more A composition, wherein the one or more compositions comprise: i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and ii) one or more options exogenous interleukins from IL-4, IL-7, and IL-15; and culturing the cells for a period of time sufficient to expand T cells; thereby generating a population of expanded T cells.

In some aspects, the invention provides an in vitro method for generating an expanded T cell population, comprising: providing a starting population of mononuclear spheroids in a cell culture; adding to the cell culture one or more A composition, wherein the one or more compositions comprise: i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; or ii) one or more options exogenous interleukins from IL-4, IL-7, and IL-15; and culturing the cells for a period of time sufficient to expand T cells; thereby generating a population of expanded T cells.

In some embodiments, the expanded T cells comprise T cells specific for at least one target antigen. In some embodiments, the expanded T cells are enriched with T cells specific for at least one target antigen.

In some embodiments, the expanded T cell population includes T cells specific for at least one target antigen. In some embodiments, the expanded T cell population is enriched with T cells specific for at least one target antigen.

In some embodiments, a composition comprising: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen is added to the cell culture; and (ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15.

In some embodiments, a first composition comprising: (i) at least one target antigen or a portion of a target antigen or a peptide corresponding to at least one target antigen or a portion of a target antigen is added to the cell culture; a peptide mixture; and a second composition comprising: (ii) one or more exogenous interleukins selected from the group consisting of IL-4, IL-7 and IL-15.

In some embodiments, the first composition and the second composition are added to the cell culture simultaneously.

In some embodiments, the second composition is added to the cell culture after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 1 minute to about 30 minutes after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 30 minutes to about 60 minutes after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 1 hour to about 2 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 2 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 6 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 12 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 18 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 24 hours after the first composition is added to the cell culture.

In some embodiments, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; two compositions, comprising IL-4 and IL-7; and a third composition, comprising IL-15.

In some embodiments, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; Two compositions, comprising IL-4; a third composition, comprising IL-7; and a fourth composition, comprising IL-15.

In some embodiments, to the cell culture is simultaneously added: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and a second composition comprising IL-4 and IL-7.

In some embodiments, to the cell culture is added in parallel: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; and a second composition comprising IL-4 and IL-7.

In some embodiments, the first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen is added to the cell culture. A second composition comprising IL-4 and IL-7 is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 1 minute to about 30 minutes after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 30 minutes to about 60 minutes after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 1 hour to about 2 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 2 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 6 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 12 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 18 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 24 hours after the first composition is added to the cell culture.

In some embodiments, the second composition comprising IL-4 and IL-7 and the third composition comprising IL-15 are added simultaneously. In some embodiments, the second composition comprising IL-4 and IL-7 and the third composition comprising IL-15 are added in parallel.

In some embodiments, the third composition comprising IL-15 is added to the cell culture after the second composition comprising IL-4 and IL-7 is added to the cell culture. In some embodiments, the third composition is added to the cell culture approximately 12 hours after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture approximately 24 hours after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture approximately 48 hours after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture approximately 72 hours after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture about 1 day after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture about 2 days after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture about 3 days after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture about 4 days after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture about 5 days after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture about 6 days after the second composition is added to the cell culture. In some embodiments, the third composition is added to the cell culture about 7 days after the second composition is added to the cell culture.

In some embodiments, to the cell culture is added: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen, IL -4 and IL-7; and a second composition comprising IL-15. In some embodiments, the first composition and the second composition are added simultaneously. In some embodiments, the second composition is added to the cell culture after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 0 hours to about 1 hour after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 0 hours to about 6 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 0 hours to about 12 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 6 hours to about 12 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 6 hours to about 18 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 12 hours to about 18 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 12 hours to about 24 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 18 hours to about 24 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 18 hours to about 36 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 24 hours to about 36 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 24 hours to about 48 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 36 hours to about 48 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 24 hours to about 72 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 48 hours to about 72 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 1 hour after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 2 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 6 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 12 hours after the first composition is added to the cell culture.

In some embodiments, the second composition is added to the cell culture approximately 18 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 24 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 36 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 48 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture approximately 72 hours after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 1 day after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 2 days after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 3 days after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 4 days after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 5 days after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 6 days after the first composition is added to the cell culture. In some embodiments, the second composition is added to the cell culture about 7 days after the first composition is added to the cell culture.

In some embodiments, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; two compositions, comprising IL-4; and a third composition, comprising IL-7 and IL-15.

In some embodiments, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; two compositions, comprising IL-7; and a third composition, comprising IL-4 and IL-15.

In some embodiments, adding to the cell culture: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen; Two compositions, comprising IL-4; a third composition, comprising IL-7; and a fourth composition, comprising IL-15.

In some embodiments, the first, second, third and fourth compositions are added in parallel. In some embodiments, the first, second, third and fourth compositions are added sequentially. In some embodiments, the first, second and third compositions are added in parallel and the fourth composition is added at a later time. In some embodiments, the fourth composition is added after 0 to 2 days. In some embodiments, the fourth composition is added 1 to 2 days later. In some embodiments, the fourth composition is added after 0 to 6 days. In some embodiments, the fourth composition is added after 1 to 6 days.

In some embodiments, to the cell culture is added: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and IL -4; a second composition comprising IL-7; and a third composition comprising IL-15.

In some embodiments, to the cell culture is added: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and IL -7; a second composition comprising IL-4; and a third composition comprising IL-15.

In some embodiments, to the cell culture is added: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and IL -4; and a second composition comprising IL-7 and IL-15.

In some embodiments, to the cell culture is added: a first composition comprising at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a portion of a target antigen and IL -7; and a second composition comprising IL-4 and IL-15.

In some embodiments, the cells are cultured for about 18 days or less, optionally about 14 days. In some embodiments, the cells are cultured for about 6 days to about 14 days. In some embodiments, the cells are cultured for about 6 days to about 10 days. In some embodiments, the cells are cultured for about 8 days to about 10 days. In some embodiments, the cells are cultured for about 8 days to about 14 days. In some embodiments, the cells are cultured for about 8 days to about 18 days. In some embodiments, the cells are cultured for about 1 week to about 2 weeks. In some embodiments, the cells are cultured for about 1 week to about 3 weeks. In some embodiments, the cells are cultured for about 1 week to about 4 weeks. In some embodiments, cells are cultured for about 14 days or less. In some embodiments, cells are cultured for about 10 days or less. In some embodiments, cells are cultured for about 8 days or less. In some embodiments, cells are cultured for more than about 4 weeks. In some embodiments, cells are cultured for an amount of time sufficient to produce cells that have completed logarithmic phase growth. In some embodiments, the cells are cultured for an amount of time sufficient to produce cells that have completed log phase growth, wherein the cells are cultured for about 8 days to about 18 days.

In some embodiments, the cell culture period includes a startup period and an expansion period. In some embodiments, priming involves adding antigen (eg, mixed peptides). In some embodiments, priming includes adding antigen (eg, mixed peptides), IL-4, and IL-7 to the cell culture. In some embodiments, priming includes adding antigen (eg, mixed peptides), IL-4, IL-7, and IL-15 to the cell culture. In some embodiments, activation begins on day 0.

In some embodiments, the amplification period includes a period after the initiation period. In some embodiments, the expansion period includes adding IL-15 to the cell culture. In some embodiments, the expansion period includes adding IL-15 to the cell culture to expand the cells.

In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 4 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 5 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 6 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 7 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 8 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 9 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 10 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 11 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 12 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 13 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 14 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 15 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 16 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 17 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by an expansion period of about 18 days.

In some embodiments, the cell culture time includes an initiation period of about day 0 of culture, followed by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17 or 18 days of expansion period. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 1 of culture, followed by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Expansion period of 12, 13, 14, 15, 16, 17 or 18 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 2 of culture, followed by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Expansion period of 12, 13, 14, 15, 16, 17 or 18 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 3 of culture, followed by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Expansion period of 12, 13, 14, 15, 16, 17 or 18 days. In some embodiments, the cell culture time includes an initiation period from about day 0 to about day 4 of culture, followed by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Expansion period of 12, 13, 14, 15, 16, 17 or 18 days.

In some embodiments, the cell culture period begins when the cells first come into contact with the antigen or fragment thereof (eg, mixed peptide). In some embodiments, the cells are isolated from the donor before the cell culture period begins. In some embodiments, cells are isolated from the donor and cryopreserved prior to the start of the cell culture period.

In some embodiments, cells are cultured in the presence of T cell expansion medium. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4 and IL-7. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4, IL-7, and IL-15. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4, IL-7, and mixed peptides. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4, IL-7, IL-15, and mixed peptides. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL of IL-4 and 20 ng/mL of IL-7. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL of IL-4, 20 ng/mL of IL-7 and 5 ng/mL. Culture was carried out in Il-15 of mL. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL of IL-4, 20 ng/mL of IL-7, 5 ng/ mL of Il-15 and 2 ng/peptide/mL of mixed peptides were cultured. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL IL-4, 20 ng/mL IL-7 and 2 nanograms /peptide/ml mixed peptides were cultured. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4 and IL-7; and IL-15 is subsequently added to the cells. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4, IL-7, and mixed peptides; and IL-15 is then added to in cells. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4 and IL-7; and IL-15 is added to the cells in parallel. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4, IL-7, and mixed peptides; and IL-15 is added in parallel to in cells. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4 and IL-7; and IL-15 is then cultured at 5 ng/mL Add to cells. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4, IL-7, and mixed peptides; and IL-15 is subsequently treated with 5 ng/mL was added to the cells. In some embodiments, cells are cultured in T cell medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4 and IL-7; and IL-15 in parallel at 5 ng/mL Add to cells. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX, and 10% human AB serum, IL-4, IL-7, and mixed peptides; and IL-15 is 5 ng /mL was added to cells in parallel. In some embodiments, the cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL of IL-4 and 20 ng/mL of IL-7, And IL-15 was then added to the cells at 5 ng/mL. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL IL-4, 20 ng/mL IL-7 and 2 nanograms /peptide/ml of mixed peptides, and IL-15 was subsequently added to the cells at 5 ng/ml. In some embodiments, the cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL of IL-4 and 20 ng/mL of IL-7, And IL-15 was added to the cells in parallel at 5 ng/mL. In some embodiments, cells are cultured in T cell culture medium containing 90% TexMACS GMP medium, 2 mM GlutaMAX and 10% human AB serum, 800 U/mL IL-4, 20 ng/mL IL-7 and 2 nanograms /peptide/ml of mixed peptides, and IL-15 was added to the cells in parallel at 5 ng/ml.

In some embodiments, seeding densities for methods for expanding T cells as provided herein include seeding densities in the range of 2 × 10 ⁶ cells per square centimeter to 4 × 10 ⁶ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 1 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 1.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 2 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 2.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 3 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 3.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 4 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes 5.5 × ¹⁰ cells per square centimeter. In some embodiments, the seeding density of methods for expanding T cells as provided herein includes ⁶ × 10 cells per square centimeter.

In some embodiments, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in a cell culture; (b) adding to the cell culture a or a plurality of compositions, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and (ii) one or more substances selected from the group consisting of IL- 4. Exogenous interleukins such as IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to expand VST.

In some embodiments, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in a cell culture; (b) adding to the cell culture a or a plurality of compositions, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, or (ii) one or more compositions selected from IL- 4. Exogenous interleukins such as IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to expand VST.

In some embodiments, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) performing a priming step, wherein the priming The steps comprise adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and ( ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to expand VST.

In some embodiments, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) performing a priming step, wherein the priming The step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, (ii) ) IL-4 and IL-7, and (iii) IL-15; wherein the cells are cultured for a period of time sufficient to expand VST during the expansion period.

In some embodiments, the invention provides an in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) performing a priming step, wherein the priming The steps comprise adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, or ( ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to expand VST.

In some embodiments, the duration of the initiating step is from about 1 to about 5 minutes. In some embodiments, the duration of the initiating step is from about 1 to about 30 minutes. In some embodiments, the duration of the activation step is from about 1 to about 45 minutes. In some embodiments, the duration of the activation step is from about 5 to about 10 minutes. In some embodiments, the duration of the activation step is from about 10 to about 30 minutes. In some embodiments, the duration of the activation step is from about 20 to about 30 minutes. In some embodiments, the duration of the activation step is from about 30 to about 40 minutes. In some embodiments, the duration of the initiating step is from about 1 hour to about 2 hours. In some embodiments, the duration of the initiating step is from about 2 hours to about 5 hours. In some embodiments, the duration of the initiating step is from about 0 to about 4 days. In some embodiments, the duration of the initiating step is from about 0 to about 3 days. In some embodiments, the duration of the initiating step is from about 0 to about 2 days. In some embodiments, the duration of the initiating step is from about 0 to about 1 day. In some embodiments, the duration of the activation step is from about 1 to about 2 days. In some embodiments, the activation step lasts for more than about 24 hours. In some embodiments, the duration of the activation step is less than 24 hours. In some embodiments, the duration of the activation step is from about 0 to about 12 hours. In some embodiments, the duration of the initiating step is from about 0 to about 24 hours. In some embodiments, the duration of the initiating step is from about 0 to about 48 hours. In some embodiments, the duration of the activation step is from about 24 to about 48 hours. In some embodiments, the duration of the priming step is from about 24 to about 72 hours. In some embodiments, the duration of the activation step is less than about 144 hours. In some embodiments, the duration of the priming step is sufficient to produce at least a 2-fold greater amplification of the number of VSTs.

In some embodiments, adding IL-15 to the cell culture (step c) occurs after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 12 hours after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 24 hours after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 48 hours after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 72 hours after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs about 1 day after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 2 days after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 3 days after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 4 days after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 5 days after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 6 days after the priming step. In some embodiments, adding IL-15 to the cell culture (step c) occurs approximately 7 days after the priming step.

In some embodiments, the addition of IL-15 to the cell culture occurs after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 0 hours to about 12 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 0 hours to about 24 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 0 hours to about 48 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 12 hours to about 24 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 12 hours to about 48 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 24 hours to about 48 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 12 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 24 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 48 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 72 hours after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 1 day after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 2 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 3 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 4 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 5 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 6 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs approximately 7 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 0 days to about 2 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 0 days to about 4 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 0 days to about 7 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 1 day to about 2 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 1 day to about 4 days after the priming step. In some embodiments, the addition of IL-15 to the cell culture occurs from about 1 day to about 7 days after the priming step.

In some embodiments, the period of time sufficient to expand the VST is about 18 days or less, optionally about 14 days. In some embodiments, the period of time sufficient to expand VST is from about 8 days to about 10 days. In some embodiments, the period of time sufficient to expand VST is from about 8 days to about 14 days. In some embodiments, the period of time sufficient to expand VST is from about 8 days to about 18 days. In some embodiments, the period of time sufficient to expand VST is about 1 week to about 2 weeks. In some embodiments, the period of time sufficient to expand VST is from about 1 week to about 3 weeks. In some embodiments, the period of time sufficient to expand VST is from about 1 week to about 4 weeks. In some embodiments, the period of time sufficient to expand VST is about 14 days or less. In some embodiments, the period of time in culture for amplifying VST is about 18 days or less. In some embodiments, the period of time in culture for amplifying VST is about 14 days or less. In some embodiments, the period of time in culture for amplifying VST is about 13 days or less. In some embodiments, the period of time in culture for amplifying VST is about 12 days or less. In some embodiments, the period of time in culture for amplifying VST is about 11 days or less. In some embodiments, the period of time in culture for amplifying VST is about 10 days or less. In some embodiments, the period of time sufficient to expand the VST is about 9 days or less. In some embodiments, the period of time in culture for amplifying VST is about 8 days or less. In some embodiments, the period of time in culture for amplifying VST is about 7 days or less. In some embodiments, the period of time in culture for amplifying VST is about 6 days or less. In some embodiments, the period of time in culture for amplifying VST is about 5 days or less. In some embodiments, the period of time in culture for amplifying VST is about 4 days or less. In some embodiments, the period of time in culture for amplifying VST is about 3 days or less. In some embodiments, the period of time used to expand VST in culture exceeds about 3 weeks. In some embodiments, the period of time in culture for amplifying VST exceeds about 4 weeks. In some embodiments, the period of time in culture for amplification of VST is the amount of time in culture to produce cells that have completed log phase growth.

In some embodiments, in vitro methods for amplifying VST result in expansion of VST but not NK cells.

In some embodiments, the present invention provides a two-step method for selective expansion of VST in a heterogeneous cell population, comprising: a first step of culturing VST in the presence of a stimulating antigen and IL-4 and IL-7; and the second step of culturing VST in the presence of IL-15.

In some embodiments, the present invention provides a composition comprising VST expanded in a two-step process, the two-step process comprising: a first step of culturing VST in the presence of IL-4 and IL-7; and The second step of culturing VST in the presence of -15.

In some aspects, the invention provides a method for expanding T cells in vitro, comprising the step of contacting a starting population of mononuclear spheres with one or more compositions, the one or more compositions comprising: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; ii) one or more interleukins comprising at least IL-4 and IL-7, wherein the interleukins do not comprise IL-15; and iii) IL-15, wherein contact with the composition in iii) is optionally followed by contact with the composition in i) and ii), thereby producing an amplified T cell population. In some embodiments, contacting with the composition of iii) occurs less than 6 days after initiating the culture with the mononuclear spheres and the composition of i) and ii). In some embodiments, contacting with the composition of iii) occurs less than 3 days after initiating the culture with the mononuclear spheres and the composition of i) and ii). In some embodiments, contacting the composition of iii) occurs simultaneously with contacting the mononuclear spheres with the composition of i) and ii). In some embodiments, contacting with the composition of iii) occurs between 1 and 3 days after initiating the culture with the mononuclear spheres and the composition of i) and ii).

In some embodiments, the monocytes are peripheral blood monocytes (PBMC).

In some aspects, the invention provides a method for expanding T cells ex vivo, comprising contacting a starting population of mononuclear spheres with one or more compositions, the one or more compositions comprising: i) at least a target antigen or a part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a part of a target antigen; and ii) one or more interleukins comprising IL-4, IL-7 and IL-15 ; thereby generating an expanded T cell population. In some embodiments, the mononuclear spheres are PBMCs.

In some embodiments, culture of mononuclear cells (MNCs) can be performed in a container containing a gas-permeable culture surface. In some embodiments, the container can be an infusion bag with a breathable portion. In some embodiments, the container may be a rigid container. In some embodiments, the vessel may be a GRex bioreactor. In some embodiments, culturing PBMC to construct the first donor minibank of the antigen-specific T cell line of the present invention can be performed in the presence of one or more interleukins. In some embodiments, culturing PBMC to construct multiple antigen-specific T cell lines of the present invention can be performed in the presence of one or more interleukins. In one embodiment, the one or more interleukins may include IL-4. In one embodiment, the one or more interleukins may include IL-7. In one embodiment, the one or more interleukins may include IL-4 and IL-7. In one embodiment, the one or more interleukins may include IL-4 and IL-7, but not IL-2. In one embodiment, the one or more interleukins may include IL-4 and IL-7, but not IL-15. In some embodiments, the one or more interleukins include IL-4. In some embodiments, the one or more interleukins include IL-7. In some embodiments, the one or more interleukins include IL-4 and IL-7. In some embodiments, the one or more interleukins include IL-4 and IL-7, but not IL-2. In some embodiments, the one or more interleukins include IL-4 and IL-7, but not IL-15. In some embodiments where the one or more interleukins include IL-4 and IL-7 but does not include IL-15, IL-15 is then added to the culture at a later time during the culture. In some embodiments, the one or more interleukins include IL-4, IL-7, and IL-15. In some embodiments, the method does not include adding exogenous IL-2. In some embodiments, the method does not include adding IL-2. In some embodiments, the one or more interleukins include IL-4. In some embodiments, the one or more interleukins include IL-7. In some embodiments, the one or more interleukins include IL-4 and IL-7. In some embodiments, the one or more interleukins include IL-4 and IL-7, but not IL-2. In some embodiments, the one or more interleukins include IL-4 and IL-7, but not IL-15. In some embodiments where the one or more interleukins include IL-4 and IL-7 but does not include IL-15, IL-15 is then added to the culture at a later time during the culture. In some embodiments, the one or more interleukins include IL-4, IL-7, and IL-15. In some embodiments, the one or more interleukins are IL-4. In some embodiments, the one or more interleukins are IL-7. In some embodiments, the one or more interleukins are IL-4 and IL-7. In some embodiments, the one or more interleukins are IL-4 and IL-7, but not IL-2. In some embodiments, the one or more interleukins are IL-4 and IL-7, but not IL-15. In some embodiments where the one or more interleukins are IL-4 and IL-7 but not IL-15, IL-15 is then added to the culture at a later time during the culture. In some embodiments, the one or more interleukins are IL-4, IL-7, and IL-15.

In some embodiments, the invention provides an in vitro method for generating an expanded population of T cells, comprising: providing a starting population of mononuclear spheroids in a cell culture; adding to the culture: i) a first composition comprising at least one target antigen or a part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a part of a target antigen; ii) a second composition comprising IL-4 and IL-7; and iii) a third composition comprising IL-15; and a period of time during which the cells are cultured to expand the T cells; thereby producing a population of expanded T cells.

In some embodiments, the invention provides an in vitro method for generating an expanded population of T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; adding to the culture: i) a first composition comprising at least one target antigen or a part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a part of a target antigen, IL-4 and IL-7; and iii) a second composition comprising IL-15; and a period of time during which the cells are cultured to expand the T cells; thereby producing a population of expanded T cells.

In some embodiments, the invention provides an in vitro method for generating an expanded population of T cells, comprising: providing a starting population of mononuclear spheroids in a cell culture; adding to the culture: i) a first composition comprising at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) a second composition comprising IL-4 and IL-7, wherein the cells are cultured with the first and second compositions for about 0 days to about 2 days before adding iii) a third composition comprising IL-15; A period during which cells are cultured to expand T cells; thereby producing a population of expanded T cells. In some embodiments, the cells are cultured with the first, second, and third compositions for about 8 to about 14 days.

In some embodiments, the invention provides an in vitro method for generating an expanded population of T cells, comprising: providing a starting population of mononuclear spheres in a cell culture; adding to the culture: i) A composition comprising at least one target antigen or a part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or a part of a target antigen, IL-4 and IL-7, wherein the cells are Cultivate with the first and second compositions for about 0 days to about 2 days ii) compositions containing IL-15; A period during which cells are cultured to expand T cells; thereby producing a population of expanded T cells. In some embodiments, the cells are cultured with the first, second, and third compositions for about 8 to about 14 days.

In some embodiments, the present invention provides an in vitro method for generating an expanded T cell population, comprising: culturing a cell culture with about 2 × 10 ⁶ cells per square centimeter to about 4 × 10 ⁶ cells per square centimeter. providing a starting population of mononuclear spheres in the culture; adding to the culture: i) a first composition comprising at least one target antigen or a part of a target antigen or corresponding to at least one target antigen or a part of a target antigen a peptide or peptide mixture; ii) a second composition comprising IL-4 and IL-7, wherein the cells are cultured with the first and second compositions for about 0 days to about 2 days before adding iii) a third A composition comprising IL-15; and wherein the cells are cultured for a period of time to expand T cells; thereby producing a population of expanded T cells. In some embodiments, the cells are cultured with the first, second, and third compositions for about 8 to about 14 days.

In some embodiments, the present invention provides an in vitro method for generating an expanded T cell population, comprising: culturing a cell culture with about 2 × 10 ⁶ cells per square centimeter to about 4 × 10 ⁶ cells per square centimeter. Provide a starting population of mononuclear spheres in the culture; add to the culture: i) a peptide or peptide mixture comprising at least one target antigen or a part of a target antigen or corresponding to at least one target antigen or a part of a target antigen, IL -4 and IL-7, wherein the cells are cultured with the first and second compositions for about 0 days to about 2 days before adding ii) a composition comprising IL-15; and wherein the cells are cultured with A period of time to expand T cells; thereby generating a population of expanded T cells. In some embodiments, the cells are cultured with the first, second, and third compositions for about 8 to about 14 days. Exemplary in vitro stimulation of amplification

In some embodiments, the present invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in a cell culture; (b) adding to the cell culture One or more compositions are added, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and (ii) one or more optional exogenous interleukins from IL-4, IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to stimulate expansion of VST.

In some embodiments, the present invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in a cell culture; (b) adding to the cell culture Add one or more compositions, wherein the one or more compositions comprise: (i) at least one target antigen or a portion of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, or (ii) one or more options exogenous interleukins from IL-4, IL-7 and IL-15; and (c) culturing the cells for a period of time sufficient to stimulate expansion of VST.

In some embodiments, the present invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in cell culture; (b) performing a priming step, wherein The priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to stimulate expansion of the VST.

In some embodiments, the present invention provides an in vitro method for stimulating expansion of VST, comprising: (a) providing an initial population of mononuclear spheres in cell culture; (b) performing a priming step, wherein The priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, or (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; wherein the cells are cultured for a period of time sufficient to stimulate expansion of VST.

In some embodiments, the period used to stimulate expansion of the VST is about 25 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 20 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 19 days or less. In some embodiments, the period of time sufficient to stimulate expansion of the VST is about 18 days or less, optionally about 14 days. In some embodiments, the period used to stimulate expansion of the VST is about 18 days or less, optionally about 14 days. In some embodiments, the period used to stimulate expansion of the VST is about 18 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 17 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 16 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 15 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 14 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 13 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 12 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 11 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 10 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 9 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 8 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 7 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 6 days or less. In some embodiments, the period used to stimulate expansion of the VST is about 5 days or less. In some embodiments, the period used to stimulate expansion of the VST is from about 8 days to about 10 days. In some embodiments, the period of time used to stimulate expansion of the VST is from about 8 days to about 14 days.

In some embodiments, the period of time sufficient to stimulate expansion of the VST is from about 8 days to about 18 days. In some embodiments, the period of time sufficient to stimulate expansion of the VST is about 1 week to about 2 weeks. In some embodiments, the period of time sufficient to stimulate expansion of the VST is from about 1 week to about 3 weeks. In some embodiments, the period of time sufficient to stimulate expansion of the VST is from about 1 week to about 4 weeks. In some embodiments, the period of time sufficient to stimulate expansion of the VST is about 14 days or less. In some embodiments, the period of time sufficient to stimulate expansion of the VST is about 10 days or less. In some embodiments, the period of time sufficient to stimulate expansion of the VST is about 8 days or less. In some embodiments, the period of time sufficient to stimulate expansion of the VST exceeds about 4 weeks. In some embodiments, the period of time sufficient to stimulate expansion of the VST is an amount of time sufficient to produce cells that have completed log phase growth.

In some embodiments, in vitro methods for stimulating expansion of VST result in expansion of VST but not NK cells. Exemplary in vitro selective amplification

In some aspects, the present invention provides an in vitro method for stimulating the selective expansion of virus-specific T (VST) cells, comprising contacting a starting population of mononuclear spheres in culture with at least one target antigen or Peptides or peptide mixtures corresponding to at least one target antigen are contacted first in the presence of IL-4 and IL-7 and then in the presence of IL-15. In some embodiments, the mononuclear spheres are PBMCs.

In some aspects, the present invention provides an in vitro method for stimulating the selective expansion of virus-specific T (VST) cells, comprising contacting a starting population of mononuclear spheres in culture with at least one target antigen or Peptides or peptide mixtures corresponding to at least one target antigen are contacted in the presence of IL-4, IL-7 and IL-15. In some embodiments, the mononuclear spheres are PBMCs.

In some embodiments, an in vitro method for stimulating the selective expansion of virus-specific T (VST) cells, comprising contacting a starting population of monocytes in culture with at least one target antigen or corresponding to at least one The peptide or peptide mixture of the target antigen is contacted for a first period of time in the presence of IL-4 and IL-7 and for a second period of time in the presence of IL-15.

In some embodiments, the first period of time is less than about 24 hours. In some embodiments, the first period exceeds about 24 hours. In some embodiments, the first period of time is about 24 hours. In some embodiments, the first period is 24 hours. In some embodiments, the first period of time is between about 24 and about 36 hours. In some embodiments, the first period of time is between about 24 and about 48 hours. In some embodiments, the first period of time is between about 24 and about 60 hours. In some embodiments, the first period is between about 24 and about 72 hours. In some embodiments, the first period of time is less than about 144 hours. In some embodiments, the first period exceeds about 144 hours. In some embodiments, the first period is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the first period of time is about 1 day. In some embodiments, the first period is about 2 days. In some embodiments, the first period is about 3 days. In some embodiments, the first period is about 4 days. In some embodiments, the first period is about 5 days. In some embodiments, the first period is about 6 days. In some embodiments, the first period is about 7 days. In some embodiments, the first period is about 8 days. In some embodiments, the first period is about 9 days. In some embodiments, the first period is about 10 days. In some embodiments, the first period is about 11 days. In some embodiments, the first period is about 12 days. In some embodiments, the first period is less than 24 hours. In some embodiments, the first period exceeds 24 hours. In some embodiments, the first period of time is from about 24 hours to about 36 hours. In some embodiments, the first period of time is from about 24 hours to about 48 hours. In some embodiments, the first period of time is from about 24 hours to about 60 hours. In some embodiments, the first period of time is from about 24 hours to about 72 hours. In some embodiments, the first period is less than 144 hours. In some embodiments, the first period exceeds 144 hours. In some embodiments, the first period is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In some embodiments, contact with IL-4 and IL-7 results in selective expansion of VST but not NK cells.

In some embodiments, the second period of time is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In some embodiments, the second period of time is about 1 to about 2 days. In some embodiments, the second period of time is from about 1 to about 5 days. In some embodiments, the second period of time is from about 1 to about 10 days. In some embodiments, the second period exceeds about 10 days.

In some embodiments, contact of cells with IL-15 results in accelerated expansion of cells in culture. In some embodiments, contacting cells with IL-4 and IL-7 prior to subsequent contact with IL-15 produces an expanded population of VSTs that comprise an expanded population of VSTs obtainable by contacting the cells with Population compared to broader spectrum of enriched VST for: (i) IL-4 and IL-7, no IL-15; (ii) IL-7 and IL-15, no IL-4; or (iii) both IL -4, IL-7 and IL-15.

In some embodiments, contact of cells with IL-15 results in accelerated expansion of cells in culture. In some embodiments, contacting cells with IL-4 and IL-7 prior to subsequent contact with IL-15 produces an expanded population of VSTs that comprise an expanded population of VSTs obtainable by contacting the cells with Populations compared to a broader spectrum of enriched VST for: (i) IL-4 and IL-7, without IL-15; or (ii) IL-7 and IL-15, without IL-4.

In some aspects, the invention provides an in vitro method for selectively amplifying virus-specific T (VST) cells, comprising: a) contacting a starting population of mononuclear spheres with one or more compositions; For a period of time, the one or more compositions comprise: (i) at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen; and (ii) IL-4 and IL-7; and b) from (a) ) cells are exposed to IL-15 for the second period.

In some embodiments, the first period does not induce higher levels of NK cell expansion than would be expected if a population of mononuclear spheres were simultaneously exposed to IL-4, IL-7, and IL-15.

In some embodiments, VST targets one or more antigens from latent viruses. As used herein, the term "latent virus" is used to describe viruses that can remain dormant within cells. In some embodiments, latent viruses undergo the lysogenic portion of the viral life cycle. In some embodiments, VST targets one or more antigens from lytic virus. As used herein, the term "lytic virus" is used to describe viruses that do not remain latent within cells. In some embodiments, VST targets one or more latent antigens of the virus, wherein the one or more antigens correlate with the latency of the virus. In some embodiments, VST targets one or more lytic antigens of the virus, wherein the one or more antigens are associated with the lytic cycle of the virus. In some embodiments, VST targets one or more latent antigens and one or more lytic antigens of the virus.

In some aspects, the present invention provides a two-step method for selectively expanding VST in a heterogeneous cell population, comprising: a first step of culturing VST in the presence of a stimulating antigen and IL-4 and IL-7; and the second step of culturing VST in the presence of IL-15. In some embodiments, the present invention provides a two-step method for selective expansion of VST in a heterogeneous cell population, comprising: culturing VST in the presence of mixed peptides and IL-4 and IL-7 as described herein. a first step; and a second step of culturing VST in the presence of IL-15.

In some aspects, the present invention provides a composition comprising VST expanded in a two-step process, the two-step process comprising: a first step of culturing VST in the presence of IL-4 and IL-7; and The second step of culturing VST in the presence of -15. expanded T cell population

In some aspects, the invention provides expanded T cells obtained by the methods of the invention. In some aspects, the invention provides expanded T cells.

In some embodiments, the expanded T cells comprise T cells specific for at least one target antigen. In some embodiments, the expanded T cells are enriched with T cells specific for at least one target antigen.

In some embodiments, the expanded T cells comprise antigen-specific T cells. In some embodiments, the antigen-specific T cells are pathogen-specific T cells (PST). In some embodiments, the antigen-specific T cells are virus-specific T cells (VST). In some embodiments, the antigen-specific T cells are tumor-specific T cells (TST).

In some embodiments, the expanded T cells comprise VSTs that recognize at least one antigen from hepatitis B virus (HBV). In some embodiments, the expanded T cells comprise VSTs that recognize at least one antigen from human herpesvirus 8 (HHV-8). In some aspects, the invention provides an expanded T cell population obtained by the methods of the invention. In some aspects, the invention provides a population of expanded T cells.

In some embodiments, the expanded T cell population includes T cells specific for at least one target antigen. In some embodiments, the expanded T cell population is enriched with T cells specific for at least one target antigen.

In some embodiments, the expanded T cell population includes antigen-specific T cells. In some embodiments, the antigen-specific T cells are pathogen-specific T cells (PST). In some embodiments, the antigen-specific T cells are virus-specific T cells (VST). In some embodiments, the antigen-specific T cells are tumor-specific T cells (TST).

In some embodiments, the T cells of the expanded T cell population have reduced alloreactivity. In some embodiments, the T cells of the expanded T cell population have negligible alloreactivity. In some embodiments, the T cells of the expanded T cell population have reduced reactivity against an allogeneic target. In some embodiments, the T cells of the expanded T cell population have negligible reactivity against the allogeneic target. In some embodiments, the T cells of the expanded T cell population have reduced autoreactivity. In some embodiments, the T cells of the expanded T cell population have negligible autoreactivity. In some embodiments, the alloreactivity of the T cells of the expanded T cell population is reduced compared to an unenriched T cell population. In some embodiments, the T cell reactivity of the expanded T cell population against an allogeneic target is reduced compared to an unenriched T cell population. In some embodiments, the autoreactivity of the T cells of the expanded T cell population is reduced compared to an unenriched T cell population. i. Antigen specificity

In some embodiments, the expanded T cell population includes virus-specific T cells (VST). In some embodiments, the expanded T cell population comprises virus-specific T cells (VST), wherein the virus is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus tumor virus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex Virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human Herpesvirus 8 (HHV-8), Human Herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), Respiratory Synthetic Virus (RSV), Influenza virus, Parainfluenza virus, Bocavirus, Coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV) ), Zika virus, Ebola virus and human T-lymphotropic virus. In some embodiments, the expanded T cell population comprises virus-specific T cells (VST), wherein the viral lineage is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus tumor virus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex Virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human Herpesvirus 8 (HHV-8), Human Herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), Respiratory Synthetic Virus (RSV), Influenza virus, Parainfluenza virus, Bocavirus, Coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV) ), Zika virus, Ebola virus and human T-lymphotropic virus. In some embodiments, the expanded T cell population includes virus-specific T cells (VST) specific for hepatitis B virus (HBV). In some embodiments, the expanded T cell population includes virus-specific T cells (VST) specific for human herpesvirus 8 (HHV-8). In some embodiments, the expanded T cell population includes virus-specific T cells (VST) having specificity for hepatitis B virus (HBV). In some embodiments, the expanded T cell population includes virus-specific T cells (VST) with specificity for human herpesvirus 8 (HHV-8).

In some embodiments, the expanded T cells comprise VSTs that recognize at least one antigen, at least two antigens, or at least three antigens from hepatitis B virus (HBV). In some embodiments, the expanded T cells comprise VSTs that recognize at least one antigen from hepatitis B virus (HBV). In some embodiments, the expanded T cells comprise VSTs that recognize at least one, at least two, or at least three antigens from human herpesvirus 8 (HHV-8). In some embodiments, the expanded T cells comprise VSTs that recognize at least one antigen from human herpesvirus 8 (HHV-8).

In some embodiments, the expanded T cell population includes VSTs that recognize at least one antigen from hepatitis B virus (HBV). In some embodiments, the expanded T cell population includes VSTs that recognize at least one antigen from human herpesvirus 8 (HHV-8).

In some embodiments, at least one T cell recognizes one or more tumor-associated antigens, or portions thereof. In some embodiments, the one or more tumor-associated antigens are selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu, MAGE A3, p53 non-mutant, NY- ESO-1, PSMA, GD2, melanin A/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl , mesothelin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fas-related antigen 1. In some embodiments, the one or more tumor-associated antigens are selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu, MAGE A3, p53 non-mutant, NY- ESO-1, PSMA, GD2, melanin A/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl , mesothelin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fas-related antigen 1.

In some embodiments, at least one T cell recognizes one or more target antigens, or portions thereof, from at least one pathogen. In some embodiments, at least one pathogen is a virus. In some embodiments, the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), Hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 ( HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV) , mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T lymphocyte Cellular viruses.

In some embodiments, the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), Hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 ( HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV) , mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T lymphocyte Cellular viruses.

In some embodiments, the pathogen is HBV, and the at least one target antigen is selected from the group consisting of surface antigen (HBsAg), core antigen (HBcAg), pre-core antigen (HBeAg), DNA polymerase, and combinations thereof. In some embodiments, the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), core antigen (HBcAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or a combination thereof. In some embodiments, the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or a combination thereof. In some embodiments, the pathogen is HBV, and the at least one target antigen is a surface antigen (HBsAg) and/or a core antigen (HBcAg).

In some embodiments, at least one T cell recognizes one or more target antigens from at least one virus, or a portion thereof. In some embodiments, the at least one virus is a latent virus. In some embodiments, the at least one virus is a lytic virus. In some embodiments, the at least one virus is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), Hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 ( HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV) , mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T lymphocyte Cellular viruses.

In some embodiments, the virus is HBV, and the at least one target antigen is surface antigen (HBsAg), core antigen (HBcAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or a combination thereof. In some embodiments, the virus is HBV and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or a combination thereof. In some embodiments, the virus is HBV, and the at least one target antigen is a surface antigen (HBsAg) and/or a core antigen (HBcAg).

HBsAg (also known as "LE") is the surface antigen of the HBV virus. Its presence in the blood indicates current HBV infection. HBsAg can be recognized by antibodies that specifically bind to the surface protein or a portion thereof. HBsAg is also the target antigen used in HBV vaccines.

HBcAg (core antigen, also known as "C") is an HBV protein that, together with the pre-core antigen, indicates active viral replication. HBcAg can be present on the surface of the nucleocapsid core of HBV. Although HBcAg and HBeAg are produced from the same open reading frame (ORF), HBcAg is not secreted. When the gene ORF core and PreC (pre-core) are translated together, HBeAg is produced. HBeAg is secreted as an immunologically distinct soluble antigen and accumulates in the serum.

HBP is a multifunctional HBV enzyme with RNA-dependent and DNA-dependent polymerase functions. HBP acts on HBV pregenomic RNA (pgRNA) to reverse-transcribe it, thereby forming new rcDNA (relaxed circular DNA) molecules within the new capsid.

HBX is a multifunctional HBV protein that promotes efficient replication of HBV by stimulating HBV gene expression from cccDNA (covalently closed circular DNA) template. Research has shown its role in host-virus interactions.

In some embodiments, the target antigen includes HBsAg. In some embodiments, the target antigen includes HBcAg. In some embodiments, the target antigen includes HBeAg. In some embodiments, the target antigen includes HBP. In some embodiments, the target antigen includes HBX. In some embodiments, the target antigen is HBsAg. In some embodiments, the target antigen is HBcAg. In some embodiments, the target antigen is HBeAg. In some embodiments, the target antigen is HBP. In some embodiments, the target antigen is HBX.

In some embodiments, target antigens include HBsAg and HBcAg. In some embodiments, target antigens include HBsAg and HBeAg. In some embodiments, target antigens include HBsAg and HBP. In some embodiments, target antigens include HBsAg and HBX. In some embodiments, target antigens include HBcAg and HBeAg. In some embodiments, target antigens include HBcAg and HBP. In some embodiments, target antigens include HBcAg and HBX. In some embodiments, target antigens include HBeAg and HBP. In some embodiments, target antigens include HBeAg and HBX. In some embodiments, target antigens include HBP and HBX.

