Methods for selecting T cell lines and donors for adoptive cell therapy

Abstract

This article discloses a method for selecting allogeneic T cell lines for therapeutic administration to patients who carry / have or are suspected of carrying / having the pathogen or cancer. It also discloses a method for selecting donors from which allogeneic T cell lines for therapeutic administration to patients who carry / have or are suspected of carrying / having the pathogen or cancer.

Description

[0001] Related applications

[0002] This application is a divisional application of Chinese Patent Application No. 202111030031.8, filed on November 4, 2015, entitled "Method for Selecting T Cell Lines and Donors for Adoptive Cell Therapy".

[0003] Cross-reference of related applications

[0004] This application claims the benefit of provisional application number 62 / 075,856, filed November 5, 2014, which is incorporated herein by reference in its entirety.

[0005] Government Rights Statement

[0006] This invention was developed with government support from the National Institutes of Health (NIH) under license numbers NCI Ca23766, SR21 CA 162002, SP30-Ca08748-40, P01 Ca 106450, P01 Ca 52477-13, and P01Ca54350. The government holds certain rights in this invention. Invention Field

[0007] This article discloses a method for selecting allogeneic T cell lines for therapeutic administration to patients who carry / have or are suspected of carrying / having pathogens or cancer. It also discloses a method for selecting donors from which allogeneic T cell lines for therapeutic administration to patients who carry / have or are suspected of carrying / having pathogens or cancer are obtained. Background of the Invention

[0008] Antiviral CD8+ T cells respond to a cluster of potential peptides encoded by the viral genome. Cytotoxic T cells recognize infected cells through the interaction of the T cell receptor (TCR) with an 8–11 amino acid antigenic peptide complexed with major histocompatibility (MHC) class I molecules. These MHC-peptide complexes are generated from the intracellular processing of endogenously synthesized viral proteins (Saveanu, L. et al., Immunol Rev., 2005. 207: 42–59; Strehl, B. et al., Immunol Rev., 2005. 207: 19–30).

[0009] Peptide determinants bind to predicted binding motifs within specific HLA molecules. Although multiple peptide epitopes can be generated, T cell responses are concentrated on multiple selected epitopes, a phenomenon known as immunodominance (Sercarz, EE et al., Annu Rev Immunol, 1993. 11: 729-66; Yewdell, JW and JR Bennink, Annu Rev Immunol, 1999. 17: 51-88). The highly focused nature of CD8-T cell responses to pathogens suggests that each epitope differs in its ability to induce a T cell response (Yewdell, JW and JR Bennink, Annu Rev Immunol, 1999. 17: 51-88).

[0010] Peptide epitopes that induce the most significant T-cell response in any given individual can be further classified based on their proportional contribution to the overall T-cell response to any particular viral peptide. "Immunodominant" epitopes are recognized by the most abundant associated T-cell populations, while "subdominant" epitopes are recognized by less abundant T-cell populations. Therefore, based on their relative contribution to the overall T-cell response, each epitope can be classified as dominant, codominant, or subdominant, thus establishing an immune dominance stratification.

[0011] In cases of influenza virus infection in mice, the CD8 T cell response typically targets only a few specific epitopes (LaGruta, NL, et al., Proc Natl Acad Sci USA, 2006. 103: 994-999). In particularly extreme cases, the entire CD8- T cell response to mouse parainfluenza virus (Sendai virus) targets a single epitope (Cole, GA, et al., Int Immunol, 1994. 6: 1767-1775; Kast, WM, et al., Proc Natl Acad Sci USA, 1991. 88: 2283-2287).

[0012] Human T-cell responses to several viral infections have been characterized. Studies of T-cell responses to human immunodeficiency virus (HIV) have led to the identification of several epitopes in different proteins of the virus, and these studies have also shown that immunodominant epitopes can be presented by human leukocyte antigen (HLA) alleles common in individuals who co-inherit these HLA alleles, such as HLA A0301, B0702, or A0201 (Day, CL et al., J Virol, 2001. 75: 6279-6291). Furthermore, multiple epitopes can be presented by these same HLA alleles at different stages of infection (Yu, XG et al., J Virol, 2002. 76: 8690-8701). Evaluation of T-cell responses to human cytomegalovirus (CMV) has led to the identification of several immunodominant epitopes and their presenting HLA alleles in the most immunogenic proteins of the virus, namely CMVpp65 and IE1. This was subsequently identified in individuals who inherited specific HLA alleles (e.g., HLA B0702 and HLA A0201), and the epitopes presented by these alleles constituted dominant immune epitopes. When these alleles are co-inherited, the epitopes presented by HLA B0702 constituted a dominant immune T-cell response, while the epitopes presented by HLA A0201 were subdominant (Lacey, SF et al., Hum Immunol, 2003. 64: 440-452).

[0013] Immunodominance reflects the final product of multiple positive and negative factors controlling antigen processing and presentation, as well as T cell activation and T cell receptor affinity (Yewdell, JW and JR Bennink, Annu Rev Immunol, 1999.17: 51-88). Among these, the main factors evaluated in most studies to date include the genetic HLA class background of the infected individual, the sequence of viral proteins and the kinetics of viral infection, as well as binding affinity in the HLA groove and TCR affinity of the peptide-MHC complex.

[0014] Following allogeneic hematopoietic stem cell transplantation (HSCT), adoptive immunotherapy using donor-derived virus-specific T cells can be effective in clearing viral infections such as Epstein-Barr virus (EBV) and CMV. The lack of timely availability of donor-derived virus-specific T cells is a major limitation to the successful application of this treatment. Furthermore, these cells cannot be generated from seroreactive donors or umbilical cord blood donors. In such cases, pre-generated third-party donor-derived virus-specific T cells can be readily obtained for the treatment of severe viral infections in these patients. Several cohorts have demonstrated the safety and potential efficacy of third-party donor-derived virus-specific cytotoxic T lymphocyte (CTL) lines selected based on empirical input of two or more HLA alleles for the treatment of EBV, CMV, and adenovirus (ADV) infections (Haque, T et al., Lancet, 2002. 360: 436-442; Barker, JN et al., Blood, 2010. 116: 5045-5049; Doubrovina, E. et al., Blood, 2012. 119: 2644-2656; Uhlin, M. et al., Clinical Infectious Diseases, 2012. 55: 1064-1073; Leen, AM et al., Blood, 2013. 121: 5113-5123). A method for selecting CTL lines is needed to ensure high and consistent efficacy of CTL therapy.

[0015] The citation of references in this document should not be construed as an admission that the references are prior art to this disclosure. Invention Overview

[0016] The present invention provides a method for selecting an allogeneic T cell line for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, and a method for selecting an allogeneic T cell donor from which the allogeneic T cell line is derived.

[0017] In various respects, methods for selecting allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer include: using (i) identifying multiple HLA alleles and optional combinations of HLA alleles, and (ii) plotting indicators of the relative activity of T cell lines of different HLA alleles or combinations of HLA alleles that reveal at least one epitope of each antigen recognizing the pathogen or cancer and limited to a large number of HLA alleles or combinations of HLA alleles (hereinafter “Representation of Activity”). Activity”), selecting a patient who is an allogeneic T cell line that recognizes at least one epitope of an antigen of a pathogen or cancer; wherein each identified HLA allele or HLA allele combination in the diagram is associated with a corresponding indicator of the relative activity of the T cell line restricted to the HLA allele or HLA allele combination, said relative activity being a relative measure of the known activity of the T cell line against the pathogen or against the cancer; wherein (A) the selected T cell line has the same HLA allele or HLA allele combination as the patient or the patient’s diseased cells (e.g., cancer or diseased cells associated with the presence of a pathogen) identified in the diagram that is restricted to the recognition of the T cell line therein; and (B) the selected T cell line’s restricted HLA allele or HLA allele combination in the diagram is associated with an indicator of the highest relative activity among the HLA alleles and HLA allele combinations known in the diagram to be the same as those of the patient or the patient’s diseased cells (based on the HLA type of the patient or the patient’s diseased cells) and not otherwise disqualified.

[0018] In some embodiments, the method for selecting allogeneic T cell lines further includes a step of creating a viability map prior to the selection step. In some embodiments, the method for selecting allogeneic T cell lines further includes a step of measuring relative viability prior to the creation step. In some embodiments, the method for selecting allogeneic T cell lines further includes a step of determining the HLA type of the patient or the patient's diseased cells prior to the selection step. In a specific embodiment, the determination step includes typing at least four HLA loci.

[0019] In various aspects, the method of selecting allogeneic T cell lines from which allogeneic T cell lines are obtained for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer includes: selecting T cell donors who are allogeneic to the patient using an activity map, said activity map (i) identifying multiple HLA alleles and optional HLA allele combinations, and (ii) revealing an index of the relative activity of T cell lines that recognize at least one epitope of an antigen of a pathogen or cancer and are limited to a large number of HLA alleles or HLA allele combinations; wherein the individual identified HLA alleles or HLA allele combinations in the map are compared with those limited to HLA alleles. This relates to a corresponding indicator of the relative activity of a T cell line with a combination of HLA alleles, said relative activity being a relative measure of the known activity of the T cell line against a pathogen or against cancer; wherein (A) the selected T cell donor has at least one HLA allele or HLA allele combination that is identical to the patient or the patient's diseased cells (e.g., cancer or diseased cells associated with the presence of a pathogen); and (B) one of the at least one HLA allele or HLA allele combination that is identical to the patient or the patient's diseased cells is associated with an indicator of the highest relative activity among the HLA alleles and HLA allele combinations known in the activity plot to be identical to the patient or the patient's diseased cells and not otherwise disqualified.

[0020] In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of creating an activity map prior to the selection step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of measuring relative activity prior to the creation step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of determining the HLA type of the patient or the patient's diseased cells prior to the selection step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of determining the HLA type of the T-cell donor prior to the selection step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of determining the HLA type of the patient or the patient's diseased cells and the HLA type of the T-cell donor prior to the selection step. In a specific embodiment, the determination step includes typing at least four HLA loci.

[0021] In some embodiments, the activity plot is a list of multiple HLA alleles and optional HLA allele combinations ordered by relative activity. In some embodiments, the activity plot is a database overview of multiple HLA alleles and optional HLA allele combinations, each associated with a score indicating relative activity. In some embodiments, the activity plot is a scatter plot. In one specific aspect of such embodiments, the first axis of the scatter plot represents different HLA alleles and optional HLA allele combinations from a large number of HLA alleles and optional HLA allele combinations, and the second axis of the scatter plot represents the percentage of interferon-γ-secreting CD3+ cells from each T cell line that reveal an indicator of relative activity in the plot when stimulated with antigen-presenting cells that are autologous to each T cell line and loaded with one or more peptides displaying antigenicity to a pathogen or cancer, as an indicator of said relative activity. In a preferred embodiment, relative activity is the in vivo clinical efficacy of the T cell line in the treatment of patients carrying / having a pathogen or cancer. In some embodiments, the activity plot is stored in a database.

[0022] In various aspects, the method of selecting allogeneic T cell lines from which allogeneic T cell lines are obtained for therapeutic administration to human patients who carry / have or are suspected of carrying / having pathogens or cancer includes: using a diagram (hereinafter “Representation of Frequency”), selecting patients who have one or more HLA alleles identical to those of the patient or the patient’s diseased cells (e.g., cancer or diseased cells associated with the presence of pathogens) as allogeneic T cell donors, the diagram (i) identifying multiple HLA alleles, and (ii) revealing an index of the relative frequency generated by T cell lines of different HLA alleles that recognize at least one epitope of an antigen of pathogen or cancer and are limited to the multiple HLA alleles; wherein in the diagram, each identified HLA allele is associated with a corresponding index of the relative frequency generated by the T cell lines limited to the HLA alleles, wherein: the selected T cell donor has at least one HLA allele identical to those of the patient or the patient’s diseased cells that are associated with a higher frequency index compared to the donor’s HLA alleles that are different from those of the patient or the patient’s diseased cells in the diagram.

[0023] In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of creating a frequency map prior to the selection step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of measuring relative frequencies prior to the creation step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of determining the HLA type of the patient or the patient's diseased cells prior to the selection step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of determining the HLA type of the T-cell donor prior to the selection step. In some embodiments, the method for selecting allogeneic T-cell donors further includes a step of determining the HLA type of the patient or the patient's diseased cells and the HLA type of the T-cell donor prior to the selection step. In a specific embodiment, the determination step includes typing at least four HLA loci.

[0024] In some implementations, the frequency map is a list of multiple HLA alleles ordered by relative frequency. In some implementations, the frequency map is a database overview of multiple HLA alleles, each associated with a score representing relative frequency. In some implementations, the frequency map is stored in a database.

