Cancer immunotherapy using injectable transfer of allogeneic, tumor-specific CD4+ T cells

Abstract

The present invention provides methods and compositions for administering allogeneic lymphocytes as an exogenous source of CD4+ T cell help for endogenous tumor-reactive CD8+ T cells.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. patent application Ser. No. 16 / 786,761, filed February 10, 2020, which is a continuation-in-part of U.S. patent application Ser. No. 15 / 939,059, filed March 28, 2018, which is a continuation-in-part of U.S. patent application Ser. No. 14 / 398,724, filed November 3, 2014 (now issued as U.S. Patent No. 9,931,359); U.S. patent application Ser. No. 14 / 398,724 is a continuation-in-part of International Application No. PCT / US2013 / 032129, filed March 15, 2013, which ... which is a continuation-in-part of International Application No. PCT / US2013 / 032129, which is a continuation-in-part of International Application No. PCT / US2013 / 032129, which is a continuation-in-part of International Application No. PCT / US2013 / 032129 §371 national stage application (now expired); International Application No. PCT / US2013 / 032129 claims the benefit under 35 U.S.C. §119(e) to U.S. Patent Application No. 61 / 644,126 (now expired), filed May 8, 2012. The disclosures of each prior application are considered part of this application and are incorporated by reference into the disclosure of this application.

[0002] [Statement regarding federally funded research and development] This invention was made with government support under CA105148 and CA015396 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] [Import sequence list] The material in the accompanying Sequence Listing is incorporated herein by reference. The attached Sequence Listing text file, JHU3680_3WO_Sequence_Listing.txt, was created on February 8, 2021, and is 12kb in size. The file may be reviewed using Microsoft Word on a computer using the Windows OS.

[0004] FIELD OF THE INVENTION The present invention relates generally to immunology, and more specifically to methods and compositions comprising allogeneic lymphocytes for treating cancer. [Background technology]

[0005] [ Background information] The host immune system provides the means to rapidly and specifically mount defensive responses against pathogenic microorganisms and also contributes to the rejection of malignant tumors. The immune response is generally described as comprising humoral responses, in which differentiated B lymphocytes produce antigen-specific antibodies, and cell-mediated responses, in which various types of T lymphocytes eliminate antigens through various mechanisms. For example, CD4 (also called CD4+) helper T cells, which can recognize specific antigens, may respond by releasing soluble mediators, such as cytokines, to recruit additional immune system cells involved in the immune response. CD8 (also called CD8+) cytotoxic T cells can also recognize specific antigens and bind to and destroy or damage antigen-bearing cells or microparticles. In particular, cell-mediated immune responses, including cytotoxic T lymphocyte (CTL) responses, can be important for eliminating tumor cells and cells infected with microorganisms (e.g., viruses, bacteria, or parasites).

[0006] Cancer comprises a wide range of diseases, affecting approximately one in four people worldwide. CTL responses are a key feature of effective cancer vaccines; effective CD4 T cell help plays a critical role in maintaining the cytotoxic activity of CD8 T cells, and therefore provides clinical benefit.

[0007] Regarding microbial infections, malaria, tuberculosis, HIV-AIDS, and other viral infections, such as Epstein-Barr virus, hepatitis B and C viruses, herpes simplex virus (HSV), and human papillomavirus (HPV), remain global health concerns. Viruses are estimated to cause approximately 15% of all human cancers. Human papillomaviruses, including both oncogenic and non-oncogenic serotypes, are the most common sexually transmitted diseases worldwide. While most immunocompetent individuals can clear the virus, a subset of healthy individuals are unable to clear oncogenic strains of HPV, which can then establish persistent infection in epithelial cells and induce malignant transformation.

[0008] Emerging evidence suggests that cancer induces a state of unresponsiveness in lymphocytes specific to antigens uniquely expressed by the cancer. However, it should be possible to reverse this unresponsiveness. Some human tumors are infiltrated with CD8+ T cells, and the degree of CD8+ T cell infiltration often correlates with the absence of metastasis and improved survival. However, these CD8+ T cells may not eliminate the cancer due to functional paralysis of tumor-specific CD4+ T cells.

[0009] Immunological checkpoint inhibitors (CIs), such as ipilimumab, nivolumab, and pembrolizumab, have been successful in treating a variety of cancers, and it is now conclusively established that T cells from the immune system can induce tumor regression, leading to prolonged survival and improved quality of life. However, the success of any cancer immunotherapy is limited by T cell exhaustion, a phenomenon characterized by impaired proliferation, cytokine secretion, and cytotoxic activity of tumor-specific T cells. Therefore, there is considerable interest in developing strategies to restore T cell exhaustion in anti-cancer immunotherapy.