In some embodiments, target antigens include HBsAg, HBcAg, and HBeAg. In some embodiments, target antigens include HBsAg, HBcAg, and HBP. In some embodiments, target antigens include HBsAg, HBcAg, and HBX. In some embodiments, target antigens include HBsAg, HBeAg, and HBP. In some embodiments, target antigens include HBsAg, HBeAg, and HBX. In some embodiments, target antigens include HBsAg, HBP, and HBX. In some embodiments, target antigens include HBcAg, HBeAg, and HBP. In some embodiments, target antigens include HBcAg, HBeAg, and HBX. In some embodiments, target antigens include HBcAg, HBP, and HBX. In some embodiments, target antigens include HBeAg, HBP, and HBX.

In some embodiments, target antigens include HBsAg, HBcAg, HBeAg, and HBP. In some embodiments, target antigens include HBsAg, HBcAg, HBeAg, and HBX. In some embodiments, target antigens include HBsAg, HBcAg, HBP, and HBX. In some embodiments, target antigens include HBsAg, HBeAg, HBP, and HBX. In some embodiments, target antigens include HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the target antigen includes each of HBsAg, HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the expanded T cells target the antigens HbsAg (LE) and HBcAg (C). In some embodiments, VST targets the antigens HbsAg (LE) and HBcAg (C). In some embodiments, the expanded T cells target the antigen HbsAg (LE). In some embodiments, VST targets the antigen HbsAg (LE). In some embodiments, the expanded T cells target the antigen HBcAg(C). In some embodiments, VST targets the antigen HBcAg(C).

In some embodiments, at least two HBV antigen lines are selected from different HBV genotypes. HBV is divided into multiple genotypes based on genome sequence. At least ten genotypes (A to J) of the HBV genotype have been identified. Some HBV genotypes are further classified into subgenotypes, and at least 30 subgenotypes have been identified. HBV sequences are characterized by genotypes with >8% nucleotide differences, and subgenotypes with 4% to 8% nucleotide differences (Sanbul, World J Gstroenterol , 2014. 20(18): 5427-5434). In some embodiments, at least one antigenic line is selected from HBV genotype A and at least one antigenic line is selected from HBV genotype C. In some embodiments, the amino acid sequence of each HBV antigen differs from the amino acid sequence of each other HBV antigen by at least one amino acid.

In some embodiments, at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO :3). In some embodiments, at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4), EYLVSFGVWIRTPPA (SEQ ID NO: 5), ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO : 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15) or its functional variant. In some embodiments, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are peptide sequences derived from HBsAg. In some embodiments, SEQ ID NO: 4 and SEQ ID NO: 5 are peptide sequences derived from HBeAg. In some embodiments, SEQ ID NOs: 6-12 are peptide sequences derived from HHV8 LANA1. In some embodiments, SEQ ID NOs: 13-15 are peptide sequences derived from HHV8 gB.

In some embodiments, at least a portion of the T cell response is HLA-DR, HLA-DQ or HLA-DP restricted. In some embodiments, at least a portion of the T cell reactivity is restricted by HLA-DR, HLA-DQ, or HLA-DP.

In some embodiments, the pathogen is EBV, and the at least one target antigen is selected from EBNAl, LMP2, BZLF1, and combinations thereof. In some embodiments, the pathogen is EBV, and the at least one target antigen is selected from EBNAl, LMP2, and BZLF1. In some embodiments, the pathogen is CMV and the at least one target antigen is selected from IEl and pp65, and combinations thereof. In some embodiments, the pathogen is CMV and the at least one target antigen is selected from IEl and pp65.

In some embodiments, the pathogen is Adv and the at least one target antigen is selected from the group consisting of hexons, pentons, and combinations thereof. In some embodiments, the pathogen is Adv and the at least one target antigen is selected from hexon and penton. In some embodiments, the pathogen is BK virus, and the at least one target antigen is selected from LT, VP-1, and combinations thereof. In some embodiments, the pathogen is BK virus and the at least one target antigen is selected from LT and VP-1. In some embodiments, the pathogen is HHV6 and the at least one target antigen is selected from U14, U11, U71, U54, U90, and combinations thereof. In some embodiments, the pathogen is HHV6 and the at least one target antigen is selected from U14, U11, U71, U54, and U90. In some embodiments, the pathogen is RSV and the at least one target antigen is selected from N, F, and combinations thereof. In some embodiments, the pathogen is RSV and the at least one target antigen is selected from N and F. In some embodiments, the pathogen is influenza virus, and the at least one target antigen is selected from MP1, NP1, and combinations thereof. In some embodiments, the pathogen is influenza virus, and the at least one target antigen is selected from MP1 and NP1. In some embodiments, the pathogen is a parainfluenza virus (PIV), and the at least one target antigen is selected from the group consisting of F, N, M, M2-1, HN, and combinations thereof. In some embodiments, the pathogen is a parainfluenza virus (PIV) and the at least one target antigen is selected from F, N, M, M2-1, and HN. In some embodiments, the pathogen is hMPV and the at least one target antigen is selected from the group consisting of F, N, M2-1, M, and combinations thereof. In some embodiments, the pathogen is hMPV and the at least one target antigen is selected from F, N, M2-1, and M.

In some embodiments, the pathogen is HHV8 and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), card Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) and their combinations. In some embodiments, the pathogen is HHV8 and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), card Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof. In some embodiments, the pathogen is HHV8 and one of the target antigens is selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8 and the two target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8 and the two target antigens are LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the pathogen is HHV8 and the two target antigens are LANA1 (ORF3) and gB (ORF8). In some embodiments, the pathogen is HHV8 and the two target antigens are LANA2 (ORF3) and gB (ORF8). In some embodiments, the pathogen is HHV8 and the at least two target antigens are LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the pathogen is HHV8 and the at least two target antigens are LANA1 (ORF3) and gB (ORF8). In some embodiments, the pathogen is HHV8 and the at least two target antigens are LANA2 (ORF3) and gB (ORF8).

In some embodiments, the pathogen is HHV8, and the three target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least three target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the four target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least four target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the five target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least five target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the six target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least six target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the seven target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least seven target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the eight target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least eight target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the nine target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71) , Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least nine target antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71 ), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the pathogen is HHV8, and the ten target antigens are LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), card Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the pathogen is HHV8, and the at least ten target antigens are LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the target antigen includes LANA-1 (ORF3). In some embodiments, the target antigen includes LANA-2 (vIRF3, K10.5). In some embodiments, the target antigen includes vCYC (ORF72). In some embodiments, the target antigen includes RTA (ORF50). In some embodiments, the target antigen includes vFLIP (ORF71). In some embodiments, the target antigen includes Kaposi proteins (ORF12, K12). In some embodiments, the target antigen includes gB (ORF8). In some embodiments, the target antigen includes MIR1 (K3). In some embodiments, the target antigen includes SSB (ORF6). In some embodiments, the target antigen includes TS (ORF70).

In some embodiments, the target antigen is LANA-1 (ORF3). In some embodiments, the target antigen is LANA-2 (vIRF3, K10.5). In some embodiments, the target antigen is vCYC (ORF72). In some embodiments, the target antigen is RTA (ORF50). In some embodiments, the target antigen is vFLIP (ORF71). In some embodiments, the target antigen is Kaposi protein (ORF12, K12). In some embodiments, the target antigen is gB (ORF8). In some embodiments, the target antigen is MIR1 (K3). In some embodiments, the target antigen is SSB (ORF6). In some embodiments, the target antigen is TS (ORF70).

In some embodiments, the pathogen is SARS-CoV2, and at least one target antigen is selected from the group consisting of spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14 and combinations thereof.

In some embodiments, the pathogen is SARS-CoV2, and at least one target antigen is selected from the group consisting of spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B and Y14.

In some embodiments, the pathogen is SARS-CoV2, and at least one target antigen is selected from the group consisting of spike protein (S), envelope protein (E), matrix protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B and Y14. ii.Cell phenotype

In some embodiments, the expanded T cell population generates a T cell response upon activation. In some embodiments, at least a portion of the T cell response is CD4+. In some embodiments, at least a portion of the T cell response is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted.

In some embodiments, the T cell population includes about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% CD3+ T cells. In some embodiments, the T cell population includes at least 70% CD3+ T cells. In some embodiments, the T cell population includes about 70% CD3+ T cells. In some embodiments, the T cell population includes up to 70% CD3+ T cells. In some embodiments, the T cell population includes 70% CD3+ T cells.

In some embodiments, no more than about 30% of the cells in the population are natural killer (NK) cells. In some embodiments, no more than 30% of the cells in the population are natural killer (NK) cells. In some embodiments, approximately 30% of the cells in the population are natural killer (NK) cells. In some embodiments, more than 30% of the cells in the population are natural killer (NK) cells. In some embodiments, no more than about 20% of the cells in the population are natural killer (NK) cells. In some embodiments, no more than 20% of the cells in the population are natural killer (NK) cells. In some embodiments, approximately 20% of the cells in the population are natural killer (NK) cells. In some embodiments, more than 20% of the cells in the population are natural killer (NK) cells. In some embodiments, no more than about 10% of the cells in the population are natural killer (NK) cells. In some embodiments, no more than 10% of the cells in the population are natural killer (NK) cells. In some embodiments, about 10% of the cells in the population are natural killer (NK) cells. In some embodiments, more than 10% of the cells in the population are natural killer (NK) cells. In some embodiments, no more than about 40% of the cells in the population are natural killer (NK) cells. In some embodiments, no more than 40% of the cells in the population are natural killer (NK) cells. In some embodiments, approximately 40% of the cells in the population are natural killer (NK) cells. In some embodiments, more than 40% of the cells in the population are natural killer (NK) cells.

As used herein, the term "natural killer cells" or "NK cells" is used to refer to cells that are cytotoxic lymphocytes, which constitute a major component of the innate immune system. In humans, natural killer cells typically express the surface markers CD16 (FCyRIII) and CD56. NK cells are cytotoxic; their cytoplasm contains small particles of specific proteins (such as perforin) and proteases called granzymes. NK cells provide a rapid response to virus-infected cells and respond to transformed cells. When released in close proximity to the cell to be killed, perforin forms pores in the cell membrane of the target cell through which granzymes and related molecules can enter, inducing apoptosis. Thus, NK cells may serve as effectors of lymphocyte populations in anti-tumor and anti-infectious immunity.

In some embodiments, the natural killer cells are characterized as CD3- CD56+ CD45+ CD94+ CD122+/IL-2Rβ+ CD127/IL-7Rα- FcγRIII/CD16+ KIR+ NKG2A+ NKG2D+ NKp30+ NKp44+ NKP46+ and/or NKp80+ cells.

In some embodiments, the T cell population includes CD4+ and CD8+ T cells.

In some embodiments, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cell population comprise CD4+ and/or CD8+ T cells cells. In some embodiments, about 70% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, about 70% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, more than 70% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, more than 70% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, less than 70% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, less than 70% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, about 75% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, less than 70% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, more than 75% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, more than 75% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, less than 75% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, less than 75% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, about 80% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, about 80% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, more than 80% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, more than 80% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, less than 80% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, less than 80% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, about 85% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, about 85% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, more than 85% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, more than 85% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, less than 85% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, less than 85% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, approximately 90% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, about 90% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, more than 90% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, more than 90% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, less than 90% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, less than 90% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, about 95% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, about 95% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, more than 95% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, more than 95% of the T cell population comprises CD4+ and/or CD8+ T cells. In some embodiments, less than 95% of the T cell population consists of CD4+ and/or CD8+ T cells. In some embodiments, less than 95% of the T cell population comprises CD4+ and/or CD8+ T cells.

In some embodiments, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cell population comprises CD4+ T cells. In some embodiments, about 70% of the T cell population consists of CD4+ T cells. In some embodiments, about 70% of the T cell population comprises CD4+ T cells. In some embodiments, more than 70% of the T cell population consists of CD4+ T cells. In some embodiments, more than 70% of the T cell population comprises CD4+ T cells. In some embodiments, less than 70% of the T cell population consists of CD4+ T cells. In some embodiments, less than 70% of the T cell population comprises CD4+ T cells. In some embodiments, about 75% of the T cell population consists of CD4+ T cells. In some embodiments, less than 75% of the T cell population comprises CD4+ T cells. In some embodiments, more than 75% of the T cell population consists of CD4+ T cells. In some embodiments, more than 75% of the T cell population comprises CD4+ T cells. In some embodiments, less than 75% of the T cell population consists of CD4+ T cells. In some embodiments, less than 75% of the T cell population comprises CD4+ T cells. In some embodiments, about 80% of the T cell population consists of CD4+ T cells. In some embodiments, about 80% of the T cell population comprises CD4+ T cells. In some embodiments, more than 80% of the T cell population consists of CD4+ T cells. In some embodiments, more than 80% of the T cell population comprises CD4+ T cells. In some embodiments, less than 80% of the T cell population consists of CD4+ T cells. In some embodiments, less than 80% of the T cell population comprises CD4+ T cells. In some embodiments, about 85% of the T cell population consists of CD4+ T cells. In some embodiments, about 85% of the T cell population comprises CD4+ T cells. In some embodiments, more than 85% of the T cell population consists of CD4+ T cells. In some embodiments, more than 85% of the T cell population comprises CD4+ T cells. In some embodiments, less than 85% of the T cell population consists of CD4+ T cells. In some embodiments, less than 85% of the T cell population comprises CD4+ T cells. In some embodiments, approximately 90% of the T cell population consists of CD4+ T cells. In some embodiments, about 90% of the T cell population comprises CD4+ T cells. In some embodiments, more than 90% of the T cell population consists of CD4+ T cells. In some embodiments, more than 90% of the T cell population comprises CD4+ T cells. In some embodiments, less than 90% of the T cell population consists of CD4+ T cells. In some embodiments, less than 90% of the T cell population comprises CD4+ T cells. In some embodiments, about 95% of the T cell population consists of CD4+ T cells. In some embodiments, about 95% of the T cell population comprises CD4+ T cells. In some embodiments, more than 95% of the T cell population consists of CD4+ T cells. In some embodiments, more than 95% of the T cell population comprises CD4+ T cells. In some embodiments, less than 95% of the T cell population consists of CD4+ T cells. In some embodiments, less than 95% of the T cell population comprises CD4+ T cells.

In some embodiments, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cell population comprises CD8+ T cells. In some embodiments, about 70% of the T cell population consists of CD8+ T cells. In some embodiments, about 70% of the T cell population comprises CD8+ T cells. In some embodiments, more than 70% of the T cell population consists of CD8+ T cells. In some embodiments, more than 70% of the T cell population comprises CD8+ T cells. In some embodiments, less than 70% of the T cell population consists of CD8+ T cells. In some embodiments, less than 70% of the T cell population comprises CD8+ T cells. In some embodiments, about 75% of the T cell population consists of CD8+ T cells. In some embodiments, less than 75% of the T cell population comprises CD8+ T cells. In some embodiments, more than 75% of the T cell population consists of CD8+ T cells. In some embodiments, more than 75% of the T cell population comprises CD8+ T cells. In some embodiments, less than 75% of the T cell population consists of CD8+ T cells. In some embodiments, less than 75% of the T cell population comprises CD8+ T cells. In some embodiments, about 80% of the T cell population consists of CD8+ T cells. In some embodiments, about 80% of the T cell population comprises CD8+ T cells. In some embodiments, more than 80% of the T cell population consists of CD8+ T cells. In some embodiments, more than 80% of the T cell population comprises CD8+ T cells. In some embodiments, less than 80% of the T cell population consists of CD8+ T cells. In some embodiments, less than 80% of the T cell population comprises CD8+ T cells. In some embodiments, about 85% of the T cell population consists of CD8+ T cells. In some embodiments, about 85% of the T cell population comprises CD8+ T cells. In some embodiments, more than 85% of the T cell population consists of CD8+ T cells. In some embodiments, more than 85% of the T cell population comprises CD8+ T cells. In some embodiments, less than 85% of the T cell population consists of CD8+ T cells. In some embodiments, less than 85% of the T cell population comprises CD8+ T cells. In some embodiments, approximately 90% of the T cell population consists of CD8+ T cells. In some embodiments, about 90% of the T cell population comprises CD8+ T cells. In some embodiments, more than 90% of the T cell population consists of CD8+ T cells. In some embodiments, more than 90% of the T cell population comprises CD8+ T cells. In some embodiments, less than 90% of the T cell population consists of CD8+ T cells. In some embodiments, less than 90% of the T cell population comprises CD8+ T cells. In some embodiments, about 95% of the T cell population consists of CD8+ T cells. In some embodiments, about 95% of the T cell population comprises CD8+ T cells. In some embodiments, more than 95% of the T cell population consists of CD8+ T cells. In some embodiments, more than 95% of the T cell population comprises CD8+ T cells. In some embodiments, less than 95% of the T cell population consists of CD8+ T cells. In some embodiments, less than 95% of the T cell population comprises CD8+ T cells.

In some embodiments, the T cells are polyline. As used herein, the term "multiple strains" is used to describe a population derived from multiple pure lines, as opposed to an oligostrain or a single strain, in which the population is derived from several pure lines or one pure line, respectively.

In some embodiments, the T cells are activated or display characteristics of activation. In some embodiments, activated T cells are referred to as CD3+ T cells. In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of CD3+ T cells produce tumor necrosis factor-alpha (TNFa). In some embodiments, at least about 1% of CD3+ T cells produce TNFα. In some embodiments, at least 1% of CD3+ T cells produce TNFα. In some embodiments, at least about 10% of CD3+ T cells produce TNFα. In some embodiments, at least about 10% of CD3+ T cells produce TNFα and IFNγ.

In some embodiments, the T cells are activated or display characteristics of activation. In some embodiments, activated T cells are referred to as CD3+ T cells. In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of the CD3+ T cells produce tumor necrosis factor-alpha (TNFa). In some embodiments, at least about 1% of CD3+ T cells produce TNFα. In some embodiments, at least 1% of CD3+ T cells produce TNFα. In some embodiments, at least about 10% of the CD3+ T cells produce TNFα. In some embodiments, at least about 10% of CD3+ T cells produce TNFα and IFNγ.

In some embodiments, the expanded T cell population is responsive upon stimulation. In some embodiments, T cell reactivity includes upregulation of activation markers (eg, CD25, CD69, etc.), production of effector molecules (eg, IFNg, TNFa, granzyme B, etc.), killing of target cells, or a combination thereof.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of CD3+ T cells produce interferon gamma (IFNγ). In some embodiments, at least about 1% of CD3+ T cells produce IFNγ. In some embodiments, at least 1% of CD3+ T cells produce IFNγ. In some embodiments, at least about 10% of CD3+ T cells produce IFNγ. In some embodiments, at least about 20% of CD3+ T cells produce IFNγ. In some embodiments, at least about 30% of CD3+ T cells produce IFNγ.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of the CD3+ T cells produce interferon gamma (IFNγ). In some embodiments, at least about 1% of the CD3+ T cells produce IFNγ. In some embodiments, at least 1% of the CD3+ T cells produce IFNγ. In some embodiments, at least about 10% of the CD3+ T cells produce IFNγ. In some embodiments, at least about 20% of the CD3+ T cells produce IFNγ. In some embodiments, at least about 30% of the CD3+ T cells produce IFNγ.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of CD3+ T cells produce granzyme B (GrB). In some embodiments, at least about 1% of CD3+ T cells produce GrB. In some embodiments, at least 1% of CD3+ T cells produce GrB.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of the CD3+ T cells produce granzyme B (GrB). In some embodiments, at least about 1% of CD3+ T cells produce GrB. In some embodiments, at least 1% of CD3+ T cells produce GrB.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of CD3+ T cells produce TNFα and IFNγ. In some embodiments, at least about 1% of CD3+ T cells produce TNFα and IFNγ. In some embodiments, at least 1% of CD3+ T cells produce TNFα and IFNγ.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of the CD3+ T cells produce TNFα and IFNγ. In some embodiments, at least about 1% of CD3+ T cells produce TNFα and IFNγ. In some embodiments, at least 1% of CD3+ T cells produce TNFα and IFNγ.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of CD3+ T cells produce GrB and IFNγ. In some embodiments, at least about 1% of CD3+ T cells produce GrB and IFNγ. In some embodiments, at least 1% of CD3+ T cells produce GrB and IFNγ.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of the CD3+ T cells produce GrB and IFNγ. In some embodiments, at least about 1% of CD3+ T cells produce GrB and IFNγ. In some embodiments, at least 1% of CD3+ T cells produce GrB and IFNγ.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of the CD3+ T cells produce GrB, IFNγ, and TNFα. In some embodiments, at least about 1% of CD3+ T cells produce GrB, IFNγ, and TNFα. In some embodiments, at least 1% of CD3+ T cells produce GrB, IFNγ, and TNFα.

In some embodiments, about 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% of the CD3+ T cells produce GrB, IFNγ, and TNFα. In some embodiments, at least about 1% of CD3+ T cells produce GrB, IFNγ, and TNFα. In some embodiments, at least 1% of CD3+ T cells produce GrB, IFNγ, and TNFα.

In some embodiments, the expanded T cell populations of the invention are multifunctional. As used herein, "multifunctionality" is used to describe cells that are capable of producing a variety of effector molecules upon antigen exposure. In some embodiments, the expanded T cell population is capable of producing IFNγ. In some embodiments, the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, the expanded T cell population is capable of producing IFNγ, TNFα, and GrB. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the expanded T cell population is capable of producing IFNγ . In some embodiments, at least about 10% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 20% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 30% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 40% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 50% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 60% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 70% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 80% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 90% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 95% of the expanded T cell population is capable of producing IFNγ. In some embodiments, at least about 99% of the expanded T cell population is capable of producing IFNγ.

In some embodiments, the expanded T cell population produces IFNγ. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the expanded T cell population produces IFNγ. In some embodiments, at least about 10% of the expanded T cell population produces IFNγ. In some embodiments, at least about 20% of the expanded T cell population produces IFNγ. In some embodiments, at least about 30% of the expanded T cell population produces IFNγ. In some embodiments, at least about 40% of the expanded T cell population produces IFNγ. In some embodiments, at least about 50% of the expanded T cell population produces IFNγ. In some embodiments, at least about 60% of the expanded T cell population produces IFNγ. In some embodiments, at least about 70% of the expanded T cell population produces IFNγ. In some embodiments, at least about 80% of the expanded T cell population produces IFNγ. In some embodiments, at least about 90% of the expanded T cell population produces IFNγ. In some embodiments, at least about 95% of the expanded T cell population produces IFNγ. In some embodiments, at least about 99% of the expanded T cell population produces IFNγ.

In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 10% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 13% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 20% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 30% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 40% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 50% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 60% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 70% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 80% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 90% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 95% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, at least about 99% of the expanded T cell population is capable of producing IFNγ and TNFα.

In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 10% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 13% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 20% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 30% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 40% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 50% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 60% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 70% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 80% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 90% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 95% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, at least about 99% of the expanded T cell population produces IFNγ and TNFα.

In some embodiments, about 10% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 15% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 20% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 25% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 30% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 35% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 36% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 37% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 38% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 39% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 40% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 45% of the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, about 50% of the expanded T cell population is capable of producing IFNγ and TNFα.

In some embodiments, about 10% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 13% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 15% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 20% of the expanded T cells produce IFNγ and TNFα. In some embodiments, about 25% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 30% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 35% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 36% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 37% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 38% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 39% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 40% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 45% of the expanded T cell population produces IFNγ and TNFα. In some embodiments, about 50% of the expanded T cell population produces IFNγ and TNFα.

In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 10% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 20% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 30% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 40% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 50% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 60% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 70% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 80% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 90% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 95% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 99% of the expanded T cell population is capable of producing IFNγ and GrB.

In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 10% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 20% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 30% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 40% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 50% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 60% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 70% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 80% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 90% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 95% of the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 99% of the expanded T cell population produces IFNγ and GrB.

In some embodiments, about 20% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 25% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 30% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 35% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 40% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 45% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 50% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 55% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 56% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 57% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 58% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 59% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 60% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 65% of the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, about 70% of the expanded T cell population is capable of producing IFNγ and GrB.

In some embodiments, about 20% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 25% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 30% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 35% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 40% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 45% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 50% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 55% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 56% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 57% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 58% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 59% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 60% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 65% of the expanded T cell population produces IFNγ and GrB. In some embodiments, about 70% of the expanded T cell population produces IFNγ and GrB.

In some embodiments, the expanded T cell population is capable of producing IFNγ and TNFα. In some embodiments, the expanded T cell population is capable of producing IFNγ, TNFα, and GrB. In some embodiments, the expanded T cell population is capable of producing IFNγ and GrB. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells of the expanded T cell population are polypeptides. Functional, in which the T cells are capable of producing one or more of IFNγ, TNFα, and GrB. In some embodiments, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% of the expanded T cell population , 70%, 75%, 80%, 85%, 90%, 95% or 99% of the cells are multifunctional, and the T cells are capable of producing one or more of IFNγ, TNFα and GrB. In some embodiments, about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of cells are multifunctional, with T cells capable of producing one or more of IFNγ, TNFα and GrB. In some embodiments, at least about 66% of the cells of the expanded T cell population are multifunctional, wherein the T cells are capable of producing one or more of IFNγ, TNFα, and GrB. In some embodiments, more than 66% of the cells of the expanded T cell population are multifunctional, wherein the T cells are capable of producing one or more of IFNγ, TNFα, and GrB. In some embodiments, at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of the cells of the expanded T cell population are polypeptides. Functional, in which the T cells are capable of producing IFNγ as well as one or more of TNFα and GrB. In some embodiments, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of the cells of the expanded T cell population are multifunctional Sexual, in which T cells can produce IFNγ as well as one or more of TNFα and GrB. In some embodiments, at least about 69% of the cells of the expanded T cell population are multifunctional, wherein the T cells are capable of producing IFNγ and one or more of TNFα and GrB. In some embodiments, about 69% of the cells of the expanded T cell population are multifunctional, wherein the T cells are capable of producing IFNγ and one or more of TNFα and GrB. In some embodiments, at least about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the cells of the expanded T cell population are polypeptides. Functional, among which T cells can produce IFNγ, TNFα and GrB. In some embodiments, about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the cells of the expanded T cell population are multifunctional Sexual, among which T cells can produce IFNγ, TNFα and GrB. In some embodiments, about 25% of the cells of the expanded T cell population are multifunctional, wherein the T cells are capable of producing one or more of IFNγ, TNFα, and GrB. In some embodiments, less than about 66% of the cells of the expanded T cell population are multifunctional, wherein the T cells are capable of producing one or more of IFNγ, TNFα, and GrB. In some embodiments, an expanded T cell is multifunctional if it produces two or more of IFNγ, TNF-α, GM-CSF, MIP-1a, MIP-1b, GrB, and perforin. of.

In some embodiments, the expanded T cell population produces IFNγ and TNFα. In some embodiments, the expanded T cell population produces IFNγ, TNFα, and GrB. In some embodiments, the expanded T cell population produces IFNγ and GrB. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells of the expanded T cell population are polypeptides. Functional, in which the T cells produce one or more of IFNγ, TNFα, and GrB. In some embodiments, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% of the expanded T cell population , 70%, 75%, 80%, 85%, 90%, 95% or 99% of cells are multifunctional, with T cells producing one or more of IFNγ, TNFα and GrB. In some embodiments, about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of cells are multifunctional, with T cells producing one or more of IFNγ, TNFα and GrB. In some embodiments, at least about 66% of the cells of the expanded T cell population are multifunctional, wherein the T cells produce one or more of IFNγ, TNFα, and GrB. In some embodiments, more than 66% of the cells of the expanded T cell population are multifunctional, wherein the T cells produce one or more of IFNγ, TNFα, and GrB. In some embodiments, at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of the cells of the expanded T cell population are polypeptides. Functional, in which the T cells produce IFNγ and one or more of TNFα and GrB. In some embodiments, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of the cells of the expanded T cell population are multifunctional Sexual, in which T cells produce IFNγ and one or more of TNFα and GrB. In some embodiments, at least about 69% of the cells of the expanded T cell population are multifunctional, wherein the T cells produce IFNγ and one or more of TNFα and GrB. In some embodiments, about 69% of the cells of the expanded T cell population are multifunctional, with the T cells producing IFNγ and one or more of TNFα and GrB. In some embodiments, at least about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the cells of the expanded T cell population are polypeptides. Functional, in which T cells produce IFNγ, TNFα and GrB. In some embodiments, about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the cells of the expanded T cell population are multifunctional Sexual, in which T cells produce IFNγ, TNFα and GrB. In some embodiments, less than about 66% of the cells of the expanded T cell population are multifunctional, wherein the T cells produce one or more of IFNγ, TNFα, and GrB. In some embodiments, about 25% of the cells of the expanded T cell population are multifunctional, wherein the T cells produce one or more of IFNγ, TNFα, and GrB. In some embodiments, an expanded T cell is multifunctional if it produces two or more of IFNγ, TNF-α, GM-CSF, MIP-1a, MIP-1b, GrB, and perforin. of.

In some embodiments, VSTs specific for HHV8 antigen are multifunctional upon antigen exposure. In some embodiments, a VST specific for HHV8 antigen is multifunctional upon exposure to the antigen of one or more of LANA1 and LANA2. In some embodiments, VSTs specific for HHV8 antigens are multifunctional upon antigen exposure of LANA1 and LANA2. In some embodiments, VST specific for HHV8 antigen expresses IFNγ and/or TNF-α upon antigen exposure of LANA1 and LANA2.

In some embodiments, the expanded T cell populations of the invention exhibit activation markers. As used herein, "activation marker" refers to a cellular phenotype indicative of T cell activation. Examples of T cell activation markers include, but are not limited to, CD4+ HLA-DR+ CD38+ cells.

In some embodiments, the expanded T cell populations of the invention exhibit minimal exhaustion. As used herein, "exhaustion" refers to a state of abnormal T cell function (e.g., progressive loss of effector function) caused by chronic antigen stimulation, such as during chronic infection and/or cancer. Exhaustion may include an adverse effector functional state, persistent expression of inhibitory receptors, and/or a transcriptional state that is distinct from functional effector or memory T cells. In some embodiments, T cell exhaustion markers include Lag3, TIM-3, PD-1 and/or TIGIT. In some embodiments, the T cell exhaustion marker includes Lag3. In some embodiments, the T cell exhaustion marker includes TIM-3. In some embodiments, T cell exhaustion markers include PD-1. In some embodiments, the T cell exhaustion marker includes TIGIT. In some embodiments, T cell exhaustion markers include TIM-3 and PD-1.

In some embodiments, the antigen-specific T cells are Th 1 polarized. In some embodiments, antigen-specific T cells are capable of lysing target cells expressing viral antigens. In some embodiments, antigen-specific T cells are capable of lysing target cells expressing other suitable types of antigens. In some embodiments, the antigen-specific T cells in the composition do not significantly lyse uninfected autologous target cells. In some embodiments, the antigen-specific T cells in the composition do not significantly lyse uninfected autologous allogeneic target cells.

In some embodiments, the expanded T cell populations of the invention exhibit greater specificity and enrichment for specific antigens.

In some aspects, the invention provides a plurality of expanded T cell populations, each of which is an expanded T cell according to the invention, wherein each population includes T cells generated from donor monocytes obtained from a different donor. . As used herein, the term "donor" refers to the organism from which the biological sample is generated. In some embodiments, the donor is human.

In some embodiments, each donor's HLA type differs from at least one of the other donors by at least one HLA allele.

In some embodiments, the expanded T cell population has a cell number that is at least 1.1 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.2 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.3 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.4 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.6 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.7 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.8 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 1.9 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 2-fold greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 2.5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 3 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 3.5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is at least 4 times greater than the starting cell population.

In some embodiments, the expanded T cell population has a cell number that is about 1.1 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 1.2 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 1.3 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 1.4 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 1.5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 1.6 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 1.7 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is about 1.8 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is approximately 1.9 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is approximately 2-fold greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is approximately 2.5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is approximately 3 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is approximately 3.5 times greater than the starting cell population. In some embodiments, the expanded T cell population has a cell number that is approximately 4 times greater than the starting cell population.

In some embodiments, a multi-strain population of VST has reduced alloreactivity. In some embodiments, a multi-strain population of VST has negligible alloreactivity. In some embodiments, a multi-strain population of VST has reduced reactivity against an allogeneic target. In some embodiments, a multi-strain population of VST has negligible reactivity against an allogeneic target. In some embodiments, a multi-strain population of VST has reduced autoreactivity. In some embodiments, a multi-strain population of VST has negligible autoreactivity.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, wherein the expanded T cell population is at least 1000-fold enriched compared to monocytes prior to culture with: i) at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a portion of a target antigen; a portion of a peptide or a mixture of peptides; and ii) one or more exogenous interleukins selected from the group consisting of IL-4, IL-7 and IL-15. In some embodiments, the invention provides an expanded population of VST cells produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, wherein the expanded VST cell population is at least 1000-fold enriched compared to monocytes prior to culture with: i) at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a portion of a target antigen; a portion of a peptide or a mixture of peptides; and ii) one or more exogenous interleukins selected from the group consisting of IL-4, IL-7 and IL-15.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15 but excluding IL-2, wherein the expanded T cell population is at least 1000-fold enriched compared to monocytes prior to culture with: i) at least one target antigen or a portion of a target antigen or corresponding to at least one target antigen or a portion of a target antigen; a portion of a peptide or a mixture of peptides; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15 but excluding IL-2.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, The expanded T cell population contains at least 70% CD3+ T cells.

In some embodiments, the invention provides an expanded population of VST cells produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, The expanded VST cell population contains at least 70% CD3+ T cells.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15 but excluding IL-2, The expanded T cell population contains at least 70% CD3+ T cells.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, The expanded T cell population includes CD4+ and CD8+ T cells.

In some embodiments, the invention provides an expanded population of VST cells produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, The expanded VST cell population includes CD4+ and CD8+ T cells.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) One or more exogenous interleukins selected from IL-4, IL-7 and IL-15 but not including IL-2 The expanded T cell population includes CD4+ and CD8+ T cells.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, Among them, the expanded T cell population produces IFNγ and TNFα.

In some embodiments, the invention provides an expanded population of VST cells produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, Among them, the expanded VST cell population produces IFNγ and TNFα.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15 but excluding IL-2, Among them, the expanded T cell population produces IFNγ and TNFα.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, wherein the expanded T cell population produces GrB.

In some embodiments, the invention provides an expanded population of VST cells produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, wherein the expanded VST cell population produces GrB.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) One or more exogenous interleukins selected from IL-4, IL-7 and IL-15 but not including IL-2 wherein the expanded T cell population produces GrB.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, No more than 20% of the expanded T cell population exhibits markers of T cell exhaustion.

In some embodiments, the invention provides an expanded population of VST cells produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, No more than 20% of the expanded VST cell population exhibits markers of T cell exhaustion.

In some embodiments, the invention provides an expanded T cell population produced by a method comprising: culturing mononuclear spheres with i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) One or more exogenous interleukins selected from IL-4, IL-7 and IL-15 but not including IL-2 No more than 20% of the expanded T cell population exhibits markers of T cell exhaustion. In some embodiments, T cell exhaustion markers include PD1+ and Tim3+. In some embodiments, the T cell exhaustion marker includes PD1+ or Tim3+. In some embodiments, the T cell exhaustion marker comprises PD1+. In some embodiments, the T cell exhaustion marker includes Tim3+. Amplified virus-specific T cell compositions

In some aspects, the invention includes amplified virus-specific T cell compositions specific for one or more viruses. In some embodiments, the one or more viruses include latent viruses. In some embodiments, the one or more viruses include lytic viruses. i.Antigen _

In some embodiments, viruses include, but are not limited to, EBV, CMV, adenovirus, BK virus, JC virus, HHV6, RSV, influenza virus, parainfluenza virus, Boca virus, coronavirus, LCMV, mumps virus, Measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, HSV, HBV, HCV, HDV, HPV, HIV, HTLVl, HHV8, West Nile virus, Zika virus and Ebola virus . In some embodiments, the virus includes HBV. In some embodiments, the virus is HBV. In some embodiments, the virus includes HHV8. In some embodiments, the virus is HHV8. In some embodiments, the virus-specific T cell composition is specific for HBV. In some embodiments, the virus-specific T cell composition is specific for HHV8.

The present invention provides, at least in part, a composition comprising an expanded population of virus-specific T cells. In some embodiments, the expanded population of virus-specific T cells recognizes a plurality of viral antigens. In some embodiments, the expanded population of virus-specific T cells recognizes two or more or a plurality of viral antigens from a single virus. For example, in some embodiments, the expanded population of antigen-specific T cells can recognize two or more or a plurality of viral antigens from: EBV, CMV, adenovirus, BK virus, JC virus, HHV6, RSV, influenza virus, parainfluenza virus, Boca virus, coronavirus, LCMV, mumps virus, measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, HSV, HBV, HCV, HDV, HPV, HIV, HTLVl, HHV8, West Nile virus, Zika virus and/or Ebola virus.

In some embodiments, the population of amplified VSTs includes VSTs specific for HBV antigens. In some embodiments, the population of amplified VSTs comprises HBV antigen surface antigen (HBsAg), core antigen (HBcAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or combinations thereof. Specificity VST. In some embodiments, HBeAg includes pre-core and core antigens. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for HBV antigen surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or combinations thereof. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBV antigen surface antigen (HBsAg) and core antigen (HBcAg).

In some embodiments, the multi-strain population of VSTs includes VSTs specific for the HBV antigens E antigen (HBeAg), P antigen (HBP), and LE antigen (HBsAg). In some embodiments, HBeAg (E antigen) includes pre-core (PreC) and core (HBcAg) antigens.

In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HBV antigen HBsAg. In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HBV antigen HBcAg. In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HBV antigen HBeAg. In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HBV antigen HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HBV antigen HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for the HBV antigen HBsAg. In some embodiments, the population of amplified VSTs includes VSTs specific for the HBV antigen HBcAg. In some embodiments, the population of amplified VSTs includes VSTs specific for the HBV antigen HBeAg. In some embodiments, the population of amplified VSTs includes VSTs specific for the HBV antigen HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for the HBV antigen HBX.

In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg and HBcAg. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg and HBeAg. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg and HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBcAg and HBeAg. In some embodiments, the population of amplified VSTs includes VSTs specific for HBcAg and HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for HBcAg and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBeAg and HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for HBeAg and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBP and HBX.

In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBcAg, and HBeAg. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBcAg, and HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBcAg, and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBeAg, and HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBeAg, and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBP, and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBcAg, HBeAg, and HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for HBcAg, HBeAg, and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBcAg, HBP, and HBX. In some embodiments, target antigens include HBeAg, HBP, and HBX.

In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBcAg, HBeAg, and HBP. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBcAg, HBeAg, and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBcAg, HBP, and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBsAg, HBeAg, HBP, and HBX. In some embodiments, the population of amplified VSTs includes VSTs specific for HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the population of amplified VSTs includes VSTs specific for each of HBsAg, HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the population of amplified VSTs includes VSTs specific for HHV8 antigen.

In some embodiments, the population of amplified VSTs includes responses to the HHV8 antigens LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), card VST with specificity for Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or their combination. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD A VST specific for HHV8 antigen, one of Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD At least one HHV8 antigen of Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) has a specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD Two HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD VST specific for at least two HHV8 antigens of Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and gB (ORF8). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigens LANA2 (ORF3) and gB (ORF8). In some embodiments, the pathogen is HHV8 and the population of amplified VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and gB (ORF8). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigens LANA2 (ORF3) and gB (ORF8).

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD The three HHV8 antigens of Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) have specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD VST specific for at least three HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD Four HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD VST specific for at least four HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD Five HHV8 antigens, namely Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD VST has specificity for at least five HHV8 antigens including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD Six HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD At least six HHV8 antigens, including Posi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD Seven HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD At least seven HHV8 antigens, including Posi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD Eight HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD At least eight HHV8 antigens, including Posi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD VST specific for nine HHV8 antigens including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD At least nine HHV8 antigens, including Posi proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD Ten HHV8 antigens, including Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs. In some embodiments, the population of amplified VSTs includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), CARD At least ten HHV8 antigens of Posi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) have specific VST.

In some embodiments, the population of amplified VSTs includes responses to the HHV8 antigens LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), card Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) have specific VSTs.

In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen LANA-1 (ORF3). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen LANA-2 (vIRF3, K10.5). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen vCYC (ORF72). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen RTA (ORF50). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen vFLIP (ORF71). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen Kaposi protein (ORF12, K12). In some embodiments, the population of amplified VSTs includes VSTs specific for at least HHV8 antigen gB (ORF8). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen MIR1 (K3). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen SSB (ORF6). In some embodiments, the population of amplified VSTs includes VSTs specific for at least the HHV8 antigen TS (ORF70).