[0025] This document also provides a method for treating human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising: (a) selecting an allogeneic T cell line for therapeutic administration to the patient according to the method described in this disclosure; and (b) administering a population of T cells derived from the selected allogeneic T cell line to the patient.

[0026] This document also describes a method for obtaining an allogeneic T cell line for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising: (a) selecting an allogeneic T cell donor in accordance with the method for selecting an allogeneic T cell donor described herein; and (b) obtaining an allogeneic T cell line from the selected allogeneic T cell donor, the allogeneic T cell line recognizing at least one epitope of an antigen of the pathogen or cancer.

[0027] In various embodiments, the patient carries or is suspected of carrying a pathogen, wherein the T cell lineage recognizes at least one epitope of the pathogen's antigen. In different embodiments, the pathogen is a virus, bacteria, fungus, parasite, or protozoa. In some embodiments, the pathogen is a virus.

[0028] In some implementations, the virus is cytomegalovirus (CMV). In specific implementations, the patient carries or is suspected of carrying CMV infection after undergoing HSCT. In specific implementations, the antigen is CMV pp65. In specific implementations, the antigen is CMV IE1.

[0029] In some implementations, the virus is Epstein-Barr virus (EBV). In specific implementations, the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, or LMP2.

[0030] In some implementations, the virus is BKV, JCV, herpesvirus, adenovirus, human immunodeficiency virus, influenza virus, Ebola virus, poxvirus, rod-shaped virus, or paramyxovirus.

[0031] In some implementations, the virus is human herpesvirus-6 (HHV-6) or human herpesvirus-8 (HHV-8).

[0032] In various embodiments, a patient has or is suspected of having cancer, in which T-cell lines recognize at least one epitope of a cancer antigen. In some embodiments, the cancer is a cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain, or skin. In some embodiments, the cancer is a blood cancer. In a specific embodiment, the cancer is a lymphoproliferative disorder.

[0033] In some implementations, the cancer is WT1-positive cancer. In some implementations, the antigen is WT1.

[0034] In some implementations, the cancer is EBV-positive post-transplant lymphoproliferative disorder (EBV-PTLD). In specific implementations, the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B, or EBNA3C. In specific implementations, the antigen is LMP1 or LMP2.

[0035] In some implementations, the cancer is EBV-positive nasopharyngeal carcinoma. In specific implementations, the antigen is EBNA1, LMP1, or LMP2.

[0036] In various embodiments, the method for selecting allogeneic T cell lines as described in this disclosure is performed by computer. In various embodiments, the method for selecting allogeneic T cell donors as described in this disclosure is performed by computer.

[0037] This document also provides a computer system for selecting allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, comprising: a central processing unit; and memory connected to the central processing unit for executing steps of any method for selecting an allogeneic T cell line or any method for selecting an allogeneic T cell donor as described in this disclosure, and memory storage instructions.

[0038] This document also provides a computer-readable medium having computer-executable instructions having steps for any method of selecting an allogeneic T cell line or any method of selecting an allogeneic T cell donor as described in this disclosure.

[0039] In different implementation schemes, the patient has previously been a recipient of a hematopoietic stem cell transplant (HSCT). In specific implementation schemes, the HSCT may be a bone marrow transplant, a peripheral blood stem cell transplant, or a cord blood transplant. In different implementation schemes, the patient has previously been a recipient of a solid organ transplant (SOT).

[0040] The patients mentioned in this disclosure are human patients. Brief description of the attached diagram

[0041] Figure 1 This is a graph representing the percentage of interferon-γ-secreting CD3+ cells in each T cell lineage of a library of 119 CMV-specific CTL lineages, limited to presenting HLA alleles or HLA allele combinations that exhibit immunodominant epitopes through their individual HLA alleles or HLA allele combinations clusters, as described in the examples in Section 6.2.3. Invention Details

[0043] This invention provides a method for selecting allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, and a method for selecting allogeneic T cell donors from which said allogeneic T cell lines are derived. According to the invention, there exists a stratification of HLA alleles that presents immunodominant epitopes that result in preferential expansion of epitope-specific T cells limited to specific HLA alleles relative to other genetically and expressed cells. The invention utilizes a diagram reflecting this expansion stratification (reflected by antipathogenic or anticancer activity) to select allogeneic T cell lines for therapeutic use and to select donors from which allogeneic T cell lines are derived.

[0044] There is also a stratification of HLA alleles that present epitope-specific T cells with specific HLA alleles, leading to preferential production of immunodominant epitopes over other genetically and expressively expressed cells. This invention utilizes a diagram reflecting this production stratification (reflected by the frequency of production) to select donors from which T cell lines are derived.

[0045] Selection of T cell lines for adoptive cell therapy

[0046] This article provides a method for selecting allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having pathogens or cancer.

[0047] In various aspects, the method of selecting allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer includes: using a plot (hereinafter, “activity plot”) to select T cell lines that are allogeneic to the patient and recognize at least one epitope of an antigen of the pathogen or cancer, said plot (i) identifying multiple HLA alleles and optional HLA allele combinations, and (ii) revealing an indicator of the relative activity of T cell lines that recognize at least one epitope of an antigen of the pathogen or cancer and are limited to a large number of HLA alleles or HLA allele combinations; wherein in the plot, each identified HLA allele or HLA allele combination and the limited HLA allele or HLA allele combination are indicators of the relative activity of different HLA alleles or HLA allele combinations. The relative activity of the T cell line with the gene combination is related to a corresponding indicator, said relative activity being a relative measure of the known activity of the T cell line against a pathogen or against cancer; wherein (A) the selected T cell line has the same HLA allele or HLA allele combination as the patient or the patient's diseased cells (e.g., cancer or diseased cells associated with the presence of a pathogen) identified by the diagram, and (B) the selected T cell line's restricted HLA allele or HLA allele combination is associated with an indicator of the highest relative activity among the HLA alleles and HLA allele combinations known in the diagram to be the same as the patient or the patient's diseased cells (based on the HLA matching of the patient or the patient's diseased cells) and not otherwise disqualified. Regardless of the reason, if the T cell line restricted to this HLA allele or HLA allele combination is known to be unsuitable for therapeutic administration, then the HLA allele or HLA allele combination is considered "otherwise disqualified". For example, if an experimentally selected T cell line is observed to have no or too few viable cells in a cell line sample, the HLA allele or HLA allele combination (which restricts such T cell lines) may be considered unqualified. As another example, if the relative activity in an activity plot is based on an in vitro or ex vivo assay of activity, and it is known that the relative in vivo activity of a T cell line restricted to a particular HLA allele or HLA allele combination is independent of the relative in vitro or ex vivo assay used to generate the activity plot, such that the highest relative activity in the activity plot is not the highest relative in vivo activity, then the particular HLA allele or HLA allele combination (which restricts such T cell lines) may be considered unqualified.For example, it has been observed that HLA-B35-restricted T cell lines exhibit clinically ineffective in vivo activity against CMV infection in human patients (and therefore negligible relative in vivo activity), despite the percentage of interferon-γ secreting CD3+ T cells derived from HLA-B35-restricted T cell lines indicating a much higher relative activity; therefore, in the case of treating CMV infection, if an HLA-B35-restricted T cell line is experimentally selected, HLA-B35 may be "disqualified in other ways." By using the claimed method, the selected T cell line is specific to epitopes of pathogens or cancers, said epitopes being presented by patient-shared HLA alleles or HLA allele combinations that are associated with the highest activity in a patient's HLA allele or HLA allele combination. In a relatively specific embodiment, if a T cell line known to be clinically ineffective in treating patients carrying / having pathogens or cancer, then the HLA allele or HLA allele combination is considered disqualified.

[0048] In another embodiment, the method provided by the present invention is a method for selecting candidate allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising: selecting, using an activity plot, an allogeneic T cell line that recognizes at least one epitope of an antigen of a pathogen or cancer, the patient being an allogeneic T cell line, the activity plot (i) identifying multiple HLA alleles and optional HLA allele combinations, and (ii) revealing an index of the relative activity of T cell lines that recognize at least one epitope of an antigen of a pathogen or cancer and are limited to a large number of HLA alleles or HLA allele combinations; wherein, in the plot, each identified HLA allele or HLA allele combination and limited to HLA alleles or HLA allele combinations are an index of the relative activity of different HLA alleles or HLA allele combinations. The relative activity of a T cell line with an allele or HLA allele combination is associated with a corresponding indicator of the relative activity of the T cell line against a pathogen or against cancer; wherein (A) the selected T cell line has an HLA allele or HLA allele combination that is restricted to identification by the diagram, which is the same as that of the patient or the patient's diseased cells (e.g., cancer or diseased cells associated with the presence of a pathogen); and (B) the selected T cell line has an HLA allele or HLA allele combination that is restricted to identification by the diagram, which is associated with an indicator of the highest relative activity among the HLA alleles and HLA allele combinations known in the diagram to be the same as those of the patient or the patient's diseased cells (based on the HLA type of the patient or the patient's diseased cells).

[0049] In some embodiments, the method further includes a step of creating an activity map prior to the selection step. Methods for creating an activity map are described below. In some embodiments, the method further includes a step of measuring relative activity prior to the creation step. In some embodiments, the method further includes a step of determining the HLA type of the patient or the patient's diseased cells prior to the selection step.

[0050] In a specific implementation, the selected T cell line recognizes at least one epitope of an antigen of a pathogen or cancer, said at least one epitope being presented by an HLA allele or combination of HLA alleles identical to those in the patient or diseased cells of the patient, wherein the HLA allele or combination of HLA alleles is associated with an indicator of the highest relative activity of the HLA alleles and HLA allele combinations in the patient or diseased cells of the patient (and which have not been otherwise disqualified as described above). In a preferred aspect of such implementations, relative activity is the in vivo clinical efficacy of the T cell line in treating patients carrying / having the pathogen or cancer.

[0051] In a specific implementation of the method described herein, at least one epitope is at least one immunodominant epitope.

[0052] In some embodiments of the method of the present invention, the selected T cell line has the same HLA alleles or HLA allele combinations as identified by the viability profile of the patient or the patient's diseased cells (e.g., cancerous or pathogen-associated diseased cells). In some embodiments, the patient is a transplant recipient. In a specific embodiment where the patient is a transplant recipient, the same HLA alleles or HLA allele combinations as the patient or the patient's diseased cells (e.g., cancerous or pathogen-infected cells) refers to the same HLA alleles or HLA allele combinations as the patient before and / or after transplantation. In some embodiments, the patient's diseased cells are derived from the graft given to the patient and therefore express the graft's HLA alleles; in such embodiments, determining the HLA typing of the patient's diseased cells can be performed by HLA allele typing in the graft given to the patient. In other embodiments, the patient's diseased cells are not derived from the graft given to the patient and therefore have the patient's pre-transplant HLA typing. In specific embodiments, the transplant is HSCT or solid organ transplantation.

[0053] The emergence of T cell lineages

[0054] T cell lines can be prepared as described herein and selected for therapeutic administration and / or use to obtain mapping information. T cell lines that recognize at least one epitope of an antigen of a pathogen or cancer can be generated by any method known in the art or described herein. Non-restricted exemplary methods for T cell lines producing at least one epitope of an antigen of a pathogen or cancer can be found in Trivedi, D. et al., Blood, 2005. 105: 2793-2801; Koehne, G. et al., Blood, 2000. 96: 109-117; Koehne, G. et al., Blood, 2002. 99: 1730-1740; Doubrovina, E. et al., Blood, 2012. 119: 2644-2656; Barker, JN et al., Blood, 2010. 116: 5045-5049; O'Reilly, RJ et al., Immunol Res, 2007. 38: 237-250; and O'Reilly, RJ et al., Best Practice & Research Clinical Haematology, 2011. 24: 381-391.

[0055] In some embodiments, T cell lines are generated by stimulating T cells from seropositive donors with antigen-presenting cells that present one or more peptides of antigens displaying the antigenicity of a pathogen or cancer (in the patient's case). Preferably, the antigen-presenting cells are autologous to the T cells (and thus derived from the T cell donor). In specific embodiments, T cells are stimulated with dendritic cells loaded with a peptide library of one or more antigens of a pathogen or cancer. In some embodiments, the dendritic cells are derived from a T cell donor. In specific embodiments, T cells are stimulated with cytokine-activated monocytes (CAMS) loaded with a peptide library of one or more antigens of a pathogen or cancer. In some embodiments, the CAMS are derived from a T cell donor. In specific embodiments, T cells are stimulated with peripheral blood mononuclear cells (PBMCs) loaded with a peptide library of one or more antigens of a pathogen or cancer. In some embodiments, the PBMCs are derived from a T cell donor. In some embodiments, T cell lines are generated by stimulating T cells with a B lymphocyte cell line (BLCL) loaded with a peptide library of one or more antigens of a pathogen or cancer. In some embodiments, the BLCLs are derived from a T cell donor. In specific implementations, BLCLs are EBV-converted BLCLs derived from T cell donors. In some implementations, T cell lines are generated by stimulating T cells with artificial antigen-presenting cells (AAPCs) loaded with a peptide library of one or more antigens of pathogens or cancer.