[0010] Recent evidence suggests that CIs are unable to restore T cells from exhaustion. In contrast, allogeneic cell therapy, especially CD4 T cells derived from major histocompatibility complex (MHC)-mismatched donors, + It has been found that injecting T cells can induce regression of advanced cancers that are resistant to immune checkpoint blockade, even though the donor cells are ultimately rejected. +Cell depletion eliminated the risk of persistent donor cell engraftment and fatal graft-versus-host disease (GVHD) without compromising the antitumor efficacy of the injection. Vaccination of the donor against the E7 antigen of human papillomavirus (HPV) serotype 16 (HPV16) enhanced the antitumor efficacy of CD8-depleted, non-engrafting donor lymphocyte infusion (NEDLI) against the E7-expressing lung cancer TC-1, when the donor and recipient were MHC-haploidentical (e.g., parent to child, or vice versa). The antitumor efficacy of E7-primed donor-derived NEDLI may be enhanced and recipients cured by co-culturing the primed cells with host- or donor-derived dendritic cells pulsed with HPV16 E7-derived peptides before injection. Mice cured of advanced TC-1 tumors by E7-primed NEDLI showed no evidence of donor chimerism but expanded populations of host-derived E7-specific memory CD4+ and CD8+ T cells, indicating that NEDLI imprinted antitumor immunity in the recipient. These results demonstrate that virus-induced tumors can be treated by injecting lymphocytes from partially or fully HLA-matched donors vaccinated against viral antigens, and suggest the possibility that sporadic tumors can be treated by injecting lymphocytes from donors vaccinated against tumor neoantigens.

[0011] Furthermore, mice cured of TC-1 by NEDLI resisted challenge with the same tumor, and spleen cells from the cured animals transferred antitumor immunity to TC-1-bearing, MHC-haploidentical recipients. Vaccination of healthy donors against tumor-specific antigens followed by incubation of vaccinated donor lymphocytes with peptides derived from tumor-specific antigens significantly enhanced the antitumor effect of injection of non-engrafted donor lymphocytes into allogeneic recipients bearing antigen-expressing tumors. Summary of the Invention [Problem to be solved by the invention]

[0012] [ Summary of the Invention The present invention demonstrates that injecting allogeneic lymphocytes containing CD4+ T cells can break host anti-tumor CD8+ T cell tolerance, even though the donor cells do not survive long-term in the recipient; and that alloreactive and neoantigen-specific CD4 + Injecting T cells stimulates endogenous tumor-specific CD8 + The present invention is based on the seminal discovery that T cells can be mobilized to recover from exhaustion and thereby induce tumor regression. + Allogeneic virus-specific and / or tumor neoantigen (neoAg)-specific CD4 T cells in subjects with or susceptible to cancer to restore T cell exhaustion. + and methods and compositions relating to administering T cells by injection. [Means for solving the problem]

[0013] In one embodiment, the present invention provides a method for producing a lymphocyte composition, the method comprising: a) obtaining a peripheral blood cell composition from a donor, wherein the donor has optionally been vaccinated against an antigen present in the recipient, and wherein the peripheral blood composition comprises CD8+ T cells, CD4+ T cells, and natural killer cells; b) depleting the CD8+ T cells in the peripheral blood cell composition, wherein depleting the CD8+ T cells in the peripheral blood cell composition reduces the number of CD8+ T cells in the peripheral blood cell composition by at least one order of magnitude; and c) expanding CD4+ T cells specific for the antigen by culturing CD4+ T cells with the antigen, wherein the donor is HLA-matched, partially HLA-matched, or HLA-haploidentical to the recipient, thereby producing a lymphocyte composition.

[0014] In another embodiment, the present invention provides a method of treating cancer in a subject, wherein the method comprises: a) administering lymphocyte-depleting chemotherapy to the subject; and b) administering a lymphocyte cell composition to the subject, wherein the lymphocyte cell composition is obtained from the peripheral blood cell composition of an HLA-matched, partially HLA-mismatched, or HLA-haploidentical donor, who has optionally been vaccinated against a viral antigen and / or tumor neo-antigen present in the subject, wherein the composition is depleted of CD8+ T cells, and wherein the composition comprises an expanded population of CD4+ T cells specific for a viral and / or tumor neo-antigen present in the subject. In one aspect, a partially HLA-matched or HLA-haploidentical donor has at least one human leukocyte antigen (HLA) class II allele match to the recipient, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1.