In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen LANA-1 (ORF3). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen LANA-2 (vIRF3, K10.5). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen vCYC (ORF72). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen RTA (ORF50). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen vFLIP (ORF71). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen Kaposi protein (ORF12, K12). In some embodiments, the population of amplified VSTs includes VSTs specific for HHV8 antigen gB (ORF8). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen MIR1 (K3). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen SSB (ORF6). In some embodiments, the population of amplified VSTs includes VSTs specific for the HHV8 antigen TS (ORF70).

In some embodiments, the population of amplified VSTs includes VSTs specific for EBV antigens. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one EBV antigen selected from EBNAl, LMP2, and BZLF1.

In some embodiments, the population of amplified VSTs includes VSTs specific for CMV antigens. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one CMV antigen selected from IEl and pp65.

In some embodiments, the population of amplified VSTs includes VSTs specific for the Adv antigen. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one Adv antigen selected from the group consisting of hexons and pentons.

In some embodiments, the population of amplified VSTs includes VSTs specific for BK virus antigens. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one BK virus antigen selected from LT and VP-1. In some embodiments, VST specific for BK virus antigens is reactive against JCV antigens. In some embodiments, a population of amplified VSTs comprising VSTs specific for BK virus antigens is reactive against JCV antigens.

In some embodiments, the population of amplified VSTs includes VSTs specific for HHV6 antigen. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one HHV6 antigen selected from U14, U11, U71, U54, and U90.

In some embodiments, the population of amplified VSTs includes VSTs specific for HHV6 antigen. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one HHV6 antigen selected from U14, U11, and U90.

In some embodiments, the population of amplified VSTs includes VSTs specific for RSV antigens. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one RSV antigen selected from N and F.

In some embodiments, the population of amplified VSTs includes VSTs specific for influenza virus antigens. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one influenza virus antigen selected from MP1 and NP1.

In some embodiments, the population of amplified VSTs includes VSTs specific for parainfluenza virus (PIV) antigens. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one parainfluenza virus (PIV) antigen selected from F, N, M, M2-1, and HN.

In some embodiments, the population of amplified VSTs includes VSTs specific for hMPV antigens. In some embodiments, the population of amplified VSTs includes VSTs specific for at least one hMPV antigen selected from F, N, M2-1, and M.

In some embodiments, the population of amplified VSTs includes VSTs specific for SARS-CoV2 antigens. In some embodiments, the population of amplified VSTs includes pairs selected from the group consisting of spike protein (S), envelope protein (E), matrix protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, VST specific to at least one SARS-CoV2 antigen of nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B and Y14.

In some embodiments, the population of amplified VSTs includes VSTs specific for SARS-CoV2 antigens. In some embodiments, the population of amplified VSTs includes pairs selected from the group consisting of spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, VST specific to at least one SARS-CoV2 antigen of nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B and Y14. ii.Cell phenotype

In some embodiments, the expanded T cell population is responsive upon stimulation. In some embodiments, T cell reactivity includes upregulation of activation markers (eg, CD25, CD69, etc.), production of effector molecules (eg, IFNg, TNFa, granzyme B, etc.), killing of target cells, or a combination thereof.

In some embodiments, at least a portion of the T cell reactivity is restricted by HLA-DR, HLA-DQ, or HLA-DP.

In some embodiments, the specificity of VST for two or more antigens is measured by IFNγ ELISpot analysis. In some embodiments, the virus-specific T cells have a specificity for the target antigen of at least 10 SFC/2 × 10 ⁵ cells, at least 20 SFC/2 × 10 ⁵ cells, at least 30 SFC/2 × 10 ⁵ cells , at least 40 SFC/2 × 10 ⁵ cells, at least 50 SFC/2 × 10 ⁵ cells, at least 60 SFC/2 × 10 ⁵ cells, at least 70 SFC/2 × 10 ⁵ cells, at least 80 SFC/2 × 10 ⁵ cells, at least 90 SFC/2 × 10 ⁵ cells, or at least 100 SFC/2 × 10 ⁵ cells. In some embodiments, the virus-specific T cells have a specificity for the target antigen of about 10 SFC/2 × 10 ⁵ cells to about 1000 SFC/2 × 10 ⁵ cells. In some embodiments, the virus-specific T cells have a specificity for the target antigen of about 10 SFC/2 × 10 ⁵ cells to about 500 SFC/2 × 10 ⁵ cells. In some embodiments, the virus-specific T cells have a specificity for the target antigen of about 10 SFC/2 × 10 ⁵ cells to about 100 SFC/2 × 10 ⁵ cells.

In some embodiments of the expanded population of virus-specific T cells of the invention, the virus-specific T cells have a specificity for two or more antigens as measured by an IFNγ ELISpot assay of at least about 1,000 SFC /2 × 10 ⁵ cells, or at least about 1,500 SFC/2 × 10 ⁵ cells, or at least about 2,000 SFC/2 × 10 ⁵ cells, or at least about 2,500 SFC/2 × 10 ⁵ cells, or at least about 3,000 SFC/2 × ¹⁰ cells.

In some embodiments, the population of expanded VSTs includes at least 65% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 70% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 75% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 80% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 85% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 90% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 95% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 96% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 97% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 98% CD3+ T cells. In some embodiments, the population of expanded VSTs includes at least 99% CD3+ T cells.

In some embodiments, the population of expanded VSTs includes no more than 30% CD56+ cells. In some embodiments, the population of expanded VSTs includes no more than 25% CD56+ cells. In some embodiments, the population of expanded VSTs includes no more than 20% CD56+ cells. In some embodiments, the population of expanded VSTs includes no more than 15% CD56+ cells. In some embodiments, the population of expanded VSTs includes no more than 10% CD56+ cells. In some embodiments, the population of expanded VSTs includes no more than 5% CD56+ cells.

In some embodiments, the population of expanded VSTs includes CD4+ and CD8+ T cells.

In some embodiments, no more than 20% of the VSTs in the population of expanded VSTs exhibit T cell exhaustion markers. In some embodiments, no more than 15% of the VSTs in the population of expanded VSTs exhibit T cell exhaustion markers. In some embodiments, no more than 10% of the VSTs in the population of expanded VSTs exhibit T cell exhaustion markers. In some embodiments, no more than 5% of the VSTs in the population of expanded VSTs exhibit T cell exhaustion markers. In some embodiments, the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT. In some embodiments, the T cell exhaustion marker is a co-expression of PD1 and TIM3.

In some embodiments, the VSTs of the population of expanded VSTs express one or more markers associated with T cell activation. In some embodiments, one or more markers associated with T cell activation are selected from CD25 and CD69.

In some embodiments, the VSTs of the population of expanded VSTs express one or more markers associated with central or effector cell memory. In some embodiments, one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

In some embodiments, after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST-expanded population has a VST enrichment of at least 1000% compared to the cell population before expansion. times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST-amplified population has a VST enrichment of at least 3000 compared to the cell population before amplification. times. In some embodiments, after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the VST-expanded population has a VST enrichment of at least 6000 compared to the cell population before expansion. times.

In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, the VST-amplified population has a VST enrichment of at least 1000% compared to the cell population before amplification. times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, the VST-amplified population has a VST enrichment of at least 3000 compared to the cell population before amplification. times. In some embodiments, after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, the VST-amplified population has a VST enrichment of at least 6000 compared to the cell population before expansion times.

In some embodiments, the VSTs of the population of expanded VSTs are multifunctional. In some embodiments, the VSTs of the population of expanded VSTs produce two or more effector molecules when stimulated with two or more antigens. In some embodiments, the VSTs of the population of expanded VSTs produce two or more effector molecules when stimulated with three or more antigens. In some embodiments, the VSTs of the population of expanded VSTs produce two or more effector molecules when stimulated with four or more antigens. In some embodiments, the VSTs of the population of expanded VSTs produce two or more effector molecules when stimulated with five or more antigens. In some embodiments, the effector molecule is selected from the group consisting of IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a, and MIP-1b. In some embodiments, the VSTs of the population of amplified VSTs produce IFNγ. In some embodiments, the VSTs of the population of expanded VSTs produce TNF-α. In some embodiments, the VSTs of the population of amplified VSTs produce granzyme B. In some embodiments, the VSTs of the population of expanded VSTs produce IFNγ and TNFα. In some embodiments, the VSTs of the amplified population of VSTs produce IFNγ and granzyme B. In some embodiments, the VSTs of the population of amplified VSTs produce IFNγ, TNFα, and granzyme B. In some embodiments, the VSTs of the population of amplified VSTs produce perforin. In some embodiments, the VSTs of the population of expanded VSTs produce GM-CSF. In some embodiments, the VSTs of the population of amplified VSTs produce MIP-1a. In some embodiments, the VSTs of the population of amplified VSTs produce MIP-1b.

In some embodiments, the VSTs of the population of amplified VSTs produce stimulatory molecules. In some embodiments, the VSTs of the population of expanded VSTs produce GM-CSF. In some embodiments, the VSTs of the population of expanded VSTs produce IL-5.

In some embodiments, the VSTs of the population of amplified VSTs produce chemoattractive molecules. In some embodiments, the VSTs of the population of amplified VSTs produce MIP-1b.

In some embodiments, the VSTs of the population of expanded VSTs have reduced alloreactivity. In some embodiments, the VSTs of the population of amplified VSTs have negligible alloreactivity. In some embodiments, the population of expanded VSTs has VSTs that have reduced reactivity against an allogeneic target. In some embodiments, the VSTs of the population of expanded VSTs have negligible reactivity against the allogeneic target. In some embodiments, the VSTs of the population of expanded VSTs have reduced autoreactivity. In some embodiments, the VSTs of the population of expanded VSTs have negligible autoreactivity. In some embodiments, the alloreactivity of the VST is reduced compared to an unenriched T cell population. In some embodiments, VST reactivity against an allogeneic target is reduced compared to an unenriched T cell population. In some embodiments, VST has reduced autoreactivity compared to an unenriched T cell population.

In some embodiments, the present invention provides a composition comprising VST expanded in a two-step process, the two-step process comprising: a first step of culturing VST in the presence of IL-4 and IL-7; and The second step of culturing VST in the presence of -15. Multi-strain virus-specific T cell composition

In some aspects, the invention includes a multi-strain virus-specific T cell composition specific for one or more viruses. In some embodiments, viruses include, but are not limited to, EBV, CMV, adenovirus, BK virus, JC virus, HHV6, RSV, influenza virus, parainfluenza virus, Boca virus, coronavirus, LCMV, mumps virus, Measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, HSV, HBV, HCV, HDV, HPV, HIV, HTLVl, HHV8, West Nile virus, Zika virus and Ebola virus . In some embodiments, the virus includes HBV. In some embodiments, the virus is HBV. In some embodiments, the virus includes HHV8. In some embodiments, the virus is HHV8. In some embodiments, the multi-strain virus-specific T cell composition is specific for HBV. In some embodiments, the multi-strain virus-specific T cell composition is specific for HHV8. i.Antigen _

The present invention provides, at least in part, compositions comprising a multi-strain population of virus-specific T cells. In some embodiments, a multi-strain population of virus-specific T cells recognizes a plurality of viral antigens. In some embodiments, a multi-strain population of virus-specific T cells can recognize two or more or a plurality of viral antigens from a single virus. For example, in some embodiments, a multi-strain population of antigen-specific T cells can recognize two or more or a plurality of viral antigens from: EBV, CMV, adenovirus, BK, JC virus, HHV6 , RSV, influenza virus, parainfluenza virus, Boca virus, coronavirus, LCMV, mumps virus, measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, HSV, HBV, HCV , HDV, HPV, HIV, HTLVl, HHV8, West Nile virus, Zika virus and/or Ebola virus.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for HBV antigens. In some embodiments, the population of VSTs includes those specific for HBV antigen surface antigen (HBsAg), core antigen (HBcAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or combinations thereof VST. In some embodiments, the multi-strain population of VSTs includes VSTs specific for the HBV antigens E antigen (HBeAg), P antigen (HBP), and LE antigen (HBsAg). In some embodiments, HBeAg includes pre-core (PreC) and core (HbcAg) antigens.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for HBV antigen surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX), or combinations thereof. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBV antigen surface antigen (HBsAg) and core antigen (HBcAg). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HBV antigen HBsAg. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HBV antigen HBcAg. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HBV antigen HBeAg. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HBV antigen HBP. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HBV antigen HBX. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HBV antigen HBsAg. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HBV antigen HBcAg. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HBV antigen HBeAg. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HBV antigen HBP. In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HBV antigen HBX.

In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg and HBcAg. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg and HBeAg. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg and HBP. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBcAg and HBeAg. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBcAg and HBP. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBcAg and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBeAg and HBP. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBeAg and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBP and HBX.

In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBcAg, and HBeAg. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBcAg, and HBP. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBcAg, and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBeAg, and HBP. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBeAg, and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBP, and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBcAg, HBeAg, and HBP. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBcAg, HBeAg, and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBcAg, HBP, and HBX. In some embodiments, target antigens include HBeAg, HBP, and HBX.

In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBcAg, HBeAg, and HBP. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBcAg, HBeAg, and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBcAg, HBP, and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBsAg, HBeAg, HBP, and HBX. In some embodiments, the multi-strain population of VSTs includes VSTs specific for HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the multi-strain population of VSTs includes VSTs specific for each of HBsAg, HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for HHV8 antigen.

In some embodiments, the multi-strain population of VST includes responses to the HHV8 antigens LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo VST specific for Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or their combination. In some embodiments, the multi-strain population of VST includes responses to the HHV8 antigens LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo VST is a specific HHV8 antigen, one of Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least one HHV8 antigen of Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) has a specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Two HHV8 antigens, namely Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least two HHV8 antigens of Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) have specific VST. In some embodiments, the multi-strain population of VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the multi-strain population of VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and gB (ORF8). In some embodiments, the multi-strain population of VSTs includes VSTs specific for the HHV8 antigens LANA2 (ORF3) and gB (ORF8). In some embodiments, the pathogen is HHV8 and the multi-strain population of VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the multi-strain population of VSTs includes VSTs specific for the HHV8 antigens LANA1 (ORF3) and gB (ORF8). In some embodiments, the multi-strain population of VSTs includes VSTs specific for the HHV8 antigens LANA2 (ORF3) and gB (ORF8).

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Three HHV8 antigens, namely Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least three HHV8 antigens, including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST.

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo The four HHV8 antigens of Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least four HHV8 antigens, including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST.

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Five HHV8 antigens, including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo VST has specificity for at least five HHV8 antigens including Western proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Six HHV8 antigens, including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least six HHV8 antigens, including Western proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Seven HHV8 antigens, including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least seven HHV8 antigens, including Western proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Eight HHV8 antigens, namely Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least eight HHV8 antigens, including Western proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Nine HHV8 antigens, including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least nine HHV8 antigens, including Western proteins (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VSTs.

In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo Ten HHV8 antigens, including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70), have specific VST. In some embodiments, the multi-strain population of VST includes pairs selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kapo At least ten HHV8 antigens including Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) have specific VST.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen LANA-1 (ORF3). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen LANA-2 (vIRF3, K10.5). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen vCYC (ORF72). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least HHV8 antigen RTA (ORF50). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen vFLIP (ORF71). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen kaposin (ORF12, K12). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least HHV8 antigen gB (ORF8). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen MIR1 (K3). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen SSB (ORF6). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for at least the HHV8 antigen TS (ORF70).

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen LANA-1 (ORF3). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen LANA-2 (vIRF3, K10.5). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen vCYC (ORF72). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen RTA (ORF50). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen vFLIP (ORF71). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen Kaposi protein (ORF12, K12). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for HHV8 antigen gB (ORF8). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen MIR1 (K3). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen SSB (ORF6). In some embodiments, the multi-strain population of VSTs comprises VSTs specific for the HHV8 antigen TS (ORF70).

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for EBV antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one EBV antigen selected from EBNAl, LMP2, and BZLF1.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for CMV antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one CMV antigen selected from IEl and pp65.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for Adv antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one Adv antigen selected from the group consisting of hexons and pentons.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for BK virus antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one BK virus antigen selected from LT and VP-1. In some embodiments, a multi-strain population of VSTs comprising VSTs specific for BK virus antigens is reactive against JCV antigens.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for HHV6 antigen. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one HHV6 antigen selected from U14, U11, U71, U54, and U90.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for RSV antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one RSV antigen selected from N and F.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for influenza virus antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one influenza virus antigen selected from MP1 and NP1.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for parainfluenza virus (PIV) antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one parainfluenza virus (PIV) antigen selected from F, N, M, M2-1, and HN.

In some embodiments, the multi-strain population of VSTs comprises VSTs specific for hMPV antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for at least one hMPV antigen selected from F, N, M2-1, and M.

In some embodiments, the multi-strain population of VSTs includes VSTs specific for BKV, Adv, CMV, HHV-6 and EBV antigens. In some embodiments, the multi-strain population of VSTs includes VSTs specific for BKV, Adv, CMV, HHV-6, JCV and EBV antigens. In some embodiments, the multi-strain population of VST includes response to at least one BKV antigen selected from large T and VP1, at least one AdV antigen selected from hexon and penton, at least one CMV antigen selected from IE1 and pp65 , VST with specificity for at least one HHV-6 antigen selected from U11, U14 and U90 and at least one EBV antigen selected from LMP2, EBNAl and BZLF1. In some embodiments, the multi-strain population of VST includes response to at least one BKV antigen selected from large T and VP1, at least one AdV antigen selected from hexon and penton, at least one CMV antigen selected from IE1 and pp65 , at least one HHV-6 antigen selected from U11, U14 and U90 and at least one EBV antigen selected from LMP2, EBNAl and BZLF1 have specific VSTs, and these VSTs are reactive against JCV. In some embodiments, the multi-strain population of VSTs includes VSTs specific for RSV, influenza virus, PIV, and hMPV. In some embodiments, the multi-strain population of VST includes response to at least one RSV antigen selected from F and N, at least one influenza virus antigen selected from NP1 and MP1, at least one PIV antigen selected from M, HN, N, and F and a VST specific for at least one hMPV antigen selected from F, N, M2-1 and M.

In some embodiments, the multi-strain population of VSTs includes VSTs specific for SARS-CoV2 antigens. In some embodiments, the multi-strain population of VST includes pairs selected from the group consisting of spike protein (S), envelope protein (E), matrix protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5 , nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B and Y14, at least one SARS-CoV2 antigen has a specific VST.

In some embodiments, the multi-strain population of VSTs includes VSTs specific for SARS-CoV2 antigens. In some embodiments, the multi-strain population of VST includes pairs selected from the group consisting of spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5 , nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B and Y14, at least one SARS-CoV2 antigen has a specific VST.

In some embodiments, the multi-strain population of VST includes responses to the SARS-CoV2 antigens spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a (AP7a), and non-structural protein 4 ( nsp4) VST with specificity. ii.Cell phenotype

In some embodiments, the multi-strain population of VST includes CD4+ and CD8+ cells.

In some embodiments, a multi-strain population of VST produces two or more effector molecules. In some embodiments, a multi-strain population of VST produces two or more effector molecules when stimulated with two or more HBV antigens. In some embodiments, the effector molecule is selected from the group consisting of IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a, and MIP-1b.

In some embodiments, the multi-strain system of VST is generated in vitro.

In some embodiments, multiple strains of VST are produced ex vivo.

In some embodiments, a multi-strain population of VST is multifunctional.

In some embodiments, a multi-strain population of VST comprises at least 70% CD3+ T cells. In some embodiments, the multi-strain population of VST contains no more than 30% CD56+ cells.

In some embodiments, the multi-strain population of VSTs includes CD4+ and CD8+ T cells.

In some embodiments, no more than 20% of the multi-strain population of VST exhibits a T cell exhaustion marker. In some embodiments, the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT. In some embodiments, the T cell exhaustion markers are PD1 and TIM3.

In some embodiments, a population of strains of VST expresses one or more markers associated with T cell activation. In some embodiments, one or more markers associated with T cell activation are selected from CD25 and CD69.

In some embodiments, a multi-strain population of VST expresses one or more markers associated with central or effector cell memory. In some embodiments, one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

In some embodiments, the multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in medium containing cytokines. In some embodiments, a multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in media containing IL-4. In some embodiments, a multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in media containing IL-7. In some embodiments, a multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in media containing IL-15. In some embodiments, a multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in media containing IL-4 and IL-7. In some embodiments, a multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in media containing IL-4 and IL-15. In some embodiments, a multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in media containing IL-7 and IL-15. In some embodiments, a multi-strain population of VST is enriched 500- to 7000-fold in VST after expansion in culture medium comprising IL-4, IL-7, and IL-15.

In some embodiments, the multi-strain population of VST is enriched in VST by 500 to 7000 after amplification in IL-4, IL-7 and IL-15 in the presence of viral antigen compared to the cell population before amplification times. In some embodiments, the multi-strain population of VST is at least 500-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. . In some embodiments, the multi-strain population of VST is at least 1000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. . In some embodiments, the multi-strain population of VST is at least 2000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. . In some embodiments, the multi-strain population of VST is at least 3000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. . In some embodiments, the multi-strain population of VST is at least 4000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. . In some embodiments, the multi-strain population of VST is at least 5000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. . In some embodiments, the multi-strain population of VST is at least 6000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. . In some embodiments, the multi-strain population of VST is at least 7000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of viral antigen compared to the cell population prior to amplification. .

In some embodiments, the multi-strain population of VST is enriched in VST by 500 after amplification in IL-4, IL-7 and IL-15 in the presence of latent viral antigen compared to the cell population before amplification. to 7000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 500 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 1000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 2000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 3000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 4000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 5000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 6000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of latent viral antigen, the VST enrichment of the multi-strain population of VST is at least 7000 times.

In some embodiments, the multi-strain population of VST is enriched in VST by 500 after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen compared to the cell population before amplification. to 7000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 500 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 1000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 2000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 3000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 4000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 5000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 6000 times. In some embodiments, after amplification in IL-4, IL-7, and IL-15 in the presence of lytic viral antigen, the VST enrichment of the multi-strain population of VST is at least 7000 times.

In some embodiments, the multi-strain population of VST is enriched by 500 to 7000 VST after expansion in IL-4, IL-7 and IL-15 in the presence of HBV antigen compared to the cell population before expansion. times. In some embodiments, the multi-strain population of VST is at least 500-fold enriched in VST after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. . In some embodiments, the multi-strain population of VST is at least 1000-fold enriched in VST after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. . In some embodiments, the multi-strain population of VST is at least 2000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. . In some embodiments, the multi-strain population of VST is at least 3000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. . In some embodiments, the multi-strain population of VST is at least 4000-fold enriched in VST after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. . In some embodiments, the multi-strain population of VST is at least 5000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. . In some embodiments, the multi-strain population of VST is at least 6000-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. . In some embodiments, the multi-strain population of VST is at least 7000-fold enriched in VST after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen compared to the cell population before expansion. .

In some embodiments, the multi-strain population of VST is enriched in VST by 500 to 7000 after expansion in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before expansion times. In some embodiments, the multi-strain population of VST is at least 500-fold enriched in VST after amplification in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen compared to the cell population prior to expansion. . In some embodiments, the multi-strain population of VST is at least 1000-fold enriched in VST after amplification in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before amplification . In some embodiments, the multi-strain population of VST is at least 2000-fold enriched in VST after amplification in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before amplification . In some embodiments, the multi-strain population of VST is at least 3000-fold enriched in VST after amplification in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before amplification . In some embodiments, the multi-strain population of VST is at least 4000-fold enriched in VST after amplification in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before amplification . In some embodiments, the multi-strain population of VST is at least 5000-fold enriched in VST after amplification in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before amplification . In some embodiments, the multi-strain population of VST is at least 6000-fold enriched in VST after amplification in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before amplification . In some embodiments, the multi-strain population of VST is at least 7000-fold enriched in VST after amplification in IL-4, IL-7 and IL-15 in the presence of HHV8 antigen compared to the cell population before amplification .

In some embodiments, the multi-strain population of VST is an antigen-specific multi-strain population of VST.

In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HBV antigens. In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg. In some embodiments, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBeAg. In some embodiments, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg and one or more peptide sequences derived from HBeAg. In some embodiments, the antigen-specific multistrain population of VST pairs at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO. At least one peptide sequence of NO: 5 is specific.

In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HHV8 antigens. In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1. In some embodiments, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NOs: 6-12. In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from gB. In some embodiments, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NOs: 13-15. In some embodiments, the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1 and one or more peptide sequences derived from gB. In some embodiments, the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 6-12 and at least one peptide sequence selected from SEQ ID NO: 13-15.

In some embodiments, the multi-strain population of VSTs has reduced alloreactivity. In some embodiments, the VST of the multi-strain population of VST has negligible alloreactivity. In some embodiments, the multi-strain population of VSTs has reduced reactivity against an allogeneic target. In some embodiments, the multi-strain population of VSTs has negligible reactivity against an allogeneic target. In some embodiments, the VST of the multi-strain population of VST has reduced autoreactivity. In some embodiments, the VST of the multi-strain population of VST has negligible autoreactivity.

In some embodiments, the VST of the multi-strain population of VST has negligible autoreactivity. In some embodiments, the alloreactivity of a multi-strain population of VST is reduced compared to an unenriched T cell population. In some embodiments, the multi-strain population of VST has reduced reactivity against an allogeneic target compared to an unenriched T cell population. In some embodiments, the multi-strain population of VST has reduced autoreactivity compared to an unenriched T cell population. Universal Antigen-Specific T Cell Compositions

In one aspect, the invention provides a universal antigen-specific T cell therapy product comprising an expanded T cell population or a plurality of expanded T cell populations according to the invention, wherein the product comprises a population obtained from different suppliers. T cells generated from donor monocytes.

In some embodiments, each donor's HLA type differs from at least one of the other donors by at least one HLA allele.

In some aspects, the invention provides a universal antigen-specific T cell therapy product comprising an expanded T cell population or a plurality of expanded T cell populations according to the invention, wherein the product comprises a population obtained from a different supplier. T cells generated from donor monocytes. A method of generating a universal antigen-specific T cell therapy product comprising a population of antigen-specific T cells may comprise: (i) culturing cells from a plurality of donors (e.g., wherein each donor has an HLA type that is the same as that of at least one of the other donors) There are a plurality of donors who differ in at least one HLA allele, and/or a single spheroid from each donor included in a plurality of suitable donors in a donor mini-library as described herein, each of which is in a or cultured separately in the presence of multiple interleukins and one or more antigens to generate individual cell lines of a plurality of amplified antigen-specific T cells; and (ii) pooling individual cell lines together to generate universal antigen-specific T cells Cell therapy products. In some embodiments, the monocytes are peripheral blood monocytes (PBMC).

In some embodiments, the method further includes one or more freeze-thaw steps. For example, in some embodiments, each cell line is cryopreserved and subsequently thawed prior to pooling in (ii). In other embodiments, the cell lines are pooled together as freshly prepared cell lines without any freeze-thaw steps prior to pooling in (ii). In some embodiments, the methods include freezing the pool of cell lines obtained in (ii). In some embodiments, the methods include generating a plurality of individual cell lines as provided in (i), determining cell line identity, viability, sterility, phenotype, potency and/or alloreactivity, freezing the individual cells strains, followed by thawing individual cell lines, and pooling the cell lines together to form a universal antigen-specific T cell therapy product. Alternatively, such methods include generating a plurality of individual cell lines as provided in (i), freezing the individual cell lines, thawing the individual cell lines and subsequently determining cell line identity, viability, sterility, phenotype, potency and/or Alloreactivity, individual cell lines are then pooled together to form a universal antigen-specific T cell therapy product, or individual cell lines are refrozen and subsequently thawed and pooled together to form a universal antigen-specific T cell therapy product. In some embodiments, the invention provides a method for generating a universal antigen-specific T cell therapy product comprising a population of antigen-specific T cells, the method comprising: (i) pooling data from a plurality of donors (e.g., wherein A plurality of donors whose HLA type differs from that of at least one of the other donors by at least one HLA allele, and/or a plurality of suitable donors included in a donor mini-library as described herein) monocytes from each donor; and (ii) culturing a pool of monocytes in the presence of one or more interleukins and one or more antigens to generate a population of expanded antigen-specific T cells. In some embodiments, the monocytes are peripheral blood monocytes (PBMC). In some embodiments, the cell line identity, viability, sterility, phenotype, potency, and/or alloreactivity of the pooled cells is determined as provided herein. In some embodiments, the pooled cells can be frozen after steps (i) and/or (ii). Universal antigen-specific T cell products and analogs thereof are discussed in PCT/US2021/016266, which is incorporated by reference in its entirety.

In some embodiments, methods provided herein include freezing individual antigen-specific T cell lines generated as provided herein and/or pooled universal antigen-specific T cell therapy products in cryopreservation medium. In some embodiments, the cryopreservation medium includes human serum albumin, Hank's balanced salt solution (Hank's balanced salt solution; HBSS), and dimethylsulfoxide (DMSO). In some embodiments, the culture medium contains about 10% (v/v) DMSO. In some embodiments, the culture medium includes about 50% (v/v) 25% human serum albumin and about 40% (v/v) HBSS. In some embodiments, the pooled universal antigen-specific T cell therapy products are cryopreserved and stored until selected for use in a method of treating a disease or condition in a patient. Accordingly, the present invention provides compositions comprising antigen-specific T cell lines and/or pooled universal antigen-specific T cell therapy products in cryopreservation medium. In some embodiments, the methods further include one or more filtering steps. In some embodiments, the methods further comprise filtering each cell line obtained in step (i) of the previous paragraph. In some embodiments, the methods further comprise filtering the pooled universal antigen-specific T cell therapy product obtained in (ii) of the preceding paragraph. In some embodiments, the methods further comprise filtering each cell line before and/or after the freeze-thaw step and/or filtering the pooled universal antigen-specific T cell therapy product.

In some embodiments, the methods further comprise transfecting one or more individual cell lines obtained in (i) of the first two paragraphs with a transfection gene. In some embodiments, the methods further comprise transfecting the pooled cell lines obtained in (ii) of the first two paragraphs with a transfection gene. In some embodiments, the transgene encodes a chimeric antigen receptor (CAR), T cell receptor (TCR), or NK cell receptor.

In some embodiments, the culturing step of the production methods provided herein is performed in a container that includes a gas-permeable culturing surface. In some embodiments, the vessel is a GRex bioreactor. In some embodiments, the one or more interleukin systems cultured with the monocytes and the antigen are selected from the group consisting of: IL-1, IL-2, IL-4, IL-6, IL-7, IL -12, IL-15 and IL-21. In some embodiments, the one or more interleukins incubated with the monocytes and antigen are IL-4 and/or IL-7. In some embodiments, the interleukins include IL-4 and IL-7 and do not include IL-15. In some embodiments where the one or more interleukins cultured with the monocytes and antigen are IL-4 and/or IL-7 and do not include IL-15, IL-15 is then added later in the culture.

In some embodiments, each donor's HLA type differs from at least one of the other donors by at least one HLA allele. In some embodiments, the product is not alloreactive to partially HLA matched and/or to HLA mismatched target cells. In some embodiments HLA type is assessed by the abundance of HLA-A, HLA-B, HLA-C, HLA-DPBl, HLA-DQB1 and/or DRB-1 phenotypic characteristics.

In some embodiments, one or more antigens are in the form of: (a) a whole protein; (b) a mixed peptide comprising a series of overlapping peptides covering a portion or the entire sequence of each antigen; or (c) (a) combined with (b) combination. In some embodiments, the antigens comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more Different mixtures of peptides. Mixed peptides can be specific for the specific desired antigen.

In some embodiments, the one or more antigens are viral antigens or tumor-associated antigens. In some embodiments, each antigen in the culture is a viral antigen. In some embodiments, the viral antigen is from a virus selected from: EBV, CMV, adenovirus, BK virus, JC virus, HHV6, RSV, influenza virus, parainfluenza virus, bocavirus, coronavirus, LCMV, parotid virus inflammatory virus, measles virus, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, HSV, HBV, HCV, HDV, HPV, HIV, HTLVl, HHV8, West Nile virus, Zika virus and Ebola virus. In some embodiments, the viral antigen is from HBV. In some embodiments, the viral antigen is derived from HHV8.

In some embodiments, the antigen is selected from one or more of large T and VP1 from BKV. In some embodiments, the antigen is selected from one or more of the hexon and penton from Adv. In some embodiments, the antigen is selected from one or more of IEl and pp65 from CMV. In some embodiments, the antigen is selected from one or more of U11, U14, and U90 from HHV-6. In some embodiments, the antigen is selected from one or more of LMP2, EBNA, and BZLF1 from EBV. In some embodiments, the antigen is selected from one or more of F and N from RSV. In some embodiments, the antigen is selected from one or more of NP1 and MP1 from influenza virus. In some embodiments, the antigen is selected from one or more of M, HN, N, and F from PIV. In some embodiments, the antigen is selected from one or more of F, N, M2-1, and M from hMPV.

In some embodiments, the antigen is selected from any combination of i. Big T and VP1 from BKV; ii. Hexon and penton from Adv; iii. IE1 and pp65 from CMV; iv. U11, U14 and U90 from HHV-6; and v. LMP2, EBNA and BZLF1 from EBV.

In some embodiments, the antigen is selected from any combination of i.F and N from RSV; ii. NP1 and MP1 from influenza virus; iii.M, HN, N and F from PIV; and iv. F, N, M2-1 and M from hMPV.

In some embodiments, the antigen includes HBsAg. In some embodiments, the antigen includes HBcAg. In some embodiments, the antigen includes HBeAg. In some embodiments, the antigen includes HBP. In some embodiments, the antigen includes HBX. In some embodiments, the antigen is HBsAg. In some embodiments, the antigen is HBcAg. In some embodiments, the antigen is HBeAg. In some embodiments, the antigen is HBP. In some embodiments, the antigen is HBX.

In some embodiments, the antigens include HBsAg and HBcAg. In some embodiments, the antigens include HBsAg and HBeAg. In some embodiments, the antigens include HBsAg and HBP. In some embodiments, the antigens include HBsAg and HBX. In some embodiments, the antigens include HBcAg and HBeAg. In some embodiments, the antigens include HBcAg and HBP. In some embodiments, the antigens include HBcAg and HBX. In some embodiments, the antigens include HBeAg and HBP. In some embodiments, the antigens include HBeAg and HBX. In some embodiments, the antigens include HBP and HBX.

In some embodiments, the antigens include HBsAg, HBcAg, and HBeAg. In some embodiments, the antigens include HBsAg, HBcAg, and HBP. In some embodiments, the antigens include HBsAg, HBcAg, and HBX. In some embodiments, the antigens include HBsAg, HBeAg, and HBP. In some embodiments, the antigens include HBsAg, HBeAg, and HBX. In some embodiments, the antigens include HBsAg, HBP, and HBX. In some embodiments, the antigens include HBcAg, HBeAg, and HBP. In some embodiments, the antigens include HBcAg, HBeAg, and HBX. In some embodiments, the antigens include HBcAg, HBP, and HBX. In some embodiments, the antigens include HBeAg, HBP, and HBX.

In some embodiments, the antigens include HBsAg, HBcAg, HBeAg, and HBP. In some embodiments, the antigens include HBsAg, HBcAg, HBeAg, and HBX. In some embodiments, the antigens include HBsAg, HBcAg, HBP, and HBX. In some embodiments, the antigens include HBsAg, HBeAg, HBP, and HBX. In some embodiments, the antigens include HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the antigen includes each of HBsAg, HBcAg, HBeAg, HBP, and HBX.

In some embodiments, the at least one antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof. In some embodiments, an antigen is selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12 , K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the two antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the two antigens are LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the two antigens are LANA1 (ORF3) and gB (ORF8). In some embodiments, the two antigens are LANA2 (ORF3) and gB (ORF8). In some embodiments, the at least two antigens are LANA1 (ORF3) and LANA2 (vIRF3, K10.5). In some embodiments, the at least two antigens are LANA1 (ORF3) and gB (ORF8). In some embodiments, the at least two antigens are LANA2 (ORF3) and gB (ORF8).

In some embodiments, the three antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12 , K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least three antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the four antigen lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least four antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the five antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least five antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the six antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least six antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the seven antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least seven antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the eight antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least eight antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the nine antigenic lines are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least nine antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the ten antigens are LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). In some embodiments, the at least ten antigens are LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12 , K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

In some embodiments, the antigen includes LANA-1 (ORF3). In some embodiments, the antigen includes LANA-2 (vIRF3, K10.5). In some embodiments, the antigen includes vCYC (ORF72). In some embodiments, the antigen includes RTA (ORF50). In some embodiments, the antigen includes vFLIP (ORF71). In some embodiments, the antigen includes Kaposi protein (ORF12, K12). In some embodiments, the antigen includes gB (ORF8). In some embodiments, the antigen includes MIR1 (K3). In some embodiments, the antigen includes SSB (ORF6). In some embodiments, the antigen includes TS (ORF70).

In some embodiments, the antigen is LANA-1 (ORF3). In some embodiments, the antigen is LANA-2 (vIRF3, K10.5). In some embodiments, the antigen is vCYC (ORF72). In some embodiments, the antigen is RTA (ORF50). In some embodiments, the antigen is vFLIP (ORF71). In some embodiments, the antigen is Kaposi protein (ORF12, K12). In some embodiments, the antigen is gB (ORF8). In some embodiments, the antigen is MIR1 (K3). In some embodiments, the antigen is SSB (ORF6). In some embodiments, the antigen is TS (ORF70).

In some embodiments, each antigen in the culture is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is one or more of the following: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, MAGE A3, p53 non-mutant, NY-ESO- 1. PSMA, GD2, melanin A/MARTI, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML- IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl, meta Cortin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY- TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fos Related antigen l.

In some embodiments, the product includes virus-specific T cells (VST) that recognize one or more target antigens from at least one pathogen disclosed herein, or a portion thereof. In some embodiments, the product includes virus-specific T cells (VST) that recognize one or more target antigens from at least one latent virus, or portions thereof. In some embodiments, the product includes virus-specific T cells (VST) that recognize one or more target antigens from at least one lytic virus, or a portion thereof. Pharmaceutical composition

In one aspect, the invention provides a pharmaceutical composition comprising an expanded T cell population, a plurality of expanded T cell populations or a universal antigen-specific T cell therapy product of the invention. In some aspects, the present invention provides a pharmaceutical composition comprising an expanded T cell population, a plurality of expanded T cell populations, or a universal antigen-specific T cell therapy product, each of which is an expanded population according to the present invention. T cells. In some embodiments, the invention provides pharmaceutical compositions comprising an expanded population of T cells. In some embodiments, the invention provides pharmaceutical compositions comprising a plurality of expanded T cell populations. In some embodiments, the invention provides pharmaceutical compositions comprising universal antigen-specific T cell therapy products.

In some embodiments, the invention provides pharmaceutical compositions comprising VST. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for HBV. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for HHV8. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for BKV. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for AdV. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for CMV. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for HHV-6. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for EBV. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for RSV. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for influenza virus. In some embodiments, the present invention provides pharmaceutical compositions comprising VST specific for PIV. In some embodiments, the invention provides pharmaceutical compositions comprising VST specific for hMPV.

In some embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients or diluents. As used herein, the term "pharmaceutical composition" refers to a pharmaceutically acceptable composition, wherein the composition includes a pharmaceutically active agent, and in some embodiments further includes a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition can be a combination of a pharmaceutically active agent and a carrier.

In some embodiments, pharmaceutical compositions comprise the universal antigen-specific T cell therapy products of the invention. In some embodiments, the universal antigen-specific T cell therapy product of the present invention further includes one or more pharmaceutically acceptable excipients or diluents. In some embodiments, a pharmaceutical composition includes an expanded population of VSTs of the invention. In some embodiments, the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable excipients or diluents. In some embodiments, a pharmaceutical composition is administered to an individual without knowledge of the individual's HLA type.

In some embodiments, a pharmaceutical composition includes a population of multiple strains of VST of the invention. In some embodiments, the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable excipients or diluents. In some embodiments, a pharmaceutical composition is administered to an individual without knowledge of the individual's HLA type.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopoeia, other recognized pharmacopeias, and in animals and more particularly in humans and/or Other formulations safe for use in non-human mammals.