[0056] In different embodiments, the peptide library is an overlapping peptide library spanning antigens of pathogens or cancers. In different embodiments, the peptide library is an overlapping peptide library spanning more than one antigen of a pathogen or cancer. In one specific embodiment, the overlapping peptide library is an overlapping 15-peptide library.

[0057] In some embodiments, T cell lines are generated by stimulating T cells with AAPCs that have been genetically engineered to express at least one immunogenic peptide or protein of a pathogen. In some embodiments, T cell lines are generated by stimulating T cells with BLCLs transformed with a virus, wherein the virus is a pathogen.

[0058] In some embodiments, T cells are stimulated in culture for a period of 28-40 days. In a specific embodiment, T cells are stimulated in the presence of IL-2. In different embodiments, the T cell line is cryopreserved after stimulation. In one specific embodiment, the T cell line is selected according to the required conservation method, cryopreserved, and then thawed prior to therapeutic administration. In yet another specific embodiment, the thawed T cell line is optionally expanded in culture prior to therapeutic administration.

[0059] In different embodiments, the T cells used to generate the T cell line are purified using methods known in the art. In some embodiments, T cells are enriched from peripheral blood lymphocytes isolated from PBMCs. In some embodiments, T cells are enriched from peripheral blood lymphocytes isolated from PBMCs by consuming adherent monocytes followed by consuming natural killer cells.

[0060] Dendritic cells of a T cell line that can be used to stimulate T cells to produce at least one epitope that recognizes an antigen of a pathogen or cancer can be derived from cytokine-activated monocytes (CAMS). In some embodiments, CAMS are generated by incubating PBMCs with cytokines such as GM-CSF, IL-4, TNF-α, IL-1β, IL-6, and / or prostaglandin E2.

[0061] BLCLs can be generated from PBMCs using any method known in the art, such as those described in Koehne, G. et al., Blood, 2000. 96: 109-117 or Koehne, G. et al., Blood, 2002. 99: 1730-1740, which can be used to stimulate T cells to produce at least one epitope that recognizes an antigen of a pathogen or cancer.

[0062] Each HLA allele or combination of HLA alleles of the resulting T cell line that recognizes at least one epitope of an antigen of a pathogen or cancer can be determined by any method known in the art, such as those described in Trivedi, D. et al., Blood, 2005. 105: 2793-2801; Barker, JN et al., Blood, 2010. 116: 5045-5049; Hasan, AN et al., J Immunol, 2009. 183: 2837-2850 or Doubrovina, E. et al., Blood, 2012. 120: 1633-1646.

[0063] HLA matching determination

[0064] The steps for determining HLA typing (i.e., HLA locus typing) can be performed by any method known in the art. Non-limiting exemplary methods for determining HLA typing can be found in Lange, V. et al., BMC Genomics, 2014. 15: 63; Erlich, H., Tissue Antigens, 2012. 80:1-11; Bontadini, A., Methods, 2012. 56:471-476; Dunn, PP, Int J Immunogenet, 2011 38:463-473; and Hurley, CK, “DNA-based typing of HLA for transplantation.” in Leffell, MS et al. (eds.), Handbook of Human Immunology, 1997. Boca Raton: CRC Press. In some embodiments, the step of determining HLA matching includes typing at least four HLA loci, preferably HLA-A, HLA-B, HLA-C, and HLA-DRB1. In some embodiments, the step of determining HLA matching includes typing at least four HLA loci, preferably HLA-A, HLA-B, HLA-C, and HLA-DRB1. In some embodiments, the step of determining HLA matching includes typing at least six HLA loci. In some embodiments, the step of determining HLA matching includes typing at six HLA loci. In some embodiments, the step of determining HLA matching includes typing at least eight HLA loci. In some embodiments, the step of determining HLA matching includes typing at eight HLA loci. In some embodiments, the step of determining HLA matching includes typing at all known HLA loci. In some embodiments, the step of determining HLA matching includes typing at fewer than all known HLA loci.

[0065] Generally, it is preferable to perform HLA typing on a larger number of HLA loci when carrying out the present invention, because the more HLA loci that are typed, the more likely the selected allogeneic T cell line is to have the highest activity relative to other allogeneic T cell lines that have the same HLA alleles or HLA allele combinations as the patient or the patient's diseased cells.

[0066] Creating activity maps for selecting T cell lines

[0067] Activity mapping identifies multiple HLA alleles and optional combinations of HLA alleles, and reveals (i) at least one epitope of each antigen that recognizes a pathogen or cancer (in the patient's case), and (ii) an indicator of the relative activity of T cell lines limited to a large number of HLA alleles or combinations of HLA alleles or HLA allele genomes. In activity mapping, each identified HLA allele or combination of HLA allele is associated with a corresponding indicator of the relative activity of the T cell line limited to that HLA allele or combination of HLA alleles or combinations of HLA alleles, said relative activity being a relative measure of the known activity of the T cell line against the pathogen or against the cancer.

[0068] The relative activity of T cell lines can be obtained by any in vitro, ex vivo, or in vivo method known in the art.

[0069] In a preferred embodiment, relative activity is measured by the in vivo clinical efficacy of the T-cell line in treating patients carrying / having the pathogen or cancer. In a specific aspect of this embodiment, relative activity can be measured by the percentage of patients carrying / having or suspected of carrying / having the pathogen or cancer who achieve complete remission (CR) after treatment with the T-cell line. In a specific embodiment, relative activity is measured by the percentage of patients carrying / having or suspected of carrying / having the pathogen or cancer who achieve CR or partial remission (PR) after treatment with the T-cell line.

[0070] In some embodiments, relative activity is measured as the percentage of interferon-γ-producing CD3+ cells derived from each T cell line upon stimulation with antigen-presenting cells presenting one or more peptides displaying antigenicity of a pathogen or cancer. In specific embodiments, the antigen is CMV or EBV, and relative activity is measured according to methods modified and described from Koehne, G. et al., Blood, 2002. 99: 1730-1740 or Waldrop, SL et al., J Clin Invest, 1997. 99: 1739-1750.

[0071] In some embodiments, relative activity is measured as the percentage of cells expressing pathogen or cancer antigens that lyse when exposed to each T cell line in a cytotoxic assay performed according to methods known in the art.

[0072] According to the present invention, relative activity is not measured by the binding affinity between the epitope recognized by each T cell line and the HLA allele of the presented epitope.

[0073] In some aspects, activity mapping is achieved through a list of multiple HLA alleles and optional HLA allele combinations ordered by relative activity. In some embodiments, the selection of allogeneic T cell lines is performed by examining a list of numerous HLA alleles and optional HLA allele combinations ordered by relative activity, where the highest stratum in the list indicates the highest relative activity; identifying the highest-ranked HLA allele or HLA allele combination known to be identical to the patient or the patient's diseased cells; and selecting allogeneic T cell lines limited to the HLA allele or HLA allele combination. For example, in one specific embodiment, the activity mapping is the list shown in Table 6.

[0074] In some aspects, the activity map is a database (e.g., a table) listing multiple HLA alleles and optional HLA allele combinations, each associated with a score indicating relative activity. In some embodiments, the selection of allogeneic T cell lines is performed by examining a database of multiple HLA alleles and optional HLA allele combinations, each associated with a score indicating relative activity, with the highest score in the database being the indicator of highest relative activity; determining the highest-scoring HLA allele or HLA allele combination known to be the same as the patient or the patient's diseased cells; and selecting an allogeneic T cell line limited to the HLA allele or HLA allele combination. In one specific implementation, the process of selecting allogeneic T cell lines using an activity map (i.e., such a database) can be performed by first filtering out (excluding) all HLA alleles and HLA allele combinations in the database that are different from the patient or the patient's diseased cells, then determining the HLA alleles or HLA allele combinations associated with the highest relative activity among the remaining ones, and then selecting allogeneic T cell lines that are limited to that HLA allele or HLA allele combination.

[0075] In some aspects, the activity plot is a scatter plot. In some embodiments, the first axis of the scatter plot represents different HLA alleles and optional HLA allele combinations from a large number of HLA alleles and optional HLA allele combinations. In some embodiments, the second axis of the scatter plot represents relative activity. In one specific embodiment, the second axis of the scatter plot represents the percentage of interferon-γ-secreting CD3+ cells from each T cell line that reveal an indicator of relative activity against which the activity plot reveals when stimulated with antigen-presenting cells that present one or more peptides of one or more antigens displaying antigenicity to a pathogen or cancer. In one specific embodiment, the stimulation is with antigen-presenting cells that are autologous to each T cell line and loaded with one or more peptides displaying antigenicity to a pathogen or cancer, as an indicator of said relative activity. For example, in one specific embodiment, the activity plot is Figure 1 The scatter plot shown.

[0076] In some implementations, the activity patterns are stored in a database.

[0077] In different embodiments, the method for selecting allogeneic T cell lines is performed by a computer. In some embodiments, the method for selecting allogeneic T cell lines is performed by a computer using the computer system described in Section 5.6. In some embodiments, the method for selecting allogeneic T cell lines is performed by a computer using a readable medium described in Section 5.6.

[0078] Once other data becomes available, it can be used to update the activity plot.

[0079] Therapeutic applications of selected T cell lines

[0080] This invention also provides a method for treating human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising: (a) selecting an allogeneic T cell line for therapeutic administration to the patient according to any of the methods for selecting an allogeneic T cell line as described in Section 5.1; and (b) administering a population of T cells derived from the selected allogeneic T cell line to the patient. Thus, in patients with cancer, the invention provides a method for treating cancer; in patients carrying a pathogen, the invention provides a method for treating a disease, symptom, or condition related to the presence of the pathogen.

[0081] In some embodiments, administration is performed via infusion of a population of T cells derived from a selected allogeneic T cell line. In some embodiments, administration is performed via intravenous bolus injection of a population of T cells derived from a selected allogeneic T cell line. The amount to be administered may be determined based on the patient's condition and the physician's knowledge. In some embodiments, administration includes administering at least about 1 x 10 5The patient is given 1 x 10 T cells / kg / dose / week, wherein the T cell population is derived from a selected allogeneic T cell line. In some embodiments, administration includes administering approximately 1 x 10 T cells / kg / dose / week. 6 -2 x 10 6 The patient is given 1 x 10 T cells / kg / dose / week, wherein the T cell population is derived from a selected allogeneic T cell line. In some embodiments, administration includes administering approximately 1 x 10 T cells / kg / dose / week. 6 Administering cells / kg / dose / week to the patient, wherein the T cell population is derived from a selected allogeneic T cell line. In some embodiments, administration includes delivering approximately 2 x 10 cells / kg / dose / week. 6 The patient is given T cells / kg / dose / week, wherein the T cell population is derived from a selected allogeneic T cell line. In some embodiments, the above dosing regimen is performed for at least 3 weeks, resulting in at least 3 doses. In some embodiments, the above dosing regimen is performed for 3 weeks, resulting in at least 3 doses. In some embodiments, the above dosing regimen is performed for 6 weeks, resulting in 6 doses. In some embodiments, the above dosing regimen is performed for 3 weeks, resulting in at least 3 doses, and a T cell population derived from a selected allogeneic T cell line is administered for at least one week via another dosing regimen, wherein the second dosing regimen is approximately 1 x 102 T cells / kg / dose / week. 7 One T cell / kg / dose / week. In some embodiments, the above dosing regimen is administered for 3 weeks, resulting in at least 3 doses, followed by administration of a T cell population derived from a selected allogeneic T cell line for 3 weeks via another dosing regimen, wherein the second dosing regimen is approximately 1 x 10⁻⁶ T cells / kg / dose / week. 7 1 T cells / kg / dose / week. In some implementations, where the patient has cancer, approximately 1 x 10 T cells / kg is given. 8- 1 x 10 9 Five repeated infusions per T cells / kg / dose / week.

[0082] Selection of T-cell donors for adoptive cell therapy

[0083] This article also provides a method for selecting allogeneic T cell lines from which to therapeutically administer allogeneic T cell donors to human patients who carry / have or are suspected of carrying / having pathogens or cancer.