[0015] In a further embodiment, the present invention provides a cell bank comprising CD4+ T cells specific for a viral antigen, a tumor neoantigen, or a combination thereof, wherein the cell bank comprises distinct lines of CD4+ T cells, each line collected from a single donor of different human leukocyte antigen (HLA) types. In one aspect, the viral antigen is derived from HPV or Epstein-Barr virus. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows the hematocrits of five patients who experienced graft rejection after HLA-haploidentical bone marrow transplantation for myelodysplastic syndromes; the jagged areas reflect the effect of blood transfusions (graph). [Figure 2] FIG. 2 shows (graph) that non-engrafting DLI induces anti-tumor immunity. [Figure 3] Figure 3A shows donor cell engraftment (graph) as vaccine plus allogeneic CD4s prolongs survival. Figure 3B shows donor CD4+ T cell chimerism versus days post-transplant. [Figure 4] Figure 4 shows the results of flow cytometry analysis of CD8 reduction using leukapheresis preparations and bloodletting specimens. [Figure 5] FIG. 5 shows a model illustrating the anti-tumor efficacy of injecting transiently engrafting, MHC-mismatched donor lymphocytes. [Figure 6] Figure 6A shows the protocol used to obtain the results in Figure 6B. C57BL / 6 x C3H (B6 x C3H; B6C3) F1 mice or BALB / c x C57BL / 6 (BALB / c x B6; CB6) F1 mice were vaccinated weekly for three doses with 25 μg of pcDNA-3-CRT / E7 (a DNA vaccine against human papillomavirus serotype 16 E7). One week later, splenocytes from naive or vaccinated mice were either undepleted or depleted in CD8+ T cells. 20 million cells were then injected into B6 x C3H F1 mice (which had received 50,000 E7-expressing TC1 lung cancer cells two weeks earlier and cyclophosphamide at 200 mg / kg IP one day earlier). Survival of tumor-bearing mice is shown on the left for recipients of syngeneic B6 x C3H F1 cells and on the right for recipients of MHC-haploidentical BALB / cx B6 F1 cells. [Figure 7]Figure 7A shows the protocol used to obtain the results shown in Figure 7B. Figure 7B is a graph showing that ex vivo culturing of spleen cells from tumor Ag-primed donors enhances the anti-tumor efficacy of NEDLI. Spleen cells from an E7-primed CB6 F1 donor were cultured with CB6 F1 (syn) or B6C3 F1 (haplo) dendritic cells (DCs) pulsed with overlapping pentadecamers of E7 from HPV16 (JPT Peptide Tech.). [Figure 8]Figures 8A–H show the percentage of recipient CD8+ T cells obtained by flow cytometry. Figure 8A shows the percentage of CD8+ T cells that were reactive to the E7 immunodominant, H-2Kb-restricted peptide, as measured by staining with H-2Kb-tetramers pulsed with the peptide in naive B6C3 F1 mice. Figure 8B shows the percentage of CD8+ T cells that were reactive to the E7 immunodominant, H-2Kb-restricted peptide, as measured by staining with H-2Kb-tetramers pulsed with the peptide in mice cured by haplo-DLI. Figure 8C shows the percentage of CD8+ T cells that were reactive to the E7 immunodominant, H-2Kb-restricted peptide, as measured by staining with H-2Kb-tetramers pulsed with the peptide in mice cured by haplo-DLI 14 days after TC-1 challenge. Figure 8D shows the percentage of CD8+ T cells that were reactive to the immunodominant H-2Kb-restricted peptide of E7, as measured by staining with H-2Kb tetramer pulsed with the peptide, in mice cured by haplo-DLI 60 days after TC-1 challenge. Figure 8E shows the cell surface expression of CD127 and PD-1 on E7-specific CD8+ T cells, gated under the same conditions as Figure 8A. Figure 8F shows the cell surface expression of CD127 and PD-1 on E7-specific CD8+ T cells, gated under the same conditions as Figure 8B. Figure 8G shows the cell surface expression of CD127 and PD-1 on E7-specific CD8+ T cells, gated under the same conditions as Figure 8C. FIG. 8H shows cell surface expression of CD127 and PD-1 on E7-specific CD8+ T cells gated under the same conditions as in FIG. 8D. [Figure 9]Figure 9A shows dot plots depicting intracellular interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) staining for CD4+ T cells from untreated B6 × C3H F1 naive mice 5 days after stimulation with unpulsed B6 × C3H F1 dendritic cells. Figure 9B shows intracellular interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) staining for CD4+ T cells from untreated B6 × C3H F1 naive mice 5 days after stimulation with E7 peptide-pulsed DCs. Figure 9C shows intracellular interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) staining for CD4+ T cells from mice cured by injection of non-engrafted donor lymphocytes 5 days after stimulation with E7 peptide-pulsed DCs. FIG. 9D shows a graph of the results quantified by ELISPOT assay for IFNγ secretion from the same cells shown in FIGS. 9A-C. [Figure 10] Figure 10A shows the protocol used to obtain the results in Figure 10B. TC1-bearing B6 × C3H F1 mice were cured with cyclophosphamide plus CD8-depleted lymphocytes (derived from E7-vaccinated donors and expanded ex vivo with E7 peptide). 300 days after lymphocyte injection, spleen cells from the cured mice were depleted of CD8+ cells, cultured with E7 peptide for 1 week, and transferred to TC1-bearing BALB / c × B6 F1 mice that had been treated with cyclophosphamide 1 day prior (20 million cells per recipient). Alternatively, TC1-bearing BALB / c × B6 F1 mice received spleen cells from naive B6 × C3H F1 donors that had been depleted of CD8+ cells and cultured with E7 for 1 week prior to adoptive transfer. The survival rate is shown in Figure 10B. [Figure 11]Figure 11A shows a schematic diagram of biparental bone marrow chimeras to determine the necessity of APC licensing in tumor immunity. Figure 11B shows a schematic diagram of F1 bone marrow chimeras to determine the necessity of APC licensing in tumor immunity. [Figure 12] Figure 12 shows a schematic diagram illustrating how CD4+ T cell help rescues exhausted CD8+ T cells (eCTLs) specific for neoantigens B, C, and E, which are released from dying tumor cells. DETAILED DESCRIPTION OF THE INVENTION