As used herein, the term "pharmaceutically acceptable diluent or excipient" or "pharmaceutically acceptable carrier" refers to an excipient, diluent, or excipient that is administered with the T cells of the invention. Preservatives, solubilizers, emulsifiers, adjuvants and/or vehicles. Such carriers may be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycol, glycerol, Propylene glycol or other synthetic solvent. The following may also be carriers: antimicrobial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and tonicity regulators, such as chloride sodium or dextrose. Methods for producing compositions in combination with carriers are known to those skilled in the art. In some embodiments, the term "pharmaceutically acceptable diluent or excipient" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration, as well as the Similar. The use of such media and agents for pharmaceutical active substances is well known in the art. See, for example, Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Such use in the compositions is contemplated unless any conventional media or agent is incompatible with the active compound.

Formulations suitable for administration of pharmaceutical compositions generally generally comprise the active ingredient in combination with a pharmaceutically acceptable diluent or excipient, such as sterile water or sterile isotonic saline. Such formulations may be suitable for preparation, packaging or sale in a form suitable for bolus injection or continuous administration. Injectable formulations may be prepared, packaged or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing or dispersing agents. Formulations may also include aqueous solutions, which may contain excipients such as salts, carbohydrates and buffers or sterile pyrogen-free water. Exemplary administration forms may include solutions or suspensions in sterile aqueous solutions, such as propylene glycol or aqueous dextrose solutions. Where necessary, such dosage forms may be appropriately buffered.

The compositions of the present invention may additionally contain other adjuvant components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials, such as antipruritic, astringent, local anesthetic or anti-inflammatory agents, or may contain additional materials suitable for physical formulation of the various dosage forms of the compositions of the present invention. Such as dyes, preservatives, antioxidants, opacifiers, thickeners and stabilizers. However, such materials should not be added so as to unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations can be sterilized and optionally mixed with auxiliaries such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants and/or aromatics. family of substances and their analogues that do not interact deleteriously with the formulation. In some embodiments, pharmaceutical compositions include the T cells in combination with other therapeutically active agents. In some embodiments, pharmaceutical compositions comprise the T cells in combination with antibodies specific for the disease cell phenotype. In some embodiments, the disease cell phenotype is that of a malignant tumor cell. In some embodiments, the disease cell phenotype is that of viral infection. In some embodiments, the disease cell phenotype is that of pathogen infection.

The term "combination" means a fixed combination in a unit dosage form, or a set of portions for combined administration, of one or more active compounds and a combination partner (such as another drug as explained below, also (referred to as "therapeutic agents" or "adjuvants") may be administered independently at the same time or administered separately over time intervals. In some cases, combining items exhibits cooperative effects, such as synergy. As used herein, the terms "co-administration" or "combination administration" or the like are intended to encompass the administration of a selected combination of agents to a single individual in need thereof (e.g., a patient), and are intended to include where the agent is not necessarily administered by Administer the same treatment regimen by the same route or at the same time. The term "pharmaceutical combination" as used herein means a product resulting from mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients (eg, compounds and combination partners) are all administered simultaneously to a patient in a single entity or dosage form. The term "non-fixed combination" means that the active ingredients (e.g., compounds and combination partners) are administered as separate entities to a patient simultaneously, concurrently, or sequentially (without any particular time limit), where such administration is provided in the patient's body Two compounds with therapeutically effective levels. The latter also applies to mixture therapies, for example, the administration of three or more active ingredients.

Depending on whether local or systemic treatment is desired, the immune cells or pharmaceutical compositions of the invention can be administered in a variety of ways. Antigen-specific T cells or pharmaceutical compositions of expanded T cells according to the present invention are generally suitable for parenteral administration, wherein the administration includes administration characterized by physically breaking the tissue of the individual and administering through a breach in the tissue. Any route of administration of a pharmaceutical composition, thereby typically resulting in direct administration into the bloodstream, into muscles, or into internal organs. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injecting the composition, by applying the composition through a surgical incision, by applying the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated including, but not limited to, subcutaneous, intraperitoneal, intramuscular, intrabrainstem, intravenous, intranasal, intratracheal, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, Intratumoral, intraocular, intradermal, intrasynovial injection or infusion, intratumoral; and renal dialysis infusion technology. In some embodiments, the immune cells or pharmaceutical compositions of the invention comprise intravenous administration. In some embodiments, the immune cells or pharmaceutical compositions of the invention comprise intratumoral administration. In some embodiments, pharmaceutical compositions are formulated for intravenous delivery. In some embodiments, pharmaceutical compositions are formulated for intravenous administration. In some embodiments, pharmaceutical compositions are formulated for intravenous infusion. In some embodiments, the pharmaceutical composition is formulated for administration via a peripheral catheter. In some embodiments, pharmaceutical compositions are formulated for administration via a central catheter.

In some embodiments, the compositions include a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a cryopreservation medium. Suitable pharmaceutically acceptable carriers will be known to those skilled in the art.

The present invention provides, at least in part, pharmaceutical compositions comprising any composition formulated for intravenous delivery (e.g., antigen-specific T cells from a donor minibank described herein formulated for intravenous delivery) strains of pharmaceutical compositions).

The invention provides, at least in part, a pharmaceutical composition comprising any composition formulated for intravenous delivery (eg, a pharmaceutical composition comprising a virus-specific T cell strain of the invention formulated for intravenous delivery).

In some embodiments, the composition is maintained in culture for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , negative for bacteria and fungi within 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In some embodiments, the composition is negative for bacteria and fungi for at least about 1 day in culture. In some embodiments, the composition is negative for bacteria and fungi for at least about 2 days in continued culture. In some embodiments, the composition is negative for bacteria and fungi for at least about 3 days in continued culture. In some embodiments, the composition is negative for bacteria and fungi for at least about 4 days in continued culture. In some embodiments, the composition is negative for bacteria and fungi for at least about 5 days in continued culture. In some embodiments, the composition is negative for bacteria and fungi for at least about 6 days in continued culture. In some embodiments, the composition is negative for bacteria and fungi for at least about 7 days in continued culture. In some embodiments, the composition is negative for bacteria and fungi during continued culture for at least 8 days.

In some embodiments, the composition exhibits less than about 20 EU/mL endotoxins. In some embodiments, the composition exhibits less than about 10 EU/mL endotoxins. In some embodiments, the composition exhibits less than about 7.5 EU/mL endotoxin. In some embodiments, the composition exhibits less than about 5 EU/mL of endotoxin. In some embodiments, the composition exhibits less than 5 EU/mL of endotoxin. In some embodiments, the composition is negative for Mycoplasma species. Instructions

The invention provides a method of lysing a target cell, comprising contacting the target cell with an antigen-specific composition or a pharmaceutical composition as described herein (e.g., an expanded T cell according to the invention formulated for intravenous delivery). T cell lines or pharmaceutical compositions containing such T cell lines). In some embodiments, contact between the target cells and the composition or pharmaceutical composition occurs in vivo in the individual. In some embodiments, contact between the target cells and the composition or pharmaceutical composition occurs in vivo via administration of antigen-specific T cells to the individual. In some embodiments, contact between the target cells and the composition or pharmaceutical composition occurs in vivo via administration of virus-specific T cells to the individual. In some embodiments, the individual is a human.

In some embodiments, the antigen-specific T cells of the invention are virus specific. In some embodiments, virus-specific T cells of the invention are antigen-specific. In some embodiments, the antigen-specific T cells of the invention are virus-specific T cells. In some embodiments, T cells generated using the methods of the invention are antigen-specific. In some embodiments, T cells generated using the methods of the invention are virus specific. In some embodiments, T cells generated using the methods of the invention are antigen-specific and virus-specific.

In some embodiments, virus-specific T cells comprise VST specific for one or more latent viruses. In some embodiments, virus-specific T cells comprise VST specific for one or more lytic viruses. In some embodiments, the virus-specific T cells comprise VSTs specific for one or more of: CMV, Adv, BKV, HPV, HAV, HBV, HCV, HDV, HSV-1, HSV-2, HIV, VZV, EBV, CMV, JCV, HHV-6, HHV-8, HHV-7, KSHV, RSV, influenza virus, PIV, Bocavirus, coronavirus, LCMV, mumps virus, measles virus, hMPV, small Virus B, rotavirus, Merkel cell virus, WNV, Zika virus, Ebola virus, SARS-CoV and human T-lymphotropic virus. In some embodiments, the virus-specific T cells comprise VST specific for HBV. In some embodiments, the virus-specific T cells comprise VST specific for HHV8. In some embodiments, the virus-specific T cells comprise VST specific for CMV. In some embodiments, virus-specific T cells comprise VST specific for AdV. In some embodiments, virus-specific T cells comprise VST specific for BKV. In some embodiments, the virus-specific T cells comprise VST specific for HPV. In some embodiments, the virus-specific T cells comprise VST specific for HCV. In some embodiments, the virus-specific T cells comprise VST specific for HDV. In some embodiments, the virus-specific T cells comprise VST specific for HSV-1. In some embodiments, the virus-specific T cells comprise VST specific for HSV-2. In some embodiments, the virus-specific T cells comprise VST specific for HIV. In some embodiments, virus-specific T cells comprise VST specific for VZV. In some embodiments, virus-specific T cells comprise VST specific for EBV. In some embodiments, the virus-specific T cells comprise VST specific for HHV-6. In some embodiments, the virus-specific T cells comprise VST specific for HHV-7. In some embodiments, the virus-specific T cells comprise VST specific for KSHV. In some embodiments, the virus-specific T cells comprise VST specific for RSV. In some embodiments, the virus-specific T cells comprise VST specific for influenza virus. In some embodiments, the virus-specific T cells comprise VST specific for parainfluenza virus. In some embodiments, the virus-specific T cells comprise VST specific for Bocavirus. In some embodiments, the virus-specific T cells comprise VST specific for coronavirus. In some embodiments, the virus-specific T cells comprise VST specific for LCMV. In some embodiments, the virus-specific T cells comprise VST specific for mumps virus. In some embodiments, the virus-specific T cells comprise VST specific for measles virus. In some embodiments, the virus-specific T cells comprise VST specific for hMPV. In some embodiments, the virus-specific T cells comprise VST specific for parvovirus B. In some embodiments, the virus-specific T cells comprise VST specific for rotavirus. In some embodiments, the virus-specific T cells comprise VST specific for Merkel cell virus. In some embodiments, virus-specific T cells comprise VST specific for WNV. In some embodiments, the virus-specific T cells comprise VST specific for Zika virus. In some embodiments, the virus-specific T cells comprise VST specific for Ebola virus. In some embodiments, the virus-specific T cells comprise VST specific for human T-lymphotropic virus. In some embodiments, the virus-specific T cells comprise VST specific for SARS-CoV.

The invention provides methods of treating a disease or condition by administering to a patient one or more suitable antigen-specific T cell lines of expanded T cells according to the invention (e.g., two or more such T cells cell lines) and/or universal antigen-specific T cell products described herein. As used herein, the term "treatment" includes prophylactic and therapeutic treatment. In some embodiments, prophylactic treatment refers to treating an individual who is at risk of infection with a pathogen, such as a virus. In some embodiments, therapeutic treatment refers to treating an individual infected with a pathogen (eg, virus).

In some embodiments, an individual may suffer from one or more medical conditions. In some embodiments, the individual receives a low-intensity conditioning matched related donor transplant prior to receiving antigen-specific T cells. In some embodiments, the individual receives a myeloablatively primed matched unrelated donor transplant prior to receiving antigen-specific T cells. In some embodiments, the individual receives a low-intensity conditioning haploidentical transplant before receiving antigen-specific T cells. In some embodiments, the individual receives a myeloablatively primed matched related donor transplant prior to receiving antigen-specific T cells. In some embodiments, the individual has received a solid organ transplant. In some embodiments, the individual has received chemotherapy. In some embodiments, the individual has an HIV infection. In some embodiments, the individual suffers from a genetic immunodeficiency. In some embodiments, the individual has received an allogeneic stem cell transplant. In some embodiments, an individual suffers from more than one medical condition as described in this paragraph.

In some aspects, the invention provides a method of treating or preventing a disease or condition in an individual in need thereof, comprising administering to the individual a T cell of the invention or a pharmaceutical composition of the invention. In some aspects, the invention provides a method of treating or preventing a disease or condition in an individual in need thereof, comprising administering to the individual a T cell expanded T cell according to the invention or a pharmaceutical composition of the invention . In some embodiments, the invention provides a method of treating or preventing a disease or condition in an individual in need thereof, comprising administering to the individual an expanded T cell of the invention or a pharmaceutical composition of the invention. In some embodiments, two or more cell lines of donor minipools generated as described by any of the methods provided herein can be pooled together to produce a universal antigen-specific T cell product. In some embodiments, two or more cell lines of a donor minipool generated as described herein can be used as a universal antigen-specific T cell product, for example, by administration to a patient in a single administration session. In some embodiments, the disease or condition is malignancy. In some embodiments, the malignant tumor contains a tumor-associated antigen. In some embodiments, the disease or condition is a viral infection. In some embodiments, the viral infection is from a virus containing an antigen associated with viral infection. In some embodiments, the viral infection comprises viral infection-associated antigens.

In some embodiments, the T cells or pharmaceutical compositions are administered by parenteral administration. In some embodiments, T cells or pharmaceutical compositions are administered by injection or infusion. In some embodiments, the antigen-specific T cells expanded T cells according to the invention or the pharmaceutical compositions of the invention can be administered by parenteral administration. In some embodiments, antigen-specific T cells or pharmaceutical compositions of expanded T cells according to the invention are administered by injection or infusion. In some embodiments, antigen-specific T cells or pharmaceutical compositions comprising antigen-specific T cells are administered by injection. In some embodiments, the antigen-specific T cells or pharmaceutical compositions comprising antigen-specific T cells are administered by infusion.

In some embodiments, virus-specific T cells or pharmaceutical compositions are administered by parenteral administration. In some embodiments, virus-specific T cells or pharmaceutical compositions are administered by injection or infusion. In some embodiments, virus-specific T cells expanded T cells according to the invention or pharmaceutical compositions of the invention can be administered by parenteral administration. In some embodiments, expanded T cells, virus-specific T cells or pharmaceutical compositions according to the invention are administered by injection or infusion. In some embodiments, virus-specific T cells or pharmaceutical compositions comprising virus-specific T cells are administered by injection. In some embodiments, virus-specific T cells or pharmaceutical compositions comprising virus-specific T cells are administered by infusion.

In some embodiments, the invention provides a method for treating or preventing viral infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some embodiments, the invention provides a method for treating or preventing viral infection in an individual in need thereof by one or more of: CMV, Adv, BKV, HPV, HAV, HBV, HCV, HDV, HSV -1, HSV-2, HIV, VZV, EBV, CMV, JCV, HHV-6, HHV-8, HHV-7, KSHV, RSV, influenza virus, PIV, Boca virus, coronavirus, LCMV, mumps virus , measles virus, hMPV, parvovirus B, rotavirus, Merkel cell virus, WNV, Zika virus, Ebola virus, SARS-CoV and human T-lymphotropic virus, comprising administering to the individual an effective amount of Compositions comprising a multi-strain population of VST of the invention.

In some embodiments, the invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some embodiments, the invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises Specificity for two or more HBV antigens. In some embodiments, the invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some embodiments, the invention provides a method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of the VST of the invention. In some embodiments, a multi-strain population of VST comprises specificity for two or more HBV antigens. In some embodiments, HBV antigens include E antigen (HBeAg), P antigen (HBP), and LE antigen (HBsAg). In some embodiments, HBeAg (E antigen) includes pre-core (PreC) and core (HBcAg) antigens.

In some embodiments, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some embodiments, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises Specificity for two or more HHV8 antigens. In some embodiments, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some embodiments, the invention provides a method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of the VST of the invention. In some embodiments, a multi-strain population of VST comprises specificity for two or more HHV8 antigens. In some embodiments, the HHV8 antigens include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12 ), two or more of gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70). In some embodiments, the HHV8 antigens include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12 ), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70).

In some embodiments, the invention provides a method for treating or preventing SARS-CoV infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some embodiments, the invention provides a method for treating or preventing SARS-CoV infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST Populations contain specificities for two or more SARS-CoV antigens. In some embodiments, the invention provides a method for treating or preventing SARS-CoV infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of the VST of the invention. In some embodiments, the invention provides a method for treating or preventing SARS-CoV infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of the VST of the invention. In some embodiments, a multi-strain population of VSTs includes specificity for two or more SARS-CoV antigens. In some embodiments, the SARS-CoV antigen includes two of spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a (AP7a), and non-structural protein 4 (nsp4) or more. In some embodiments, SARS-CoV antigens include spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a (AP7a), and nonstructural protein 4 (nsp4).

In some embodiments, a composition comprising a population of VSTs of the invention is administered to an individual in need thereof. In some embodiments, about 5 × 10 ⁶ to about 5 × 10 ⁸ cells/square meter are administered to the individual. In some embodiments, 5 × 10 ⁶ to 5 × 10 ⁸ cells/square meter are administered to the subject. In some embodiments, between about 5 × 10 ⁶ and about 5 × 10 ⁷ cells/square meter are administered to the individual. In some embodiments, about 5 × 10 ⁶ to about 5 × 10 ⁷ cells/square meter are administered to the individual. In some embodiments, between 5 × 10 ⁶ and 5 × 10 ⁷ cells/square meter are administered to the subject. In some embodiments, 5 × 10 ⁶ to 5 × 10 ⁷ cells/square meter are administered to the subject. In some embodiments, approximately 5 × ¹⁰ cells/square meter are administered to the subject. In some embodiments, approximately 5 × ¹⁰ cells/square meter are administered to the subject. In some embodiments, approximately 5 × ¹⁰ cells/square meter are administered to the subject. In some embodiments, 5 × 10 ⁸ cells/square meter are administered to the subject. In some embodiments, less than about 5 × ¹⁰ cells/square meter are administered to the subject. In some embodiments, more than about 5 × ¹⁰ cells/square meter is administered to an individual. In some embodiments, more than about 5 × ¹⁰ cells/square meter is administered to an individual.

In some embodiments, the composition is administered to the subject once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the composition is administered to an individual multiple times. In some embodiments, the method includes administering to the individual a population of antigen-specific T cells or a pharmaceutical composition thereof a plurality of times. In some embodiments, the method includes administering to the individual a population of antigen-specific T cells or a pharmaceutical composition thereof once weekly, once every two weeks, once every three weeks, or once every four weeks.

In some embodiments, antigen-specific T cells according to the expanded T cells of the invention or pharmaceutical compositions of the invention are administered to an individual multiple times. In some embodiments, a composition as described herein is administered to an individual more than once. In some embodiments, a composition as described herein is administered to an individual more than twice. In some embodiments, a composition as described herein is administered to an individual more than three times. In some embodiments, a composition as described herein is administered to an individual more than four times. In some embodiments, a composition as described herein is administered to an individual more than five times. In some embodiments, a composition as described herein is administered to an individual more than six times. In some embodiments, a composition as described herein is administered to an individual more than seven times. In some embodiments, a composition as described herein is administered to an individual more than eight times. In some embodiments, a composition as described herein is administered to an individual more than nine times. In some embodiments, a composition as described herein is administered to an individual more than ten times. In some embodiments, a composition as described herein is administered to an individual a number of times appropriate for the individual.

In some embodiments, antigen-specific T cells according to the expanded T cells of the invention or pharmaceutical compositions of the invention are administered to an individual multiple times. In some embodiments, a composition as described herein is administered to an individual once. In some embodiments, a composition as described herein is administered to an individual twice. In some embodiments, a composition as described herein is administered to an individual three times. In some embodiments, a composition as described herein is administered to an individual four times. In some embodiments, a composition as described herein is administered to an individual five times. In some embodiments, a composition as described herein is administered to an individual six times. In some embodiments, a composition as described herein is administered to an individual seven times. In some embodiments, a composition as described herein is administered to an individual eight times. In some embodiments, a composition as described herein is administered to an individual nine times. In some embodiments, a composition as described herein is administered to an individual ten times. In some embodiments, a composition as described herein is administered to an individual a number of times appropriate for the individual.

In some embodiments, administration to a patient can be used to treat viral infections. In some embodiments, administration to a patient can be used to treat latent viral infection. In some embodiments, administration to a patient can be used to treat lytic viral infections. In some embodiments, administration to a patient can be used to treat chronic viral infections, such as chronic HBV infection. In some embodiments, administration to a patient can be used to treat a disease associated with reactivation of infection. For example, treatment may be applied to patients who have been diagnosed with a disease associated with reactivation of HHV8, such as Kaposi's sarcoma, primary effusion lymphoma, or multicentric Castleman's disease. In some embodiments, administration to a patient can be used to treat tumors. In some embodiments, administration to patients may be for primary immunodeficiency prior to transplantation. In some embodiments, methods as described herein can comprise administering to a patient a plurality of antigen-specific T cell strains, wherein the second antigen-specific T cell strain can be selected from the same strain as the first antigen-specific T cell strain. Micro library. In some embodiments, the second antigen-specific T cell line can be selected from a different mini-bank than the mini-bank from which the first antigen-specific T cell line was obtained. In some embodiments, the second antigen-specific T cell line can be selected from a mini-library or from a mini-library contained in a library as described herein by repeated selection of the first antigen-specific T cell line. The mini library contains all the remaining antigen-specific T cell lines in the donor library except the first antigen-specific T cell line. In some embodiments, methods as described herein can comprise administering to a patient a universal antigen-specific T cell product, wherein the product comprises a population of antigen-specific T cells, wherein the population of antigen-specific T cells comprises at least 2 Cell lines, wherein each cell line is generated from a separate donor, wherein the HLA type of each donor is different from at least one of the other donors by at least one HLA allele. Universal antigen-specific T cells can be obtained by pooling cell lines from one or more donor mini-banks, and/or can be administered as individual antigen-specific T cells from donor mini-banks in a single administration session .

In some aspects, the invention provides a method of preventing or treating a pathogenic infection, comprising administering to an individual a T cell or pharmaceutical composition of the invention. In some aspects, the invention provides a method of preventing or treating a pathogenic infection, comprising administering to an individual an antigen-specific T cell expanded T cell according to the invention or a pharmaceutical composition of the invention.

In some aspects, the invention provides a method of preventing or treating a pathogenic infection comprising administering to an individual a population of expanded VSTs of the invention or a pharmaceutical composition of the invention.

In some aspects, the invention provides a method of preventing or treating a pathogenic infection, comprising administering to an individual a population of multi-strain VSTs of the invention or a pharmaceutical composition of the invention.

In some aspects, the invention provides a method of preventing or treating viral infection, which comprises administering to an individual a T cell or pharmaceutical composition of the invention. In some aspects, the invention provides a method of preventing or treating a viral infection, comprising administering to an individual an antigen-specific T cell expanded T cell according to the invention or a pharmaceutical composition of the invention. The invention provides, at least in part, a method for treating or preventing a viral infection comprising administering to an individual in need thereof an antigen-specific T cell expanded T cell according to the invention or a pharmaceutical composition as described herein (e.g., , antigen-specific T cell lines from the donor minibanks described herein, or pharmaceutical compositions comprising such T cell lines, formulated for intravenous delivery).

In some aspects, the present invention provides a method of preventing or treating viral infection, comprising administering to an individual a population of expanded VSTs of the present invention or a pharmaceutical composition of the present invention.

In some aspects, the invention provides a method of preventing or treating viral infection, which comprises administering to an individual a population of multiple strains of VST of the invention or a pharmaceutical composition of the invention. In some aspects, the present invention provides a method of preventing or treating latent viral infection, comprising administering to an individual a population of multi-strain VSTs of the present invention or a pharmaceutical composition of the present invention. In some aspects, the invention provides a method of preventing or treating lytic viral infection, comprising administering to an individual a population of multi-strain VSTs of the invention or a pharmaceutical composition of the invention. In some embodiments, the individual is infected or at risk of becoming infected with a virus. In some embodiments, the individual has or is believed to have a viral infection. In some embodiments, the viral infection is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), type B Hepatitis virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella-zoster virus ( VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV- 7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV), parotid gland inflammatory virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus, SARS-CoV and human Tropical Virus T-lymphovirus. In some embodiments, the individual has or is believed to have hepatitis B virus (HBV). In some embodiments, the individual suffers from or is at risk of developing cirrhosis or liver cancer. In some embodiments, the individual has or is believed to have human herpesvirus 8 (HHV-8). In some embodiments, the individual has or is at risk of developing Kaposi's sarcoma, primary effusion lymphoma, or multicentric Castleman's disease.

In some embodiments, administration of a pharmaceutical composition is effective to treat or prevent viral infection in an individual. In some embodiments, administration of antigen-specific T cells according to the invention or pharmaceutical compositions of the invention is effective in treating or preventing viral infection in an individual. In some embodiments, administration of antigen-specific T cells according to the invention or pharmaceutical compositions of the invention is effective in treating or preventing a disease or disorder associated with a viral infection. In some embodiments, the viral infection is HBV. In some embodiments, the viral infection is HHV8.

In some aspects, the present invention provides a method of preventing or treating malignant tumors, comprising administering to an individual an antigen-specific T cell expanded T cell according to the present invention or a pharmaceutical composition of the present invention.

In some aspects, the invention provides a method of preventing or treating malignant tumors, which includes administering to an individual a T cell or pharmaceutical composition of the invention.

In some embodiments, the malignant tumor comprises an antigen selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu, MAGE A3, p53 non-mutant, NY-ESO- 1. PSMA, GD2, melanin A/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML- IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl, meta Cortin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY- TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fas Related antigen 1.

In some embodiments, the individual has or is believed to have a malignant tumor. In some embodiments, the malignant tumor comprises an antigen selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu, MAGE A3, p53 non-mutant, NY-ESO- 1. PSMA, GD2, melanin A/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML- IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl, meta Cortin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY- TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fas Related antigen 1.

In some embodiments, a pharmaceutical composition is administered to an individual without knowledge of the individual's HLA type. In some embodiments, a pharmaceutical composition is administered to an individual with knowledge of the individual's HLA type.

In some embodiments, the invention provides an antigen-specific T cell for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides an antigen-specific T cell for use in treating a disease or condition in an individual. In some embodiments, the invention provides an antigen-specific T cell for use in preventing a disease or condition in an individual. In some embodiments, the invention provides a population of antigen-specific T cells for use in the treatment or prevention of a disease or condition in an individual. In some embodiments, the invention provides a population of antigen-specific T cells for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of antigen-specific T cells for use in preventing a disease or condition in an individual. In some embodiments, the invention provides one or more suitable antigen-specific T cell lines of expanded T cells according to the invention (eg, two or more such T cell lines) and/or as described herein A universal antigen-specific T cell product by administering to a patient one or more suitable antigen-specific T cell lines of expanded T cells according to the invention (e.g., two or more such T cell lines ) and/or the universal antigen-specific T cell products described herein for use in treating a disease or condition. In some embodiments, the invention provides one or more suitable antigen-specific T cell lines of expanded T cells according to the invention (eg, two or more such T cell lines) and/or as described herein A universal antigen-specific T cell product by administering to a patient one or more suitable antigen-specific T cell lines of expanded T cells according to the invention (e.g., two or more such T cell lines ) and/or the universal antigen-specific T cell products described herein for use in preventing disease or conditions.

In some embodiments, the invention provides a population of virus-specific T cells for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells for use in preventing a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of a latent virus for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of a latent virus for use in treating a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of a latent virus for use in preventing disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of a lytic virus for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of a lytic virus for use in treating a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of a lytic virus for use in preventing a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of HBV for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of HBV for use in treating a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of HBV for use in preventing disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of HHV-8 for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of HHV-8 for use in treating a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen of HHV-8 for use in preventing a disease or condition in an individual.

In some embodiments, the invention provides a population of virus-specific T cells for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells for use in preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of a latent virus for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of a latent virus for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of a latent virus for use in preventing disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of a lytic virus for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of a lytic virus for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of a lytic virus for use in preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of HBV for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of HBV for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of HBV for use in preventing disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of HHV-8 for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of HHV-8 for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen of HHV-8 for use in preventing a disease or condition in an individual.

In some embodiments, the invention provides virus-specific T cells specific for at least one antigen from each of BKV, AdV, CMV, HHV6, and EBV for use in treating or preventing a disease or disorder in an individual. status. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen from each of BKV, AdV, CMV, HHV6, and EBV for use in treating a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen from each of BKV, AdV, CMV, HHV6, and EBV for use in preventing disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen from each of RSV, IFV, PIV, and hPMV for use in treating or preventing a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen from each of RSV, IFV, PIV, and hPMV for use in treating a disease or condition in an individual. In some embodiments, the invention provides virus-specific T cells specific for at least one antigen from each of RSV, IFV, PIV, and hPMV for use in preventing disease or condition in an individual.

In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen from each of BKV, AdV, CMV, HHV6 and EBV for use in treating or preventing disease in an individual or symptoms. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen from each of BKV, AdV, CMV, HHV6, and EBV for use in treating a disease or disorder in an individual. status. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen from each of BKV, AdV, CMV, HHV6, and EBV for use in preventing disease or illness in an individual. status. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen from each of RSV, IFV, PIV, and hPMV for use in treating or preventing a disease or disease in an individual. status. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen from each of RSV, IFV, PIV, and hPMV for use in treating a disease or condition in an individual. In some embodiments, the invention provides a population of virus-specific T cells specific for at least one antigen from each of RSV, IFV, PIV, and hPMV for use in preventing disease or condition in an individual. individual

In some embodiments, the invention provides a method of administering a T cell or pharmaceutical composition described herein. In some embodiments, an individual is administered a population of expanded virus-specific T cells (VST) as described herein.

In some embodiments, the individual is a mammal. In some embodiments, the individual is a human, a domestic animal, a farm animal, a dog, a horse, a cat, a cow, a sheep, a pig, a goat, a rat, a guinea pig, or a non-human primate, such as a monkey, a chimpanzee, a baboon, or a ganglion. monkey. In some embodiments, the individual is a human.

In some embodiments, the individual is an adult. In some embodiments, the individual is a child.

In some embodiments, the individual has one or more of the following: a solid organ transplant, receiving chemotherapy, HIV infection, a genetic immunodeficiency, a chronic viral infection, and has received an allogeneic or autologous stem cell transplant. In some embodiments, the individual has received a solid organ transplant. In some embodiments, the individual has received chemotherapy. In some embodiments, the individual has an HIV infection. In some embodiments, the individual suffers from a genetic immunodeficiency. In some embodiments, the individual suffers from a chronic viral infection. In some embodiments, the individual has received an allogeneic stem cell transplant. In some embodiments, the individual receives an autologous stem cell transplant. In some embodiments, the subject is an allogeneic hematopoietic stem cell transplant recipient.

In some embodiments, the individual is immunocompromised. In some embodiments, the subject is immunocompetent. In some embodiments, patients treated with one or more suitable populations of expanded VSTs of the invention may be immunocompromised. In some embodiments, patients treated with a population of one or more suitable multi-strain VSTs of the invention may be immunocompromised. In some embodiments, the patient is treated with one or more suitable antigen-specific T cell lines as described herein (eg, two or more such T cell lines) and/or a universal antigen-specific T cell product. Patients may be immunocompromised. In some embodiments, the patient is immunocompromised as a result of treatments the patient received to treat the disease or condition or another disease or condition. In some embodiments, the patient is immunocompromised due to age. In one embodiment, the patient is immunocompromised due to young age. In some embodiments, the patient is immunocompromised due to young age. In one embodiment, the patient is immunocompromised due to age. In some embodiments, the patient is immunocompromised due to age. In some embodiments, the condition treated may be immunodeficiency. In one embodiment, the immunodeficiency is primary immunodeficiency. In some embodiments, the immunodeficiency is primary immunodeficiency. In some embodiments, the patient is in need of transplant therapy. In some embodiments, the individual is immunocompetent or immunocompromised. In some embodiments, the individual: a. has received a solid organ transplant; b. has received chemotherapy; c. has an HIV infection; d. has a genetic immunodeficiency; e. has a chronic viral infection; and/or f .Have received allogeneic or autologous stem cell transplantation. In some embodiments, the individual has received a solid organ transplant. In some embodiments, the subject has received chemotherapy. In some embodiments, the individual has an HIV infection. In some embodiments, the individual suffers from a genetic immunodeficiency. In some embodiments, the individual suffers from a chronic viral infection. In some embodiments, the individual has received an allogeneic or autologous stem cell transplant.

In some embodiments, transplanted material received by a patient as described herein can comprise stem cells. In some embodiments, the transplanted material received by a patient as described herein may comprise a solid organ. In some embodiments, the solid organ is a kidney. In some embodiments, the transplanted material received by a patient as described herein may comprise bone marrow. In some embodiments, transplanted material received by a patient as described herein may include stem cells, solid organs, and bone marrow.

In some embodiments, the individual is infected with a virus. In some embodiments, the individual is infected with a latent virus. In some embodiments, the individual is infected with a lytic virus. In some embodiments, an individual is at risk of contracting a virus. In some embodiments, the individual is at risk for infection with a latent virus. In some embodiments, the individual is at risk of latent viral reactivation. In some embodiments, the individual is at risk of infection with a lytic virus. In some embodiments, the individual is infected with one or more of the following: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), type B Hepatitis virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella-zoster virus ( VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV- 7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, pocavirus, coronavirus, lymphocytic choriomeningitis virus (LCMV), parotid gland inflammatory virus, measles virus, human metapneumovirus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T-lymphotropic virus . In some embodiments, the individual is at risk for infection with one or more of the following: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV) ), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zone Herpes zoster virus (VZV), Estin-Barr virus (EBV), cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), human herpes virus 8 (HHV-8), human herpes virus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, bocavirus, coronavirus, lymphocytic choriomeningitis virus ( LCMV), mumps virus, measles virus, human metapneumovirus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T-lymphovirus.

In some embodiments, the individual is infected with or at risk of infection with cytomegalovirus (CMV). In some embodiments, the individual is infected or at risk of infection with an adenovirus (Adv). In some embodiments, the individual is infected or at risk of infection with BK virus. In some embodiments, the individual is infected with or at risk of infection with human papillomavirus (HPV). In some embodiments, the individual is infected or at risk of infection with hepatitis A virus (HAV). In some embodiments, the individual is infected with or at risk of infection with hepatitis B virus (HBV). In some embodiments, the individual is infected with or at risk of infection with hepatitis C virus (HCV). In some embodiments, the individual is infected with or at risk of infection with hepatitis D virus (HDV). In some embodiments, the individual is infected with or at risk of infection with herpes simplex virus 1 (HSV-1). In some embodiments, the individual is infected with or at risk of infection with herpes simplex virus 2 (HSV-2). In some embodiments, the individual is infected or at risk of infection with HIV. In some embodiments, the individual is infected or at risk of infection with varicella zoster virus (VZV). In some embodiments, the individual is infected or at risk of infection with Estén-Barr virus (EBV). In some embodiments, the individual is infected with or at risk of infection with cytomegalovirus (CMV). In some embodiments, the individual is infected with or at risk of infection with JC virus. In some embodiments, the individual is infected with or at risk of infection with human herpesvirus 6 (HHV-6). In some embodiments, the individual is infected with or at risk of infection with human herpesvirus 8 (HHV-8). In some embodiments, the individual is infected with or at risk of infection with human herpesvirus 7 (HHV-7). In some embodiments, the individual is infected with or at risk of infection with Kaposi's sarcoma-associated herpesvirus (KSHV). In some embodiments, the individual is infected with or at risk of infection with respiratory syncytial virus (RSV). In some embodiments, the individual is infected or at risk of infection with an influenza virus. In some embodiments, the individual is infected with or at risk of infection with a parainfluenza virus. In some embodiments, the individual is infected with or at risk of infection with Bocavirus. In some embodiments, the individual is infected or at risk of infection with a coronavirus. In some embodiments, the individual is infected with or at risk of infection with lymphocytic choriomeningitis virus (LCMV). In some embodiments, the individual is infected or at risk of infection with the mumps virus. In some embodiments, the individual is infected with or at risk of infection with measles virus. In some embodiments, the individual is infected or at risk of infection with human metapneumovirus (hMPV). In some embodiments, the individual is infected with or at risk of infection with parvovirus B. In some embodiments, the individual is infected with or at risk of infection with rotavirus. In some embodiments, the individual is infected or at risk of infection with Merkel cell virus. In some embodiments, the individual is infected or at risk of infection with West Nile virus (WNV). In some embodiments, the individual is infected or at risk of infection with Zika virus. In some embodiments, the individual is infected with or at risk of infection with Ebola virus. In some embodiments, the individual is infected with or at risk of infection with human T-lymphotropic virus. set

In some embodiments, compositions described herein (eg, expanded T cells, virus-specific T cells, antigen-specific T cells, etc.) are provided in vials. In some embodiments, the compositions include a pharmaceutically acceptable carrier.

In some embodiments, the invention provides a kit comprising the expanded T cells of the invention or a composition thereof. In some embodiments, the invention provides a kit comprising the expanded T cells of the invention or a composition thereof and instructions for use. In some embodiments, the invention provides a kit comprising the expanded T cells of the invention or a composition thereof as described herein, and a package insert containing instructions for use of the kit and/or any component thereof. In some embodiments, a kit includes an expanded T cell of the invention or a composition thereof, one or more controls, and various buffers, reagents, enzymes, and other standard ingredients well known in the art in a suitable container. . In some embodiments, a container includes at least one vial, well, test tube, flask, bottle, syringe, or other container member to hold and, in some cases, appropriately aliquot the expanded T cells of the invention or compositions thereof. In some embodiments where additional components are provided, the kit contains additional containers for placing such components. The kit may also include components to contain the expanded T cells of the invention or compositions thereof and any other reagents for commercial sale. Such containers may include injection molded or blow molded plastic containers that hold the desired vials. The container and/or kit may include labels with instructions for use and/or warnings.

In some embodiments, the invention provides a kit comprising the antigen-specific T cells of the invention or a composition thereof. In some embodiments, the invention provides a kit comprising the antigen-specific T cells of the invention or a composition thereof and instructions for use. In some embodiments, the invention provides a kit comprising the antigen-specific T cells of the invention or a composition thereof as described herein, and a package insert containing instructions for use of the kit and/or any component thereof. In some embodiments, a kit includes an antigen-specific T cell of the invention or a composition thereof, one or more controls, and various buffers, reagents, enzymes, and other standard ingredients well known in the art in a suitable container. . In some embodiments, a container includes at least one vial, well, test tube, flask, bottle, syringe, or other container member for housing and, in some cases, appropriately aliquoting the antigen-specific T cells of the invention or compositions thereof. In some embodiments where additional components are provided, the kit contains additional containers for placing such components. The kit may also include components that tightly contain the antigen-specific T cells of the invention or compositions thereof and any other reagents for commercial sale. Such containers may include injection molded or blow molded plastic containers that hold the desired vials. The container and/or kit may include labels with instructions for use and/or warnings.

In some embodiments, the present invention provides a kit comprising the VST of the present invention or a composition thereof. In some embodiments, the present invention provides a kit comprising the VST of the present invention or a composition thereof and instructions for use. In some embodiments, the invention provides a kit comprising a VST of the invention or a composition thereof as described herein, and a package insert containing instructions for use of the kit and/or any component thereof. In some embodiments, a kit includes a VST of the invention or a composition thereof, one or more controls, and various buffers, reagents, enzymes, and other standard ingredients well known in the art in suitable containers. In some embodiments, the container includes at least one vial, well, test tube, flask, bottle, syringe, or other container member that holds and, in some cases, appropriately aliquots the VST of the present invention or a composition thereof. In some embodiments where additional components are provided, the kit contains additional containers for placing such components. The kit may also include components that tightly contain the VST of the invention or compositions thereof and any other reagents for commercial sale. Such containers may include injection molded or blow molded plastic containers that hold the desired vials. The container and/or kit may include labels with instructions for use and/or warnings.

In some embodiments, a panel includes a VST of the invention that includes specificity for at least one antigen from one or more of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human Papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), simplex Herpes virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), Human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, Bocavirus , coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus ( WNV), Zika virus, Ebola virus and human T-lymphotropic virus.