[0084] Selection of T-cell donors based on activity mapping

[0085] In various aspects, the method of selecting allogeneic T cell lines from which allogeneic T cell lines are obtained for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer includes: selecting T cell donors who are allogeneic to the patient using an activity map, said activity map (i) identifying multiple HLA alleles and optional HLA allele combinations, and (ii) revealing an index of the relative activity of T cell lines that recognize at least one epitope of an antigen of a pathogen or cancer and are limited to a large number of HLA alleles or HLA allele combinations; wherein the individual identified HLA alleles or HLA allele combinations in the map are compared with those limited to HLA alleles. This relates to a corresponding indicator of the relative activity of a T cell line with a combination of HLA alleles, said relative activity being a relative measure of the known activity of the T cell line against a pathogen or against cancer; wherein (A) the selected T cell donor has at least one HLA allele or HLA allele combination identical to the patient or the patient's diseased cells (e.g., cancer or diseased cells associated with the presence of a pathogen); and (B) one of the at least one HLA allele or HLA allele combination identical to the patient or the patient's diseased cells is associated with an indicator of the highest relative activity among the HLA alleles and HLA allele combinations known in the activity plot to be identical to the patient or the patient's diseased cells and not otherwise disqualified. Regardless of the reason, if a T cell line known to be restricted to that HLA allele or HLA allele combination is unsuitable for therapeutic administration, then the HLA allele or HLA allele combination is considered "otherwise disqualified". For example, if the relative activity in an activity plot is based on an in vitro or ex vivo assay of activity, and it is known that the relative in vivo activity of T cell lines restricted to a specific HLA allele or combination of HLA alleles is independent of the relative in vitro or ex vivo assay used to generate the activity plot, such that the highest relative activity in the activity plot is not the highest relative in vivo activity, then the specific HLA allele or combination of HLA alleles (to which such T cell lines are restricted) may be considered unqualified. For example, it has been observed that in vivo activity against CMV infection in human patients is clinically ineffective for HLA-B35-restricted T cell lines (and therefore negligible relative in vivo activity), even though the percentage of interferon-γ secreting CD3+ T cells derived from HLA-B35-restricted T cell lines indicates a much higher relative activity; therefore, in the case of treating CMV infection, if an experimental selection of T cell donors with HLA-B35 is made, the preference for HLA-B35 may be "unqualified in other ways."In a relatively specific implementation, an HLA allele or HLA allele combination is considered ineligible if it is known that a T cell line limited to an HLA allele or combination of HLA alleles is clinically ineffective in treating patients carrying / having a pathogen or cancer.

[0086] In another embodiment, the method provided by the present invention is a method for selecting candidate allogeneic T cell donors from which allogeneic T cell lines are obtained for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising: selecting T cell donors who are allogeneic to the patient using an activity map, the activity map (i) identifying a plurality of HLA alleles and optional HLA allele combinations, and (ii) revealing an index of the relative activity of T cell lines of different HLA alleles or HLA allele combinations that recognize at least one epitope of each antigen of a pathogen or cancer and are limited to a plurality of HLA alleles or HLA allele combinations; wherein in the map, each identified HLA allele or HLA allele... Allele combinations are associated with a corresponding indicator of the relative activity of a T cell line limited to an HLA allele or HLA allele combination, said relative activity being a relative measure of the known activity of the T cell line against a pathogen or against cancer; wherein (A) the selected T cell donor has at least one HLA allele or HLA allele combination identical to the patient or the patient's diseased cells (e.g., cancer or diseased cells associated with the presence of a pathogen); and (B) one of the at least one HLA allele or HLA allele combination identical to the patient or the patient's diseased cells is associated in the diagram with an indicator of the highest relative activity among the HLA alleles and HLA allele combinations known to be identical to the patient or the patient's diseased cells in the activity diagram.

[0087] In some embodiments, the method further includes a step of creating an activity map prior to the selection step. Methods for creating an activity map are described in Section 5.1.3. In some embodiments, the method further includes a step of measuring relative activity prior to the creation step. In some embodiments, the method further includes a step of determining the HLA type of the patient or the patient's diseased cells prior to the selection step. In some embodiments, the method further includes a step of determining the HLA type of the T cell donor prior to the selection step. In some embodiments, the method further includes a step of determining the HLA type of the patient or the patient's diseased cells and the HLA type of the T cell donor prior to the selection step.

[0088] In a specific implementation, the selected T-cell donor has at least one HLA allele or combination of HLA alleles identical to the patient or the patient's diseased cells, wherein one of the at least one HLA allele or combination of HLA alleles is associated with an indicator of the highest relative activity among the patient's HLA alleles and HLA allele combinations (and which are not otherwise disqualified as described above). In a preferred aspect of this type of implementation, relative activity is the in vivo clinical efficacy of the T-cell line in treating patients carrying / having pathogens or cancer.

[0089] In a specific implementation of the method described herein, at least one epitope is at least one immunodominant epitope.

[0090] In some embodiments of the method of the present invention, the selected T-cell donor has at least one HLA allele or HLA allele combination that is identical to the patient or the patient's diseased cells (e.g., cancerous or pathogen-associated diseased cells). In some embodiments, the patient is the transplant recipient. In one specific embodiment where the patient is the transplant recipient, the identical HLA allele or HLA allele combination to the patient or the patient's diseased cells (e.g., cancerous or pathogen-infected cells) refers to the same HLA allele or HLA allele combination as the patient before and / or after transplantation. In some embodiments, the patient's diseased cells are derived from the graft given to the patient and therefore express the graft's HLA alleles; in such embodiments, determining the HLA typing of the patient's diseased cells can be performed by HLA allele typing of the graft given to the patient. In other embodiments, the patient's diseased cells are not derived from the graft given to the patient and therefore have the patient's pre-transplant HLA typing. In specific embodiments, the transplant is HSCT or solid organ transplantation.

[0091] T cell lines for creating activity maps can be prepared according to Section 5.1.1.

[0092] The steps for determining HLA typing can be performed as described in Section 5.1.2. Generally, for the purpose of carrying out this invention, it is preferable to type more HLA loci, because the more HLA loci are typed, the more likely the selected T cell donor will yield an allogeneic T cell line with the highest activity relative to other allogeneic T cell lines derived from other T cell donors having at least one HLA allele or combination of HLA alleles identical to the patient or the patient's diseased cells.

[0093] Generation of activity maps for donor selection

[0094] Activity patterns can be prepared in accordance with the discussion in Section 5.1.3 and as described herein.

[0095] In some aspects, activity mapping is a list of multiple HLA alleles and optional HLA allele combinations ordered by relative activity. In some embodiments, the steps for selecting allogeneic T cell donors are as follows: examining a list of numerous HLA alleles and optional HLA allele combinations ordered by relative activity, wherein the highest stratum in the list is an indicator of the highest relative activity; identifying the highest-ranked HLA allele or HLA allele combination known to be identical to the patient or the patient's diseased cells; and selecting allogeneic T cell donors possessing that HLA allele or HLA allele combination. For example, in one specific embodiment, activity mapping is the list shown in Table 6.

[0096] In some aspects, the activity map is a database (e.g., a table) listing multiple HLA alleles and optional HLA allele combinations, each associated with a score indicating relative activity. In some embodiments, the steps for selecting allogeneic T cell donors are as follows: by examining a database of multiple HLA alleles and optional HLA allele combinations, each associated with a score indicating relative activity, with the highest score in the database being the indicator of the highest relative activity; determining the highest-scoring HLA allele or HLA allele combination known to be identical to the patient or the patient's diseased cells; and selecting an allogeneic T cell donor possessing that HLA allele or HLA allele combination. In one specific implementation, the steps for selecting allogeneic T cell donors using activity maps (i.e., such databases) can be performed as follows: first, filter out (exclude) all HLA alleles and HLA allele combinations in the database that are different from the patient or the patient's diseased cells; then, among the remaining donors, determine the HLA alleles or HLA allele combinations associated with the highest relative activity; and then select allogeneic T cell donors who possess that HLA allele or HLA allele combination.

[0097] In some aspects, the activity plot is a scatter plot. In some embodiments, the first axis of the scatter plot represents different HLA alleles and optional HLA allele combinations from a large number of HLA alleles and optional HLA allele combinations. In some embodiments, the second axis of the scatter plot represents relative activity. In one specific embodiment, the second axis of the scatter plot represents the percentage of interferon-γ-secreting CD3+ cells from each T cell line that, when stimulated with antigen-presenting cells presenting one or more peptides of one or more antigens displaying antigenicity to a pathogen or cancer, as indicated by the relative activity against them in the activity plot. In one specific embodiment, the stimulation is with antigen-presenting cells that are autologous to each T cell line and loaded with one or more peptides displaying antigenicity to a pathogen or cancer, as an indicator of said relative activity. For example, in a specific embodiment, the activity plot is Figure 1 The scatter plot shown.

[0098] In some implementations, the activity patterns are stored in a database.

[0099] In various embodiments, the method for selecting the allogeneic T-cell donor described herein is performed by a computer. In some embodiments, the method for selecting the allogeneic T-cell donor described herein is performed by a computer using the computer system described in Section 5.6. In some embodiments, the method for selecting the allogeneic T-cell donor described in Section 5.3.1 is performed by a computer using the readable medium described in Section 5.6.

[0100] Once other data becomes available, it can be used to create an activity plot.

[0101] T-cell donor selection based on frequency mapping

[0102] In various respects, the method of selecting allogeneic T cell donors from which allogeneic T cell lines are obtained for therapeutic administration to human patients who carry / have or are suspected of carrying / having pathogens or cancer includes: using a plot (hereinafter “frequency plot”) to select patients who have the same one or more HLA alleles as the patient or the patient’s diseased cells (e.g., cancer or diseased cells associated with the presence of pathogens) as allogeneic T cell donors, said plot (i) identifying multiple HLA alleles, and (ii) revealing an index of the relative frequency generated by T cell lines of different HLA alleles that recognize at least one epitope of an antigen of pathogen or cancer and are limited to the multiple said HLA alleles; wherein in the plot, each identified HLA allele is associated with a corresponding index of the relative frequency generated by said HLA alleles, wherein: the selected T cell donor has at least one HLA allele that is the same as the patient or the patient’s diseased cells as the HLA alleles of donors who are different from those of the patient or the patient’s diseased cells in the plot, which is associated with an index of higher frequency of generation.

[0103] In another embodiment, the present invention provides a method for selecting an allogeneic T cell line from which a candidate allogeneic T cell donor is obtained for therapeutic administration to a human patient who carries / has or is suspected of carrying / having a pathogen or cancer, the method comprising: using a frequency plot to select a patient who is an allogeneic T cell donor and has one or more HLA alleles identical to those of the patient or the patient’s diseased cells (e.g., cancer or diseased cells associated with the presence of a pathogen), the frequency plot (i) identifying a plurality of HLA alleles, and (ii) revealing an index of the relative frequency generated by T cell lines of different HLA alleles that recognize at least one epitope of an antigen of a pathogen or cancer and are limited to the plurality of said HLA alleles; wherein in the plot, each identified HLA allele is associated with a corresponding index of the relative frequency generated by said HLA alleles, wherein: the selected T cell donor has at least one HLA allele identical to those of the patient or the patient’s diseased cells that are associated with a higher frequency index compared to donors whose HLA alleles are different from those of the patient or the patient’s diseased cells in the plot.

[0104] In some embodiments, the method further includes a step of creating a frequency plot before the selection step. Methods for creating a frequency plot can be described below. In some embodiments, the method further includes a step of measuring relative frequencies before the plotting step. In some embodiments, the method further includes a step of determining the HLA type of the patient or the patient's diseased cells before the selection step. In some embodiments, the method further includes a step of determining the HLA type of the T cell donor before the selection step. In some embodiments, the method further includes a step of determining the HLA type of the patient or the patient's diseased cells and the HLA type of the T cell donor before the selection step.

[0105] In a specific implementation of the method described herein, at least one epitope is at least one immunodominant epitope.

[0106] In some embodiments of the method of the present invention, the selected T-cell donor has at least one HLA allele that is the same as that of the patient or the patient's diseased cells (e.g., cancerous or pathogen-associated diseased cells) as the donor's HLA alleles in the frequency diagram that are different from those of the patient or the patient's diseased cells. In some embodiments, the patient is the transplant recipient. In one specific embodiment in which the patient is the transplant recipient, the HLA allele that is the same as that of the patient or the patient's diseased cells (e.g., cancerous or pathogen-infected cells) refers to the same HLA allele as that of the patient before and / or after transplantation. In some embodiments, the patient's diseased cells are derived from the graft given to the patient and therefore express the graft's HLA alleles; in such embodiments, determining the HLA typing of the patient's diseased cells can be performed by HLA allele typing in the graft given to the patient. In other embodiments, the patient's diseased cells are not derived from the graft given to the patient and therefore have the patient's pre-transplant HLA typing. In specific embodiments, the transplant is HSCT or solid organ transplantation.