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[0029] [Detailed Description of the Invention] The present invention stems, at least in part, from the seminal discovery that immune responses against cancer are hindered by functional defects in a patient's CD4+ T cells. Injection of allogeneic lymphocytes can provide an exogenous source of CD4+ T cell help for endogenous, tumor-reactive CD8+ T cells. Depletion of CD8+ T cells from injected donor lymphocytes reduces the risk of persistent engraftment and graft-versus-host disease. Depletion of regulatory T cells from the injected population enhances the ability of non-regulatory T cells to provide help to endogenous effectors of anti-tumor immunity. Allogeneic T cell therapy is typically performed in conjunction with allogeneic stem cell transplantation, in which the patient undergoes highly immunosuppressive conditioning followed by injection of a stem cell graft containing an unselected population of mature T cells. In the treatment described in this application, the graft is engineered to minimize the likelihood of persistent donor cell engraftment, and anti-tumor effector T cells are derived from the host. Thus, this treatment requires a unique partnership between host and donor lymphocytes during transient donor cell engraftment.

[0030] Before the compositions and methods of the present invention are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as, for example, compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0031] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes a plurality of methods, and / or steps of the type described herein that would become apparent to those of ordinary skill in the art upon reading this disclosure and so forth.

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into this application to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0033] Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, although it will be understood that modifications and variations are within the spirit and scope of the present invention. Preferred methods and materials are described herein.

[0034] Prescribing chemotherapy prior to injecting allogeneic cells can enhance the antitumor activity of transiently engrafted lymphocytes by promoting homeostatic proliferation of the transplanted lymphocytes and / or by depleting host regulatory T cells and myeloid-derived suppressor cells. Injecting allogeneic lymphocytes can provide an exogenous source of CD4+ T cell help for endogenous, tumor-reactive CD8+ T cells. Depleting CD8+ T cells from the injected donor lymphocytes reduces the risk of persistent engraftment and graft-versus-host disease. Removing regulatory T cells from the injected population enhances the ability of non-regulatory T cells to provide help to endogenous effectors of antitumor immunity.

[0035] Allogeneic T cell therapy is typically performed in the context of allogeneic stem cell transplantation, in which the patient undergoes highly immunosuppressive conditioning followed by infusion of a stem cell graft containing an unselected population of mature T cells. The goal of alloSCT is to achieve durable engraftment of donor cells, which carries the risk of mortality due to graft-versus-host disease. In the treatment described herein, the graft is engineered to minimize the likelihood of persistent donor cell engraftment, and anti-tumor effector T cells are derived from the host. Thus, this treatment requires a unique partnership between host and donor lymphocytes during transient donor cell engraftment.

[0036] This is a treatment that can be applied to any human or animal cancer. Variations of the present invention include: 1) variations in the chemotherapy regimen prescribed before injecting allogeneic lymphocytes (which may include cyclophosphamide, fludarabine, 5-fluorouracil, gemcitabine, dasatinib, or combinations thereof); 2) variations in the source of donor lymphocytes (which may be from related or unrelated donors and may include defined mismatches in HLA class I or class II alleles); and 3) variations in the type of cells selected for injection (e.g., depletion of CD4+CD25+ regulatory T cells, depletion of CD8+ T cells, etc.). The donor may be depleted of type I (IFN-γ producing) or type 17 (IL-17 producing) CD4 + To enrich for T cells, lymphocytes may be immunized against a defined antigen prior to injection, or may be polarized ex vivo with cytokines or drugs.