In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from HBV. In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from HHV8. In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from CMV. In some embodiments, a kit includes a VST of the invention that includes specificity for at least one antigen from Adv. In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from BK virus. In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from HBV. In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from HHV6. In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from EBV. In some embodiments, a kit includes a VST of the invention that includes specificity for at least one antigen from RSV. In some embodiments, a kit includes a VST of the invention that includes specificity for at least one antigen from influenza virus. In some embodiments, the kit includes a VST of the invention that includes specificity for at least one antigen from a parainfluenza virus. In some embodiments, a kit includes a VST of the invention that includes specificity for at least one antigen from hMPV. In some embodiments, the kit includes a VST of the invention that is reactive against at least one antigen from JCV.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for delaying the progression of a disease, disorder, or condition in an individual that is associated with one or more of the following: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human Papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), simplex Herpes virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), Human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, Bocavirus , coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus ( WNV), Zika virus, Ebola virus and human T-lymphotropic virus.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual's disease, disease or condition related to HBV or delaying its progression.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for individuals with HHV8-related diseases, disorders or conditions or delaying their progression.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual to have a disease, disorder or condition related to BK virus or to delay the progression thereof.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual to have an AdV-related disease, disorder or condition or to delay the progression thereof.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual's disease, disease or condition related to CMV or delaying its progression.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for individuals with HHV6-related diseases, disorders or conditions or delaying their progression.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual to have an EBV-related disease, disorder or condition or to delay the progression thereof.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual to have an AdV-related disease, disorder or condition or to delay the progression thereof.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual to have a disease, disorder or condition associated with RSV or to delay the progression thereof.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for preventing or delaying the progression of an influenza virus-related disease, illness or condition in an individual.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual to have a parainfluenza virus-related disease, illness or condition or to delay the progression thereof.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual's disease, disease or condition related to JCV or delaying its progression.

In some embodiments, the kit includes: a VST of the invention described herein or a composition thereof and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a multi-strain population of VST; and for treatment in need Instructions for an individual's disease, disease or condition related to hMPV or delaying its progression.

In some embodiments, any of the compositions of the invention are provided in a kit. In some embodiments, kits may be provided that include one or more of the following: mixed peptide libraries, cells of any type (e.g., monocytes), interleukins, and/or methods for processing mixed peptides and/or cells. Reagents. In some embodiments, the components of the kit are provided in suitable containers. In some embodiments, the kit of the present invention includes instructions for use.

In some embodiments, the invention provides a kit comprising one or more of the following: a mixed peptide library, any type of cell (eg, monocytes, PBMC, etc.), interleukins, and/or reagents. In some embodiments, the present invention provides a kit comprising one or more of the following: a mixed peptide library, any type of cell (e.g., monocytes, PBMC, etc.), interleukins, and/or reagents; and Instructions for use. In some embodiments, the present invention provides a kit comprising one or more of the following: a mixed peptide library, any type of cell (e.g., monocytes, PBMC, etc.), interleukins, and/or reagents; and A package insert containing instructions for use of the kit and/or any of its components. In some embodiments, the kit includes one or more of the following in one or more suitable containers: a mixed peptide library, any type of cell (e.g., monocytes, PBMC, etc.), an interleukin, a or Various controls and various buffers, reagents, enzymes and other standard components well known in the art. In some embodiments, one or more containers comprise at least one vial, well, test tube, flask, flask, syringe or other to house and, in some cases, appropriately aliquot the antigen-specific T cells of the invention or compositions thereof Container widget. In some embodiments where additional components are provided, the kit contains additional containers for placing such components. Kits may also include components that tightly contain one or more of the following for commercial sale: mixed peptide libraries, cells of any type (eg, monocytes, PBMCs, etc.), interleukins, and any other reagents. Such containers may include injection molded or blow molded plastic containers that hold the desired vials. The container and/or kit may include labels with instructions for use and/or warnings. Additional embodiments

Notwithstanding the appended claims, the following numbered examples form part of the invention.

Example 1-1. A method for expanding T cells in vitro, comprising contacting a starting population of mononuclear spheres with one or more compositions, the one or more compositions comprising: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) One or more interleukins comprising IL-4, IL-7 and IL-15; thereby generating an expanded T cell population.

Example 1-2. A method for expanding T cells in vitro, which includes the following steps: a) Contacting an initial population of mononuclear spheres with one or more compositions comprising: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and ii) one or more interleukins comprising IL-4 and IL-7, wherein the interleukins do not comprise IL-15; b) Subsequently contacting the population of cells with IL-15, thereby generating a population of expanded T cells.

Embodiment I-3. The method of Embodiment I-1 or I-2, wherein the mononuclear cells are peripheral blood mononuclear cells (PBMC).

Embodiment I-4. The method of any one of Embodiments I-1 to I-3, wherein the method does not comprise adding IL-2.

Embodiment 1-5. The method of any one of Embodiments I-1 to I-4, wherein the starting population of mononuclear spheres is contacted with a composition comprising (i) and a separate composition comprising (ii) .

Embodiment I-6. The method of any one of Embodiments I-1 to I-4, wherein the starting population of mononuclear spheres is contacted with a composition comprising (i) and (ii).

Embodiment I-7. The method of any one of Embodiments I-1 to I-6, wherein IL-4 and IL-7 are added simultaneously.

Embodiment I-8. The method of any one of Embodiments I-1 to I-6, wherein IL-4 and IL-7 are added separately.

Embodiment 1-9. The method of Embodiment 1-8, wherein IL-4 and IL-7 are added within 24 hours of each other.

Embodiment 1-10. The method of any one of Embodiments I-1 to I-9, wherein step (b) is performed within about 7 days of step (a).

Embodiment I-11. The method of any one of Embodiments I-1 to I-9, wherein step (b) is performed within about 2 days of step (a).

Embodiment 1-12. The method of any one of Embodiments I-1 to I-9, wherein step (b) is performed about 48 hours after step (a).

Embodiment 1-13. The method of any one of Embodiments I-1 to I-9, wherein step (b) is performed about 24 hours after step (a).

Embodiment I-14. The method of any one of Embodiments I-1 to I-9, wherein step (b) and step (a) are performed on the same day, and optionally within 12 hours of step (a).

Embodiment I-15. The method of any one of Embodiments I-1 to I-9, wherein step (b) is performed simultaneously with step (a), and optionally within 2 hours of step (a).

Embodiment I-16. The method of any one of Embodiments I-1 to I-15, wherein the amplification is performed for 18 days or less, optionally 14 days.

Embodiment I-17. The method of any one of Embodiments I-1 to I-15, the amplification is performed for an amount of time sufficient to produce cells that have completed logarithmic phase growth, wherein the amplification is performed for about 9 days to about 18 days sky.

Embodiment I-18. The method of any one of embodiments I-1 to I-17, wherein the amplification is performed in the presence of T cell expansion medium.

Embodiment I-19. The method of any one of Embodiments I-1 to I-18, wherein the amplification uses between 2 × 10 ⁶ cells/cm2 and 4 × 10 ⁶ cells/cm2 , depending on the situation, the seeding density is 3 × 10 ⁶ cells/cm2.

Embodiment 1-20. The method of any one of Embodiments I-1 to I-19, wherein the cell number of the expanded T cell population is at least 2 times higher than the starting cell population.

Embodiment 1-21. The method of any one of Embodiments I-1 to I-20, the expanded T cell population comprises antigen-specific T cells, wherein the antigen-specific T cells are pathogen-specific T cells cells (PST), virus-specific T cells (VST), or tumor-specific T cells (TST).

Embodiment 1-22. The method of any one of Embodiments I-1 to I-21, wherein the peptide mixture contains between 2 and 750 peptides.

Embodiment 1-23. The method of Embodiment 1-22, wherein each peptide in the peptide mixture has at least 2 amino acid overlaps with at least one other peptide.

Embodiment 1-24. The method of any one of Embodiments I-1 to I-23, wherein the peptides are at least 7 amino acids long.

Embodiment 1-25. The method of any one of Embodiments I-1 to I-24, wherein the peptide mixture comprises one or more tumor-associated antigens or a portion thereof.

Embodiment 1-26. The method of Embodiment 1-25, wherein the one or more tumor-associated antigens are selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-L1, TRP1 , CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu , MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, melanin A/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, tyrosinase, Survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl, mesothelin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fas-related antigen 1.

Embodiment 1-27. The method of any one of embodiments 1-1 to 1-26, wherein the peptide mixture comprises one or more target antigens from at least one pathogen or a portion thereof.

Embodiment 1-28. The method of Embodiment 1-27, wherein the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV) ), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV -2), HIV, varicella-zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 ( HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, Bocavirus, coronavirus, lymphoma Cellular choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonitis virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika viruses, Ebola virus and human T-lymphotropic virus.

Embodiment 1-29. The method of Embodiment 1-27, wherein the pathogen is HBV, and the at least one target antigen is selected from the group consisting of surface antigen (HBsAg), core antigen (HBcAg), pre-core antigen (HBeAg), and DNA. Polymerases and combinations thereof.

Embodiment 1-30. The method of Embodiment 1-29, wherein the peptide mixture comprises a peptide comprising or consisting of a sequence selected from the following: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2 ) and/or AKYLWEWASVRFSWL (SEQ ID NO: 3).

Embodiment I-31. The method of embodiment I-29 or I-30, wherein the one or more HBV antigens are selected from one or more HBV genotypes.

Embodiment 1-32. The method of embodiment 1-31, wherein at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

Embodiment 1-33. The method of any one of Embodiments I-29 to I-32, wherein the amino acid sequence of each HBV antigen is different from the amino acid sequence of each other HBV antigen by at least one amino acid. .

Embodiment 1-34. The method of any one of embodiments 1-29 to 1-33, wherein at least a portion of the T cell response is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted.

Embodiment 1-35. The method of Embodiment 1-27, wherein the pathogen is EBV, and the at least one target antigen is selected from the group consisting of EBNA1, LMP2, BZLF1 and combinations thereof.

Embodiment 1-36. The method of Embodiment 1-27, wherein the pathogen is CMV, and the at least one target antigen is selected from IE1, pp65 and combinations thereof.

Embodiment 1-37. The method of Embodiment 1-27, wherein the pathogen is Adv, and the at least one target antigen is selected from the group consisting of hexon, penton and combinations thereof.

Embodiment 1-38. The method of Embodiment 1-27, wherein the pathogen is BK virus, and the at least one target antigen is selected from the group consisting of LT, VP-1 and combinations thereof.

Embodiment 1-39. The method of Embodiment 1-27, wherein the pathogen is HHV6, and the at least one target antigen is selected from the group consisting of U14, U11, U71, U54, U90 and combinations thereof.

Embodiment 1-40. The method of Embodiment 1-27, wherein the pathogen is RSV, and the at least one target antigen is selected from N, F and combinations thereof.

Embodiment 1-41. The method of Embodiment 1-27, wherein the pathogen is influenza virus, and the at least one target antigen is selected from MP1, NP1 and combinations thereof.

Embodiment 1-42. The method of Embodiment 1-27, wherein the pathogen is parainfluenza virus type 3 (PIV3), and the at least one target antigen is selected from the group consisting of F, N, M, M2-1, HN and combination.

Embodiment 1-43. The method of Embodiment 1-27, wherein the pathogen is hMPV, and the at least one target antigen is selected from F, N, M2-1, M and combinations thereof.

Embodiment 1-44. The method of Embodiment 1-27, wherein the pathogen is HHV8, and the at least one target antigen is selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC ( ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70), and combinations thereof.

Embodiment 1-45. The method of Embodiment 1-27, wherein the pathogen is SARS-CoV2, and the at least one target antigen is selected from the group consisting of spike protein (S), envelope protein (E), and matrix protein (M). ), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14 and their combinations . The term "matrix (M) protein" and its analogues (eg, M protein or M antigen) may be used interchangeably herein with the term "membrane (M) protein" and its analogues (eg, membrane (M) matrix protein).

Embodiment I-46. The method of any one of Embodiments I-1 to I-45, further comprising the step of preparing a pharmaceutical composition, wherein a diluent, a stabilizer, Preservatives and/or other pharmaceutically acceptable excipients.

Embodiment I-47. An expanded T cell population obtained by the method of any one of Embodiments I-1 to I-46.

Embodiment 1-48. The expanded T cell population of embodiment 1-47, wherein the T cell population comprises at least 70% CD3+ T cells.

Embodiment 1-49. The expanded T cell population of embodiment 1-47 or 1-48, wherein the expanded T cell population comprises virus-specific T cells (VST).

Embodiment 1-50. The expanded T cell population of any one of embodiments 1-47 to 1-49, wherein no more than 30% of the cells in the population are natural killer (NK) cells.

Embodiment 1-51. The expanded T cell population of any one of embodiments 1-47 to 1-50, wherein the T cell population comprises CD4+ and CD8+ T cells.

Embodiment 1-52. The expanded T cell population of Embodiment 1-51, wherein more than 80% of the T cell population consists of CD4+ and/or CD8+ T cells.

Embodiment 1-53. The expanded T cell population of any one of embodiments 1-47 to 1-52, wherein the T cells are multilineal.

Embodiment 1-54. The expanded T cell population of any one of Embodiments 1-48 to 1-53, wherein at least 1% of the CD3+ T cells can produce TNFα.

Embodiment 1-55. The expanded T cell population of any one of Embodiments 1-48 to 1-54, wherein at least one T cell recognizes one or more tumor-associated antigens or a portion thereof.

Embodiment 1-56. The expanded T cell population of embodiment 1-55, wherein the one or more tumor-associated antigens are selected from the group consisting of: CEA, MHC, CTLA-4, gp100, mesothelin, PD-Ll, TRPl, CD40, EGFP, Her2, TCRα, trp2, TCR, MUCl, cdr2, ras, 4-lBB, CT26, GITR, OX40, TGF-a, WTl, MUCl, LMP2, HPV E6 E7, EGFRvllll, HER-2/neu, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, melanin A/MARTl, Ras mutant, gp 100, p53 mutant, proteinase 3 (PRl), bcr-abl, Tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid , MYCN, RhoC, TRP-2, GD3, fucosyl GMl, mesothelin, PSCA, MAGE Al, sLe(a), CYPlBl, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR -1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TESl, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD -CT-1, FAP, PDGFR-f3, MAD-CT-2 and Fas-related antigen 1.

Embodiment 1-57. The expanded T cell population of any one of embodiments 1-47 to 1-54, wherein at least one T cell recognizes one or more target antigens from at least one pathogen, or a portion thereof.

Embodiment 1-58. The expanded T cell population of embodiment 1-57, wherein the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human Papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), simplex Herpes virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), Human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, Bocavirus , coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus ( WNV), Zika virus, Ebola virus and human T-lymphotropic virus.

Embodiment 1-59. The expanded T cell population of Embodiment 1-57, wherein the pathogen is HBV, and the at least one target antigen is selected from the group consisting of surface antigen (HBsAg), core antigen (HBcAg), and pre-core antigen. (HBeAg), DNA polymerase, and combinations thereof.

Embodiment 1-60. The expanded T cell population of embodiment 1-59, wherein the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2) and/or AKYLWEWASVRFSWL (SEQ ID NO: 3).

Embodiment 1-61. The expanded T cell population of embodiment 1-59 or 1-60, wherein at least two HBV antigens are selected from different HBV genotypes.

Embodiment 1-62. The expanded T cell population of Embodiment 1-61, wherein at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

Embodiment 1-63. The expanded T cell population of any one of Embodiments 1-59 to 1-62, wherein the amino acid sequence of each HBV antigen has at least the same amino acid sequence as that of each other HBV antigen. One amino acid is different.

Embodiment 1-64. The expanded T cell population of any one of embodiments 1-59 to 1-63, wherein at least a portion of the T cell response is HLA-DR, HLA-DQ, or HLA-DP restricted.

Embodiment 1-65. The expanded T cell population of Embodiment 1-57, wherein the pathogen is EBV and the at least one target antigen is selected from the group consisting of EBNA1, LMP2, BZLF1 and combinations thereof.

Embodiment 1-66. The expanded T cell population of Embodiment 1-57, wherein the pathogen is CMV and the at least one target antigen is selected from IE1, pp65 and combinations thereof.

Embodiment 1-67. The expanded T cell population of Embodiment 1-57, wherein the pathogen is Adv and the at least one target antigen is selected from the group consisting of hexon, penton and combinations thereof.

Embodiment 1-68. The expanded T cell population of Embodiment 1-57, wherein the pathogen is BK virus and the at least one target antigen is selected from the group consisting of LT, VP-1 and combinations thereof.

Embodiment 1-69. The expanded T cell population of Embodiment 1-57, wherein the pathogen is HHV6, and the at least one target antigen is selected from the group consisting of U14, U11, U71, U54, U90, and combinations thereof.

Embodiment 1-70. The expanded T cell population of Embodiment 1-57, wherein the pathogen is RSV and the at least one target antigen is selected from the group consisting of N, F and combinations thereof.

Embodiment 1-71. The expanded T cell population of Embodiment 1-57, wherein the pathogen is influenza virus and the at least one target antigen is selected from MP1, NP1 and combinations thereof.

Embodiment 1-72. The expanded T cell population of Embodiment 1-57, wherein the pathogen is parainfluenza virus type 3 (PIV3), and the at least one target antigen is selected from F, N, M, M2- 1. HN and its combinations.

Embodiment 1-73. The expanded T cell population of Embodiment 1-57, wherein the pathogen is hMPV and the at least one target antigen is selected from the group consisting of F, N, M2-1, M and combinations thereof.

Embodiment 1-74. The expanded T cell population of Embodiment 1-57, wherein the pathogen is HHV8, and the at least one target antigen is selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10. 5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) and their combinations .

Embodiment 1-75. The expanded T cell population of Embodiment 1-57, wherein the pathogen is SARS-CoV2, and the at least one target antigen is selected from the group consisting of spike protein (S) and envelope protein (E) , matrix protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B , Y14 and their combinations.

Embodiment 1-76. A plurality of expanded T cell populations, each as in any one of Embodiments 1-47 to 1-75, wherein each population comprises donor mononuclear spheres obtained from a different donor of T cells.

Embodiment 1-77. The plurality of expanded T cell populations of Embodiment 1-76, wherein each donor has an HLA type that differs from at least one of the other donors by at least one HLA allele.

Embodiment 1-78. A universal antigen-specific T cell therapy product comprising an expanded T cell population or a plurality of expanded T cell populations as in any one of Embodiments 1-47 to 1-77, wherein The product includes T cells generated from donor monocytes obtained from different donors.

Embodiment 1-79. The universal antigen-specific T cell therapy product of Embodiment 1-78, wherein the HLA type of each donor is different from at least one HLA allele gene of at least one of the other donors.

Embodiment 1-80. The universal antigen-specific T cell therapy product of embodiment 1-78 or 1-79, wherein the product is not alloreactive to partially HLA-matched and/or HLA-mismatched target cells.

Embodiment 1-81. The universal antigen-specific T cell therapy product of any one of embodiments 1-78 to 1-80, wherein the product comprises a virus that recognizes one or more target antigens from at least one pathogen or a portion thereof Specific T cells (VST).

Embodiment 1-82. A pharmaceutical composition comprising an expanded T cell population, a plurality of expanded T cell populations or universal antigen-specific T cells as in any one of Embodiments I-47 to I-81 Therapeutic products.

Embodiment 1-83. The pharmaceutical composition of embodiment 1-82, wherein the pharmaceutical composition is formulated for intravenous delivery.

Embodiment I-84. The pharmaceutical composition of embodiment I-82 or I-83, wherein the composition is negative for bacteria and fungi during continuous culture for at least 7 days.

Embodiment 1-85. The pharmaceutical composition of any one of embodiments 1-82 to 1-84, wherein the composition exhibits endotoxin less than 5 EU/ml.

Embodiment I-86. The pharmaceutical composition of any one of embodiments I-82 to I-85, wherein the composition is negative for Mycoplasma.

Embodiment 1-87. A method of preventing or treating viral infection, comprising administering to an individual a pharmaceutical composition as in any one of Embodiments I-82 to I-86.

Embodiment 1-88. The method of Embodiment 1-87, wherein the individual is immunocompetent or immunocompromised.

Embodiment 1-89. The method of Embodiment 1-87 or 1-88, wherein the pharmaceutical composition is administered by injection or infusion.

Embodiment 1-90. The method of any one of Embodiments 1-87 to 1-89, wherein the pharmaceutical composition is administered to the individual multiple times.

Embodiment 1-91. The method of any one of Embodiments 1-87 to 1-90, wherein between 5×10 ⁶ and 5×10 ⁷ cells/square meter is administered to the subject.

Embodiment 1-92. The method of any one of Embodiments 1-87 to 1-91, wherein the individual is infected with or at risk of infection with a virus.

Embodiment 1-93. The method of Embodiment 1-92, wherein administration of the pharmaceutical composition is effective to treat or prevent the viral infection in the individual.

Embodiment 1-94. The method of any one of embodiments 1-87 to 1-93, wherein the individual a.Have received solid organ transplant; b.Have received chemotherapy; c. Suffering from HIV infection; d. Suffering from genetic immunodeficiency; e. Suffering from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation.

Embodiment I-95. The method of any one of embodiments I-87 to I-94, wherein the pharmaceutical composition comprises the universal antigen-specific T cells of any one of embodiments I-78 to I-81 Therapeutic products.

Embodiment 1-96. The method of Embodiment 1-95, wherein the pharmaceutical composition is administered to the individual without knowledge of the HLA type of the individual.

Embodiment 1-97. The method of any one of embodiments 1-87 to 1-96, wherein the subject is a human.

Example 1-98. An in vitro method for stimulating the selective expansion of virus-specific T (VST) cells, comprising contacting a starting population of mononuclear spheres in culture with at least one target antigen or corresponding to at least A peptide or peptide mixture of the target antigen is contacted first in the presence of IL-4 and IL-7 and then in the presence of IL-15.

Embodiment 1-99. The method of Embodiment 1-98, wherein the cells are contacted with IL-4 and IL-7 in culture for a first period in the range of between about 1 day and about 2 days.

Embodiment 1-100. The method of Embodiment 1-98 or 1-99, wherein contact with IL-4 and IL-7 results in selective expansion of the VSTs but not NK cells.

Embodiment 1-101. The method of any one of Embodiments 1-98 to 1-100, wherein contact with IL-15 causes an acceleration of expansion of the cells in culture.

Embodiment 1-102. The method of any one of Embodiments 1-98 to 1-101, wherein contacting with IL-4 and IL-7 prior to subsequent contacting with IL-15 produces an expanded population of VST, wherein Enriched VSTs comprising a broader repertoire than the expanded population of VSTs obtainable by contacting the cells with: (i) IL-4 and IL-7, without IL-15; (ii) IL-7 and IL-15, without IL-4; or (iii) both IL-4, IL-7 and IL-15.

Example 1-103. An in vitro method for selectively amplifying virus-specific T (VST) cells, comprising a) Contacting an initial population of mononuclear spheres for a first period of time with one or more compositions comprising: (i) at least one target antigen or a peptide or peptide mixture corresponding to at least one target antigen; and (ii) IL-4 and IL-7; and b) Exposing cells from (a) to IL-15 for a second period of time.

Embodiment 1-104. The method of Embodiment 1-103, wherein the first period of time exceeds about 24 hours.

Embodiment 1-105. The method of Embodiment 1-103 or 1-104, wherein the first period of time is between about 24 and about 48 hours.

Embodiment 1-106. The method of any one of Embodiments 1-103 to 1-105, wherein the first period of time is less than about 144 hours.

Embodiment 1-107. The method of any one of Embodiments 1-103 to 1-106, wherein the first period of time is sufficient to produce at least a 2-fold higher amplification of the number of VSTs.

Embodiment 1-108. The method of any one of Embodiments 1-103 to 1-107, wherein the first period of time does not result in simultaneous exposure to IL-4, IL-7, and IL- Higher levels of NK cell expansion were expected in the case of 15.

Embodiment 1-109. The method of any one of Embodiments 1-103 to 1-108, wherein the VSTs target one or more antigens from a latent virus.

Example 1-110. A two-step method for selectively expanding VSTs in a heterogeneous cell population, comprising: a first step of culturing the VSTs in the presence of a stimulating antigen and IL-4 and IL-7; and The second step is to culture the VSTs in the presence of IL-15.

Example 1-111. A composition comprising VST expanded in a two-step process comprising: a first step of culturing the VST in the presence of IL-4 and IL-7; and in IL- The second step is to culture the VST in the presence of 15.

Example II-1. An in vitro method for expanding T cells, comprising: providing a starting population of mononuclear spheres in cell culture; One or more compositions are added to the cell culture, wherein the one or more compositions comprise: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and/or ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15; and The cells are cultured for a period of time sufficient to expand the T cells; thereby expanding the T cells.

Example II-2. An in vitro method for generating an expanded T cell population, comprising: Providing a starting population of mononuclear spheres in cell culture; One or more compositions are added to the cell culture, wherein the one or more compositions comprise: i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; and/or ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15; and The cells are cultured for a period of time sufficient to expand the T cells; An expanded T cell population is thereby generated.

Embodiment II-3. The method of Embodiment II-1 or II-2, wherein the expanded T cells or the expanded T cell population comprise T cells specific for at least one target antigen.

Embodiment II-4. The method of Embodiment II-3, wherein the expanded T cells are enriched with T cells specific for at least one target antigen.

Embodiment II-5. The method of Embodiment II-3, wherein the expanded T cell population is enriched with T cells specific for at least one target antigen.

Embodiment II-6. The method of any one of Embodiments II-1 to II-5, wherein the monocytes are peripheral blood monocytes (PBMC).

Embodiment II-7. The method of any one of Embodiments II-1 to II-6, wherein the one or more exogenous interleukins comprise IL-4 and IL-7.

Embodiment II-8. The method of any one of Embodiments II-1 to II-7, wherein the one or more exogenous interleukins comprise IL-4, IL-7, and IL-15.

Embodiment II-9. The method of any one of Embodiments II-1 to II-8, wherein the method does not comprise adding exogenous IL-2.

Embodiment II-10. The method of any one of Embodiments II-1 to II-8, wherein the method does not comprise adding IL-2.

Embodiment II-11. The method of any one of Embodiments II-1 to II-10, wherein a composition comprising the components of (i) and (ii) is added to the cell culture.

Embodiment II-12. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i) and a first composition comprising the component of (ii) are added to the cell culture Second composition.

Embodiment II-13. The method of Embodiment II-12, wherein the first composition and the second composition are added to the cell culture simultaneously.

Embodiment II-14. The method of Embodiment II-12, wherein the second composition is added to the cell culture after the first composition is added to the cell culture.

Embodiment II-15. The method of Embodiment II-14, wherein the second composition is added to the cell culture from about 1 minute to about 30 minutes after the first composition is added to the cell culture. middle.

Embodiment II-16. The method of Embodiment II-14, wherein the second composition is added to the cell culture about 30 minutes to about 60 minutes after the first composition is added to the cell culture. middle.

Embodiment II-17. The method of Embodiment II-14, wherein the second composition is added to the cell culture about 1 hour to about 2 hours after the first composition is added to the cell culture. middle.

Embodiment II-18. The method of Embodiment II-14, wherein the second composition is added to the cell culture about 2 hours after the first composition is added to the cell culture.

Embodiment II-19. The method of Embodiment II-14, wherein the second composition is added to the cell culture about 6 hours after the first composition is added to the cell culture.

Embodiment II-20. The method of Embodiment II-14, wherein the second composition is added to the cell culture about 12 hours after the first composition is added to the cell culture.

Embodiment II-21. The method of Embodiment II-14, wherein the second composition is added to the cell culture about 18 hours after the first composition is added to the cell culture.

Embodiment II-22. The method of Embodiment II-14, wherein the second composition is added to the cell culture about 24 hours after the first composition is added to the cell culture.

Embodiment II-23. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i), IL-4 and IL-1 is added to the cell culture. The second composition of 7 and the third composition comprising IL-15.

Embodiment II-24. The method of Embodiment II-23, wherein the first composition and the second composition are added to the cell culture simultaneously.

Embodiment II-25. The method of Embodiment II-23, wherein the second composition is added to the cell culture after the first composition is added to the cell culture.

Embodiment II-26. The method of Embodiment II-25, wherein the second composition is added to the cell culture from about 1 minute to about 30 minutes after the first composition is added to the cell culture. middle.

Embodiment II-27. The method of Embodiment II-25, wherein the second composition is added to the cell culture about 30 minutes to about 60 minutes after the first composition is added to the cell culture. middle.

Embodiment II-28. The method of Embodiment II-25, wherein the second composition is added to the cell culture about 1 hour to about 2 hours after the first composition is added to the cell culture. middle.

Embodiment II-29. The method of Embodiment II-25, wherein the second composition is added to the cell culture about 2 hours after the first composition is added to the cell culture.

Embodiment II-30. The method of Embodiment II-25, wherein the second composition is added to the cell culture about 6 hours after the first composition is added to the cell culture.

Embodiment II-31. The method of Embodiment II-25, wherein the second composition is added to the cell culture about 12 hours after the first composition is added to the cell culture.

Embodiment II-32. The method of Embodiment II-25, wherein the second composition is added to the cell culture about 18 hours after the first composition is added to the cell culture.

Embodiment II-33. The method of Embodiment II-25, wherein the second composition is added to the cell culture about 24 hours after the first composition is added to the cell culture.

Embodiment II-34. The method of any one of Embodiments II-23 to II-33, wherein the second composition and the third composition are added simultaneously.

Embodiment II-35. The method of any one of embodiments II-23 to II-33, wherein the third composition is added to the cell culture after the second composition is added to the cell culture. among things.

Embodiment II-36. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 12 hours after the second composition is added to the cell culture.

Embodiment II-37. The method of Embodiment II-35, wherein the third composition is added to the cell culture approximately 24 hours after the second composition is added to the cell culture.

Embodiment II-38. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 48 hours after the second composition is added to the cell culture.

Embodiment II-39. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 72 hours after the second composition is added to the cell culture.

Embodiment II-40. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 1 day after the second composition is added to the cell culture.

Embodiment II-41. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 2 days after the second composition is added to the cell culture.

Embodiment II-42. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 3 days after the second composition is added to the cell culture.

Embodiment II-43. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 4 days after the second composition is added to the cell culture.

Embodiment II-44. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 5 days after the second composition is added to the cell culture.

Embodiment II-45. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 6 days after the second composition is added to the cell culture.

Embodiment II-46. The method of Embodiment II-35, wherein the third composition is added to the cell culture about 7 days after the second composition is added to the cell culture.

Embodiment II-47. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i), IL-4 and IL-7 is added to the cell culture and a second composition comprising IL-15.

Embodiment II-48. The method of Embodiment II-47, wherein the first composition and the second composition are added simultaneously.

Embodiment II-49. The method of Embodiment II-47, wherein the second composition is added to the cell culture after the first composition is added to the cell culture.

Embodiment II-50. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 12 hours after the first composition is added to the cell culture.

Embodiment II-51. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 24 hours after the first composition is added to the cell culture.

Embodiment II-52. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 48 hours after the first composition is added to the cell culture.

Embodiment II-53. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 72 hours after the first composition is added to the cell culture.

Embodiment II-54. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 1 day after the first composition is added to the cell culture.

Embodiment II-55. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 2 days after the first composition is added to the cell culture.

Embodiment II-56. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 3 days after the first composition is added to the cell culture.

Embodiment II-57. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 4 days after the first composition is added to the cell culture.

Embodiment II-58. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 5 days after the first composition is added to the cell culture.

Embodiment II-59. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 6 days after the first composition is added to the cell culture.

Embodiment II-60. The method of Embodiment II-49, wherein the second composition is added to the cell culture about 7 days after the first composition is added to the cell culture.

Embodiment II-61. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i), a second composition comprising IL-4 are added to the cell culture. compositions and a third composition comprising IL-7 and IL-15.

Embodiment II-62. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i), a second composition comprising IL-7 are added to the cell culture. compositions and a third composition comprising IL-4 and IL-15.

Embodiment II-63. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i) and a second composition comprising IL-4 are added to the cell culture A third composition comprising IL-7 and a fourth composition comprising IL-15.

Embodiment II-64. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i) and IL-4, a first composition comprising IL-4 is added to the cell culture. The second composition of 7 and the third composition comprising IL-15.

Embodiment II-65. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i) and IL-7, a first composition comprising IL-7 is added to the cell culture. The second composition of 4 and the third composition comprising IL-15.

Embodiment II-66. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i) and IL-4 and a first composition comprising IL-4 are added to the cell culture. 7 and the second composition of IL-15.

Embodiment II-67. The method of any one of Embodiments II-1 to II-10, wherein a first composition comprising the component of (i) and IL-7 and a first composition comprising IL-7 are added to the cell culture. 4 and the second composition of IL-15.

Embodiment II-68. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for about 18 days or less, optionally about 14 days.

Embodiment II-69. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for about 8 days to about 10 days.

Embodiment II-70. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for about 8 days to about 14 days.

Embodiment II-71. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for about 8 days to about 18 days.

Embodiment II-72. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for about 1 week to about 2 weeks.

Embodiment II-73. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for about 1 week to about 3 weeks.

Embodiment II-74. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for about 1 week to about 4 weeks.

Embodiment II-75. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for an amount of time sufficient to produce cells that have completed logarithmic phase growth, wherein the cells are cultured for about 8 days to about 18 days.

Embodiment II-76. The method of any one of Embodiments II-1 to II-67, wherein the cells are cultured for an amount of time sufficient to produce cells that have completed logarithmic phase growth.

Embodiment II-77. The method of any one of Embodiments II-1 to II-76, wherein the cells are cultured in the presence of T cell expansion medium.

Embodiment II-78. The method of any one of Embodiments II-1 to II-77, wherein the cells are at about 2 × 10 ⁶ cells/cm2 to about 4 × 10 ⁶ cells/cm2, Depending on the situation, the cell seeding density is about 3 × 10 ⁶ cells/cm2 for culture.

Embodiment II-79. The method of any one of Embodiments II-1 to II-78, wherein the number of expanded T cells or the number of cells in the expanded T cell population is greater than the number of cells in the starting cell population. The number of cells is at least 2 times larger.

Embodiment II-80. The method of any one of Embodiments II-1 to II-79, wherein the expanded T cells or the expanded T cell population comprise antigen-specific T cells.

Embodiment II-81. The method of Embodiment II-80, wherein the antigen-specific T cells are pathogen-specific T cells (PST).

Embodiment II-82. The method of Embodiment II-80, wherein the antigen-specific T cells are virus-specific T cells (VST).

Embodiment II-83. The method of Embodiment II-80, wherein the antigen-specific T cells are tumor-specific T cells (TST).

Embodiment II-84. The method of any one of Embodiments II-1 to II-80, wherein the expanded T cells or the expanded T cell population comprise recognition from hepatitis B virus (HBV) VST for at least one antigen.

Embodiment II-85. The method of any one of Embodiments II-1 to II-80, wherein the expanded T cells or the expanded T cell population comprise recognition from human herpesvirus 8 (HHV-8 ) VST of at least one antigen.

Embodiment II-86. The method of any one of Embodiments II-1 to II-85, wherein the peptide mixture comprises between 2 and 750 peptides.

Embodiment II-87. The method of Embodiment II-86, wherein each peptide in the peptide mixture has at least 2 amino acid overlaps with at least one other peptide.

Embodiment II-88. The method of any one of Embodiments II-1 to II-87, wherein the peptides are at least 7 amino acids long.

Embodiment II-89. The method of any one of Embodiments II-1 to II-88, wherein the peptide mixture comprises one or more target antigens from at least one pathogen or a portion thereof.

Embodiment II-90. The method of Embodiment II-89, wherein the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV) ), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV -2), HIV, varicella-zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpesvirus 6 (HHV-6), human herpesvirus 8 ( HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, Bocavirus, coronavirus, lymphoma Cellular choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonitis virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika viruses, Ebola virus and human T-lymphotropic virus.

Embodiment II-91. The method of embodiment II-89, wherein the at least one pathogen is hepatitis B virus (HBV).

Embodiment II-92. The method of embodiment II-89, wherein the at least one pathogen is human herpesvirus 8 (HHV-8).

Embodiment II-93. The method of Embodiment II-89, wherein the pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase ( HBP), X antigen (HBX) or a combination thereof.

Embodiment II-94. The method of Embodiment II-89, wherein the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen ( HBX) or a combination thereof.

Embodiment II-95. The method of Embodiment II-93 or II-94, wherein the HBeAg (E antigen) includes pre-core (PreC) and core (HBcAg) antigens.

Embodiment II-96. The method of Embodiment II-89, wherein the peptide mixture comprises a peptide comprising or consisting of a sequence selected from the following: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2 ), AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and/or EYLVSFGVWIRTPPA (SEQ ID NO: 5).

Embodiment II-97. The method of any one of Embodiments II-93 to II-96, wherein the one or more HBV antigens are selected from one or more HBV genotypes.

Embodiment II-98. The method of Embodiment II-97, wherein at least two antigens are selected from the same HBV genotype.

Embodiment II-99. The method of embodiment II-97, wherein at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first genotype and the second Genotypes are different.

Embodiment II-100. The method of embodiment II-97, wherein at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

Embodiment II-101. The method of any one of Embodiments II-93 to II-100, wherein the amino acid sequence of each HBV antigen is different in at least one amino acid.

Embodiment II-102. The method of any one of Embodiments II-93 to II-101, wherein the expanded T cells are reactive upon stimulation.

Embodiment II-103. The method of any one of Embodiments II-93 to II-101, wherein the expanded T cell population is responsive upon stimulation.

Embodiment II-104. The method of any one of Embodiments II-102 to II-103, wherein at least a portion of the T cell reactivity is restricted by CD4+ and HLA-DR, HLA-DQ or HLA-DP.

Embodiment II-105. The method of Embodiment II-89, wherein the pathogen is EBV, and the at least one target antigen is EBNAl, LMP2, BZLF1 or a combination thereof.

Embodiment II-106. The method of Embodiment II-89, wherein the pathogen is CMV, and the at least one target antigen is IEl, pp65, or a combination thereof.

Embodiment II-107. The method of Embodiment II-89, wherein the pathogen is Adv, and the at least one target antigen is hexon, penton, or a combination thereof.

Embodiment II-108. The method of Embodiment II-89, wherein the pathogen is BK virus, and the at least one target antigen is LT, VP-1, or a combination thereof.

Embodiment II-109. The method of Embodiment II-89, wherein the pathogen is HHV6, and the at least one target antigen is U14, U11, U71, U54, U90 or a combination thereof.

Embodiment II-110. The method of Embodiment II-89, wherein the pathogen is RSV, and the at least one target antigen is N, F or a combination thereof.

Embodiment II-111. The method of Embodiment II-89, wherein the pathogen is influenza virus, and the at least one target antigen is MP1, NP1 or a combination thereof.

Embodiment II-112. The method of Embodiment II-89, wherein the pathogen is a parainfluenza virus type (PIV), and the at least one target antigen is F, N, M, M2-1, HN, or a combination thereof.

Embodiment II-113. The method of Embodiment II-89, wherein the pathogen is hMPV, and the at least one target antigen is F, N, M2-1, M or a combination thereof.

Embodiment II-114. The method of Embodiment II-89, wherein the pathogen is HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72) , RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70), or combinations thereof.

Embodiment II-115. The method of Embodiment II-89, wherein the peptide mixture comprises a peptide comprising or consisting of a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7 ), TTDTHSPSALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

Embodiment II-116. The method of Embodiment II-89, wherein the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), matrix protein (M), Nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof. The term "matrix (M) protein" and its analogues (eg, M protein or M antigen) may be used interchangeably herein with the term "membrane (M) protein" and its analogues (eg, membrane (M) matrix protein).

Embodiment II-117. The method of any one of Embodiments II-1 to II-116, comprising obtaining the expanded T cells or the expanded T cell population.

Embodiment II-118. The method of any one of Embodiments II-1 to II-117, further comprising the step of preparing a pharmaceutical composition, wherein to the expanded T cells or the expanded T cell population Add diluents, stabilizers, preservatives and/or other pharmaceutically acceptable excipients.

Embodiment II-119. An expanded T cell population obtained by the method of any one of Embodiments II-2 to II-118.

Embodiment II-120. The expanded T cell population of embodiment II-119, wherein the T cell population comprises at least 70% CD3+ T cells.

Embodiment II-121. The expanded T cell population of embodiment II-119 or II-120, wherein the expanded T cell population comprises VST.

Embodiment II-122. The expanded T cell population of any one of Embodiments II-119 to II-121, wherein no more than 30% of the cells in the expanded T cell population are natural killer (NK) cells.

Embodiment II-123. The expanded T cell population of any one of Embodiments II-119 to II-122, wherein the T cell population includes CD4+ and CD8+ T cells.

Embodiment II-124. The expanded T cell population of Embodiment II-123, wherein more than 80% of the T cell population consists of CD4+ and/or CD8+ T cells.

Embodiment II-125. The expanded T cell population of any one of Embodiments II-119 to II-124, wherein the T cells are multilineal.