[0107] T cell lines for making frequency maps can be prepared as described in Section 5.1.1.

[0108] The steps for determining HLA matching can be performed as described in Section 5.1.2. Generally, it is preferable to perform HLA typing on more HLA loci for the purpose of carrying out this invention.

[0109] The steps for determining HLA matching can be performed as described in Section 5.1.2.

[0110] Generation of frequency diagrams for donor selection

[0111] Frequency plots identify multiple HLA alleles and reveal indicators of the relative frequencies of T cell lines generated, said T cell lines (i) each recognizing at least one epitope of an antigen of a pathogen or cancer (in the patient's case), and (ii) different HLA alleles restricted to HLA alleles. In the frequency plots, corresponding indicators of the relative frequencies of each identified HLA allele and the T cell lines restricted to HLA alleles are associated.

[0112] In some aspects, frequency mapping is a list of multiple HLA alleles ordered by relative frequency. In some embodiments, the step of selecting an allogeneic T cell donor is performed as follows: by examining a list of multiple HLA alleles ordered by relative frequency, the highest tier in the list is the indicator of the highest relative frequency; and selecting an allogeneic T cell donor that shares at least one HLA allele with the patient or the patient's diseased cells at a higher tier in the list compared to the HLA alleles of donors who are not the same as those of the patient or the patient's diseased cells.

[0113] In some aspects, frequency mapping is a database (e.g., a table) listing a large number of HLA alleles, each associated with a score representing a relative frequency. In some embodiments, the step of selecting an allogeneic T cell donor is performed as follows: by examining a database listing HLA alleles, each associated with a score representing a relative frequency, with the highest score in the database being the indicator of the highest relative frequency; and selecting an allogeneic T cell donor that shares at least one HLA allele with the patient or the patient's diseased cells associated with a higher score in the database compared to the HLA alleles of donors different from those of the patient or the patient's diseased cells.

[0114] In some implementations, frequency plots are stored in a database.

[0115] In various embodiments, the method for selecting the allogeneic T-cell donor described in this disclosure is performed by a computer. In some embodiments, the method for selecting the allogeneic T-cell donor described in this disclosure is performed by a computer using the computer system described in Section 5.6. In some embodiments, the method for selecting the allogeneic T-cell donor described in this disclosure is performed by a computer using a readable medium described in Section 5.6.

[0116] Once other data becomes available, the frequency chart can be updated using that data.

[0117] Obtaining T cell lines

[0118] This article also describes a method for obtaining allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising: (a) selecting an allogeneic T cell donor in accordance with the method described in Section 5.3; and (b) obtaining an allogeneic T cell line from the selected allogeneic T cell donor, the allogeneic T cell line recognizing at least one epitope of an antigen of the pathogen or cancer.

[0119] patient

[0120] The patients mentioned in this disclosure are human patients.

[0121] In different implementations, the patient was previously a transplant recipient. In one specific implementation, the transplant is a HSCT (Hodgkin's transplantation). In some implementations, the HSCT is a bone marrow transplant (BMT). In some implementations, the HSCT is a peripheral blood stem cell transplant (PBSCT). In some implementations, the HSCT is a cord blood transplant (CBT). In one specific implementation, the transplant is a solid organ transplant.

[0122] In different implementation schemes, the patient is not a recipient of the transplant. In one specific implementation scheme, the patient is not a recipient of the HSCT. In another specific implementation scheme, the patient is not a recipient of a solid organ transplant.

[0123] In different ways, a patient carries or is suspected of carrying a pathogen. In one specific implementation, the patient carries a pathogen. In another specific implementation, the patient is serologically positive for the pathogen and has symptoms of infection caused by the pathogen. The pathogen can be a virus, bacteria, fungus, parasite, or protozoa. In some implementations, the pathogen is a virus.

[0124] In some implementations, the virus is cytomegalovirus (CMV). In specific implementations, the patient carries or is suspected of carrying CMV infection after undergoing HSCT. In specific implementations, the CMV antigen is CMV pp65. In specific implementations, the CMV antigen is CMV IE1.

[0125] In some implementations, the virus is Epstein-Barr virus (EBV). In specific implementations, the EBV antigen is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, or LMP2.

[0126] In some implementations, the virus is polyomatous BK virus (BKV), John Cunningham virus (JCV), herpesvirus, adenovirus (ADV), human immunodeficiency virus (HIV), influenza virus, Ebola virus, poxvirus, rod-shaped virus, or paramyxovirus. In specific implementations, the virus is BKV. In specific implementations, the virus is JCV. In specific implementations, the virus is ADV. In specific implementations, the virus is human herpesvirus-6 (HHV-6) or human herpesvirus-8 (HHV-8).

[0127] In one implementation, the patient has a viral infection that is unresponsive to antiviral (small molecule) drug therapy.

[0128] In different contexts, the patient has or is suspected of having cancer. In one specific implementation, the patient has cancer. Cancer can include cancer of the blood, breast, lung, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain, or skin. In some implementations, the cancer is a blood cancer. In a specific implementation, the cancer is a lymphoproliferative disorder. In other implementations, the cancer is brain cancer.

[0129] In some implementations, the cancer is WT1-positive. In specific implementations, the cancer antigen is WT1.

[0130] In some implementations, the cancer is EBV-positive post-transplant lymphoproliferative disorder (EBV-PTLD). In specific implementations, the antigen for EBV-PTLD is EBNA1, EBNA2, EBNA3A, EBNA3B, or EBNA3C. In other specific implementations, the antigen is LMP1 or LMP2.

[0131] In some implementations, the cancer is EBV-positive nasopharyngeal carcinoma. In specific implementations, the antigen for EBV-positive nasopharyngeal carcinoma is EBNA1, LMP1, or LMP2.

[0132] As described in this article, cancer antigens can be cancer-specific or cancer-associated antigens, and therefore can be peptides or proteins that are expressed in higher amounts in cancerous tissues or cancer cells than in non-cancer tissues or non-cancer cells, or peptides or proteins that are expressed only in cancerous tissues or cancer cells relative to non-cancer tissues or non-cancer cells.

[0133] Computer systems and computer-readable media

[0134] In various embodiments, a computer system or computer-readable medium is configured to perform any of the methods for selecting allogeneic T cell lines and selecting allogeneic T cell donors as described in this disclosure.

[0135] This document also provides a computer system for selecting allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer. In one specific embodiment, such a computer system includes: a central processing unit (CPU); memory connected to the CPU; and memory storing instructions for performing steps of any of the methods for selecting allogeneic T cell lines or allogeneic T cell donors as described in this disclosure. In some embodiments, the computer system further includes a display device that is in effective communication with the CPU.

[0136] This document also provides a computer-readable medium having computer-executable instructions for steps of any of the methods for selecting allogeneic T cell lines or selecting allogeneic T cell donors as described in this disclosure.

[0137] In some embodiments, the computer system or computer-readable medium contains software components that are standard in the art. These software components collectively enable the computer to function according to the methods for selecting allogeneic T cell lines or allogeneic T cell donors as described in this disclosure. In some embodiments, the computer system or computer-readable medium contains software components that are standard in the art and one or more computer program products specific to this invention. In specific embodiments, one or more computer program products enable the computer system to function according to the methods for selecting allogeneic T cell lines or allogeneic T cell donors as described in this disclosure. In specific embodiments, one or more computer program products specific to this invention and software components that are standard in the art collectively enable the computer system to function according to the methods for selecting allogeneic T cell lines or allogeneic T cell donors as described herein.

[0138] In some embodiments, a computer system or computer-readable medium is configured to select an allogeneic T cell line for therapeutic administration to a patient with high and consistent efficacy. In some embodiments, a computer system or computer-readable medium is configured to select an allogeneic T cell donor from which an allogeneic T cell line is obtained for therapeutic administration to a patient with high and consistent efficacy. Example

[0139] The following non-limiting examples illustrate certain embodiments provided herein, demonstrating a technique that allows for the optimized selection of therapeutically active CMVpp65-specific T cells for the treatment of CMV infection based on a stratification of HLA alleles shared by the donor and recipient of the immunodominant viral peptide.

[0140] method:

[0141] Creating the CMV CTL library:

[0142] All cell products were processed in accordance with standard operating procedures (SOPs) and FDA compliance protocols at the GMP facility of Memorial Sloan Kettering Cancer Center (MSKCC).

[0143] Production of autologous cytokine-activated monocytes (CAMS):

[0144] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of serum-reactive donors using ficoll hypaque density gradient centrifugation.

[0145] Allow PBMCs to be suspended in RPM-1640 containing 1% autologous serum at a concentration of 10 7 At a concentration of [missing value] / ml, monocytes adhered to 6-well tissue culture plates at 37°C for 2 hours, after which unadhered monocytes were gently removed. Adhered monocytes were cultured in 2 ml serum-free IMDM / well, supplemented every other day with GM-CSF 2000 IU (50 μl) and IL-4 1000 U (25 μl) until day 5. On day 5, tumor necrosis factor-α (SIGMA, St. Louis) was added to a final concentration of 10 ng / ml, interleukin-1β to 400 IU / ml, interleukin-6 (R&D systems, Inc., Minneapolis, MN USA) to 1000 IU / ml, and prostaglandin-E2 (Calbiochem, La Jolla, CA USA) to 25 mM / ml to induce final maturation of CAMS. On day 7, mature CAMS were harvested as described below, characterized by their HLA class II expression, CD14 and co-stimulatory molecules were counted by FACS, aliquoted and used for sensitization of T cell lines.

[0146] Generation of autotransformed B lymphocyte cell lines (BLCL):

[0147] As previously described, EBV-BLCLs from various donors were generated by infecting PBMCs with EBV strain B95.8 (Koehne, G. et al., Blood, 2000. 96: 109-117; Koehne, G. et al., Blood, 2002. 99: 1730-1740). Cells were maintained in RPMI 1640 (Invitrogen, Inc., Carlsbad, CA USA) supplemented with 10% fetal bovine serum (FCS) and acyclovir.

[0148] Production of CMVpp65-specific T cells:

[0149] T cells were enriched from peripheral blood lymphocytes isolated from PBMCs by immunomagnetically separating CD56+ cells using immunomagnetically pre-coated CD56 microbeads (Miltenyi Biotech Inc.), followed by the consumption of adherent monocytes and then natural killer cells. The purified T cells were co-cultured with irradiated autologous CAMS loaded with a GMP-grade overlapping 15-peptide (PL CAM) library, as previously described (Trivedi, D. et al., Blood, 2005. 105: 2793-2801). The T cells were cultured in the presence of IL-2 (5-40 U / ml) for 28-40 days at an effector-stimulatory ratio of 20:1, as previously described, and restimulated weekly with CAMS loaded with irradiated autologous peptides (Trivedi, D. et al., Blood, 2005. 105:2793-2801).

[0150] Characterization of CMVpp65-specific T cells:

[0151] Tetramer analysis:

[0152] The proportion of CMVpp65 epitope-specific T cells was determined using commercially available CMVpp65 MHC-peptide tetramers targeting HLA A0201, A2402, and B0702 carrying the peptide sequences NLVPMVATV, QYDPVAALF, and TPRVTGGGAM, respectively (Beckman Coulter, Inc., Fullerton, CA). T cells were incubated on ice for 20 min with tetrameric complexes conjugated to CD3FITC, CD8PE, CD4PerCP (BD Bioscience, San Jose, CA), and APC, washed, and then analyzed by FACS (BD LSR II). Data were analyzed using Flowjo software (Tree Star Inc., Ashland, OR). The proportions of CD4+ and CD8+ T cells, as well as the proportions of CD3+ and HLA-peptide tetramer-bound CD8+ T cells, were determined in the cultures.

[0153] TCR Vβ library (Repertoire)

[0154] Following the manufacturer's instructions (Wei, S. et al., Immunogenetics, 1994. 40: 27-36), commercially available kits (IO Test® Beta Mark, Beckman Coulter, Inc., France) containing antibodies against 24 subfamilies of the human TCR Vβ region were used to analyze CMV peptide-HLA tetramer+ T cells by flow cytometry against the TCRVβ library.