[0037] CD8 in the immune system + T cells can destroy cancer cells by recognizing tumor neoantigens, amino acid sequences caused by mutations in cancer cells, and distinguish cancer cells from their healthy, normal counterparts. However, cancers can also destroy tumors by targeting CD4 + T cell function (CD4 + T cell "help" is neoantigen-specific CD8 + By numbing the immune system (which is necessary to prevent T cell exhaustion), immune destruction can be avoided. The allogeneic transplants of the present invention can be used to induce the production of CD4 T cells derived from healthy donors who have been vaccinated against neoantigens derived from the patient's tumor. + By providing a new source of helper T cells, neoantigen-specific CD8 + May restore T cells from exhaustion and may restore anti-cancer immune responses.

[0038] In one embodiment, the present invention provides a method for producing an allogeneic lymphocyte composition for administration to a human recipient, the method comprising: vaccinating a human donor against an antigen present in the recipient; providing a peripheral blood cell composition derived from a human donor that is allogeneic to the recipient, the peripheral blood cell composition comprising some CD4+ T cells, some CD8+ T cells, and some natural killer cells, wherein (i) the donor has CD4+ T cell immunity to an antigen present in the recipient, (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele match to the recipient, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, and (iii) the recipient has no detectable antibodies reactive to the donor's human leukocyte antigens; and Producing the allogeneic lymphocyte composition from the peripheral blood cell composition by reducing the number of T cells by at least one order of magnitude, wherein the number of CD4+ T cells in the allogeneic lymphocyte composition differs from the number of CD4+ T cells in the peripheral blood cell composition by less than about 50%, and wherein the number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition.

[0039] The present invention provides a method for producing a lymphocyte composition for administration to a recipient, the method comprising: a) obtaining a peripheral blood cell composition from a donor, wherein the donor has optionally been vaccinated against an antigen present in the recipient, and wherein the peripheral blood composition comprises CD8+ T cells, CD4+ T cells, and natural killer cells; b) depleting the CD8+ T cells in the peripheral blood cell composition, wherein depleting the CD8+ T cells in the peripheral blood cell composition reduces the number of CD8+ T cells in the peripheral blood cell composition by at least one order of magnitude; and c) expanding CD4+ T cells specific for the antigen by culturing CD4+ T cells with the antigen, wherein the donor is HLA-matched, partially HLA-matched, or haploidentical to the recipient, thereby producing a lymphocyte composition. In one aspect, a partially HLA-matched or HLA-haploidentical donor has at least one human leukocyte antigen (HLA) class II allele match to the recipient, and the HLA class II allele match is in a gene selected from HLA-DRB1, HLA-DQB1, and / or HLA-DPB1.

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[0041] Preferably, the donor and recipient are not the same human. The method includes providing a peripheral blood cell composition derived from a human donor who is allogeneic to the recipient, the peripheral blood cell composition including some CD4+ T cells, some CD8+ T cells, and some natural killer cells, some of which bear the CD8+ antigen and may be removed by a "depletion" step; however, preferred lymphocyte compositions of the present invention include at least some natural killer cells derived from the donor. In one embodiment, (i) the donor contains at least one human leukocyte antigen (HLA) class II allele mismatch with the recipient in the donor-to-recipient direction (an HLA class II allele mismatch in the donor-to-recipient direction, in the "graft-versus-host" direction), and the HLA class II allele mismatch is in a gene such as HLA-DRB1, HLA-DQB1, or HLA-DPB1. The recipient does not have detectable antibodies reactive to the donor's human leukocyte antigens ("detectable antibodies" in this context is defined using standard methods for making this determination). (For example, the recipient does not have antibodies against the donor's HLA molecules detectable by complement-dependent cytotoxicity, a positive result is undesirable in a flow cytometry cross-match assay, or a mean fluorescence intensity (MFI) of 3000 or greater is unacceptable in a solid-phase immunoassay).In one embodiment, the donor has at least one HLA class II allele mismatch to the recipient, where the donor is in an anti-recipient (graft versus host) orientation, and the HLA class II allele mismatch is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1.

[0042] "Expanding" a cell population of virus-specific or tumor neoantigen-specific T cells refers to generating a lymphocyte population that contains a higher frequency of T cells responsive to a virus or tumor neoantigen than is found in a healthy individual that has not been exposed to the virus or tumor neoantigen.

[0043] The allogeneic lymphocyte composition is produced from the peripheral blood cell composition by reducing the number of CD8+ T-cells in the peripheral blood cell composition by at least one order of magnitude, wherein (a) the number of CD4+ T-cells in the allogeneic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%. In a preferred embodiment, the ratio of the number of CD4+ T-cells / the number of CD8+ T-cells in the lymphocyte composition is preferably greater than or equal to about 30. Examples of some embodiments include, but are not limited to, the following doses per kilogram of ideal body weight of the recipient: 10 5 A lymphocyte composition containing 3.2 x 10 CD4+ cells is typically 3 10 or fewer CD8+ cells 6 A lymphocyte composition containing 3.2 x 10 CD4+ cells is typically 4 10 or fewer CD8+ cells 7 A lymphocyte composition containing 3.2 x 10 CD4+ cells is typically 510 or fewer CD8+ cells 8 A lymphocyte composition containing 3.2 x 10 CD4+ cells is typically 6 have fewer than 5 x 10 CD8+ cells, and 8 A lymphocyte composition containing 1.6 x 10 CD4+ cells is typically 7 have fewer than 100 CD8+ cells.