Embodiment II-126. The expanded T cell population of any one of Embodiments II-120 to II-125, wherein at least 1% of the CD3+ T cells can produce TNFα.

Embodiment II-127. The expanded T cell population of any of Embodiments II-119 to II-126, wherein at least one T cell recognizes one or more target antigens from at least one pathogen, or a portion thereof.

Embodiment II-128. The expanded T cell population of Embodiment II-127, wherein the at least one pathogen is selected from the group consisting of: cytomegalovirus (CMV), adenovirus (Adv), BK virus, human Papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), simplex Herpes virus 2 (HSV-2), HIV, varicella zoster virus (VZV), EBV, cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), Human herpesvirus 8 (HHV-8), human herpesvirus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, Bocavirus , coronavirus, lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus ( WNV), Zika virus, Ebola virus and human T-lymphotropic virus.

Embodiment II-129. The expanded T cell population of embodiment II-127, wherein the at least one pathogen is hepatitis B virus (HBV).

Embodiment II-130. The expanded T cell population of embodiment II-127, wherein the at least one pathogen is human herpesvirus 8 (HHV-8).

Embodiment II-131. The expanded T cell population of Embodiment II-127, wherein the pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg) , DNA polymerase (HBP), X antigen (HBX) or combinations thereof.

Embodiment II-132. The expanded T cell population of Embodiment II-127, wherein the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP) ), X antigen (HBX) or a combination thereof.

Embodiment II-133. The expanded T cell population of Embodiment II-131 or II-132, wherein the HBeAg (E antigen) comprises pre-core (PreC) and core (HBcAg) antigens.

Embodiment II-134. The expanded T cell population of Embodiment II-127, wherein the pathogen is HBV and the at least one target antigen is HBcAg, HBsAg, or a combination thereof.

Embodiment II-135. The expanded T cell population of any one of Embodiments II-131 to II-134, wherein the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4), and EYLVSFGVWIRTPPA (SEQ ID NO: 5).

Embodiment II-136. The expanded T cell population of any one of Embodiments II-131 to II-135, wherein at least two HBV antigens are selected from different HBV genotypes.

Embodiment II-137. The expanded T cell population of Embodiment II-136, wherein at least two antigens are selected from the same HBV genotype.

Embodiment II-138. The expanded T cell population of embodiment II-136, wherein at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first gene genotype is different from this second genotype.

Embodiment II-139. The expanded T cell population of embodiment II-136, wherein at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C.

Embodiment II-140. The expanded T cell population of any one of Embodiments II-131 to II-139, wherein the amino acid sequence of each HBV antigen is at least One amino acid is different.

Embodiment II-141. The expanded T cell population of any one of Embodiments II-131 to II-140, wherein the expanded T cell population is reactive upon stimulation.

Embodiment II-142. The expanded T cell population of embodiment II-141, wherein at least a portion of the T cell reactivity is restricted by HLA-DR, HLA-DQ, or HLA-DP.

Embodiment II-143. The expanded T cell population of Embodiment II-127, wherein the pathogen is EBV and the at least one target antigen is EBNA1, LMP2, BZLF1, or a combination thereof.

Embodiment II-144. The expanded T cell population of embodiment II-127, wherein the pathogen is CMV and the at least one target antigen is IE1, pp65, or a combination thereof.

Embodiment II-145. The expanded T cell population of Embodiment II-127, wherein the pathogen is Adv and the at least one target antigen is hexon, penton, or a combination thereof.

Embodiment II-146. The expanded T cell population of embodiment II-127, wherein the pathogen is BK virus and the at least one target antigen is LT, VP-1, or a combination thereof.

Embodiment II-147. The expanded T cell population of Embodiment II-127, wherein the pathogen is HHV6 and the at least one target antigen is U14, U11, U71, U54, U90, or a combination thereof.

Embodiment II-148. The expanded T cell population of embodiment II-127, wherein the pathogen is RSV and the at least one target antigen is N, F, or a combination thereof.

Embodiment II-149. The expanded T cell population of Embodiment II-127, wherein the pathogen is influenza virus and the at least one target antigen is MP1, NP1, or a combination thereof.

Embodiment II-150. The expanded T cell population of Embodiment II-127, wherein the pathogen is a parainfluenza virus type (PIV) and the at least one target antigen is F, N, M, M2-1, HN or combination thereof.

Embodiment II-151. The expanded T cell population of embodiment II-127, wherein the pathogen is hMPV and the at least one target antigen is F, N, M2-1, M, or a combination thereof.

Embodiment II-152. The expanded T cell population of Embodiment II-127, wherein the pathogen is HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5) , vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70), or combinations thereof.

Embodiment II-153. The expanded T cell population of Embodiment II-152, wherein the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO : 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15).

Embodiment II-154. The expanded T cell population of Embodiment II-127, wherein the pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), matrix Protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4, nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14 or combination thereof.

Embodiment II-155. A plurality of expanded T cell populations, each of which is as in any one of Embodiments II-119 to II-154, wherein each expanded T cell population includes donors obtained from different donors. T cells produced by body monocytes.

Embodiment II-156. The plurality of expanded T cell populations of Embodiment II-155, wherein each donor has an HLA type that differs from at least one of the other donors by at least one HLA allele.

Embodiment II-157. A universal antigen-specific T cell therapy product comprising an expanded T cell population or a plurality of expanded T cell populations as in any one of Embodiments II-119 to II-156, wherein The product includes T cells generated from donor monocytes obtained from different donors.

Embodiment II-158. The universal antigen-specific T cell therapy product of Embodiment II-157, wherein the HLA type of each donor is different from at least one of the other donors by at least one HLA allele.

Embodiment II-159. The universal antigen-specific T cell therapy product of embodiment II-157 or II-158, wherein the product exhibits reduced or negligible efficacy against partially HLA matched and/or against HLA mismatched target cells. Alloreactivity.

Embodiment II-160. The universal antigen-specific T cell therapy product of any one of Embodiments II-157 to II-159, wherein the product comprises a VST that recognizes one or more target antigens from at least one pathogen, or a portion thereof .

Embodiment II-161. A pharmaceutical composition comprising an expanded T cell population, a plurality of expanded T cell populations or universal antigen-specific T cells as in any one of Embodiments II-119 to II-160 Therapeutic products.

Embodiment II-162. The pharmaceutical composition of Embodiment II-161, wherein the pharmaceutical composition is formulated for intravenous delivery.

Embodiment II-163. The pharmaceutical composition of Embodiment II-161 or II-162, wherein the pharmaceutical composition is negative for bacteria and fungi during continuous culture for at least 7 days.

Embodiment II-164. The pharmaceutical composition of any one of Embodiments II-161 to II-163, wherein the pharmaceutical composition exhibits endotoxin less than 5 EU/ml.

Embodiment II-165. The pharmaceutical composition of any one of Embodiments II-161 to II-164, wherein the pharmaceutical composition is negative for Mycoplasma.

Example II-166. A population of amplified VSTs specific for one or more viral antigens.

Example II-167. A population of amplified VSTs specific for two or more HBV antigens selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX.

Embodiment II-168. The population of amplified VSTs of Embodiment II-166, wherein the VSTs are specific for two or more HBV antigens selected from the group consisting of HBsAg, HBeAg, HBP and HBX.

Embodiment II-169. The population of amplified VSTs as in embodiment II-166, wherein the VSTs are specific for the HBV antigens HBcAg and HBsAg.

Example II-170. A population of amplified VST pairs, wherein the VST pairs are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP Two or more HHV8 antigens (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) are specific.

Embodiment II-171. The population of any one of Embodiments II-166 to II-170, comprising at least 70% CD3+ T cells.

Embodiment II-172. The population of any one of Embodiments II-166 to II-171, comprising no more than 30% CD56+ cells.

Embodiment II-173. The population of any one of Embodiments II-166 to II-172, wherein the VSTs have specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least Approximately 50 SFC/2 × ¹⁰ cells.

Embodiment II-174. The population of any one of Embodiments II-166 to II-173, wherein the VSTs comprise CD4+ and CD8+ T cells.

Embodiment II-175. The population of any one of Embodiments II-166 to II-174, wherein no more than 20% of the VSTs express a T cell exhaustion marker.

Embodiment II-176. The population of embodiment II-175, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT.

Embodiment II-177. The population of Embodiment II-175, wherein the T cell exhaustion marker is a co-expression of PD1 and TIM3.

Embodiment II-178. The population of one of Embodiments II-167 to II-177, wherein the VSTs express one or more markers associated with T cell activation.

Embodiment II-179. The population of embodiment II-178, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

Embodiment II-180. The population of any of Embodiments II-166 to II-179, wherein the VSTs express one or more markers associated with central or effector cell memory.

Embodiment II-181. The population of embodiment II-180, wherein the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

Embodiment II-182. The population of any one of Embodiments II-166 to II-181, wherein the VSTs are multifunctional.

Embodiment II-183. The population of any of Embodiments II-166 to II-182, wherein the VSTs produce two or more effector molecules when stimulated with two or more antigens.

Embodiment II-184. The population of Embodiment II-183, wherein the effector molecules are selected from the group consisting of: IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a, and MIP -1b.

Embodiment II-185. The population of any one of Embodiments II-167 to II-184, wherein compared to the cell population before expansion, IL-4, IL-7, and IL- After 15 amplifications, the VSTs were enriched at least 1000-fold.

Embodiment II-186. The population of Embodiment II-185, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, compared to the cell population before expansion, the VST is enriched at least 3000-fold.

Embodiment II-187. The population of Embodiment II-185, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, compared to the cell population before expansion, the VST is enriched at least 6000-fold.

Embodiment II-188. The population of any one of Embodiments II-170 to II-184, wherein compared to the cell population before expansion, in the presence of HHV8 antigen, IL-4, IL-7, and IL- After 15 amplifications, the VSTs were enriched at least 1000-fold.

Embodiment II-189. The population of Embodiment II-188, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, compared to the cell population before expansion, the VST is enriched at least 3000-fold.

Embodiment II-190. The population of Embodiment II-188, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, compared to the cell population before expansion, the VST is enriched at least 6000-fold.

Embodiment II-191. The population of any of Embodiments II-166 to II-190, wherein the VSTs have reduced alloreactivity.

Embodiment II-192. The population of any of Embodiments II-166 to II-190, wherein the VSTs have negligible alloreactivity.

Embodiment II-193. The population of any of Embodiments II-166 to II-190, wherein the VSTs have reduced reactivity against the allogeneic target.

Embodiment II-194. The population of any of Embodiments II-166 to II-190, wherein the VSTs have negligible reactivity against the allogeneic target.

Embodiment II-195. The population of any one of Embodiments II-166 to II-190, wherein the VSTs have reduced autoreactivity.

Embodiment II-196. The population of any one of Embodiments II-166 to II-190, wherein the VSTs have negligible autoreactivity.

Embodiment II-197. A pharmaceutical composition comprising the population of any one of Embodiments II-166 to II-196 and a pharmaceutically acceptable carrier.

Embodiment II-198. A kit comprising a multi-strain population of amplified VSTs as in any of Embodiments II-166 to II-196.

Embodiment II-199. A method of preventing or treating a viral infection, comprising administering to an individual a population of expanded VSTs as in any one of Embodiments II-166 to II-196.

Embodiment II-200. A method of preventing or treating viral infection, comprising administering to an individual the pharmaceutical composition of any one of Embodiments II-161 to II-165 and II-197.

Embodiment II-201. The method of any one of Embodiments II-199 to II-200, wherein the individual is immunocompetent or immunocompromised.

Embodiment II-202. The method of any one of Embodiments II-200 to II-201, wherein the pharmaceutical composition is administered by intravenous injection or infusion.

Embodiment II-203. The method of any one of Embodiments II-200 to II-202, wherein the pharmaceutical composition is administered to the individual multiple times.

Embodiment II-204. The method of any one of Embodiments II-200 to II-203, wherein about 5×10 ⁶ to about 5×10 ⁸ cells/square meter are administered to the individual.

Embodiment II-205. The method of any of Embodiments II-200 to II-204, wherein the individual is infected with a virus or is at risk of infection with a virus.

Embodiment II-206. The method of any one of Embodiments II-200 to II-205, wherein administration of the pharmaceutical composition is effective to treat or prevent the viral infection in the individual.

Embodiment II-207. The method of any one of Embodiments II-200 to II-206, wherein the subject a.Have received solid organ transplant; b.Have received chemotherapy; c. Suffering from HIV infection; d. Suffering from genetic immunodeficiency; e. Suffering from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation.

Embodiment II-208. The method of any one of Embodiments II-200 to II-207, wherein the pharmaceutical composition comprises the universal antigen-specific T cells of any one of Embodiments II-157 to II-160 Therapeutic products.

Embodiment II-209. The method of Embodiment II-208, wherein the pharmaceutical composition is administered to the individual without knowledge of the individual's HLA type.

Embodiment II-210. The method of any one of Embodiments II-199 to II-209, wherein the subject is a human.

Example II-211. A method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual any one of Examples II-166 to II-169 or II-171 to II-196 The population of those who have amplified VST.

Embodiment II-212. A method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual an expanded agent of any one of Embodiment II-166 or II-170 to II-196. Increase the number of VST groups.

Example II-213. An in vitro method for stimulating expansion of VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) adding one or more compositions to the cell culture, wherein the one or more compositions comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and/or (ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15; and (c) The cells are cultured for a period of time sufficient to stimulate expansion of VST.

Example II-214. An in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) adding one or more compositions to the cell culture, wherein the one or more compositions comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and/or (ii) one or more exogenous interleukins selected from IL-4, IL-7 and IL-15, and (c) Culturing the cells for a period of time sufficient to expand the VSTs.

Example II-215. An in vitro method for stimulating expansion of VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) performing a priming step, wherein the priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and/or (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; The cells are cultured for a period of time sufficient to stimulate expansion of VST.

Example II-216. An in vitro method for amplifying VST, comprising: (a) providing a starting population of mononuclear spheres in cell culture; (b) performing a priming step, wherein the priming step comprises adding to the cell culture one or more compositions, wherein the composition(s) comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen, and/or (ii) IL-4 and IL-7; and (c) adding IL-15 to the cell culture; The cells are cultured for a period of time sufficient to expand the VSTs.

Embodiment II-217. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate amplification of VST is about 18 days or less, optionally about 14 days.

Embodiment II-218. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate amplification of VST is from about 8 days to about 10 days.

Embodiment II-219. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate amplification of VST is from about 8 days to about 14 days.

Embodiment II-220. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate amplification of VST is from about 8 days to about 18 days.

Embodiment II-221. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate amplification of VST is from about 1 week to about 2 weeks.

Embodiment II-222. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate amplification of VST is from about 1 week to about 3 weeks.

Embodiment II-223. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate amplification of VST is from about 1 week to about 4 weeks.

Embodiment II-224. The method of Embodiment II-213 or II-215, wherein the period of time sufficient to stimulate expansion of VST is an amount of time sufficient to produce cells that have completed logarithmic phase growth.

Embodiment II-225. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is about 18 days or less, optionally about 14 days.

Embodiment II-226. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is from about 8 days to about 10 days.

Embodiment II-227. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is from about 8 days to about 14 days.

Embodiment II-228. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is from about 8 days to about 18 days.

Embodiment II-229. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is about 1 week to about 2 weeks.

Embodiment II-230. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is from about 1 week to about 3 weeks.

Embodiment II-231. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is from about 1 week to about 4 weeks.

Embodiment II-232. The method of Embodiment II-214 or II-216, wherein the period of time sufficient to amplify the VSTs is an amount of time sufficient to generate cells that have completed logarithmic phase growth.

Embodiment II-233. The method of any one of Embodiments II-215 to II-232, wherein step (c) is performed after step (b).

Embodiment II-234. The method of Embodiment II-233, wherein step (c) is performed about 12 hours after step (b).

Embodiment II-235. The method of Embodiment II-233, wherein step (c) is performed about 24 hours after step (b).

Embodiment II-236. The method of Embodiment II-233, wherein step (c) is performed about 48 hours after step (b).

Embodiment II-237. The method of Embodiment II-233, wherein step (c) is performed approximately 72 hours after step (b).

Embodiment II-238. The method of Embodiment II-233, wherein step (c) is performed about 1 day after step (b).

Embodiment II-239. The method of Embodiment II-233, wherein step (c) is performed about 2 days after step (b).

Embodiment II-240. The method of Embodiment II-233, wherein step (c) is performed about 3 days after step (b).

Embodiment II-241. The method of Embodiment II-233, wherein step (c) is performed about 4 days after step (b).

Embodiment II-242. The method of Embodiment II-233, wherein step (c) is performed about 5 days after step (b).

Embodiment II-243. The method of Embodiment II-233, wherein step (c) is performed about 6 days after step (b).

Embodiment II-244. The method of Embodiment II-233, wherein step (c) is performed about 7 days after step (b).

Embodiment II-245. The method of any one of Embodiments II-213 to II-244, wherein the method causes expansion of the VSTs but not NK cells.

Embodiment II-246. The method of any one of Embodiments II-213 to II-245, wherein the presence of IL-15 causes an acceleration of expansion of the cells in culture.

Embodiment II-247. The method of any of Embodiments II-213 to II-246, wherein the method generates an expanded population of VSTs that comprise a broader lineage than when the cells are contacted with Enriched VST of: (i) IL-4 only; (ii) IL-7 only; (iii) IL-15 only; (iv) IL-7 and IL-15, without IL-4; or (v) IL-4 and IL-15, no IL-7.

Embodiment II-248. The method of any one of Embodiments II-213 to II-246, wherein the method produces an expanded population of VSTs comprising an increased number of VSTs compared to when the cells are contacted with VST: (i) IL-4 only; (ii) IL-7 only; or (iii) IL-4 and IL-7 without IL-15.

Embodiment II-249. The method of any one of Embodiments II-215 to II-248, wherein the duration of the priming step is from about 1 to about 2 days.

Embodiment II-250. The method of any one of Embodiments II-215 to II-248, wherein the initiation step lasts for more than about 24 hours.

Embodiment II-251. The method of any one of Embodiments II-215 to II-248, wherein the duration of the initiation step is less than 24 hours.

Embodiment II-252. The method of any one of Embodiments II-215 to II-248, wherein the duration of the priming step is from about 0 to about 12 hours.

Embodiment II-253. The method of any one of Embodiments II-215 to II-248, wherein the duration of the priming step is from about 0 to about 24 hours.

Embodiment II-254. The method of any one of Embodiments II-215 to II-248, wherein the duration of the priming step is from about 24 to about 48 hours.

Embodiment II-255. The method of any one of Embodiments II-215 to II-248, wherein the duration of the priming step is from about 24 to about 72 hours.

Embodiment II-256. The method of any one of Embodiments II-215 to II-248, wherein the duration of the initiation step is less than about 144 hours.

Embodiment II-257. The method of any one of Embodiments II-215 to II-256, wherein the duration of the priming step is sufficient to produce at least a 2-fold higher amplification of the number of VSTs.

Embodiment II-258. The method of any one of Embodiments II-215 to II-257, wherein the method does not result in a greater than Higher levels of NK cell expansion would be expected in this scenario.

Embodiment II-259. The method of any of Embodiments II-213 to II-258, wherein the VSTs target one or more antigens from a latent virus.

Embodiment II-260. The method of any one of Embodiments II-214 or II-216 to II-259, comprising obtaining the amplified VSTs.

Example II-261. A two-step method for selectively expanding VSTs in a heterogeneous cell population, comprising: a first step of culturing the VSTs in the presence of a stimulating antigen and IL-4 and IL-7; and The second step is to culture the VSTs in the presence of IL-15.

Example II-262. A composition comprising VST expanded in a two-step process comprising: a first step of culturing the VST in the presence of IL-4 and IL-7; and in IL- The second step is to culture the VST in the presence of 15.

Example II-263. A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for two or more HBV antigens.

Embodiment II-264. The composition of embodiment II-263, wherein the population of VSTs comprises CD4+ and CD8+ cells.

Embodiment II-265. The composition of any of Embodiments II-263 to II-264, wherein the population of VSTs produces two or more effector molecules.

Embodiment II-266. The composition of any one of Embodiments II-263 to II-265, wherein the composition is produced in vitro.

Embodiment II-267. The composition of any one of Embodiments II-263 to II-265, wherein the composition is produced ex vivo.

Embodiment II-268. The composition of any one of Embodiments II-263 to II-267, wherein the VSTs produce two or more effector molecules when stimulated with the two or more HBV antigens .

Embodiment II-269. The composition of any one of Embodiments II-263 to II-268, wherein the two or more HBV antigens are selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX.

Embodiment II-270. The composition of any one of Embodiments II-263 to II-268, wherein the two or more HBV antigens are selected from the group consisting of HBsAg, HBeAg, HBP and HBX.

Embodiment II-271. The composition of any one of Embodiments II-263 to II-268, wherein the two or more HBV antigens are HBcAg and HBsAg.

Embodiment II-272. The composition of any one of Embodiments II-263 to II-271, wherein the effector molecules are selected from the group consisting of: IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b.

Embodiment II-273. The composition of any one of Embodiments II-263 to II-272, wherein the VSTs are multifunctional.

Embodiment II-274. The composition of any one of Embodiments II-263 to II-273, wherein the population of VSTs comprises at least 70% CD3+ T cells.

Embodiment II-275. The composition of any one of Embodiments II-263 to II-274, comprising no more than 30% CD56+ cells.

Embodiment II-276. The composition of any one of Embodiments II-263 to II-275, wherein the VSTs have specificities for the two or more antigens as measured by IFNγ ELISpot analysis. At least ~50 SFC/2 × 10e5 cells.

Embodiment II-277. The composition of any one of Embodiments II-263 to II-276, wherein the VSTs comprise CD4+ and CD8+ T cells.

Embodiment II-278. The composition of any one of Embodiments II-263 to II-277, wherein no more than 20% of the VSTs exhibit a T cell exhaustion marker.

Embodiment II-279. The composition of Embodiment II-278, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT.

Embodiment II-280. The composition of Embodiment II-278, wherein the T cell exhaustion markers are PD1 and TIM3.

Embodiment II-281. The composition of any one of Embodiments II-263 to II-280, wherein the VSTs express one or more markers associated with T cell activation.

Embodiment II-282. The composition of Embodiment II-281, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

Embodiment II-283. The composition of any one of Embodiments II-263 to II-282, wherein the VSTs express one or more markers associated with central or effector cell memory.

Embodiment II-284. The composition of embodiment II-283, wherein the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

Embodiment II-285. The composition of any one of Embodiments II-263 to II-284, wherein compared to the cell population before expansion, the concentration of IL-4, IL-7, and IL in the presence of HBV antigen is After amplification in -15, the VSTs were enriched at least 1000-fold.

Embodiment II-286. The composition of Embodiment II-285, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the Wait until VST is enriched at least 3000-fold.

Embodiment II-287. The composition of Embodiment II-285, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, the Wait until VST is enriched at least 6000-fold.

Embodiment II-288. The composition of any one of Embodiments II-263 to II-287, wherein the multi-strain population of VST is an antigen-specific multi-strain population of VST.

Embodiment II-289. The composition of Embodiment II-288, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HBV antigens.

Embodiment II-290. The composition of embodiment II-289, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg.

Embodiment II-291. The composition of Embodiment II-290, wherein the antigen-specific multi-strain population of VST pairs at least one peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 Sequence is specific.

Embodiment II-292. The composition of Embodiment II-289, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBeAg.

Embodiment II-293. The composition of Embodiment II-292, wherein the antigen-specific multi-strain population of VST is specific for at least one peptide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.

Embodiment II-294. The composition of Embodiment II-289, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from HBsAg and one or more peptide sequences derived from HBeAg sex.

Embodiment II-295. The composition of Embodiment II-294, wherein the antigen-specific multi-strain population of VST pairs at least one peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 The sequence and at least one peptide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5 are specific.

Example II-296. A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for two or more HHV8 antigens.

Embodiment II-297. The composition of embodiment II-296, wherein the population of VSTs comprises CD4+ and CD8+ cells.

Embodiment II-298. The composition of any of Embodiments II-296 to II-297, wherein the population of VSTs produces two or more effector molecules.

Embodiment II-299. The composition of any one of Embodiments II-296 to II-298, wherein the composition is produced in vitro.

Embodiment II-300. The composition of any one of Embodiments II-296 to II-299, wherein the composition is produced ex vivo.

Embodiment II-301. The composition of any one of Embodiments II-296 to II-300, wherein the VSTs produce two or more effector molecules when stimulated with the two or more HHV8 antigens .

Embodiment II-302. The composition of any one of embodiments II-296 to II-301, wherein the two or more HHV8 antigens are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

Embodiment II-303. The composition of any one of Embodiments II-296 to II-302, wherein the effector molecules are selected from the group consisting of: IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b.

Embodiment II-304. The composition of any one of Embodiments II-296 to II-303, wherein the VSTs are multifunctional.

Embodiment II-305. The composition of any one of Embodiments II-296 to II-304, wherein the population of VSTs comprises at least 70% CD3+ T cells.

Embodiment II-306. The composition of any one of Embodiments II-296 to II-305, comprising no more than 30% CD56+ cells.

Embodiment II-307. The composition of any one of Embodiments II-296 to II-306, wherein the specificity of the VSTs for the two or more antigens as measured by IFNγ ELISpot analysis is At least approximately 50 SFC/2 × ¹⁰ cells.

Embodiment II-308. The composition of any one of Embodiments II-296 to II-307, wherein the VSTs comprise CD4+ and CD8+ T cells.

Embodiment II-309. The composition of any one of Embodiments II-296 to II-308, wherein no more than 20% of the VSTs exhibit a T cell exhaustion marker.

Embodiment II-310. The composition of Embodiment II-309, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT.

Embodiment II-311. The composition of Embodiment II-310, wherein the T cell exhaustion marker is a co-expression of PD1 and TIM3.

Embodiment II-312. The composition of any one of Embodiments II-296 to II-311, wherein the VSTs express one or more markers associated with T cell activation.

Embodiment II-313. The composition of Embodiment II-312, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

Embodiment II-314. The composition of any one of Embodiments II-296 to II-313, wherein the VSTs express one or more markers associated with central or effector cell memory.

Embodiment II-315. The composition of embodiment II-314, wherein the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

Embodiment II-316. The composition of any one of Embodiments II-296 to II-315, wherein compared to the cell population before expansion, the concentration of IL-4, IL-7, and IL in the presence of HHV8 antigen is After amplification in -15, the VSTs were enriched at least 1000-fold.

Embodiment II-317. The composition of Embodiment II-316, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, the Wait until VST is enriched at least 3000-fold.

Embodiment II-318. The composition of Embodiment II-316, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, the Wait until VST is enriched at least 6000-fold.

Embodiment II-319. The composition of any one of Embodiments II-296 to II-318, wherein the multi-strain population of VST is an antigen-specific population of VST.

Embodiment II-320. The composition of embodiment II-319, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HHV8 antigens.

Embodiment II-321. The composition of embodiment II-320, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1.

Embodiment II-322. The composition of Embodiment II-321, wherein the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 6-12.

Embodiment II-323. The composition of embodiment II-320, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from gB.

Embodiment II-324. The composition of Embodiment II-323, wherein the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 13-15.

Embodiment II-325. The composition of Embodiment II-320, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1 and one or more peptide sequences derived from gB sex.

Embodiment II-326. The composition of Embodiment II-325, wherein the antigen-specific multi-strain population of VST pairs at least one peptide sequence selected from SEQ ID NO: 6-12 and selected from SEQ ID NO: 13- At least one of 15 peptide sequences is specific.

Embodiment II-327. The composition of any one of Embodiments II-263 to II-326, wherein the multi-strain population of VST has reduced alloreactivity.

Embodiment II-328. The composition of any one of Embodiments II-263 to II-326, wherein the multi-strain population of VST has negligible alloreactivity.

Embodiment II-329. The composition of any one of Embodiments II-263 to II-326, wherein the multi-strain population of VST has reduced reactivity against an allogeneic target.

Embodiment II-330. The composition of any one of Embodiments II-263 to II-326, wherein the multi-strain population of VST has negligible reactivity against the allogeneic target.

Embodiment II-331. The composition of any one of Embodiments II-263 to II-326, wherein the multi-strain population of VST has reduced autoreactivity.

Embodiment II-332. The composition of any one of Embodiments II-263 to II-326, wherein the multi-strain population of VST has negligible autoreactivity.

Embodiment II-333. The composition of any one of Embodiments II-263 to II-332, comprising a pharmaceutically acceptable diluent.

Embodiment II-334. The composition of Embodiment II-333 formulated for intravenous administration.

Example II-335. A method for treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises Specificity for two or more HBV antigens.

Embodiment II-336. The method of embodiment II-335, wherein the population of VSTs comprises CD4+ and CD8+ cells.

Embodiment II-337. The method of any of Embodiments II-335 to II-336, wherein the population of VSTs produces two or more effector molecules.

Embodiment II-338. The method of any one of Embodiments II-335 to II-337, wherein the composition comprising the multi-strain population of VST is produced in vitro.

Embodiment II-339. The method of any one of Embodiments II-335 to II-337, wherein the composition comprising a multi-strain population of VST is produced ex vivo.

Embodiment II-340. The method of any one of Embodiments II-335 to II-339, wherein the VSTs produce two or more effector molecules when stimulated with the two or more HBV antigens.

Embodiment II-341. The method of any one of Embodiments II-335 to II-340, wherein the two or more HBV antigens are selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX.

Embodiment II-342. The method of any one of Embodiments II-335 to II-340, wherein the two or more HBV antigens are selected from the group consisting of HBsAg, HBeAg, HBP and HBX.

Embodiment II-343. The method of any one of Embodiments II-335 to II-340, wherein the two or more HBV antigens are HBcAg and HBsAg.

Embodiment II-344. The method of any one of Embodiments II-337 to II-343, wherein the effector molecules are selected from the group consisting of: IFNγ, TNF-α, granzyme B, perforin, GM -CSF, MIP-1a and MIP-1b.

Embodiment II-345. The method of any of Embodiments II-335 to II-344, wherein the VSTs are multifunctional.

Embodiment II-346. The method of any one of Embodiments II-335 to II-345, wherein the population of VSTs comprises at least 70% CD3+ T cells.

Embodiment II-347. The method of any one of Embodiments II-335 to II-346, wherein the population of VSTs comprises no more than 30% CD56+ cells.

Embodiment II-348. The method of any one of Embodiments II-335 to II-347, wherein the VSTs have specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least Approximately 50 SFC/2 × 10e5 cells.

Embodiment II-349. The method of any one of Embodiments II-335 to II-348, wherein the VSTs comprise CD4+ and CD8+ T cells.

Embodiment II-350. The method of any one of Embodiments II-335 to II-349, wherein no more than 20% of the VSTs express a T cell exhaustion marker.

Embodiment II-351. The method of Embodiment II-350, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT.

Embodiment II-352. The method of Embodiment II-350, wherein the T cell exhaustion markers are PD1 and TIM3.

Embodiment II-353. The method of any one of Embodiments II-335 to II-352, wherein the VSTs express one or more markers associated with T cell activation.

Embodiment II-354. The method of Embodiment II-353, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

Embodiment II-355. The method of any of Embodiments II-335 to II-354, wherein the VSTs express one or more markers associated with central or effector cell memory.

Embodiment II-356. The method of embodiment II-355, wherein the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

Embodiment II-357. The method of any one of Embodiments II-335 to II-356, wherein compared to the cell population before expansion, in the presence of HBV antigen, IL-4, IL-7, and IL- After 15 amplifications, the VSTs were enriched at least 1000-fold.

Embodiment II-358. The method of Embodiment II-357, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, compared to the cell population before expansion, the VST is enriched at least 3000-fold.

Embodiment II-359. The method of Embodiment II-357, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HBV antigen, compared to the cell population before expansion, the VST is enriched at least 6000-fold.

Embodiment II-360. The method of any one of Embodiments II-335 to II-359, wherein the multi-strain population of VST is an antigen-specific multi-strain population of VST.

Embodiment II-361. The method of Embodiment II-360, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HBV antigens.

Embodiment II-362. The method of Embodiment II-361, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBsAg.

Embodiment II-363. The method of Embodiment II-362, wherein the antigen-specific multi-strain population of VST pairs at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 Be specific.

Embodiment II-364. The method of Embodiment II-361, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from HBeAg.

Embodiment II-365. The method of Embodiment II-364, wherein the antigen-specific multi-strain population of VST is specific for at least one peptide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5.

Embodiment II-366. The method of Embodiment II-361, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from HBsAg and one or more peptide sequences derived from HBeAg .

Embodiment II-367. The method of Embodiment II-361, wherein the antigen-specific multi-strain population of VST pairs at least one peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 And at least one peptide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5 is specific.

Embodiment II-368. The method of any one of Embodiments II-335 to II-367, wherein the multi-strain population of VST has reduced alloreactivity.

Embodiment II-369. The method of any one of Embodiments II-335 to II-367, wherein the multi-strain population of VST has negligible alloreactivity.

Embodiment II-370. The method of any of Embodiments II-335 to II-367, wherein the multi-strain population of VST has reduced reactivity against an allogeneic target.

Embodiment II-371. The method of any of Embodiments II-335 to II-367, wherein the multi-strain population of VST has negligible reactivity against the allogeneic target.

Embodiment II-372. The method of any one of Embodiments II-335 to II-367, wherein the multi-strain population of VST has reduced autoreactivity.

Embodiment II-373. The method of any one of Embodiments II-335 to II-367, wherein the multi-strain population of VST has negligible autoreactivity.

Embodiment II-374. The method of any one of Embodiments II-335 to II-373, wherein the composition is administered to the subject at a dose of about 5×10 ⁶ to about 5×10 ⁸ cells/square meter Invest.

Embodiment II-375. The method of any one of Embodiments II-335 to II-374, wherein the composition is administered to the subject once a week, once every two weeks, once every three weeks, or Give every four weeks.

Embodiment II-376. The method of any of Embodiments II-335 to II-374, wherein the composition is administered to the subject a plurality of times.

Embodiment II-377. The method of any of Embodiments II-335 to II-376, wherein the individual is immunocompromised.

Embodiment II-378. The method of any one of Embodiments II-335 to II-377, wherein the subject a.Have received solid organ transplant; b.Have received chemotherapy; c. Suffering from HIV infection; d. Suffering from genetic immunodeficiency; e. Suffering from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation.

Embodiment II-379. The method of any one of Embodiments II-335 to II-378, wherein the subject is an allogeneic hematopoietic stem cell transplant (allo-HCT) recipient.

Example II-380. A method for treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises a combination of two or more specificities for HHV8 antigens.

Embodiment II-381. The method of Embodiment II-380, wherein the population of VSTs comprises CD4+ and CD8+ cells.

Embodiment II-382. The method of any of Embodiments II-380 to II-381, wherein the population of VSTs produces two or more effector molecules.

Embodiment II-383. The method of any one of Embodiments II-380 to II-382, wherein the composition comprising the multi-strain population of VST is produced in vitro.

Embodiment II-384. The method of any one of Embodiments II-380 to II-382, wherein the composition comprising the multi-strain population of VST is produced ex vivo.

Embodiment II-385. The method of any one of Embodiments II-380 to II-384, wherein the VSTs produce two or more effector molecules when stimulated with the two or more HHV8 antigens.

Embodiment II-386. The method of any one of embodiments II-380 to II-385, wherein the two or more HHV8 antigens are selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10 .5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70).

Embodiment II-387. The method of any one of Embodiments II-385 to II-386, wherein the effector molecules are selected from the group consisting of: IFNγ, TNF-α, granzyme B, perforin, GM -CSF, MIP-1a and MIP-1b.

Embodiment II-388. The method of any of Embodiments II-380 to II-387, wherein the VSTs are multifunctional.

Embodiment II-389. The method of any one of Embodiments II-380 to II-388, wherein the population of VSTs comprises at least 70% CD3+ T cells.

Embodiment II-390. The method of any one of Embodiments II-380 to II-389, wherein the population of VSTs comprises no more than 30% CD56+ cells.

Embodiment II-391. The method of any one of Embodiments II-380 to II-390, wherein the VSTs have specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least Approximately 50 SFC/2 × ¹⁰ cells.

Embodiment II-392. The method of any one of Embodiments II-380 to II-391, wherein the VSTs comprise CD4+ and CD8+ T cells.

Embodiment II-393. The method of any one of Embodiments II-380 to II-392, wherein no more than 20% of the VSTs express a T cell exhaustion marker.

Embodiment II-394. The method of Embodiment II-393, wherein the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT.

Embodiment II-395. The method of Embodiment II-393, wherein the T cell exhaustion marker is a co-expression of PD1 and TIM3.

Embodiment II-396. The method of any one of Embodiments II-380 to II-395, wherein the VSTs express one or more markers associated with T cell activation.

Embodiment II-397. The method of Embodiment II-396, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69.

Embodiment II-398. The method of any of Embodiments II-380 to II-397, wherein the VSTs express one or more markers associated with central or effector cell memory.

Embodiment II-399. The method of embodiment II-398, wherein the one or more markers associated with central or effector cell memory are selected from CD45RO and CD62L.

Embodiment II-400. The method of any one of Embodiments II-380 to II-399, wherein compared to the cell population before expansion, in the presence of HHV8 antigen, IL-4, IL-7, and IL- After 15 amplifications, the VSTs were enriched at least 1000-fold.

Embodiment II-401. The method of Embodiment II-400, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, compared to the cell population before expansion, the VST is enriched at least 3000-fold.

Embodiment II-402. The method of Embodiment II-400, wherein after expansion in IL-4, IL-7, and IL-15 in the presence of HHV8 antigen, compared to the cell population before expansion, the VST is enriched at least 6000-fold.

Embodiment II-403. The method of any one of Embodiments II-380 to II-402, wherein the multi-strain population of VST is an antigen-specific population of VST.

Embodiment II-404. The method of Embodiment II-403, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HHV8 antigens.

Embodiment II-405. The method of Embodiment II-404, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1.

Embodiment II-406. The method of Embodiment II-405, wherein the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from the group consisting of SEQ ID NOs: 6-12.

Embodiment II-407. The method of Embodiment II-404, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from gB.

Embodiment II-408. The method of Embodiment II-407, wherein the antigen-specific multistrain population of VST is specific for at least one peptide sequence selected from the group consisting of SEQ ID NOs: 13-15.

Embodiment II-409. The method of Embodiment II-404, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from LANA1 and one or more peptide sequences derived from gB .

Embodiment II-410. The method of Embodiment II-409, wherein the antigen-specific multi-strain population of VST pairs at least one peptide sequence selected from SEQ ID NO: 6-12 and selected from SEQ ID NO: 13-15 At least one of the peptide sequences is specific.

Embodiment II-411. The method of any one of Embodiments II-380 to II-410, wherein the multi-strain population of VST has reduced alloreactivity.

Embodiment II-412. The method of any one of Embodiments II-380 to II-410, wherein the multi-strain population of VST has negligible alloreactivity.

Embodiment II-413. The method of any of Embodiments II-380 to II-410, wherein the multi-strain population of VST has reduced reactivity against an allogeneic target.

Embodiment II-414. The method of any of Embodiments II-380 to II-410, wherein the multi-strain population of VST has negligible reactivity against the allogeneic target.

Embodiment II-415. The method of any one of Embodiments II-380 to II-410, wherein the multi-strain population of VST has reduced autoreactivity.

Embodiment II-416. The method of any one of Embodiments II-380 to II-410, wherein the multi-strain population of VST has negligible autoreactivity.

Embodiment II-417. The method of any one of Embodiments II-380 to II-416, wherein the composition is administered to the subject at a dose of about 5×10 ⁶ to about 5×10 ⁸ cells/square meter Invest.

Embodiment II-418. The method of any one of Embodiments II-380 to II-417, wherein the composition is administered to the subject once a week, once every two weeks, once every three weeks, or Give every four weeks.

Embodiment II-419. The method of any of Embodiments II-380 to II-417, wherein the composition is administered to the individual a plurality of times.

Embodiment II-420. The method of any of Embodiments II-380 to II-419, wherein the individual is immunocompromised.