[0155] Quantification of CMV-specific and allogeneic reactive IFN-γ-producing T cells

[0156] At the beginning and at several points in the development of each CMV-specific T cell line, 1 × 10 6 Donor T lymphocytes at a concentration of / mL were mixed with autologous CAMS loaded with a CMVpp65 peptide library (20ug / ml) at an effector-stimulator cell ratio of 5:1. Control tubes containing effector cells and unloaded PBMCs were prepared in parallel. Brefidobacterium A was added to both the unstimulated and peptide-stimulated samples at a concentration of 10 μg / mL of cells. The tubes were incubated overnight at 37°C for 16 hours in a humidified 5% CO2 incubator.

[0157] Aliquots of both unstimulated and stimulated cultures were transferred to tubes for staining with monoclonal antibodies. Cells were stained with 5 μL of allophycocyanin (APC) and 10 μL of anti-CD8 polydinophyte-chlorophyll protein (PerCP) labeled with monoclonal anti-CD3 or anti-CD4 PerCP (BD Biosciences, San Jose, CA) and incubated at room temperature in the dark for 20 min. Cells were washed with 2 mL of phosphate-buffered saline (PBS) – bovine serum albumin (BSA) – azide (AZ) (PBS + 0.5% BSA + 0.1% AZ). Cells were centrifuged, the supernatant was discarded, and 100 μL of reagent A (Fix & Perm Cell Permeabilization Reagents A & B; Caltag Laboratories, Burlingame, CA) was added to each tube to fix the cells. These cells were then incubated for 15 min. Cells were washed with PBS + BSA + AZ and permeabilized with 100 μL of reagent B (Caltag Laboratories). Intracellular staining was performed by adding 10 μL of mouse IgG1 isotype control fluorescein isothiocyanate (FITC) or IFN-γ FITC (BD PharMingen, San Diego, CA) monoclonal antibody. Cells were incubated at room temperature in the dark for 20 minutes, washed twice, and then further fixed in 1% formalin.

[0158] Subsequently, stained and fixed cells were obtained using an LSR II flow cytometer (BD Biosciences) with a 10-color capability and three lasers, and analyzed using FlowJO software. Cells were first identified by forward and side scattering, and then by gating CD3 APCs and CD3+ cells in the side scattering dot plot. 20,000–50,000 events were obtained in the comprehensive gating. For further cell identification, gating was performed for CD3+CD8+ or CD3+CD4+ cells. Quadrant markers were established based on analysis of unstimulated and isotype control tubes.

[0159] Establishing Stratification: Quantifying Antiviral CD8+ T Cell Responses to Different CMV pp65 Epitopes

[0160] Epitope mapping using a library of overlapping 15 aa peptides

[0161] By following a modified version of the technique used by Waldrop et al. (J Clin Invest, 1997. 99: 1739-1750), modified by Koehne et al. (Koehne, G. et al., Blood, 2002. 99: 1730-1740), the T cell response to specific peptides within CMV pp65 was identified and quantified by measuring the number of IFNγ-positive T cells generated upon secondary stimulation with autologous APCs loaded with a target peptide or a peptide library (PL). A series of overlapping peptide libraries allowed for the identification of specific epitopes that induce T cell responses. PBMCs loaded with autologous peptides as T cell donors, CAMSs loaded with autologous T cell donors, or BLCLs loaded with autologous T cell donors were used as APCs to stimulate responsive T cells for epitope mapping.

[0162] In vitro cytotoxic activity

[0163] As mentioned above, the standard is adopted. 51 The chromium release assay was used to evaluate the ability of all T cell lines to lyse targets loaded with CMVpp65 (Koehne, G. et al., Blood, 2002. 99: 1730-1740; Trivedi, D. et al., Blood, 2005. 105: 2793-2801). The targets used in all experiments consisted of a set of EBV-BLCLs, each sharing a single HLA allele with the designated donor T cells. As specified in the experimental designation, these cells were loaded with the complete CMV pp65 peptide library or a specific sub-library of it, a single decapeptide, or a known CMV pp65 nonamer presented by that allele (e.g., NLVPMVATV for HLA A0201, QYDPVAALF and TPRVTGGGAM for HLA A2402, and RPHERNGFTV for HLA B0702) (Trivedi, D. et al., Blood, 2005. 105: 2793-2801; Hasan, AN et al., J Immunol, 2009. 183: 2837-2850). Targets loaded with peptides presented by non-shared HLA alleles were used as controls. HLA restriction is identified by the reactivity to targets of identified peptide epitope pulses presented with specific shared HLA alleles, and by the lack of reactivity to peptides loaded on EBV BLCLs carrying other shared alleles or completely mismatched EBV BLCLs.

[0164] data:

[0165] A GMP-grade CMV CTL library generated from a genetically heterologous donor population with a series of different HLA alleles.

[0166] Since the start of clinical trials using donor-derived CMVpp65-specific T cells to treat CMV viremia in allogeneic HSCT recipients, a total of 119 CMVpp65-specific CTL lines have been generated over a period of 7 years.

[0167] The donor pool used to generate CTL lines inherited 180 different HLA alleles, representing common HLA alleles prevalent in the multi-ethnic populations of New York. The distribution of HLA alleles in the donor CTL pool was closely correlated with the frequency of HLA alleles in various ethnic groups, including Caucasians, Asians, and Blacks, with the exception of HLA A0201 and B0702, which were overproduced; at 33% and 25% and 21% and 8.7%, respectively (Table 1). The frequency order of HLA class I alleles inherited from 119 donors is as follows: A0201 (n=39), A0301 (n=28), B0702 (n=25), B44 (n=24), HLA B0801 (n=22), B3501-11 (n=19), A1101 (n=16), A2402 (n=14), B1501-17 (n=14), B1801-07 (n=12), 3201-03 (n=11), A3301-04 (n=10), B4001-06 (n=9), A2601 (n=9), and B5701 (n=9). Other HLA class I alleles occur at lower frequencies, such as HLA B 5201 (n=8), B 3801 (n=6), A6801-09 (n=5), and B 5801 (n=5). For HLA class II alleles, there are six highly occurring HLA DRB1 alleles, as expected from their high frequency in the general population (Table 1). In frequency order, these include DRB1 1501-08, 0401-32, 0301-13, 0701-04, 1101-20, and 1301-34.

[0168] Table 1. HLA allele frequencies and characterization of 119 CMVpp65-specific CTL lines in the general population.

[0169]

[0170] Based on the epitope CMVpp65 presented by a few HLA class I and II alleles, the specific T cell response is dominant.

[0171] In 103 of the 119 (87%) CTL lines, immunodominant T cell responses were restricted by HLA class I alleles, while in 16 CTL lines, they were restricted by HLA class II alleles. In 54% of the CTL lines, immunodominant T cell responses were restricted by three HLA class I alleles: A0201 (25%), B0702 (21%), and B3501-11 (8%). Other alleles presenting immunodominant epitopes included HLA A2402, B4001, B4006, B4202, B4204, B4402, B4403, DRB1 0401 and 0404, DRB1 1101, and DRB1 1202. Therefore, although the library provides a wide range of class I and class II HLA alleles, only 19 of these alleles present epitopes that induce dominant T-cell immune responses. Furthermore, any detectable level of T-cell response is specific to only 49 of the 180 HLA alleles inherited from donors in the library, and only 49 of these presented epitopes.

[0172] HLA alleles that present immune dominant epitopes exist in a hierarchical order within individuals with co-inherited specific haplotypes.

[0173] Evaluation of T cell lines in the library also indicated that epitopes presented by specific HLA alleles are consistently dominant, as demonstrated by quantification of epitope-specific IFNγ+ T cells and determination of their HLA restriction. Previous studies have provided evidence that the CMVpp65 epitope presented by HLA B0702 is dominant in patients co-inheriting HLA A0201 and B0702 (Lacey, SF et al., Hum Immunol, 2003. 64: 440-452). In this series of examples, HLA B0702 is consistently the allele (100%) restricting immune dominant T cell responses in all 25 donors in the library that inherit this allele, including 9 co-inherited HLA A0201. Therefore, responses restricted by HLA B0702 are dominant regardless of any other inherited HLA class I and II alleles.

[0174] On the other hand, in 30 out of 39 donors who inherited HLA A0201 (77%), the dominant T-cell response was restricted by HLA A0201. The remaining 9 donors were co-inherited with both HLA A0201 and B0702, and in each of these 9 donors, the dominant T-cell response was restricted by HLA B0702. Therefore, HLA A0201 is an allele that restricts the dominant T-cell response when co-inherited with any other HLA class I or II allele (except when co-inherited with HLA B0702). For example, in 22 donors who inherited the HLA B44 allele, only 4 induced a dominant response restricted by that allele. When these alleles (B4401, B4402, B4403) were co-inherited with HLA A0201, 11 (91.6%) of the dominant CTL responses in the 12 donors were restricted by HLA A0201; other donors also co-inherited HLA B0702 and induced HLA B0702-restricted responses.

[0175] Another striking feature of T cell responses in donors with HLA B0702 or A0201 genetics is the fact that the observed responses are specific to epitopes presented by these alleles. In contrast, in T cell lines where responses to immunodominant epitopes are restricted by other HLA alleles, subdominant T cell populations specific to other epitopes and restricted by other HLA alleles are typically observed. This analysis allows for the presentation of... Figure 1 The hierarchical clustering of HLA alleles for the shown immunodominant epitopes is identified.

[0176] The stratification of HLA alleles presenting dominant immunomodulatory epitopes was based solely on their functional activity levels in response to peptide stimulation. There was no correlation between the peptide's affinity for HLA binding and its ability to induce a dominant T-cell response (Table 2).

[0177] Table 2. Characterization of HLA alleles presenting immunodominant epitopes.

[0178]

[0179] The epitope library and HLA alleles that make up the CMV CTL library can be used to treat different patient groups.

[0180] Within the 119 CTL lineages constituting the GMP library, a very limited pool of immunodominant CMVpp65 epitopes capable of inducing T-cell responses was identified, presented by a limited number of HLA loci. If T-cell responses are limited by such fine specificity, then for antigen-specific T cells to be clinically effective in a third-party context, the selected T cells should need to respond to epitopes presented via patient-shared HLA loci. Among these parameters, patients of different ethnicities who might be treated with CTLs from this library were analyzed.

[0181] A review was conducted over the past 3–5 years of consecutive T-cell depletion transplants performed at Memorial Sloan-Kattering Cancer Center from related or unrelated donors (HLA-matched or HLA-mismatched) and cord blood donors. At this center, in a series of 239 HLA-matched related or unrelated transplants, CMV T-cell responses restricted by patient-common HLA alleles and CTL lineages matching in 1–2 other HLA alleles were identified in 86% of the cases. Similarly, in a series of 137 HLA-mismatched transplants and 70 cord blood transplants, appropriately restricted CTL lineages were identified in 93% and 81% of the cases, respectively. Therefore, despite the broad presentation of HLA alleles in this CTL pool, T cells restricted by a limited HLA allele pool can be identified and used to treat the majority of patients in this racially diverse population.

[0182] Clinical activity of CMV CTLs selected for treatment using newly defined epitopes and HLA restriction criteria from transplant donors or third-party donors.

[0183] A total of 54 evaluable patients received CMV CTLs as treatment for clinical infection or persistent viremia that was unresponsive to antiviral therapy. Of these, 19 received CMVpp65-specific T cells from their HCT donor (NCTO1646645), and 35 received T cells from a third-party donor with >2 HLA allele matching. Results are summarized in Tables 3 and 4. In this analysis, CR was defined as clearance of clinical infection and / or detectable clearance of CMV from the blood. PR was defined as a reduction of >2 log10 in CMV in the blood. SD was defined as a patient with a stable clinical condition and a CMV reduction <2 log10. POD was defined as persistent progression of viremia and clinical disease.

[0184] Table 3. HLA-restricted response to CMVpp65-specific T cells.

[0185]

[0186] Table 4. Immunodominant HLA alleles detected in other CMVpp65-specific T cells.

[0187]

[0188] As observed, of the 19 patients who received T cells specific to the CMVpp65 epitope presented by HLA A201, 14 achieved CR or PR. Of the 9 patients treated with T cells specific to the immunodominant epitope presented by HLA B0702, 8 achieved CR. Similarly, in each of the 5 treated cases, immunodominant T cells were restricted by HLA A2402 (N=2), and B0801 (N=3) induced CR.

[0189] In contrast, recipients of CMVpp65-specific T cells that were specific to the HLA B35 allele variant did not respond. Similarly, immunodominant T cells that were specific to the A2601 (N=3), A2407 (N=1), and B5001 (N=1) epitopes failed to clear the infection or alleviate viremia.