[0044] The number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition. The number is reduced by one order of magnitude, preferably by about two orders of magnitude, and more preferably by about five orders of magnitude. In one embodiment, the number of CD8+ cells is reduced by about 2.5 orders of magnitude (e.g., using magnetic bead cell sorting).

[0045] "Depleted of CD8+ T cells" means that the number of CD8+ T cells is reduced in a mixed cell population by methods such as antibodies to CD8+ plus complement or by magnetic cell separation, resulting in at least a 10-fold lower number of CD8+ T cells relative to other cells, such as CD4+ T cells, compared to an undepleted population. For example, if the ratio of CD4+ T cells to CD8+ T cells in an undepleted peripheral blood population is 1.5:1, then in a CD8+ T cell-depleted population, the ratio of CD4+ T cells to CD8+ T cells is 15:1 or greater.

[0046] As used herein, the term "vaccination" refers to the administration of a vaccine, pharmaceutical preparation (composition), or product that, upon administration, induces an immune response (particularly a cellular immune response) capable of recognizing and attacking pathogens or diseased cells, such as cancer cells. Vaccines may be used to prevent or treat diseases. The term "immune response" refers to the body's integrated response to an antigen, preferably a cellular immune response, or a cellular and humoral immune response. The immune response may be protective / preventive / prophylactic and / or therapeutic. "Inducing an immune response" may mean that there was no immune response to a specific antigen before induction, but it may also mean that there was some level of immune response to a specific antigen before induction and that the immune response is enhanced after induction. Thus, "inducing an immune response" also includes "enhancing an immune response." Preferably, after inducing an immune response in a subject, the subject is protected from developing a disease such as a cancer disease or the symptoms of the disease are improved by inducing an immune response. For example, an immune response against a tumor-expressed antigen can be induced in a donor, which helps a recipient subject with a cancer disease fight the cancer disease. In this case, inducing an immune response can mean that the symptoms of the disease in the subject are improved, that the subject does not develop metastasis, or that a subject at risk of developing a cancer disease does not develop the cancer disease.

[0047] In the present invention, "vaccination against a tumor antigen" refers to a specific donor subject who has been immunized against a tumor antigen present in the tumor or in a recipient subject with cancer. Vaccination may be performed, for example, with a single neoAg, a single-dose vaccine, or a vaccine formulation to enhance anti-tumor immunity. If sufficient immunization is not achieved, donors may be vaccinated with mRNA pentatope vaccines.

[0048] The term "subject" refers to any individual or patient on whom the methods described in the present application may be performed. Generally, the subject is a human, but as will be understood by those skilled in the art, the subject may also be an animal. Thus, other animals, including mammals such as rodents (e.g., mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock (e.g., cows, horses, goats, sheep, and pigs), and primates (e.g., monkeys, chimpanzees, orangutans, and gorillas), are included in the definition of a subject.

[0049] The terms "treat," "treating," and "treatment" are used interchangeably herein and refer to reducing or ameliorating a disorder and / or its associated symptoms. It will be understood, although not excluded, that treating a disorder or condition does not require that the disorder, its associated condition, or its associated symptoms be completely eliminated.

[0050] The treatments described herein may be suitably prescribed to subjects, particularly humans, suffering from, having, susceptible to, or at risk for cancer or a disease, disorder, or symptom thereof. Determining a subject as "at risk" may be done by any objective or subjective method (e.g., genetic testing, enzyme or protein markers, Markers [as defined herein], family history, etc.), by diagnostic testing, or opinion by the subject or a health care provider.

[0051] In one embodiment, the donor and recipient are ABO blood type incompatible, and the peripheral blood cell composition includes a large number of red blood cells, and producing the allogeneic lymphocyte composition further includes reducing the number of red blood cells. "ABO blood type incompatibility," as used herein, refers to a case where the recipient has a major ABO red blood cell incompatibility with the donor, such as when the recipient is blood type O and the donor is blood type A, B, or AB, when the recipient is blood type A and the donor is blood type B or AB, or when the recipient is blood type B and the donor is blood type A or AB.

[0052] In some embodiments, the donor is a cancer-free donor, and wherein the cancer-free donor has at least one HLA class II allele match to the subject with cancer.

[0053] As used in this application, the term "donor" refers to the subject to be vaccinated and from whom cells are isolated to produce the lymphocyte composition. As further detailed throughout, the donor is an allogeneic donor that is at least partially HLA-matched to the recipient. Cancer-free means that the donor is cancer-free (at least at the time the peripheral blood cell composition is obtained from the donor).