Embodiment II-421. The method of any one of Embodiments II-380 to II-420, wherein the individual a. has received a solid organ transplant; b. has received chemotherapy; c. has an HIV infection; d. Suffer from genetic immunodeficiency; e. Suffer from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation. Embodiment II-422. The method of any one of Embodiments II-380 to II-421, wherein the subject is an allogeneic hematopoietic stem cell transplant (allo-HCT) recipient. sequence list SED ID number describe biological sequence SEQ ID NO:1 HBsAg pep-1 PLPVLQAGFFLLTRI SEQ ID NO:2 HBsAg pep-2 FFLLTRILTIPQSLD SEQ ID NO:3 HBsAg pep-3 AKYLWEWASVRFSWL SEQ ID NO:4 HBeAg pep-1 QAILCWGELMTLATW SEQ ID NO:5 HBeAg pep-2 EYLVSFGVWIRTPPA SEQ ID NO:6 LANA1 pep-1 ADSVDGRECGPHTLP SEQ ID NO:7 LANA1 pep-2 PGSPTVFTSGLPAFVSSPT SEQ ID NO:8 LANA1 pep-3 TDTHSPSPALPTQS SEQ ID NO:9 LANA1 pep-4 DNDNKDDEEEQETDE SEQ ID NO:10 LANA1 pep-5 EEPIILHGSSSEDEM SEQ ID NO:11 LANA1 pep-6 ILHGSSSEDEMEVDY SEQ ID NO:12 LANA1 pep-7 HPLAGNLQSSIVKFKKPLPLTQP SEQ ID NO:13 gB pep-1 IVPHIFKVRRYRKIATSVT SEQ ID NO:14 gB pep-2 DTCEHYFITRNETLV SEQ ID NO:15 gB pep-3 IFMLSRRTNTIAQAPVKMIYPDV SEQ ID NO: 16 HHV8 LANA1 MAPPGMRLRSGRSTGAPLTRGSCRKRNRSPERCDLGDDLHLQPRRKHVADSVDGRECGPHTLPIPGSPTVFTSGLPAFVSSPTLPVAPIPSPAPATPLPPPALLPPVTTSSSPIPPSHPVSPGTTDTHSPSPALPPTQSPESSQRPPLSSPTGRPDSSTTPMRPPPSQQTTPPHSPTTPPPEPPSKSSPDSLAPSTLRSLRKRRLSSPQGPSTLNPICQSPPVSPPRCDFANRSVYPP WATESPIYVGSSSDGDTPPRQPPTSPISIGSSSPSEGSWGDDTAMLVLLAEIAEEASKNEKECSENNQAGEDNGDNEISKESQVDKDDNDNKDDEEEQETDEEDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEEEDEEEDEEEDEEEEEDEEDDDDEDNEDEEDDEEEDKKEDEEDGGDGNKTLSIQSSQQQQEPQQQEPQQQEPQQQEPQQQEPQQQEP QQQEPS EQQDEQQQDEQQQQDEEQ LEEVEEQEQEQEEQELEEVEEQEQEEQEEQELEEVEEQEEQELEEVEEQEEQELEEVEEQEQQGVEQQEQETVEPIILHGSSSEDEMEVDYPVVSTHEQIASSPPGDNTPDDDPQPGPSREYRYVLRTSPPHRPGVRMRRVPVTHPKKPHPRYQQPPVPYRQIDDCPAKARPQHIFYRRFLGKDGRRDPKCQWKFAVIFWGNDPYGLKKLSQAF QFGGVKAGPVSCLPHPGPDQSPITYCVYVYCQNKDTSKKVQMARLAWEASHPLAGNLQSSIVKFKKPLTQPGENQGPGDSPQEMT Example

The invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the invention should in no way be construed as limited to such examples but should be construed to cover any and all variations that become apparent as a result of the teachings provided herein.

Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the compositions of the invention and practice the claimed methods. Accordingly, the following working examples particularly indicate preferred embodiments of the invention and should not be construed as limiting the remainder of the invention in any way.

The materials and methods used in these experiments are now described.

The invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the invention should in no way be construed as limited to such examples but should be construed to cover any and all variations that become apparent as a result of the teachings provided herein.

Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the compositions of the invention and practice the claimed methods. Accordingly, the following working examples particularly indicate preferred embodiments of the invention and should not be construed as limiting the remainder of the invention in any way.

The materials and methods used in these experiments are now described. Example 1: Expansion and analysis of hepatitis B virus-specific T cell populations

Some viruses can cause chronic viral diseases and/or virus-related malignancies. This example includes primary infection with the latent virus human herpesvirus 8, which is generally asymptomatic in immunocompetent individuals. However, in immunocompromised patients, initial infection can present with lymphadenopathy, acute pancytopenia, and rapid progression to disseminated disease. In fact, HHV8 is the causative agent of all types of Kaposi's sarcoma (KS). Similarly, hepatitis B virus (HBV) infection causes short-term illness in many people. But for others, hepatitis B virus can become a long-term, chronic infection that can lead to serious and even life-threatening health problems, such as cirrhosis or liver cancer. Endogenous T cells reactive against these viruses often circulate at low frequencies and exhibit features of dysfunction/exhaustion.

Indeed, patients chronically infected with hepatitis B virus (HBV) have endogenous T-cell defects, including dysfunctional and exhausted HBV-reactive cells, which can be induced by receptive transfer of HBV reactivity produced by healthy immune individuals. T cells solve it. HBsAg-positive allogeneic hematopoietic cell transplant (HCT) recipients who receive stem cells (and lymphocytes) from donors who are positive for HBc and HBs antibodies show higher post-transplant functional cure rates. The feasibility of extending this benefit to all patients with chronic HBV infection was investigated by generating a repertoire of HBV-specific T cells (HBVST) from healthy donors suitable for off-the-shelf clinical use. Evaluation of HBV immunity in donors with past infection compared to vaccinated donors showed that donors with past infection exhibited immune activity to antigens other than the vaccine target antigen HBsAg (surface antigen). Individuals with prior infection also showed activity against HBcAg (core) ( Figure 3 ). It has been previously demonstrated that incubation of monocytes with HBV viral antigen and both IL4 and IL7 results in selective enrichment of CD4+ and CD8+ virus-specific T cells. Although IL4 and IL7 cultures are antigen-specific, they do not expand well in culture, which limits the ability to generate cells sufficient to treat multiple patients. The present invention provides, at least in part, improved methods for enriching and expanding T cells that support maintenance of virus specificity and promote functional efficacy. Notably, the improved methods of the present invention provide selective expansion of antigen-specific cells (eg, VST) without expanding non-specific T cells that pose potential safety risks.

In this study, a pool of ex vivo expanded HBV-specific T cells (HBVST) from healthy donors was generated. Without being bound by theory, it is believed that these HBVSTs are suitable for ready allogeneic administration and (when administered as partially HLA matched products) will be beneficial to patients with chronic HBV infection. A representative diagram of a method for expanding a population of antigen-specific T cells is provided in Figure 1 . Additionally, a non-limiting example illustrating a method of using IL-4, IL-7, and IL-15 to expand a population of antigen-specific T cells is depicted in Figure 2A .

In some embodiments, PBMC (1.5 × 10 7 ) were prepared by mixing PBMC (1.5 × 10 ⁷ ) in G-Rex5 (Wilson Wolf Manufacturing Corporation, St. Paul, MN) supplemented with IL-7 (20 ng/mL), IL-4 ( 800 U/mL) (R&D Systems, Minneapolis, MN) and mixed peptides (2 nanograms/peptide/ml) in 50 mL VST medium [90% TexMACS GMP medium (Miltenyi Biotec, GmbH), 2 mM GlutaMAX, and 10% human AB serum] were cultured together to produce HBVST. IL15 (5 ng/ml) (R&D Systems, Minneapolis, MN) was added at startup or on day 1 or 2 of culture (different for each donor). Culture VST at 37°C, 5% _CO for 8 to 10 days. i. Identification of immunodominant HBV antigens using HBVST generated with at least three antigens

To investigate the possibility of using receptive transferred T cells to target hepatitis B, hepatitis B (HBV)-specific T cells (HBVST) were generated from healthy donors. Antigens encoded by HBV include (but are not limited to) surface antigen (HBsAg), E antigen (HBeAg: core (HBcAg) + pre-core), DNA polymerase (HBP), X antigen (HBX), or combinations thereof. To characterize the cellular immune response against HBV, T cell activity was assessed against 4 encoded viral antigens (LE, E, P and X). As used in this example, LE refers to HBsAg, E refers to HBeAg (combination of core and precore), P refers to HBP, and X refers to HBX. Briefly, a peptide library covering HBV LE, E, P and X antigens was used in the presence of IL-7, IL-4 and IL-15 (800 U/mL, 20 ng/mL and 5 ng/mL, respectively). Expand reactive T cells, thereby producing HBVST. To expand the frequency of reactive cells, a single dose was performed on day 0 by exposing PBMC to a master mixture of HBV mixed peptides (11aa overlapping 15-mers) and administering IL-4, IL-7, and IL-15. In vitro stimulation is followed by an expansion period of 8 to 10 days ( Figure 1 ). IL-4, IL-7, and IL-15 were administered at 800 U/mL, 20 ng/mL, and 5 ng/mL, respectively. Expansion resulted in an average 7.3 ± 0.6-fold increase in total cell number (n = 9; Figure 2B ).

After amplification, the specificity of the amplified cells was assessed by IFNγ ELISpot (enzyme-linked immunospot) analysis ( Figure 6A -Figure 6B ). This analysis measures the frequency of VST specific for different HBV antigens (LE, E, P and X). Figure 6A shows the frequency of T cells specific for specific HBV antigens. A cell population is defined as "reactive" when the frequency of reactive cells is >30 spot-forming cells (SFC)/2 × 10^5 input virus-specific T cells, as measured by the IFNγ ELISpot assay. Fold enrichment was determined based on cell number, and expansion resulted in a >6000-fold enrichment of total HBV-reactive T cells over an 8- to 10-day culture period ( Figure 6B ).

Cells from 9 healthy donors were tested for LE, P, E and X antigens. The LE antigen was recognized in cells from all 9 donors and induced the highest frequency of HBV-specific cells ( Fig. 7 ). Cells from all 9 donors were reactive to at least one antigen (LE, P, E, or X), and each antigen tested induced a response in at least one donor evaluated ( Figure 7 ). The HBV antigens observed to be recognized in an immunodominant manner were LE, P and E, and these three antigens were therefore selected for manufacturing and further characterization studies.

An illustrative manufacturing process is described below. Blood was collected by peripheral blood draw, and PBMC were isolated by Ficoll gradient. PBMC (3 × 10 ⁶ cells per square centimeter) were inoculated in a G-Rex 5 bioreactor (Wilson Wolf, Minneapolis, MN) and incubated in a solution containing 800 U/mL IL-4 and 20 ng/mL IL-7 ( R&D Systems, Minneapolis, MN), peptide mixture (mixed peptides) (2 ng/peptide/ml) in T cell culture medium [TexMACS (Miltenyi), 2 mM GlutaMAX™ TM-I (Life Technologies Grand Island, NY) and 10 % human serum (Hyclone)]. On days 8 to 10 after priming, T cells were harvested, counted and further analyzed. In some embodiments, the day of addition of mixed peptide is considered as day 0 of culture. In some embodiments, the day when mixed peptide, IL-4, and IL-7 are added is considered as day 0 of culture. In some embodiments, day 0 of culture is also referred to as start-up. In some embodiments, mixed peptides are added simultaneously with IL-4 and IL-7. In some embodiments, the mixed peptide is added after IL-4 and IL-7. In some embodiments, the mixed peptide is added after IL-4 and IL-7 but within 24 hours of adding IL-4 and IL-7. IL-15 (5 ng/mL) (R+D systems, Minneapolis, MN) was added at startup or on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of culture. After a total of 8 to 10 days in culture, T cells were harvested. In some embodiments, the optimized VST manufacturing process includes the T cell expansion process of the present invention. HBVST is multi-strain and multi-specific

Phenotypic characteristics of VST were assessed by flow cytometry using antibodies against: CD3, CD4, CD8, CD62L, CD69, CD25, CD45RO, CD279 (PD-1), and CD366 (TIM-3) (BioLegend, San Diego, CA) ( Figure 4 ). Intracellular interleukin staining (ICS) was used to assess the effector signature of reactive cells. The cells were significantly CD3+ T cells and showed activation signatures with minimal expression of exhaustion markers (PD1 and TIM3) ( Figure 4 ). Reactive cells (HBVST targeting LE, P, and E) were multi-strain and predominantly CD4+ T cells (mean 72±4%), with a minor CD8+ (21±2%) component ( Fig. 4 ). Cells expressed markers related to central (CD45RO+/CD62L+: 58±3%) or effector memory (CD45RO+/CD62L-: 30±3%) potential based on CD25 (59±5%) and/or CD69 (21± 3%) performance activation. These results indicate that HBVST exhibits phenotypes consistent with effector function and long-term memory.

Intracellular interleukin staining (ICS) was used to assess the effector signature of reactive cells and to determine the cellular phenotype of T cells ( Figure 5 ). ICS results from representative donors (depicted in Figure 2B ) showed that both CD4+ and CD8+ T cell subsets were virus-specific for the targeted antigens ( Figure 5 ). HBVST is Th-1 polarized and versatile

Use HBVST for polarization and multifunctionality analysis. The bulk interleukin secretome of T cells was assessed by ^Luminex® analysis of samples collected after overnight stimulation with individual HBV antigens (LE, E, or P) ( Figure 8 ). T cells demonstrated multifunctionality by producing multiple effector molecules upon antigen exposure via fluorescent spot analysis ( Figure 9 ), with T cells simultaneously producing molecules such as IFNγ, TNFα, and granzyme B ( Figures 8 and 9 ). In addition to IFNγ, HBVST also produces TNFα, GM-CSF, MIP-1α, MIP-1β and particles, as measured by ^Luminex® assay ( Figure 8 ) and fluorescent spot analysis ( Figure 9 ) following the manufacturer's recommendations. Enzyme B. Approximately two-thirds of IFNγ-secreting cells additionally produced TNFα, granzyme B, or both effector molecules ( Fig . 9 ). ii. HBVST generated using two antigens

In some embodiments, two antigens can be used to generate HBVST. In the following experiments of this example, HBsAg (LE) and HBcAg (C) antigens were used to generate HBVST. Optimized seeding density

For T cell expansion, various seeding densities of 2 × 10 ⁶ /cm ² , 2.5 × 10 ⁶ /cm ² , 3 × 10 ⁶ /cm ² , 3.5 × 10 ⁶ /cm ² and 4 × 10 ⁶ /cm ² were tested . Specifically, using the method described in Figure 2A , a peptide library covering LE and C (core) antigens (2 nanograms/peptide/ml) was used to stimulate health in the presence of IL-7, IL-4, and IL-15. Donor cells. Control cells (neg) were generated by stimulating cells with IL-7, IL-4 and IL-15 in the absence of mixed peptides. All vaccinations demonstrated a maintained fold expansion of cells ( Fig. 11A ), cell activation as measured by IFNγ ELISpot ( Fig. 11B ), and enrichment of cells reactive to LE and C antigens ( Fig. 11C ). densities were effective in generating virus-specific T cells. However, a seeding density of 3 × 10 ⁶ /cm ² produced better results in terms of amplification fold and enrichment fold ( Figure 11A and Figure 11C ). Antigen specificity for C and LE antigens was found at all vaccination densities tested. Optimized growth conditions

The use of various interleukins at various time points in the amplification method was assessed. In one example, T cells are contacted with medium containing IL-7 and IL-15 or medium containing IL-7 and IL-4, as depicted in Figure 12 . To assess the expansion fold and phenotype of cells using this culture method, 1.5 × 10 ⁷ donor PBMC were cultured with a peptide library of HBV LE and C antigens (2 nanograms/peptide/ml). While applying the peptide library to the cells, 20 ng/mL of IL-7 and 5 ng/mL of IL-15 or 20 ng/mL of IL-7 and 800 U/mL of IL-4 were administered to the cells. . On day 10 of incubation, VST in contact with IL-7 and IL-15 exhibited 9.9-fold amplification, and VST in contact with IL-7 and IL-4 had a 3.93-fold amplification ( Figure 13 ). The phenotypes of VST exposed to media containing different interleukins were analyzed, and the results showed that multi-strain phenotypic ratios were maintained in the expansion method using both types of media ( Figure 14 ). Including IL-15 and IL-7 seems to increase the proportion of CD56+CD3- cells and Tim3+CD3+ cells. For expansion methods using media supplemented with different interleukin combinations (IL-7+IL-15 or IL-7+IL-4), VST was antigen-specific with more than 3000-fold increase in HBV-reactive T cells enriched ( Figure 15B ). Cells expanded under IL-7 and IL-4 conditions exhibited higher overall antigen specificity as measured by IFNγ ELISpot compared to cells expanded under IL-7 + IL-15 conditions ( Figure 15A ).

Both IL-15 and IL-2 are used in cell culture methods to support T cell proliferation. (Rochman et al. Nat. Rev. Immunol. 9(7):480; July 2009). Therefore, it was investigated whether IL-15 or IL-2 would support the selective expansion of antigen-specific T cells (eg, VST) of the invention. Studies were conducted to assess whether the characteristics of expanded VST could be optimized by adding IL-15 to IL-7 and IL-4 conditions at different time points. Testing and optimizing an interleukin "mixture" for use in an expansion method in which T cells are initially exposed to culture medium containing 800 U/mL of IL-4 and 20 ng/mL of IL-7 on day 0, and IL-15 was added to the culture on day 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 of culture. Ten different IL-15 conditions were tested, with each IL-15 condition corresponding to a different IL-15 (5 ng/mL) addition day ( Figure 16 ). For comparison, 10 different IL-2 conditions were also tested, where each IL-2 condition corresponded to a different IL-2 (100 U/mL) addition day. Donor PBMC were cultured with a peptide library containing antigens against HBV LE and C. At the same time, IL-4 and IL-7 are administered to the cells. IL-15 was then added at various time points as described above. For comparison, the peptide pool, IL-4 and IL-7 were administered to the cultures as described above and IL-2 was administered at various time points. On day 10, phenotypic performance was measured. VST generated under each IL-15 condition and each IL-2 condition were phenotypically assessed for the expression of CD56+CD3- (NK cells), CD8/CD3, and CD4/CD3. The proportions of CD56+CD3- cells, CD3+CD4+ cells, and CD3+CD8+ cells in the resulting expanded population are provided in Figures 17A and 17B . Addition of IL-15 to IL-4 and IL-7 resulted in CD56+CD3- cell frequencies similar to those seen under IL-4 and IL-7 only conditions ( Fig. 17A ). However, the addition of IL-7 and IL-15 (without IL-4) caused an increase in the number of non-specific cells, namely CD56+CD3- (NK cells). Compared with VST produced under IL-2 conditions (more than 3-fold increase in amplification at culture days 0 to 2) ( Figure 1 8B ), VST produced under IL-15 conditions (over 3-fold increase in amplification at culture days 0 to 2 More than 4-fold to 6-fold increase in amplification at 2 days) ( Figure 18A ) showed a higher level of amplification. VST amplification was observed at all time points. However, adding IL-15 to the culture at an earlier time point resulted in greater expansion of VST than adding IL-15 at a later time point. For example, addition of IL-15 on day 0 resulted in a 6.44-fold expansion of virus-specific cells, while addition on day 5 induced a 3.77-fold increase in virus-specific cells ( Figure 18A ).

In addition, VST produced under IL-15 conditions showed greater specificity for different HBV antigens (HB core Ag or "C", HB surface Ag or "LE") compared to VST produced under IL-2 conditions. properties and enrichment folds ( Fig. 19A and Fig. 20A compared to Fig. 19B and Fig. 20B ). Although VST production was effective when IL-15 was added to IL-4 and IL-7 on each test day, when IL-15 was added on day 0, day 1, or day 2 of culture, it was expanded. Achieve improved results in terms of proliferation, phenotype and specificity.

The use of IL-15 was further evaluated by the growth kinetics of VST in culture. Lactate is used as an indicator of cell growth since it is a by-product of glucose metabolism in cell culture and was measured using a lactate meter (nova ^® biomedical) following the manufacturer's instructions. Measurement of lactate concentration showed that adding IL-15 to the cell culture at different time points (days 0, 2, and 7) resulted in different cell growth rates ( Figure 21 ). Addition of IL-7+IL-15 (without IL-4) to cell cultures resulted in faster growth and earlier reaching maximum lactate content compared to other cultures. Cells cultured with IL-4 + IL-7 and IL-15 added on day 0 caused similar cell growth to cells cultured with IL-7 + IL-15 alone. Cells cultured with IL-4+IL-7 and IL-15 added on day 2 resulted in slightly slower cell growth than IL-15 added on day 0. Cells cultured with IL-4+IL-7 without the addition of IL-15 resulted in slower cell growth. Interestingly, cell cultures with IL-4+IL-7 and IL-15 added on day 7 showed a similar growth pattern to cell cultures with only IL-4+IL-7 for the first seven days, followed by The growth rate increased after adding IL-15 on day 7. These results indicate that the addition of IL-15 to IL-4 and IL-7 affects the growth kinetics of VST in culture. Taken together, these data demonstrate that incubation of monocytes with antigens (eg, mixed peptides) and IL-4, IL-7, and IL-15 increases cell expansion and that cell phenotype and antigen specificity are maintained. In contrast, cells cultured with IL-4, IL-7, and IL-2 did not produce the same magnitude of cell expansion. When the results for enrichment folds and specificities for different HBV antigens were combined (Figure 20A-20B), it was observed that the combination of IL-4, IL-7, and IL-15 and IL-4, IL-7, and IL- The 2 combinations produced superior results compared to each other (i.e., greater fold enrichment and specificity). Symptoms of HBVST

Single cell pluripotency assay (IsoPlexis) was used following the manufacturer's instructions to characterize VST generated by stimulation with LE and C antigens using the method described above. The results showed that most multifunctional CD4 cells secreted ≥2 different interleukins, and a smaller percentage of multifunctional CD4 cells secreted at least 4 different interleukins ( Figure 10A ); most multifunctional CD8 cells secreted ≥2 different interleukins, and a smaller percentage of multifunctional CD8 cells secrete more than 3 interleukins ( Figure 10A ). Based on interleukin expression, multifunctional CD4 and CD8 cells were classified into effector cells, stimulatory cells and chemoattractant cells, most of which were effector cells ( Figure 10B ). Effector cells are defined as cells that produce granzyme B, IFNγ, MIP-1a, perforin, and TNF-a. Stimulatory cells are defined as cells that produce GM-CSF and IL-5. Chemoattracted cells are defined as cells that produce MIP-1b.

This study established the feasibility of generating an ex vivo expanded HBV-specific T cell bank suitable for clinical use. In addition, studies have confirmed that comparing cells with peptide mixtures and IL-7, IL Incubation of a combination of -4 and IL-15 (eg, added on or before day 6 (eg, on day 0, 1 or 2)) together results in improved amplification and antigen specificity. Example 2: HBVST identifies target cells expressing antigens

To assess whether the HBVST of the present invention recognizes target cells expressing the antigen, autologous targets loaded with antigen (i.e., pulsed with mixed peptides) were evaluated in a short-term killing assay (Cr ⁵¹ release assay) generated as described in Example 1 In vitro cell lysis ability of HBVST. PHA blasts were used for antigen loading, and unpulsed PHA blasts not loaded with antigen were used as controls. The amplified HBVST specifically recognized and lysed autologous target cells loaded with antigen (40:1 E:T 36% specific lysis, Figure 22A ). No significant off-target lysis was observed, ie, no reactivity to autologous or allogeneic targets that were not loaded with antigen (not pulsed) (1% specific lysis each, Figure 22B ). For allogeneic targeting, HLA-mismatched PHA blasts are used.

In another lysis assay, partially HLA-matched HepG2.2.15 cells were used as targets to demonstrate that HBVST recognizes cells endogenously expressing HBV antigens. Hep-G2/2.2.15 is a human hepatoblastoma cell line expressing HBV antigen (genotype D). As depicted in Figure 23A , three cultures were grown, one containing only HBVST ("HBVST Control") and one containing partially HLA-matched HBVST and Hep-G2/2.2.15 cells ("HBVST + HepG2"), and One contained only Hep-G2/2.2.15 cells ("HepG2 control"). The results showed that IFNγ production occurred only in HBVST + HepG2 cultures, indicating that HBVST recognizes HBV-infected HepG2 cells ( Figure 23A ). As shown in Figure 23B , HBVST was generated from each of five donors (i to v), each of which partially matched the HLA type of HepG2/2.2.15 cells. HBVST generated from each donor was co-cultured with HepG2.2.15 cells, and specificity was measured by IFNγ ELISpot. As shown in Figure 23C , HBVST produced by each donor ("E") was able to recognize HepG2/2.2.15 cells ("T") and secrete IFNγ when co-cultured, whereas the E-only and T-only controls showed less IFNγ production.

To measure off-target effects, specific lysis assays were performed using HBVST or non-HBVST mixed with Hep-G2/2.2.15 cells at various ratios. As shown in Figure 24 , partially HLA matched HBVST lysed Hep-G2/2.2.15 cells at 71% (40:1 ratio of HBVST:Hep-G2/2.2.15). In contrast, non-HBVST did not exhibit significant lysis of Hep-G2/2.2.15 cells ( Figure 24 ).

In addition to short-term in vitro cell lysis analysis (4- to 6-hour chromium release assay), Hep-G2/2.2.15 cells were also used to form long-term in vitro 3D tumor models. As shown in Figure 25 , Hep-G2/2.2.15 cells grew into 3D aggregates to simulate the structure of solid tissue. HBVST and non-HBVST were added to Hep-G2/2.2.15 aggregates at various ratios, and the results were imaged every 2 hours using the ^IncuCyte® Live Cell Analysis System following the manufacturer's instructions. As shown in Figure 26 , HBVST lysis of cells was measured as total red object integrated intensity (RCU × µm ² /image). Higher imaging intensity indicates higher lytic activity. 2000 cells/well of HBVST showed increased lytic activity compared to 500/well of HBVST, whereas the control group without HBVST did not show a significant increase in lytic activity in the first 40 hours.

Taken together, these results show that HBVST is cytolytic and can selectively eliminate autologous and partially HLA-matched hepatoblastoma cells expressing HBV antigens. Additionally, the results showed that HBVST did not respond to allogeneic PHA blasts ( Figure 22B ), indicating the potential for safe use of these HBVST as an off-the-shelf treatment. Example 3: Amplification and analysis of human herpesvirus 8 virus-specific T cell populations

Human herpesvirus 8 (HHV8) is a type of human herpes associated with multicentric Castleman's disease (MCD), primary effusion lymphoma (PEL), and Kaposi's sarcoma (KS) in immunocompromised individuals. Virus. Generating a library of ex vivo expanded HHV8-specific T cells (HHV8 VST) from healthy donors suitable for off-the-shelf allogeneic administration may benefit patients with HHV8 infection or related diseases.

In order to identify immunogenic and protective HHV8 antigens, we analyzed 6 latent antigens (Kaposi protein, LANA-1, LANA-2, vFLIP, vCyc and RTA) and 4 soluble antigens (MIR-I , SSB, gB and TS) to identify immunodominant targets. The functions associated with these antigens are summarized in Figure 27 .

Use a peptide library (mixed peptide) covering 6 latent antigens (kaposin, LANA-1, LANA-2, vFLIP, vCyc and RTA) and 4 soluble antigens (MIR-I, SSB, gB and TS) ) expands reactive T cells in the presence of IL-7, IL-4, and IL-15, thereby generating HHV8 VST. As used in this example, a mixed peptide is a peptide mixture containing a 15-mer with 11 amino acid overlaps. The optimized VST manufacturing process outlined in Figure 28 was used to generate reactive T cell lines reactive against this antigen combination. In this example, PBMC were obtained from 9 healthy human donors.

For fabrication, blood was collected by peripheral blood draw and PBMCs were isolated by Ficoll gradient. PBMC (3 × 10 ⁶ cells per square centimeter) were inoculated in a G-Rex 5 bioreactor (Wilson Wolf, Minneapolis, MN) and incubated in a solution containing 800 U/mL IL-4 and 20 ng/mL IL-7 ( R&D Systems, Minneapolis, MN), peptide mixture (mixed peptides) (2 ng/peptide/ml) in T cell culture medium [TexMACS (Miltenyi), 2 mM GlutaMAX™] TM-I (Life Technologies Grand Island, NY) and 10% human serum (Hyclone)]. On days 8 to 10 after priming, T cells were harvested, counted and further analyzed. In some embodiments, the day of addition of IL-4 and IL-7 is considered as day 0 of culture, which is also referred to as priming. IL-15 (5 ng/mL) (R+D systems, Minneapolis, MN) was added at priming or on day 1 or 2 of culture, and after a total of 10 days in culture, T cells were harvested. In some embodiments, the optimized VST manufacturing process includes the in vitro T cell expansion method of the present invention.

Following stimulation with mixed peptides containing latent or lytic antigens, IL-4 and IL-7 (added on day 0), IL-15 (added for each donor) was added to the culture at approximately days 0 to 2 Different days), followed by culture expansion for 10 to 12 days, the average expansion fold of cells from each of the 9 donors ( Figure 29 ).

Antigen specificity analysis was performed by IFNγ enzyme-linked immunospot (ELISpot) assay to assess the frequency of IFNγ-producing T cells following exposure to individual HHV8 antigens. In some embodiments, a cell population is defined as when the frequency of reactive cells, as measured by the IFNγ ELISpot assay, is >30 spot-forming cells (SFC)/2 × ¹⁰ input virus-specific T cells. reactive". The antigen specificity measured for each population of donor-derived VST is shown in Figure 30 .

The phenotypic characteristics of these cells were assessed by flow cytometric analysis using antibodies against: CD3, CD4, CD8, CD25, CD69, CD16, TEM, TCM, PD-1 and TIM3. After 10 days in culture, intracellular interleukin staining (ICS), ^Luminex® and/or IsoPlexis was used to assess the effector signature of the reactive cells and determine the cellular phenotype of the T cells ( Figure 31 ). Phenotypically, each generated cell line contained both CD4+ and CD8+ populations, exhibited central markers (CD45RO+) and effector memory markers (CD45RO+/CD62L-), had upregulated activation markers and minimal depletion.

The expression of IFNγ and TNFα in HHV8-VST derived from healthy donors was determined by flow cytometric analysis. HHV8 VST displays multifunctionality through the production of multiple effector molecules upon antigen exposure (LANA1 and LANA2), with T cells simultaneously producing molecules such as IFNγ and TNFα ( Figure 32 ).

In vitro killing assay of HHV8 VST reactive to LANA1 and LANA2 using autologous PHA blasts loaded with mixed peptides as targets (as described in Example 2 ), and autologous loading of HHV8 antigens (LANA1 and LANA2) Specific dissolution of the target ( Figure 33 ).

A portion of the epitopes were mapped for the LANA1 and gB antigens, and the peptide sequences are revealed in Figures 34 and 35 .

Expanded HHV8 VST contains a mixture of cytotoxic (CD8+) and helper (CD4+) T cells with phenotypes consistent with effector function and memory potential, such as upregulation of activation markers CD25, CD69 and CD28 and central (CD45RO+/CD62L+) and effector memory (CD45RO+/CD62L-) markers. As demonstrated by phenotypic and functional characterization of these HHV8 VSTs (IFNγ and granzyme B ELISpot assays, intracellular interleukin staining (ICS), and in vitro killing assays), the expanded cells demonstrated cytolytic potential The secretion of multiple effector interleukins demonstrates its ability to control HHV8 infection in immunocompromised patients. Example 4 : Treatment with virus - specific T cells targeting HBV to prevent HBV infection

To evaluate the efficacy of HBV-targeting virus-specific T cells (HBVST), mice were exposed to HBV virus and treated with HBVST. Specifically, HBV was used to treat PXB mice (a chimeric mouse in which most of the mouse liver cells are replaced by transplanted h-hep expressing h-cytochrome P450 enzymes (CYP), phase II enzymes and transporters). and therefore has CYP enzyme induction potential) (Tateno et al. PLOS ONE, DOI: 10.1371/journal.pone.0142145, November 2015, which is incorporated herein by reference), and HBVST was administered 4 weeks later. The reason for selecting this time point is that it is believed to be the earliest time point at which HBV infection can be reliably quantified by serum HBV DNA measurement. Twelve PXB mice were vaccinated with HBV on day 0 and received two intravenous (tail vein) infusions of the following on days 28 and 56: (1) placebo; (2) control unrelated VST (Irr VST), which is a VST product produced by the same donor but specific for adenovirus; (3) HBVST, 1×10 ⁶ cells/mouse; or (4) HBVST, 1×10 ⁷ cells/mouse. Three PXB mice were not vaccinated with HBV and received HBVST (1×10 ⁷ cells/mouse) on days 28 and 56 as a safety control. Blood human albumin (h-Alb) was measured every two weeks. Human alanine aminotransferase 1 (h-ALT1) was measured weekly from day 21 to day 84 and then on day 98 and at the end. Serial sera for HBV DNA and HBsAg were collected weekly until day 84 and then on day 98 and at the end of day 112. Two RNAlater liver samples and formalin fixed paraffin embedded (FFPE) liver blocks (left lobe) were collected from all animals at the end. Serum HBV parameters h-ALT1 and h-Alb were measured. A diagram of the study design and sample collection is shown in Figure 36 .

Figure 1 shows an illustrative, non-limiting example of a schematic of a method for differentiating and expanding monocytes into an expanded population of antigen-specific T cells. As used in this figure, LE is HBsAg, E is HBeAg (combination of precore (PreC) and core (HBcAg) antigens), P is HBV polymerase, and X is HBV X antigen.

Figure 2A provides a schematic diagram illustrating a non-limiting example of a method of using IL-4, IL-7, and IL-15 to expand a population of antigen-specific T cells, wherein at days 0, 1, 3, Add IL-15 on day 5, 7 or 9.

Figure 2B shows the fold amplification of VST after using the amplification method of the present invention, in which a single in vitro stimulation was performed on day 0 by exposing PBMC to a master mixture of HBV mixed peptides (11aa overlapping 15-mers), followed by The expansion period is 8 to 10 days. In this scheme, IL-4, IL-7 and IL-15 are added on day 0.

Figure 3 shows the abundance of virus-specific T cells (SFC/2×10 ⁵ VST) measured by ELISpot in vaccinated and previously infected individuals. SFC refers to spot-forming cells. Identification markers include HBsAg and HBcAg.

Figure 4 shows the cell markers (CD3+, CD8+ CD3+, CD4+ CD3+, CD56+ CD3-, CD56+ CD3+, CM/CD3, EM/CD3, EMRA/CD3, CD25+ CD3+, CD69+ CD3+, PD1+ CD3+, Tim3+ CD3+, PD1+ Tim3+) Phenotypic analysis based on percent performance following methods used to amplify VST in vitro. (n=6)

Figure 5 shows phenotypic analysis of cellular markers (CD4+ and CD8+) and IFNγ as they relate to VST specific for different HBV antigens (LE, E and P). IFNγ was measured by intracellular interleukin staining (ICS) in cells from representative donors.

Figure 6A to Figure 6B show the results of VST specific for different HBV antigens (HBeAg (PreC+HBcAg) or ‘E’, HB surface Ag or ‘LE’, polymerase or ‘P’ and HB X protein ‘X’) IFNγ performance of VST measured by ELISpot (SFC/2×10 ⁵ VST) and enrichment fold. The left panel shows the frequency of reactive cells ( Figure 6A ), and the right panel shows the enrichment fold of reactive cells ( Figure 6B ).

Figure 7 shows the response and cell count (SFC/2×10 ⁵ VST) of VST from test donors according to HBV antigen type (LE, P, E and X).

Figure 8 shows a diagram demonstrating the production of effector molecules (IFNγ, TNF-α, GM-CSF, MIP-1a and MIP-1b) as these effector molecules are specific for different HBV antigens (P, E and LE) Sexual VST related. VST was incubated overnight with HBV antigen, and the secretome was measured by Luminex analysis. (n=3)

Figure 9 shows the versatility of VST demonstrated by the production of effector molecules (IFNγ, IFNγ+TNF-α, IFNγ+GrB, IFNγ+TNF-α+GrB). Effector molecules were measured via fluorescent spot analysis when VST generated against HBV antigens P, E and LE were exposed to the antigens.

Figures 10A - 10B provide a pair of graphs demonstrating the production of various types of interleukins in pluripotent cells. VST were stimulated with LE and C antigens and characterized using single cell pluripotency assay (IsoPlexis). The number of different interleukins expressed by CD4+ T cells and CD8+ T cells was measured ( Figure 10A ), and the polyfunctionality strength index (PSI) was used to classify the cells into effector cells, stimulatory cells, and chemoattractant cells ( Figure 10A) . 10B ). Effector cells are defined as cells that produce granzyme B, IFNγ, MIP-1a, perforin, and TNF-a. Stimulatory cells are defined as cells that produce GM-CSF and IL-5. Chemoattracted cells are defined as cells that produce MIP-1b. neg = cells produced in the absence of antigenic stimulation.

Figures 11A - 11C provide graphs demonstrating seeding density optimization for T cell expansion. The amplification fold ( Fig. 11A ), the antigen specificity of LE and C antigens measured by SFC ( Fig. 11B ) and the enrichment fold ( Fig. 11C ) were measured.

Figure 12 is a schematic diagram showing a T cell expansion process using a mixture of IL-7 and IL-15 or a mixture of IL-7 and IL-4 to generate virus-specific T cells.

Figure 13 is a graph showing the expansion fold of HBV VST cells (which are specific for HBV LE and C antigens) in contact with medium containing IL-7 and IL-15 or medium containing IL-7 and IL-4 for 10 days. Schema. T cells exposed to IL-7 and IL-15 exhibited significantly higher expansion folds.

Figure 14 shows the phenotypic analysis of cell markers (CD3+, CD56+ CD3-, CD8+ CD3+, CD4+ CD3+, CD25+ CD3+, CD69+ CD3+, PD1+ CD3+, Tim3+ CD3+, PD1+ Tim3+) based on in vitro amplification and IL-7 + IL-15 or IL-7 + IL-4 after 10 days of exposure to cultured VST (which is specific for HBV antigens LE and C).

Figures 15A - 15B provide a pair of graphs showing the antigen specificity ( Figure 15A ) and fold enrichment ( Figure 15B ) of VSTs measured by ELISpot for VSTs specific for different HBV antigens ( C and LE) Mode. VST was generated by culturing cells for 10 days in medium containing IL-7 and IL-15 or IL-7 and IL-4. neg = cells produced in the absence of antigenic stimulation.

Figure 16 shows different experimental conditions in which IL-15 was added to T cell cultures containing IL-7 and IL-4 at various time points.

Figures 17A - 17B provide a pair of graphs showing the different phenotypes (expression of CD56, CD8 or CD4) of VST produced by adding IL-15 or IL-2 at different times. Figure 17A shows the addition of IL-15 to a culture containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 of culture, and Figure 17B shows IL-2 is added to cultures containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of culture. The control group in which IL-15 was added together with IL-7 on day 0 is shown at the end of the X-axis in Figure 17A and Figure 17B.

Figures 18A - 18B provide a pair of graphs showing the fold amplification of VST measured at approximately day 12 of culture. Figure 18A shows the addition of IL-15 to a culture containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of culture. The control group in which IL-15 was added together with IL-7 on day 0 is shown at the end of the X-axis. Figure 18B shows the addition of IL-2 to a culture containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of culture. The control group in which IL-15 was added together with IL-7 on day 0 is shown at the end of the X-axis.

Figures 19A - 19B provide a pair of graphs demonstrating the antigen specificity of VST cultures generated by contact with IL-15 or IL-2. ELISPOT was used to measure VST specific for different HBV antigens (HB core Ag or ‘C’, HB surface Ag or ‘LE’ and negative control antigen). Figure 19A shows the addition of IL-15 to a culture containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 of culture, and Figure 19B shows IL-2 is added to cultures containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of culture. The control group in which IL-15 and IL-7 were added together is shown at the end of the X-axis in Figure 19A and Figure 19B.

Figures 20A - 20B provide a pair of graphs showing the fold enrichment of VST cultures specific for different antigens. ELISPOT was used to measure VST specific for different HBV antigens (HB core Ag or ‘C’ and HB surface Ag or ‘LE’). Figure 20A shows the addition of IL-15 to a culture containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 of culture, and Figure 20B shows IL-2 is added to cultures containing IL-4 and IL-7 on day 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of culture. The control group in which IL-15 and IL-7 were added together is shown at the end of the X-axis in Figure 20A and Figure 20B.

Figure 21 provides a graph showing the growth kinetics of VST cultures supplemented with IL-15 on different days as measured by lactate concentration. A control without added IL-15 is also shown. IL-4 and IL-7 alone or in combination with IL-15 were administered to donor PBMC, and lactate performance was measured over a period of 10 to 13 days.