[0190] These anticipated results provide evidence that immune dominance is induced in vivo by epitopes presented by specific HLA alleles, resulting in T cells with better therapeutic activity.

[0191] Patients who received grafts from donors who also consented to having their CMVpp65-specific T cells included in the library for use in individuals to whom they also donated HLA-compatible HCTs were not recipients. This is because T cells from such donors deplete grafts, which typically contain 2–8 x 10⁻⁸ cells. 3 The number of T cells per kg of recipient body weight may also provide a small number of immunodominant CMV-specific T cells, as the frequency of IFNγ+ CMV-specific T cells in the blood of seropositive donors ranges from 0.1% to 1% of circulating T cells. Table 5 provides the results of this preliminary analysis.

[0192] Table 5. Analysis of CMV reactivation, disease, and final response to CMV-targeted therapy in patients who received grafts from HLA-compatible donors who contributed cells from the return library as third-party donors.

[0193]

[0194] Furthermore, as observed in patients treated with CMVpp65-specific T cells, recipients of grafts from donors sharing the HLA B0702 and A0201 alleles had a lower risk of developing CMV disease and maintained a sustained response to treatment with viremia, while those receiving grafts from donors lacking these alleles had a significantly higher incidence of infection. This was again particularly observed in patients carrying variants of HLA B35 lacking either HLA B0702 or A0201.

[0195] Clinical data allowed us to identify the stratification of certain HLA alleles of the immunodominant epitope of CMVpp65 that presents an inducible peptide-specific T cell response (see Table 6), an example of an activity map.

[0196] Table 6. Stratification of HLA alleles of the immunodominant epitope of CMVpp65 that presents an inducible peptide-specific T cell response.

[0197]

[0198] a Higher grades correspond to better clinical outcomes in treating HLA-restricted T-cell lines in patients with CMV infection or persistent viremia that are unresponsive to antiviral drugs.

[0199] Our data suggest that the HLA-B35 variant should not be used (and can therefore be considered "unqualified" among the HLA alleles present).

[0200] The data shows that:

[0201] 1. A limited CMVpp65 epitope library induces functional CMV-specific cytotoxic T cell responses, and 49 of the 181 alleles appearing in this CTL library present these immunodominant CMVpp65 epitopes.

[0202] 2. The identified peptide epitopes can be used to develop effective peptide vaccines or as a limited peptide library for generating highly functional CMV CTLs for adoptive immunotherapy in the future.

[0203] 3. Incorporating HLA stratification analysis into the selection of CTL lineages for treatment can promote greater and more consistent efficacy of third-party CTL therapies.

[0204] Summary of clinical and experimental results

[0205] Effective adoptive transfer of virus-specific T cells must be specific for viral peptide epitopes expressed by virus-infected host cells and presented by infected host cells and HLA-restricted donor T cells through the HLA alleles expressed by the donor T cells that recognize the viral epitopes.

[0206] Following initial CMV infection, the initially large pool of responsive T cells shrinks, leaving only T cells specific to a few immunodominant epitopes. Therefore, CMVpp65-specific T cell lines expanded from latently infected CMV seropositive normal donors are typically specific to only one or two immunodominant CMVpp65 peptides presented by only one or two HLA alleles expressed by the donor.

[0207] We found a stratification of HLA alleles presenting the immunodominant epitope of CMVpp65, which induces peptide-specific T cell responses. This resulted in preferential proliferation of CMVpp65-specific T cells presented by a specific HLA allele compared to all other genetically and expressively expressed cells. This stratification is not based on the binding affinity of the peptide to the presenting allele. As a result of this HLA-restricted stratification:

[0208] 1. The immunodominant epitopes that induce CMVpp65-specific T cells detected in in vitro expanded T cell lines are only presented by a small number of HLA alleles.

[0209] 2. Furthermore, in individuals with two or more HLA alleles inherited from this limited pool of HLA alleles that present immunodominant peptide epitopes, certain HLA alleles (e.g., HLA B0702 or HLA A0201) consistently present epitopes that induce exclusive responses.

[0210] Early results from the phase II trial further indicate that in patients with CMV infection or persistent viremia who have failed antiviral therapy and are subsequently treated with CMVpp65-specific T cells from a third-party donor that are restricted by HLA alleles expressed by the infected cells of the host, those who receive CMVpp65-specific T cells restricted by the same highest HLA allele in that stratum (e.g., HLA B0702 or HLAA0201) consistently respond, while treatment with CMVpp65-specific T cells restricted by lower HLA alleles in the stratum clears the infection and the response is less consistent.

[0211] Citing Join

[0212] This article cites various publications, the contents of which are incorporated into the article in their entirety through citation. Detailed Implementation

[0213] A method for selecting an allogeneic T cell line for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising:

[0214] Patients using a diagram to select and recognize at least one epitope of an antigen of a pathogen or cancer are allogeneic T cell lines, the diagram (i) identifying multiple HLA alleles and optional combinations of HLA alleles, and (ii) revealing an indicator of the relative activity of T cell lines that recognize at least one epitope of an antigen of a pathogen or cancer and are limited to a large number of HLA alleles or combinations of HLA alleles; wherein each identified HLA allele or combination of HLA allele in the diagram is associated with a corresponding indicator of the relative activity of T cell lines limited to HLA alleles or combinations of HLA alleles, the relative activity being a relative measure of the known activity of the T cell lines against pathogens or cancer; wherein

[0215] (A) The selected T cell line possesses the same HLA alleles or combinations of HLA alleles as the patient or diseased cells in the patient, as identified by the diagram; and

[0216] (B) The selected T cell line is associated with the highest relative activity of HLA alleles or HLA allele combinations in the diagram with those known in the diagram to be consistent with the patient or the patient's diseased cells and not otherwise disqualified.

[0217] The method of implementation scheme 1 further includes a step of creating a diagram prior to the selection step.

[0218] The method of implementation scheme 2 further includes a step of measuring relative activity prior to the manufacturing step.

[0219] The method of any one of embodiments 1-3, wherein the method further includes a step of determining the HLA type of the patient or the patient's diseased cells prior to the selection step.

[0220] The method of implementation scheme 4, wherein the defined steps include typing at least 4 HLA loci.

[0221] The method of any one of embodiments 1-5, wherein the diagram is a list of a large number of HLA alleles and optional combinations of HLA alleles ordered by relative activity.

[0222] The method of any one of embodiments 1-5, wherein the diagram is a database listing a large number of HLA alleles and optional combinations of HLA alleles, each associated with a score indicating relative activity.

[0223] The method of any one of Implementation Schemes 1-5, wherein the plot is a scatter plot.

[0224] The method of implementation scheme 8, wherein the first axis of the scatter plot represents different HLA alleles and optional HLA allele combinations from a large number of HLA alleles and optional HLA allele combinations; and wherein the second axis of the scatter plot represents the interferon-γ-secreting CD3 from each T cell line, based on an indicator of relative activity revealed in the plot, when stimulated with antigen-presenting cells that are autologous to each T cell line and loaded with one or more peptides displaying antigenicity to pathogens or cancer. + The percentage of cells, as an indicator of relative activity.

[0225] The method of any one of Implementation Schemes 1-8, wherein the relative activity is the in vivo clinical efficacy of the T cell line in the treatment of patients carrying / having pathogens or cancer.

[0226] The method of any one of implementation schemes 1-10, wherein the illustration is stored in a database.

[0227] The method of any one of Implementation Schemes 1-11, wherein the method is performed by a computer.

[0228] The method of any one of embodiments 1-12, wherein the patient carries or is suspected of carrying a pathogen, wherein the T cell line recognizes at least one epitope of an antigen of the pathogen, and wherein the relative activity is a relative measure of known activity against the pathogen.

[0229] The method of implementation scheme 13, wherein the pathogen is a virus, bacteria, fungus, parasite or protozoa.

[0230] The method of implementation scheme 14, wherein the pathogen is a virus.

[0231] The method of implementation scheme 15, wherein the virus is CMV.

[0232] The method of implementation scheme 16, wherein the patient has or is suspected of having CMV infection after undergoing hematopoietic stem cell transplantation (hereinafter "HSCT").

[0233] The method of implementation scheme 16 or 17, wherein the antigen is CMV pp65.

[0234] The method of implementation scheme 16 or 17, wherein the antigen is CMV IE1.

[0235] The method of implementation scheme 15, wherein the virus is EBV.

[0236] The method of implementation scheme 20, wherein the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1 or LMP2.

[0237] The method of implementation scheme 15, wherein the virus is BKV, JCV, herpesvirus, adenovirus, human immunodeficiency virus, influenza virus, Ebola virus, poxvirus, rod-shaped virus, or paramyxovirus.

[0238] The method of implementation scheme 15, wherein the virus is human herpesvirus-6 (HHV-6) or human herpesvirus-8 (HHV-8).

[0239] The method of any one of embodiments 1-12, wherein the patient has or is suspected of having cancer, wherein the T cell line recognizes at least one epitope of a cancer antigen, and wherein the relative activity is a relative measure of known activity against cancer.

[0240] The method of implementation scheme 24, wherein the cancer is a blood cancer.

[0241] The method of implementation scheme 24, wherein the cancer is cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain or skin.

[0242] The method of implementation scheme 24, wherein the antigen is WT1.

[0243] The method of implementation scheme 24, wherein the cancer is a lymphoproliferative disorder.

[0244] The method of implementation scheme 28, wherein the cancer is EBV-positive post-transplant lymphoproliferative disorder.

[0245] The method of implementation scheme 29, wherein the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B or EBNA3C.

[0246] The method of implementation scheme 29, wherein the antigen is LMP1 or LMP2.

[0247] The method of implementation scheme 24, wherein the cancer is EBV-positive nasopharyngeal carcinoma.

[0248] The method of implementation scheme 32, wherein the antigen is EBNA1, LMP1 or LMP2.

[0249] A method for selecting an allogeneic T cell line from which to therapeutically administer an allogeneic T cell donor to a human patient who carries / has or is suspected of carrying / having a pathogen or cancer, the method comprising:

[0250] Using a diagram, allogeneic T-cell donors are selected for the patient, the diagram (i) identifying multiple HLA alleles and optional HLA allele combinations, and (ii) revealing an indicator of the relative activity of T-cell lines of different HLA alleles or HLA allele combinations that recognize at least one epitope of each antigen of a pathogen or cancer and are limited to a large number of HLA alleles or HLA allele combinations; wherein each identified HLA allele or HLA allele combination in the diagram is associated with a corresponding indicator of the relative activity of T-cell lines limited to HLA alleles or HLA allele combinations, the relative activity being a relative measure of the known activity of the T-cell lines against pathogens or cancer; wherein

[0251] (A) The selected T-cell donor has at least one HLA allele or combination of HLA alleles identical to the patient or diseased cells in the patient; and

[0252] (B) One of the HLA alleles or combinations of HLA alleles that are identical to the patient or the patient's diseased cells is associated with the highest relative activity among the HLA alleles and HLA allele combinations known in the diagram to be identical to the patient or the patient's diseased cells and not otherwise disqualified.

[0253] The method of implementation scheme 34 further includes a step of creating a diagram prior to the selection step.

[0254] The method of embodiment 35 further includes a step of measuring relative activity prior to the manufacturing step.

[0255] The method of any one of embodiments 34-36, the method further comprising the step of determining the HLA type of the patient or the patient's diseased cells and the HLA type of the T cell donor prior to the selection step.

[0256] The method of implementation scheme 37, wherein the defined steps include typing at least 4 HLA loci.

[0257] The method of any one of embodiments 34-38, wherein the diagram is a list of a large number of HLA alleles and optional combinations of HLA alleles ordered by relative activity.

[0258] The method of any one of embodiments 34-38, wherein the diagram is a database listing a large number of HLA alleles and optional combinations of HLA alleles, each associated with a score indicating relative activity.

[0259] The method of any one of Implementation Schemes 34-38, wherein the plot is a scatter plot.

[0260] The method of implementation scheme 41, wherein the first axis of the scatter plot represents different HLA alleles and optional HLA allele combinations from a large number of HLA alleles and optional HLA allele combinations; and wherein the second axis of the scatter plot represents the interferon-γ-secreting CD3 from each T cell line when stimulated with antigen-presenting cells that are autologous and loaded with one or more peptides displaying antigenicity to pathogens or cancer, as indicated by the relative activity of these peptides in the plot. + The percentage of cells, as an indicator of relative activity.

[0261] The method of any one of embodiments 34-41, wherein the relative activity is the in vivo clinical efficacy of the T cell line in the treatment of patients carrying / having pathogens or cancer.

[0262] The method of any one of embodiments 34-43, wherein the illustration is stored in a database.