[0054] In some embodiments, the number of red blood cells is such that the packed volume is about 50 ml or less, e.g., the packed volume is about 50 ml or less, preferably the packed volume is about 30 ml or less. Furthermore, the "packed volume" should be defined, e.g., the lymphocyte composition is centrifuged to result in a packed volume of 50 ml or less of red blood cells; samples of the lymphocyte composition having a measured volume may be screened to provide a relative volume of packed blood cells.

[0055] In some embodiments, the antigen present in the recipient is selected from the group consisting of a neoplastic antigen, a neoplastic idiotype, a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a non-human animal antigen, a tumor neoantigen, and combinations thereof.

[0056] "Tumor neoepitopes" or "tumor neoantigens" are epitopes identified by methods such as comparing the whole genome or whole exome sequences of tumor tissue with those of non-tumor tissue from the same cancer patient, RNA-seq to identify expressed neoepitopes, and by methods such as prediction algorithms, single-allele purification using tagged allele constructs, or deep motif deconvolution of immunopeptidomes to identify epitopes presented by specific HLA class II alleles shared by cancer patients and cancer-free donors.

[0057] In various aspects, the antigen is a neoplastic antigen, and wherein the neoplastic antigen is a tumor antigen.

[0058] In one embodiment, the antigen present in the recipient is selected from the group consisting of neoplastic antigens (neoplastic antigens are antigens associated with neoplasms, which are defined as any new and abnormal cell growth, specifically one in which cell replication is uncontrolled and progressive). Neoplasms can be benign, pre-malignant, or malignant, and cancers are malignant neoplasms. Thus, while all cancer antigens are neoplastic antigens, not all neoplastic antigens are cancer antigens. Neoplastic idiotypes (Ids) are tumor-specific targets (e.g., in B-cell malignancies (e.g., lymphoma or multiple myeloma) that express this molecule on the cell surface), as well as viral, bacterial, fungal, parasitic, and non-human animal antigens.

[0059] In some embodiments, the viral antigen is selected from the group consisting of human papillomavirus (HPV) E6 antigen, HPV E7 antigen, and combinations thereof. In other embodiments, the viral antigen is selected from the group consisting of Epstein-Barr virus latent membrane protein 1 (LMP1), latent membrane protein 2a (LMP 2a), and combinations thereof.

[0060] In one embodiment, the tumor antigen is an antigen derived from the recipient's tumor.

[0061] In many embodiments, subjects selected from the group consisting of the recipient, the donor, and multiple potential allogeneic donors are screened for serological responsiveness to an antigen of an infectious agent selected from the group consisting of a human immunodeficiency virus (HIV) antigen, a hepatitis virus antigen, and a cytomegalovirus antigen.

[0062] In some embodiments, screening for multiple selection characteristics is performed on subjects selected from the group consisting of the recipient, the donor, and multiple potential allogeneic donors. For example, the selection characteristic is screening for serological responsiveness to an antigen of an infectious agent. The infectious agent antigen is selected from the group consisting of human immunodeficiency virus (HIV) antigen, hepatitis virus antigen, and cytomegalovirus antigen. Important subjects to screen for include, for example, HIV-1 antigen, HIV-2 antigen, hepatitis A virus antigen, hepatitis B virus antigen, hepatitis C virus antigen, CMV antigen, infectious disease, etc. If a virus or infectious agent, or its antigen, is the target of therapy, donors with the desired CD4+-mediated immune response against that subject are not excluded.

[0063] In one embodiment, the infectious agent antigen is a cytomegalovirus antigen, the recipient and the donor are screened, and there is no serological response to the cytomegalovirus antigen in the recipient or the donor. In one embodiment, the viral antigen is an influenza antigen, and the influenza antigen is a hemagglutinin antigen or a neuraminidase antigen.

[0064] In another embodiment, the selection characteristic is screening for two or more HLA class II alleles. In certain examples, a potential allogeneic donor is selected, and the potential allogeneic donor is selected as a donor, based on maximizing mismatches between the potential allogeneic donor and recipient in a potential allogeneic donor vs. recipient direction at multiple HLA class II alleles. In certain examples, the selection characteristic is screening for multiple HLA class I alleles.

[0065] A potential allogeneic donor may be selected based on minimizing mismatches between the potential allogeneic donor and the recipient in a plurality of HLA class I alleles, and the potential allogeneic donor is selected as a donor.