Figures 22A to 22B show the specificity of hepatitis B virus-specific T cells (HBVST) against HBV-infected cells, based on the specificity of PHA blasts and HBVST at different ratios of HBVST to PHA activated T cells (PHA blasts). Dissolution percentage. Figure 22A shows that HBVST specifically lyses HBV-loaded target cells (40:1 E:T 36% specific lysis). Figure 22B shows that no significant off-target lysis, ie, no reactivity to autologous or allogeneic targets not loaded with antigen, was observed (1% specific lysis each).

Figures 23A - 23C show lysis analysis using HepG2/2.2.15 cells and HBVST produced from different donors. Figure 23A provides a schematic showing lysis analysis using HepG2/2.2.15 cells and HBVST generated from donor number i, as well as an image showing the lysis results. Figure 23B is a table showing the HLA types of five different donors (i to v), each of which partially matches the HLA type of HepG2/2.2.15 cells. HBVST was generated from each of five donors. Figure 23C provides a graph showing recognition of infected HepG2/2.2.15 cells by HBVST produced by each donor as measured by IFNγ ELISpot.

Figure 24 shows killing of HepG2/2.2.15 cells by hepatitis B virus-specific T cells (HBVST) and non-HBVST as measured by percentage specific lysis at different ratios of T cells to HepG2/2.2.15 cells. The analysis results of the analysis.

Figure 25 provides a schematic showing 3D tumor analysis using HepG2/2.2.15 cells and HBVST.

Figure 26 provides a graph demonstrating lysis of Hep-G2/2.2.15 aggregated cells in a 3D tumor model. Dissolution of Hep-G2/2.2.15 aggregated cells was most pronounced at the highest VST concentration (2K/well).

Figure 27 provides a description of latent and lytic antigens used to generate human herpesvirus (HHV8) VST.

Figure 28 provides a schematic diagram of the illustrative manufacturing process of the HHV8 VST.

Figure 29 provides a graph showing the fold amplification of HHV8 VST after stimulation with latent or lytic antigen and culture for 10 to 12 days. Nine populations of HHV8 VST were generated from nine healthy human donors.

Figure 30 provides a graph showing puncta-forming cells assessed by ELISpot for the frequency of IFNγ-producing VSTs (SFC/2×10 ⁵ VSTs) against VSTs specific for different HHV8 antigens. Reactivity to LANA2, LANA1, RTA, vFLIP, kaposin, vCyc, gP, MIRI, TS and SSB was measured. Cells without antigen specificity were used as controls. The antigens summarized are those most commonly recognized by T cell lines generated from different donors.

Figure 31 provides a diagram showing phenotypic analysis of cell markers (CD3+, CD8+ CD3+, CD4+ CD3+, CD16+ CD3-, CD45RO, CD62L, CD25+, CD69+, PD1+, Tim3+, PD1+ Tim3+) based on their use in vivo Percent performance after external amplification of HHV8 VST method involving treatment of donor PBMC with a peptide library of HHV8 antigens and incubation with IL-7 (20 ng/mL), IL-4 (800 U/mL), and IL-15 (5 ng/mL) were cultured together. Generate VST specific for latent antigens (kaposin, LANA-1, LANA-2, vFLIP, vCyc and RTA) and soluble antigens (MIR-I, SSB, gB and TS). Figure 32 provides a graph showing effector molecule expression of LANA1 and LANA2-reactive HHV8 VST as measured by flow cytometric analysis.

Figure 33 provides a graph showing an in vitro killing assay of HHV8 VST reactive to LANA1 and LANA2. Specific dissolution was measured at E:T ratios of 90:1, 60:1, 40:1, 20:1 and 10:1.

Figure 34 provides an overview of LANA1 epitope mapping, including seven mapped peptide sequences according to SEQ ID NOs: 6-12.

Figure 35 provides an overview of gB epitope mapping, including 3 mapped peptide sequences according to SEQ ID NO: 13-15.

Figure 36 provides a schematic representation of the treatment protocol for PXB mice infected with HBV and treated with virus-specific T cells targeting HBV on days 28 and 56. HBV DNA and HBsAg were measured weekly, human alanine aminotransferase (ALT) was measured weekly starting from day 21 to day 84 and then on days 98 and 112, and human alanine aminotransferase (ALT) was measured every two weeks. protein. At the end of the study, liver samples were collected.

TW202340455A_111144683_SEQL.xml

Claims (228)

An ex vivo method for expanding T cells, comprising: Providing a starting population of mononuclear spheres in cell culture; The cells are contacted with one or more compositions, wherein the one or more compositions comprise: (i) at least one target antigen or part of a target antigen or a peptide or peptide mixture corresponding to at least one target antigen or part of a target antigen; (ii) Two or more interleukins including IL-4 and IL-7; and (iii) IL-15; and The cells are cultured with components of (i), (ii) and (iii) for a period of time sufficient to expand the T cells; These T cells are thereby expanded. The method of claim 1, wherein the cells are first contacted with the components of (i) and (ii) from about 0 hours to about 48 hours before being contacted with the component of (iii). Such as the method of claim 1 or 2, the restriction condition is that the two or more interleukins do not include IL-2. The method of any one of claims 1 to 3, wherein the component of (i) and the component of (ii) are provided in a composition. The method of any one of claims 1 to 3, wherein the component of (i) and the component of (ii) are provided in separate compositions. The method of any one of claims 1 to 4, wherein the component of (iii) is added to a composition containing the components of (i) and (ii). The method of any one of claims 1 to 6, wherein the cells are contacted with the components of (i) and (ii) simultaneously. Claim the method of any one of items 1 to 3 or 5, wherein the cells are contacted with the components of (i) and (ii) sequentially. Such as the method of any one of claims 1 to 8, wherein the cells are about 0 hours, about 2 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, before being contacted with the component of (iii). About 36 hours, or about 48 hours after contact with ingredients (i) and (ii). The method of any one of claims 1 to 8, wherein the cells are in contact with the component of (iii) from about 0 hours to about 48 hours, from about 1 hour to about 48 hours, from about 2 hours to about 48 hours, About 12 hours to about 48 hours, about 24 hours to about 48 hours, about 36 hours to about 48 hours, about 0 hours to about 24 hours, about 2 hours to about 24 hours, about 6 hours to about 24 hours, about 12 hours to about 24 hours, or about 18 hours to about 24 hours before being exposed to the ingredients of (i) and (ii). The method of any one of claims 1 to 3, 5, 7, 8, 9 or 10, wherein the ingredients of (i), (ii) and (iii) are provided in separate compositions. The method of any one of claims 1 to 10, wherein the components of (i) and (ii) are provided in a composition. The method of any one of claims 1 to 10, wherein the component of (iii) is added to a composition containing the components of (i) and (ii). The method of any one of claims 1 to 4, wherein the ingredients of (i), (ii) and (iii) are provided in a composition. For example, the method of any one of claims 1, 3 to 8 or 11 to 13, wherein about 0 days, about 1 day, about 2 days, about 3 days after the cells are contacted with the components of (i) and (ii) days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, so that the cells are contacted with the component of (iii). For example, the method of any one of claims 1, 3 to 8 or 11 to 13, wherein after the cells are contacted with the components of (i) and (ii), from about 0 days to about 6 days, from about 0 days to about 5 days days, about 0 days to about 4 days, about 0 days to about 3 days, about 0 days to about 2 days, about 0 days to about 1 day, so that the cells are contacted with the component of (iii). Claim the method of any one of items 1 to 16, wherein the period of time sufficient to expand the T cells is about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days. The method of claim 1 to 16, wherein the cells are cultured for about 7 days to about 14 days, about 8 days to about 10 days, about 8 days to about 14 days, or about 8 days to about 18 days. , about 14 days to about 18 days, about 1 week to about 2 weeks, about 1 week to about 3 weeks, or about 1 week to about 4 weeks. The method of any one of claims 1 to 16, wherein the cells are cultured for about 18 days or less, optionally about 14 days. The method of any one of claims 1 to 19, wherein the cells are cultured for an amount of time sufficient to produce cells that have completed logarithmic phase growth. As claimed in any one of items 1 to 20, the period during which the cells are in contact with component (i) is the priming period. Such as the method of request item 21, wherein the activation period is day 0. The method of claim 21 or 22, wherein when ingredients (i) and (ii) are added on day 0, the start-up period includes the addition of ingredient (ii). A method as claimed in any one of claims 21 to 23, wherein when ingredients (i), (ii) and (iii) are added on day 0, the start-up period includes the addition of ingredient (iii). The method of any one of claims 21 to 24, wherein priming is sufficient to stimulate T cells. A method as claimed in any one of claims 1 to 25, wherein the expansion period begins after contacting the cells with components (i) and (ii). The method of claim 26, wherein the expansion period starts on the 1st or 2nd day of culture. The method of claim 26 or 27, wherein the expansion period includes contacting the cells with component (iii). The method of any one of claims 21 to 22, wherein the expansion period begins on the 1st or 2nd day of culture. The method of claim 29, wherein when ingredient (ii) is added on day 1 or day 2, the expansion period includes the addition of ingredient (ii). The method of claim 29 or 30, wherein when ingredient (iii) is added on day 1 or day 2, the expansion period includes the addition of ingredient (iii). The method of any one of claims 26 to 31, wherein the expansion phase continues until the cells have completed logarithmic phase growth. The method of any one of claims 1 to 32, wherein the expanded T cells comprise T cells specific for at least one target antigen. The method of any one of claims 1 to 32, wherein the expanded T cells are enriched with T cells specific for at least one target antigen. The method of any one of claims 1 to 34, wherein the mononuclear cells are peripheral blood mononuclear cells (PBMC). Claim the method of any one of items 1 to 35, wherein the PBMCs are human PBMCs, donor PBMCs and/or allogeneic PBMCs. The method of any one of claims 1 to 36, wherein the T cells are human T cells, donor T cells and/or allogeneic T cells. The method of any one of claims 1 to 37, wherein the cells are cultured in the presence of VST expansion medium. The method of any one of claims 1 to 38, wherein the cells are contacted with a pepmix at a concentration of about 0.2 nanogram/peptide/ml to about 20 nanogram/peptide/ml. The method of any one of claims 1 to 38, wherein the cells are contacted with a mixed peptide at a concentration of about 2 nanograms/peptide/ml. The method of any one of claims 1 to 40, wherein the cells are contacted with IL-4 at a concentration of about 400 U/mL to about 1200 U/mL. The method of any one of claims 1 to 41, wherein the cells are contacted with IL-4 at a concentration of about 800 U/mL. The method of any one of claims 1 to 42, wherein the cells are contacted with IL-7 at a concentration of about 5 ng/mL to about 30 ng/mL. The method of any one of claims 1 to 43, wherein the cells are contacted with IL-7 at a concentration of about 20 ng/mL. The method of any one of claims 1 to 44, wherein the cells are contacted with IL-15 at a concentration of about 1 ng/mL to about 20 ng/mL. The method of any one of claims 1 to 45, wherein the cells are contacted with IL-15 at a concentration of about 5 ng/mL. The method of any one of claims 1 to 38, wherein the cells are mixed with a mixed peptide at a concentration of about 2 nanograms/peptide/ml, IL-7 at a concentration of about 20 ng/mL, and a concentration of about 800 U/mL. mL of IL-4 and IL-15 at a concentration of approximately 5 ng/mL. The method of any one of claims 1 to 47, wherein the cells are contacted with IL-15 on day 0, day 1 or day 2 of culture. The method of any one of claims 1 to 48, wherein the interleukins are recombinant interleukins. The method of any one of claims 1 to 49, wherein the interleukins are human recombinant interleukins. The method of any one of claims 1 to 48, wherein the interleukins are human interleukins. The method of any one of claims 1 to 51, wherein the cells are present at about 1.5 × 10 ⁶ cells/cm2 to about 4 × 10 ⁶ cells/cm2 or about 2 × 10 ⁶ cells/cm2 To about 4 × 10 ⁶ cells/cm2, culture at a cell seeding density of about 3 × 10 ⁶ cells/cm2 depending on the situation. The method of any one of claims 1 to 52, wherein the number of expanded T cells or the number of cells in the expanded T cell population is at least 2 times greater than the number of cells in the starting cell population. The method of any one of claims 1 to 52, wherein the number of expanded T cells or the number of cells in the expanded T cell population is about 2 times to about 7 times greater than the number of cells in the starting cell population. The method of any one of claims 1 to 54, wherein the expanded T cells or the expanded T cell population comprise antigen-specific T cells. The method of claim 55, wherein the antigen-specific T cells are pathogen-specific T cells (PST) or virus-specific T cells (VST). The method of claim 55, wherein the antigen-specific T cells are tumor-specific T cells (TST). The method of any one of claims 1 to 56, wherein the expanded T cells or the expanded T cell population comprise a VST that recognizes at least one antigen from hepatitis B virus (HBV). The method of any one of claims 1 to 56, wherein the expanded T cells or the expanded T cell population comprise a VST that recognizes at least one antigen from human herpesvirus 8 (HHV-8). The method of any one of claims 1 to 59, wherein the peptide mixture contains from about 2 to about 750 different peptides. The method of claim 60, wherein each peptide in the peptide mixture and at least one other peptide have at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 amino acids overlap. The method of any one of claims 1 to 61, wherein the peptides are at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, At least 15, at least 16, at least 17, at least 18 amino acids long. The method of any one of claims 1 to 62, wherein the peptides are about 15 amino acids long and overlap by about 11 amino acids. The method of any one of claims 1 to 62, wherein the peptides are 15 amino acids long and overlap by 11 amino acids. The method of any one of claims 1 to 64, wherein the peptide mixture contains one or more target antigens from at least one pathogen or a portion thereof. The method of claim 65, wherein the at least one pathogen is selected from the group consisting of cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B Virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella-zoster virus (VZV) ), Epstein-Barr virus (EBV), cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), human herpes virus 8 (HHV-8), human herpes virus 7 (HHV-7), Kaposi's sarcoma-associated herpes virus (KSHV), respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, bocavirus, coronavirus , Lymphocytic choriomeningitis virus (LCMV), mumps virus, measles virus, human metapneumonia virus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile West Nile virus (WNV), Zika virus, Ebola virus and human T-lymphotropic virus. The method of claim 65, wherein the at least one pathogen is hepatitis B virus (HBV). The method of claim 65, wherein the at least one pathogen is human herpesvirus 8 (HHV-8). The method of claim 65, wherein the pathogen is HBV, and the one or more target antigens include E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg). Such as the method of claim 65, wherein the pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX) or combination thereof. The method of claim 65, wherein the pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX) or a combination thereof. The method of any one of claims 69 to 71, wherein the HBeAg (E antigen) comprises pre-core (PreC) and core (HBcAg) antigens. The method of claim 65, wherein the peptide mixture includes a peptide comprising or consisting of a sequence selected from the following: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD (SEQ ID NO: 2), AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and/or EYLVSFGVWIRTPPA (SEQ ID NO: 5). The method of any one of claims 69 to 73, wherein the one or more HBV antigens are selected from one or more HBV genotypes. The method of any one of claims 69 to 74, wherein at least two antigens are selected from the same HBV genotype. The method of claim 74, wherein at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first genotype is different from the second genotype. The method of claim 74, wherein at least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C. The method of any one of claims 69 to 77, wherein the amino acid sequence of each HBV antigen is different in at least one amino acid. The method of any one of claims 69 to 78, wherein the expanded T cells or the expanded T cell population are reactive upon stimulation. The method of claim 79, wherein at least a portion of the T cell reactivity is CD4+ and HLA-DR, HLA-DQ or HLA-DP restricted. Such as the method of claim 65, wherein the pathogen is: (a) EBV, and the at least one target antigen is EBNA1, LMP2, BZLF1 or a combination thereof; (b) CMV, and the at least one target antigen is IE1, pp65 or a combination thereof; (c) Adv, and the at least one target antigen is Hexon, Penton or a combination thereof; (d) BK virus, and the at least one target antigen is LT, VP-1 or a combination thereof; (e) HHV6, and the at least one target antigen is U14, U11, U71, U54, U90 or a combination thereof; (f) RSV, and the at least one target antigen is N, F or a combination thereof; (g) Influenza virus, and the at least one target antigen is MP1, NP1 or a combination thereof; (h) Parainfluenza virus type (PIV), and the at least one target antigen is F, N, M, M2-1, HN or a combination thereof; (i) hMPV, and the at least one target antigen is F, N, M2-1, M or a combination thereof; (j) HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kaposi protein ( Kaposin) (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof; and/or (k) SARS-CoV2, and the at least one target antigen is spike protein (Spike) (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4 , nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof. Such as the method of claim 65, wherein the pathogen is HHV8, and the one or more target antigens include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70). The method of claim 65, wherein the pathogen is HHV8, and the one or more target antigens include: (a) One or more latent antigens, including Kaposi protein (ORF12, K12), LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vFLIP (ORF71), vCYC (ORF72) and RTA ( ORF50); and/or (b) One or more lytic antigens, including gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). The method of claim 65, wherein the pathogen is HHV8, and the one or more target antigens include LANA-1 (ORF3) and LANA-2 (vIRF3, K10.5). The method of claim 65, wherein the pathogen is HHV8 and the one or more target antigens include LANA-1 (ORF3) and gB (ORF8). Such as the method of claim 65, wherein the pathogen is SARS-CoV, and the one or more target antigens include spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a (AP7a) and nonstructural protein 4 (nsp4). The method of claim 65, wherein the peptide mixture includes a peptide comprising or consisting of a sequence selected from the following: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO: 7), TDTHSPSPALPPTQS (SEQ ID NO: ( SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15). The method of any one of claims 1 to 87, comprising obtaining the expanded T cells or the expanded T cell population. The method of any one of claims 1 to 88, further comprising the step of preparing a pharmaceutical composition, wherein a diluent, a stabilizer, and a preservative are added to the expanded T cells or the expanded T cell population. and/or other pharmaceutically acceptable excipients. An expanded T cell population obtained by the method of any one of claims 1 to 89. The expanded T cell population of claim 90, wherein the T cell population includes: (a) At least 70% CD3+ T cells; and/or (b) CD4+ and CD8+ T cells. The expanded T cell population of claim 90 or claim 91, wherein the expanded T cell population includes VST. The expanded T cell population of any one of claims 90 to 92, wherein (a) Approximately 30% or less of the cells in the expanded T cell population are natural killer (NK) cells; (b) More than approximately 80% of the T cell population includes CD4+ and/or CD8+ T cells; (c) The T cells are multi-strain; and/or (d) At least 1% of such CD3+ T cells can produce TNFα. The expanded T cell population of any one of claims 90 to 93, wherein at least one T cell recognizes one or more target antigens from at least one pathogen or a portion thereof. The expanded T cell population of claim 94, wherein the at least one pathogen is selected from the group consisting of cytomegalovirus (CMV), adenovirus (Adv), BK virus, human papillomavirus (HPV), hepatitis A virus (HAV) ), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), HIV, varicella zone Herpes zoster virus (VZV), Estin-Barr virus (EBV), cytomegalovirus (CMV), JC virus, human herpes virus 6 (HHV-6), human herpes virus 8 (HHV-8), human herpes virus 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV), respiratory synthetic virus (RSV), influenza virus, parainfluenza virus, bocavirus, coronavirus, lymphocytic choriomeningitis virus ( LCMV), mumps virus, measles virus, human metapneumovirus (hMPV), parvovirus B, rotavirus, Merkel cell virus, West Nile virus (WNV), Zika virus, Ebola virus and human T-lymphovirus. The expanded T cell population of claim 94, wherein the at least one pathogen is hepatitis B virus (HBV). The expanded T cell population of claim 94, wherein the at least one pathogen is human herpesvirus 8 (HHV-8). The expanded T cell population of claim 94, wherein (a) The pathogen is HBV, and the at least one target antigen is core antigen (HBcAg), surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX) or a combination thereof; (b) The pathogen is HBV, and the at least one target antigen is surface antigen (HBsAg), E antigen (HBeAg), DNA polymerase (HBP), X antigen (HBX) or a combination thereof; and/or (c) The pathogen is HBV, and the at least one target antigen is HBcAg, HBsAg or a combination thereof. The expanded T cell population of claim 98, wherein the HBeAg (E antigen) includes pre-core (PreC) and core (HBcAg) antigens. The expanded T cell population of any one of claims 98 to 99, wherein the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from the group consisting of: PLPVLQAGFFLLTRI (SEQ ID NO: 1), FFLLTRILTIPQSLD ( SEQ ID NO: 2), and/or AKYLWEWASVRFSWL (SEQ ID NO: 3), QAILCWGELMTLATW (SEQ ID NO: 4) and EYLVSFGVWIRTPPA (SEQ ID NO: 5). The expanded T cell population of any one of claims 98 to 100, wherein at least two HBV antigens are selected from different HBV genotypes. The expanded T cell population of claim 101, wherein at least two antigens are selected from the same HBV genotype. The expanded T cell population of claim 102, wherein (a) at least one antigen is selected from a first HBV genotype and at least one antigen is selected from a second HBV genotype, wherein the first genotype is different from the second genotype; and/or (b) At least one antigen is selected from HBV genotype A and at least one antigen is selected from HBV genotype C. The expanded T cell population of any one of claims 98 to 103, wherein the amino acid sequence of each HBV antigen is different from the amino acid sequence of each other HBV antigen by at least one amino acid. The expanded T cell population of any one of claims 98 to 104, wherein the expanded T cell population is reactive upon stimulation; optionally, wherein at least a portion of the T cell reactivity is affected by HLA-DR , HLA-DQ or HLA-DP restrictions. The expanded T cell population of claim 94, wherein (a) The pathogen is EBV, and the at least one target antigen is EBNA1, LMP2, BZLF1 or a combination thereof; (b) The pathogen is CMV, and the at least one target antigen is IE1, pp65 or a combination thereof; (c) The pathogen is Adv, and the at least one target antigen is hexon, penton or a combination thereof; (d) The pathogen is BK virus, and the at least one target antigen is LT, VP-1 or a combination thereof; (e) The pathogen is HHV6, and the at least one target antigen is U14, U11, U71, U54, U90 or a combination thereof; (f) The pathogen is RSV, and the at least one target antigen is N, F or a combination thereof; (g) The pathogen is influenza virus, and the at least one target antigen is MP1, NP1 or a combination thereof; (h) The pathogen is a parainfluenza virus (PIV), and the at least one target antigen is F, N, M, M2-1, HN or a combination thereof; (i) The pathogen is hMPV, and the at least one target antigen is F, N, M2-1, M or a combination thereof; (j) The pathogen is HHV8, and the at least one target antigen is LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Kapos Western protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70) or combinations thereof; and/or (k) The pathogen is SARS-CoV2, and the at least one target antigen is spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), nsp1, nsp3, nsp4 , nsp5, nsp6, nsp7a, nsp8, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, AP3A, NS7, NS8, ORF10, ORF9B, Y14, or combinations thereof. The expanded T cell population of claim 106(j), wherein the at least one target antigen comprises a peptide comprising or consisting of a sequence selected from: ADSVDGRECGPHTLP (SEQ ID NO: 6), PGSPTVFTSGLPAFVSSPT (SEQ ID NO : 7), TTDTHSPSALPPTQS (SEQ ID NO: 8), DNDNKDDEEEQETDE (SEQ ID NO: 9), EEPIILHGSSSEDEM (SEQ ID NO: 10), ILHGSSSEDEMEVDY (SEQ ID NO: 11), HPLAGNLQSSIVKFKKPLPLTQP (SEQ ID NO: 12), IVPHIFKVRRYRKIATSVT (SEQ ID NO: 13), DTCEHYFITRNETLV (SEQ ID NO: 14) and/or IFMLSRRTNTIAQAPVKMIYPDV (SEQ ID NO: 15). A plurality of ex vivo expanded T cell populations according to any one of claims 90 to 107, wherein each ex vivo expanded T cell population comprises T cells generated from donor monocytes obtained from a different donor ; Optionally, the HLA type of each donor is different from at least one HLA allele gene of at least one of the other donors. A universal antigen-specific T cell therapy product comprising an ex vivo expanded T cell population or a plurality of expanded T cell populations according to any one of claims 90 to 108, wherein the product comprises a population of T cells obtained from different suppliers. T cells generated from donor monocytes. The universal antigen-specific T cell therapy product of claim 109, wherein (a) The HLA type of each donor is different from at least one HLA allele gene of at least one of the other donors; (b) The product exhibits reduced or negligible alloreactivity to partially HLA-matched and/or HLA-mismatched target cells; and/or (c) The product contains a VST that recognizes one or more target antigens from at least one pathogen, or a portion thereof. A pharmaceutical composition comprising an ex vivo expanded T cell population, a plurality of expanded T cell populations, or a universal antigen-specific T cell therapy product according to any one of claims 90 to 110. The pharmaceutical composition of claim 111, wherein the pharmaceutical composition is formulated for intravenous delivery. Such as the pharmaceutical composition of claim 111 or claim 112, wherein (a) The pharmaceutical composition is negative for bacteria and fungi during continuous culture for at least 7 days; (b) The pharmaceutical composition exhibits endotoxin levels below 5 EU/ml; and/or (c) The pharmaceutical composition is negative for Mycoplasma species. A population of ex vivo expanded VSTs specific for two or more HBV antigens selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX. A population of ex vivo expanded VSTs specific for HBV antigens including E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg). For example, the population of VST amplified in vitro in claim 115, wherein the antigens E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg) are immunodominant antigens. A population of ex vivo expanded VSTs as claimed in claim 114, wherein the VSTs are specific for two or more HBV antigens selected from the group consisting of HBsAg, HBeAg, HBP and HBX. For example, claim 114 describes a population of ex vivo expanded VSTs, wherein the VSTs are specific for HBV antigens HBcAg and HBsAg. The ex vivo expanded VST population of any one of claims 114 to 118, wherein the E antigen (HBeAg) includes pre-core (PreC) and core (HBcAg) antigens. A population of ex vivo expanded VST pairs, wherein the VST pairs are selected from LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), Two or more HHV8 antigens of Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70) are specific. A population of ex vivo expanded VST pairs, wherein the VST pairs include LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), card The HHV8 antigens of Posyrin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), and TS (ORF70) are specific. A population of ex vivo expanded VSTs specific for HHV8 antigens including: (a) One or more latent antigens, including Kaposi protein (ORF12, K12), LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vFLIP (ORF71), vCYC (ORF72) and RTA ( ORF50); and/or (b) One or more lytic antigens, including gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). A population of ex vivo expanded VSTs specific for HHV8 antigens including LANA-1 (ORF3) and LANA-2 (vIRF3, K10.5). A population of ex vivo expanded VSTs specific for HHV8 antigens including LANA-1 (ORF3) and gB (ORF8). A population of ex vivo expanded VST pairs, wherein the VST pairs include spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a (AP7a) and non-structural protein 4 (nsp4 ) SARS-CoV antigen is specific. The population of ex vivo expanded VST cells according to any one of claims 114 to 125, wherein the VST cells are human VST cells, donor VST cells and/or allogeneic VST cells. For example, any of the groups in claim items 114 to 126 include: (a) At least 70% CD3+ T cells; (b) approximately 30% or less CD56+ cells; and/or (c) CD4+ and CD8+ T cells. The population of any one of claims 114 to 127, wherein the VST has a specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10 ⁵ cells . For example, the population of any one of claims 114 to 128, wherein no more than 20% of the VSTs express a T cell exhaustion marker; optionally, the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or TIGIT. The population of claim 129, wherein the T cell exhaustion marker is a co-expression of PD1 and/or TIM3. The population of any one of claims 114 to 130, wherein the VSTs express one or more markers associated with T cell activation; optionally, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69. Such as the population of any one of claims 114 to 131, wherein the VST expresses one or more markers related to central or effector cell memory; optionally, wherein the one or more markers related to central or effector cell memory The system is selected from CD45RO and CD62L. Such as the group of any one of claims 114 to 132, wherein the VST is multi-functional. The population of any one of claims 114 to 133, wherein the VSTs produce two or more effector molecules when stimulated with two or more antigens; optionally, wherein the effector molecules are selected from IFNγ , TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b. The population of any one of claims 114 to 119 or 126 to 134, wherein the VSTs are enriched by at least 1000% after expansion in IL4, IL7 and IL15 in the presence of HBV antigen relative to the cell population before expansion times, at least 3,000 times, or at least 6,000 times. The population of any one of claims 120 to 124 or 126 to 134, wherein the VSTs are enriched by at least 1000% after expansion in IL4, IL7 and IL15 in the presence of HHV8 antigen relative to the cell population before expansion times, at least 3,000 times, or at least 6,000 times. The population of any one of claims 114 to 136, wherein the VST has reduced alloreactivity or negligible alloreactivity. A population as claimed in any one of claims 114 to 136, wherein the VST has reduced reactivity against allogeneic targets or negligible reactivity against allogeneic targets. A population as claimed in any one of claims 114 to 136, wherein the VST has reduced autoreactivity or negligible autoreactivity. A pharmaceutical composition comprising a multi-strain population of ex vivo amplified VST according to any one of claims 114 to 139 and a pharmaceutically acceptable carrier. A kit comprising a multi-strain population of ex vivo expanded VST according to any one of claims 114 to 139. A donor minilibrary comprising a multi-strain population of amplified VST according to any one of claims 114 to 139. A method of treating or preventing viral infection comprising administering to an individual a population of ex vivo expanded VST according to any one of claims 114 to 139. A method of treating or preventing viral infection, comprising administering to an individual a pharmaceutical composition according to any one of claims 111 to 113 and 140. The method of any one of claims 143 to 144, wherein the individual is immunocompetent or immunocompromised. The method of any one of claims 144 to 145, wherein the pharmaceutical composition is administered by intravenous injection or infusion. The method of any one of claims 144 to 146, wherein the pharmaceutical composition is administered to the individual a plurality of times. The method of any one of claims 144 to 147, wherein about 5×10 ⁶ to about 5×10 ⁸ cells/cm² are administered to the subject. The method of any one of claims 144 to 148, wherein the individual is infected with or at risk of infection with a virus. The method of any one of claims 144 to 149, wherein administration of the pharmaceutical composition is effective in treating or preventing the viral infection in the individual. The method of any one of claims 144 to 150, wherein the individual a. Have received solid organ transplant; b. Have received chemotherapy; c. Suffering from HIV infection; d. Suffering from genetic immunodeficiency; e. Suffering from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation. The method of any one of claims 144 to 151, wherein the pharmaceutical composition comprises the universal antigen-specific T cell therapy product of any one of claims 75 to 76. The method of claim 152, wherein the pharmaceutical composition is administered to the individual without knowledge of the individual's HLA type. The method of any one of claims 143 to 153, wherein the individual is a human. A method of treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual a population of expanded VST according to any one of claims 114 to 119 or 126 to 139. A method of treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a population of expanded VSTs according to any one of claims 120 to 124 or 126 to 139. A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST contains specificity for two or more HBV antigens. The composition of claim 157, wherein the two or more HBV antigens are selected from the group consisting of HBcAg, HBsAg, HBeAg, HBP and HBX. The composition of any one of claims 157 to 158, wherein the two or more HBV antigens are selected from the group consisting of HBsAg, HBeAg, HBP and HBX. The composition of any one of claims 157 to 159, wherein the two or more HBV antigens are HBcAg and HBsAg. A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for HBV antigens including E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg). A composition comprising a multi-strain population of VST as claimed in claim 161, wherein the antigens E antigen (HBeAg), P antigen (HBP) and LE antigen (HBsAg) are immunodominant antigens. The composition comprising a multi-strain population of VST according to any one of claims 157 to 162, wherein the E antigen (HBeAg) comprises pre-core (PreC) and core (HBcAg) antigens. The composition of any one of claims 157 to 163, wherein the VST cells are human VST cells, donor VST cells and/or allogeneic VST cells. The composition of any one of claims 157 to 164, wherein the population of VSTs produces two or more effector molecules. The composition of any one of claims 157 to 165, wherein the VSTs produce two or more effector molecules when stimulated with the two or more HBV antigens. The composition of claim 166, wherein the effector molecules are selected from the group consisting of IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b. The composition of any one of claims 157 to 167, wherein the composition is produced in vitro or ex vivo. The composition of any one of claims 157 to 168, wherein the VST is multifunctional. The composition of any one of claims 157 to 169, wherein the population of VST comprises (a) At least 70% CD3+ T cells; (b) No more than 30% CD56+ cells; and/or (c) CD4+ and CD8+ T cells. The composition of any one of claims 157 to 170, wherein the VSTs have specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10e5 cells . The composition of any one of claims 157 to 171, wherein no more than 20% of the VSTs express a T cell exhaustion marker; optionally, the T cell exhaustion marker is selected from Tim3, PD1, Lag3 and/or Or TIGIT. The composition of claim 172, wherein the T cell exhaustion marker is PD1 and/or TIM3. The composition of any one of claims 157 to 173, wherein the VSTs express one or more markers associated with T cell activation; optionally, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69. The composition of any one of claims 157 to 174, wherein the VSTs express one or more markers associated with central or effector cell memory; optionally, wherein the one or more markers associated with central or effector cell memory The species system is selected from CD45RO and CD62L. The composition of any one of claims 157 to 175, wherein the VSTs are enriched at least 1000-fold after expansion in IL4, IL7 and IL15 in the presence of HBV antigen compared to the cell population before expansion, At least 3,000 times or at least 6,000 times. The composition of any one of claims 157 to 176, wherein the multi-strain population of VST is an antigen-specific multi-strain population of VST. The composition of claim 177, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HBV antigens. The composition of claim 178, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from HBsAg; optionally, wherein the antigen-specific multi-strain population of VST is selected from SEQ At least one peptide sequence of ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 is specific. The composition of claim 178, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from HBeAg; optionally, wherein the antigen-specific multi-strain population of VST is selected from SEQ At least one peptide sequence of ID NO: 4 and SEQ ID NO: 5 is specific. The composition of claim 178, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from HBsAg and one or more peptide sequences derived from HBeAg; optionally, wherein the VST The antigen-specific multi-strain population pairs at least one peptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and at least one peptide selected from SEQ ID NO: 4 and SEQ ID NO: 5 Sequence is specific. A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for two or more HHV8 antigens. The composition of claim 182, wherein the two or more HHV8 antigens are selected from the group consisting of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP (ORF71), kaposin (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST includes pairs of LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vCYC (ORF72), RTA (ORF50), vFLIP Specificity of HHV8 antigens (ORF71), Kaposi protein (ORF12, K12), gB (ORF8), MIR1 (K3), SSB (ORF6), TS (ORF70). A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for an HHV8 antigen comprising: (a) One or more latent antigens, including Kaposi protein (ORF12, K12), LANA-1 (ORF3), LANA-2 (vIRF3, K10.5), vFLIP (ORF71), vCYC (ORF72) and RTA ( ORF50); and/or (b) One or more lytic antigens, including gB (ORF8), MIR1 (K3), SSB (ORF6) and TS (ORF70). A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for HHV8 antigens including LANA-1 (ORF3) and LANA-2 (vIRF3, K10.5). A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST comprises specificity for HHV8 antigens including LANA-1 (ORF3) and gB (ORF8). The composition of any one of claims 182 to 187, wherein the VST cells are human VST cells, donor VST cells and/or allogeneic VST cells. The composition of any one of claims 182 to 188, wherein the population of VSTs produces two or more effector molecules. The composition of any one of claims 182 to 188, wherein the VSTs produce two or more effector molecules when stimulated with the two or more HHV8 antigens. The composition of claim 190, wherein the effector molecules are selected from the group consisting of IFNγ, TNF-α, granzyme B, perforin, GM-CSF, MIP-1a and MIP-1b. The composition of any one of claims 182 to 191, wherein the composition is produced in vitro or ex vivo. The composition of any one of claims 182 to 192, wherein the VST is multifunctional. The composition of any one of claims 182 to 193, wherein the VST comprises (a) CD4+ and CD8+ T cells; (b) At least 70% CD3+ T cells; and/or (c) About 30% or less CD56+ cells. The composition of any one of claims 182 to 194, wherein the VST has a specificity for the two or more antigens as measured by an IFNγ ELISpot assay of at least about 50 SFC/2 × 10 ⁵ cells. The composition of any one of claims 182 to 195, wherein no more than 20% of the VSTs express a T cell exhaustion marker; optionally, the T cell exhaustion marker is selected from the group consisting of Tim3, PD1, Lag3 and/or Or TIGIT. The composition of claim 196, wherein the T cell exhaustion marker is a co-expression of PD1 and/or TIM3. The composition of any one of claims 182 to 197, wherein the VSTs express one or more markers associated with T cell activation; optionally, wherein the one or more markers associated with T cell activation are selected from CD25 and CD69. The composition of any one of claims 182 to 198, wherein the VSTs express one or more markers associated with central or effector cell memory; optionally, wherein the one or more markers associated with central or effector cell memory The species system is selected from CD45RO and CD62L. The composition of any one of claims 182 to 199, wherein the VSTs are enriched at least 1000-fold after expansion in IL4, IL7 and IL15 in the presence of HHV8 antigen compared to the cell population before expansion, At least 3,000 times or at least 6,000 times. The composition of any one of claims 182 to 200, wherein the multi-strain population of VST is an antigen-specific population of VST. The composition of claim 201, wherein the antigen-specific multistrain population of VST is specific for one or more peptide sequences derived from one or more HHV8 antigens. The composition of claim 202, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from LANA1; optionally, at least one of the peptide sequences is selected from SEQ ID NOs: 6-12 . The composition of claim 202, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from gB; optionally, at least one of the peptide sequences is selected from SEQ ID NOs: 13-15 . The composition of claim 202, wherein the antigen-specific multi-strain population of VST is specific for one or more peptide sequences derived from LANA1 and one or more peptide sequences derived from gB; optionally, at least one of them The peptide sequence is selected from SEQ ID NO: 6-12 and at least one peptide sequence is selected from SEQ ID NO: 13-15. A composition comprising a multi-strain population of VST, wherein the multi-strain population of VST includes pairs of proteins including spike protein (S), membrane protein (M), nucleocapsid protein (N), accessory protein 7a (AP7a) and non- Specificity of SARS-CoV antigen of structural protein 4 (nsp4). The composition of claim 206, wherein the VST cells are human VST cells, donor VST cells and/or allogeneic VST cells. The composition of any one of claims 157 to 207, wherein the multi-strain population of VST has reduced alloreactivity or negligible alloreactivity. The composition of any one of claims 157 to 207, wherein the multi-strain population of VST has reduced reactivity against an allogeneic target or negligible reactivity against an allogeneic target. The composition of any one of claims 157 to 207, wherein the multi-strain population of VST has reduced autoreactivity or negligible autoreactivity. The composition of any one of claims 157 to 210, which contains a pharmaceutically acceptable diluent. The composition of claim 211 formulated for intravenous administration. A pharmaceutical composition comprising the composition of any one of claims 157 to 212 and a pharmaceutically acceptable carrier. A kit comprising a composition according to any one of claims 157 to 212. A method of treating a viral infection, comprising administering to an individual a composition according to any one of claims 157 to 212 or a pharmaceutical composition according to claim 158. A method of preventing viral infection, comprising administering to an individual a composition according to any one of claims 157 to 212 or a pharmaceutical composition according to claim 158. The method of claim 215, wherein the individual is immunocompetent or immunocompromised. The method of any one of claims 215 to 216, wherein the pharmaceutical composition is administered by intravenous injection or infusion. The method of any one of claims 215 to 217, wherein the pharmaceutical composition is administered to the individual a plurality of times. The method of any one of claims 215 to 218, wherein about 5×10 ⁶ to about 5×10 ⁸ cells/cm² are administered to the individual. The method of claim 215 to 219, wherein the individual is infected with a virus or is at risk of being infected with a virus. The method of any one of claims 215 to 220, wherein administration of the pharmaceutical composition is effective in treating or preventing the viral infection in the individual. The method of claim 215 to 221, wherein the individual a. Have received solid organ transplant; b. Have received chemotherapy; c. Suffering from HIV infection; d. Suffering from genetic immunodeficiency; e. Suffering from chronic viral infection; and/or f. Have received allogeneic or autologous stem cell transplantation. The method of any one of claims 215 to 222, wherein the individual is a human. A method of treating or preventing HBV infection in an individual in need thereof, comprising administering to the individual a composition according to any one of claims 157 to 181 or 207 to 212. A method of treating or preventing HHV8 infection in an individual in need thereof, comprising administering to the individual a composition according to any one of claims 182 to 205 or 207 to 212. A method of treating or preventing SARS-CoV infection in an individual in need thereof, comprising administering to the individual a composition according to any one of claims 206 to 212. The method of any one of claims 224 to 226, wherein the composition is a pharmaceutical composition.