[0263] The method of any one of embodiments 34-44, wherein the method is performed by a computer.

[0264] The method of any one of embodiments 34-45, wherein the patient carries or is suspected of carrying a pathogen, wherein the T cell line recognizes at least one epitope of an antigen of the pathogen, and wherein the relative activity is a relative measure of known activity against the pathogen.

[0265] The method of implementation scheme 46, wherein the pathogen is a virus, bacteria, fungus, parasite or protozoa.

[0266] The method of implementation scheme 46, wherein the pathogen is a virus.

[0267] The method of implementation scheme 48, wherein the virus is CMV.

[0268] The method of implementation scheme 49, wherein the patient carries or is suspected of carrying CMV infection after the patient undergoes HSCT.

[0269] The method of implementation scheme 49 or 50, wherein the antigen is CMV pp65.

[0270] The method of implementation scheme 49 or 50, wherein the antigen is CMV IE1.

[0271] The method of implementation scheme 48, wherein the virus is EBV.

[0272] The method of implementation scheme 53, wherein the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1 or LMP2.

[0273] The method of implementation scheme 48, wherein the virus is BKV, JCV, herpesvirus, adenovirus, human immunodeficiency virus, influenza virus, Ebola virus, poxvirus, rod-shaped virus, or paramyxovirus.

[0274] The method of implementation scheme 48, wherein the virus is human herpesvirus-6 (HHV-6) or human herpesvirus-8 (HHV-8).

[0275] The method of any one of embodiments 34-45, wherein the patient has or is suspected of having cancer, wherein the T cell line recognizes at least one epitope of a cancer antigen, and wherein the relative activity is a relative measure of known activity against cancer.

[0276] The method of implementation scheme 57, wherein the cancer is a blood cancer.

[0277] The method of implementation scheme 57, wherein the cancer is cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain or skin.

[0278] The method of implementation scheme 57, wherein the antigen is WT1.

[0279] The method of implementation scheme 57, wherein the cancer is a lymphoproliferative disorder.

[0280] The method of implementation scheme 61, wherein the cancer is an EBV-positive post-transplant lymphoproliferative disorder.

[0281] The method of implementation scheme 62, wherein the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B or EBNA3C.

[0282] The method of implementation scheme 62, wherein the antigen is LMP1 or LMP2.

[0283] The method of implementation scheme 57, wherein the cancer is EBV-positive nasopharyngeal carcinoma.

[0284] The method of implementation scheme 65, wherein the antigen is EBNA1, LMP1 or LMP2.

[0285] A method for selecting an allogeneic T cell line from which to therapeutically administer an allogeneic T cell donor to a human patient who carries / has or is suspected of carrying / having a pathogen or cancer, the method comprising:

[0286] The diagram is used to select allogeneic T-cell donors for patients who have one or more HLA alleles identical to those of the patient or diseased cells in the patient, the diagram (i) identifying multiple HLA alleles, and (ii) revealing an index of the relative frequency of T-cell lines generated by different HLA alleles that recognize at least one epitope of an antigen of a pathogen or cancer and are limited to a large number of said HLA alleles; wherein the corresponding index in the diagram relates to the relative frequency of each identified HLA allele to the generation of said T-cell lines limited to said HLA alleles, wherein:

[0287] The selected T-cell donors have at least one HLA allele that is the same as that of the patient or the patient's diseased cells that is associated with a higher frequency of the markers shown in the diagram compared to the donors who are different from the patient or the patient's diseased cells.

[0288] The method of implementation scheme 67 further includes a step of creating a diagram prior to the selection step.

[0289] The method of implementation scheme 68 further includes a step of measuring relative frequencies prior to the manufacturing step.

[0290] The method of any one of embodiments 67-69, the method further comprising the step of determining the HLA type of the patient or the patient's diseased cells and the HLA type of the T cell donor prior to the selection step.

[0291] The method of implementation scheme 70, wherein the defined steps include typing at least 4 HLA loci.

[0292] The method of any one of embodiments 67-71, wherein the diagram is a list of a large number of HLA alleles ordered by the relative frequencies.

[0293] The method of any one of embodiments 67-71, wherein the diagram is a database listing a large number of HLA alleles, each associated with a score representing a relative frequency.

[0294] The method of any one of embodiments 67-73, wherein the illustration is stored in a database.

[0295] The method of any one of embodiments 67-74, wherein the method is performed by a computer.

[0296] The method of any one of embodiments 67-75, wherein the patient carries or is suspected of carrying a pathogen, and wherein the T cell line recognizes at least one epitope of an antigen of the pathogen.

[0297] The method of implementation scheme 76, wherein the pathogen is a virus, bacteria, fungus, parasite or protozoa.

[0298] The method of implementation scheme 76, wherein the pathogen is a virus.

[0299] The method of implementation scheme 78, wherein the virus is CMV.

[0300] The method of implementation scheme 79, wherein the patient carries or is suspected of carrying CMV infection after the patient undergoes HSCT.

[0301] The method of implementation scheme 79 or 80, wherein the antigen is CMV pp65.

[0302] The method of implementation scheme 79 or 80, wherein the antigen is CMV IE1.

[0303] The method of implementation scheme 78, wherein the virus is EBV.

[0304] The method of implementation scheme 83, wherein the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1 or LMP2.

[0305] The method of implementation scheme 78, wherein the virus is BKV, JCV, herpesvirus, adenovirus, human immunodeficiency virus, influenza virus, Ebola virus, poxvirus, rod-shaped virus, or paramyxovirus.

[0306] The method of implementation scheme 78, wherein the virus is human herpesvirus-6 (HHV-6) or human herpesvirus-8 (HHV-8).

[0307] The method of any one of embodiments 67-75, wherein the patient has or is suspected of having cancer, and wherein the T cell line recognizes at least one epitope of the cancer antigen.

[0308] The method of implementation scheme 87, wherein the cancer is a blood cancer.

[0309] The method of implementation scheme 87, wherein the cancer is cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain or skin.

[0310] The method of implementation scheme 87, wherein the antigen is WT1.

[0311] The method of implementation scheme 87, wherein the cancer is a lymphoproliferative disorder.

[0312] The method of implementation scheme 91, wherein the cancer is EBV-positive post-transplant lymphoproliferative disorder.

[0313] The method of implementation scheme 92, wherein the antigen is EBNA1, EBNA2, EBNA3A, EBNA3B or EBNA3C.

[0314] The method of implementation scheme 92, wherein the antigen is LMP1 or LMP2.

[0315] The method of implementation scheme 87, wherein the cancer is EBV-positive nasopharyngeal carcinoma.

[0316] The method of implementation scheme 95, wherein the antigen is EBNA1, LMP1 or LMP2.

[0317] The method of any one of implementation schemes 1-96, wherein the patient was a recipient of HSCT.

[0318] The method of implementation plan 97, wherein HSCT is bone marrow transplantation, peripheral blood stem cell transplantation or umbilical cord blood transplantation.

[0319] The method of any one of implementation schemes 1-96, wherein the patient was a recipient of a solid organ transplant.

[0320] A computer system for selecting allogeneic T cell lines for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, comprising:

[0321] CPU;

[0322] A memory connected to a central processing unit, the memory storing instructions for performing the steps of the method of any one of embodiments 1-99.

[0323] A computer-readable medium having computer-executable instructions for performing the steps of the method of any one of embodiments 1-99.

[0324] A method for treating a human patient who carries / has or is suspected of carrying / having a pathogen or cancer, the method comprising:

[0325] (a) Selecting an allogeneic T cell line for therapeutic administration to the patient according to any one of the methods in Implementation Scheme 1-33; and

[0326] (b) Administer T cell populations derived from selected allogeneic T cell lines to the patient.

[0327] The method of implementation scheme 102, wherein the patient was a recipient of HSCT.

[0328] The method of implementation scheme 103, wherein HSCT is bone marrow transplantation, peripheral blood stem cell transplantation or umbilical cord blood transplantation.

[0329] The method of implementation scheme 102, wherein the patient was a recipient of a solid organ transplant.

[0330] A method for obtaining an allogeneic T cell line for therapeutic administration to human patients who carry / have or are suspected of carrying / having a pathogen or cancer, the method comprising:

[0331] (a) Selecting an allogeneic T-cell donor according to any one of the methods in Implementation Schemes 34-99; and

[0332] (b) Obtain an allogeneic T cell line from a selected allogeneic T cell donor, the allogeneic T cell line recognizing at least one epitope of an antigen of a pathogen or cancer.

Claims

1. A method for selecting an allogeneic T-cell line for therapeutic administration to human patients with or suspected of having EBV-positive post-transplant lymphoproliferative disorder (EBV-positive PTLD), comprising: A diagram is used to select allogeneic T cell lines that recognize at least one epitope of an EBV antigen from the patient, the diagram (i) listing a plurality of HLA alleles and optional HLA allele combinations, and (ii) revealing an indicator of the relative activity of T cell lines that recognize at least one epitope of an EBV antigen and are limited to different HLA alleles or HLA allele combinations among the plurality of HLA alleles and optional HLA allele combinations; wherein, in the diagram, each listed HLA allele or HLA allele combination is associated with a corresponding indicator of the relative activity of T cell lines limited to HLA alleles or HLA allele combinations, the relative activity being a relative measure of the known activity of the T cell line against EBV-positive PTLD, wherein the relative activity is the in vivo clinical efficacy of the T cell line in the treatment of human patients with EBV-positive PTLD; wherein the selection step is performed by selecting T cell lines that simultaneously possess (A) and (B): (A) Having HLA alleles or combinations of HLA alleles that are exclusive to the selected T cell lines listed in the diagram and are consistent with the patient or the patient's diseased cells; and (B) Limited to the HLA alleles or HLA allele combinations listed in the diagram that relate to the highest relative activity of the HLA alleles or HLA allele combinations known in the diagram to be consistent with the patient or the patient's diseased cells and not otherwise disqualified.

2. The method of claim 1, further comprising the step of creating the diagram prior to the selection step.

3. The method of any one of claims 1-2, wherein the diagram is a list of the plurality of HLA alleles and optional combinations of HLA alleles ordered by relative activity.

4. The method of any one of claims 1-2, wherein the diagram is a database listing a plurality of HLA alleles and optional combinations of HLA alleles, each associated with a score indicating relative activity.

5. A method for selecting an allogeneic T cell line from which to therapeutically administer an allogeneic T cell donor to a human patient with or suspected of having EBV-positive PTLD, the method comprising: Using a diagram, an allogeneic T-cell donor is selected for the patient, the diagram (i) listing multiple HLA alleles and optional HLA allele combinations, and (ii) revealing an indicator of the relative activity of T-cell lines that recognize at least one epitope of an EBV antigen and are limited to different HLA alleles or HLA allele combinations among the multiple HLA alleles and optional HLA allele combinations; wherein, the respective listed HLA alleles or HLA allele combinations in the diagram are related to a corresponding indicator of the relative activity of the T-cell lines limited to the HLA alleles or HLA allele combinations, the relative activity being the T-cell line's performance against EBV-positive PTLD. The relative measure of known activity, wherein the relative activity is the in vivo clinical efficacy of the T cell line in the treatment of human patients with EBV-positive PTLD; wherein the selection step is performed by selecting a T cell donor having at least one HLA allele or HLA allele combination consistent with the patient or the patient's diseased cells, wherein one of the at least one HLA allele or HLA allele combination consistent with the patient or the patient's diseased cells is associated with an indicator of the highest relative activity among the HLA alleles and HLA allele combinations listed in the diagram that are known to be consistent with the patient or the patient's diseased cells and have not been otherwise disqualified.

6. The method of claim 5, further comprising the step of creating the diagram prior to the selection step.

7. The method of any one of claims 5-6, wherein the diagram is a list of the plurality of HLA alleles and optional combinations of HLA alleles ordered by relative activity.

8. The method of any one of claims 5-6, wherein the diagram is a database listing a plurality of HLA alleles and optional combinations of HLA alleles, each associated with a score indicating relative activity.

9. A computer system for selecting allogeneic T cell lines for therapeutic administration to human patients with or suspected of having EBV-positive PTLD, comprising: CPU; A memory connected to a central processing unit, the memory storing instructions for performing the steps of the method according to any one of claims 1-4.

10. A computer system for selecting allogeneic T cell lines from which to therapeutically administer to human patients with or suspected of having EBV-positive PTLD, comprising: CPU; A memory connected to a central processing unit, the memory storing instructions for performing the steps of the method according to any one of claims 5-8.

11. A computer-readable medium having computer-executable instructions for performing the steps of the method according to any one of claims 1-8.

Images