[0066] In one aspect, the number of CD4+ T-cells in the allogeneic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 20%. In some embodiments, the CD4+ T-cells differ by less than about 50%, less than about 40%, preferably less than about 20%, and more preferably less than about 10%. In some embodiments, ex vivo expansion of CD4+ T-cells may be performed; in such embodiments, the number of CD4+ T-cells may significantly exceed their original number. Such expansion is an alternative embodiment. Furthermore, "differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%" refers to a difference, plus or minus, of less than 50% of the number of CD4+ T-cells in the peripheral blood cell composition. For example, when the number of CD4+ T-cells in the peripheral blood cell composition is 1×10 5 10 CD4+ cells, "differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%" means that the number of CD4+ T-cells is 1.5 x 10 5 0.5 x 10 pieces 5 It means that it is between.

[0067] In various aspects, the CD4+ T-cells obtained from the donor are not intentionally expanded or differentiated ex vivo. Intentional expansion or differentiation is distinguished from CD4+ T-cell proliferation or differentiation, which is merely a side effect (unintentional, inadvertent) of the method; for example, CD4+ T-cells may occasionally become differentiated upon contact with plastic (another example of such an inadvertent event). In another embodiment, there is a further proviso that stem cells have never been mobilized in the donor of the peripheral blood cell composition that is allogeneic to the recipient.

[0068] In some embodiments, depleting CD8+ T cells in the peripheral blood cell composition comprises using an anti-CD8+ antibody bound to a magnetic microparticle, or an anti-CD8+ antibody plus complement. The peripheral blood cell composition may be, for example, a whole blood product or an apheresis product. Furthermore, the HLA class II allele mismatch in the donor-to-recipient direction may be an HLA-DRB1 mismatch. The HLA class II allele mismatch in the donor-to-recipient direction is, for example, a "graft-versus-host direction," where at least one HLA class II allele mismatch in the allogeneic donor-to-recipient direction further includes the same HLA class II allele mismatch between the allogeneic donor and multiple first-degree relatives of the recipient, which is desirable to maintain the opportunity for bone marrow transplantation from the first-degree relatives to the recipient; ideally, all mismatches between the donor and recipient do not exist between potential family bone marrow donors and the recipient.

[0069] In one embodiment, the number of donor CD4+ T-cells based on the recipient's ideal body weight in kilograms (kg) is about 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9In the composition, the number of donor CD4+ T-cells based on ideal body weight (IBW) is based on height: for men, at 5 feet or taller, IBW = 50 + 2.3 kg / inch. For women, the recipient's IBW in kilograms (kg) at 5 feet or taller = 45.5 + 2.3 kg / inch is approximately 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9 between about 1 x 10 CD4+ T-cells / kg (in a preferred embodiment, about 1 x 10 6 CD4+ T-cells / kg and approximately 5 x 10 8 the number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition; and the allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells compared to the peripheral blood cell composition.

[0070] In other embodiments, the allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T cells compared to the peripheral blood cell composition.

[0071] In some embodiments, the donor has CD4+ T-cell immunity to an antigen not present in the recipient.

[0072] In many embodiments, the donor has CD4+ T-cell immunity to an antigen present in the recipient. As used herein, "having CD4+ T-cell immunity" refers to the effectiveness of a vaccination in inducing an immune response in the donor directed against the specific antigen used for vaccination (which translates to the expansion in the donor of new clones of CD4+ T-cells).

[0073] In other embodiments, the recipient has no detectable antibodies reactive to the donor's human leukocyte antigens.

[0074] In another embodiment, the present invention provides a method of treating cancer in a subject, the method comprising the steps of prescribing lymphocyte-depleting chemotherapy to the subject; and administering to the subject a lymphocyte composition, wherein the lymphocyte composition is obtained from a peripheral blood cell composition of an HLA-matched or HLA-haploidentical donor vaccinated against a viral antigen and / or tumor neo-antigen present in the subject, wherein the composition is depleted in CD8+ T cells, and wherein the composition comprises an expanded population of CD4+ T cells specific for the viral and / or tumor neo-antigen present in the subject.

[0075] The compositions and methods of the present invention may be used for a wide range of cancer and tumor types, including, but not limited to, bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer. More specifically, cancers that may be treated by the compositions and methods described herein include, but are not limited to, cardiac cancers, e.g., sarcomas, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyoma; fibroma; lipoma; and teratoma; lung cancers, e.g., bronchogenic carcinoma, e.g., squamous cell, small undifferentiated cell, large undifferentiated cell, and adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma; chondroitin hamartoma; and mesothelioma; gastrointestinal cancers, e.g., esophageal cancer, e.g., squamous cell carcinoma. carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma; gastric cancer, e.g., carcinoma, lymphoma, and leiomyosarcoma; pancreatic cancer, e.g., ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, and vipoma; small intestine cancer, e.g., adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma; colon cancer, e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, and leiomyoma; genitourinary tract cancer, e.g., kidney cancer, e.g., adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, and leukemia; bladder and urethral cancer, e.g., squamous cell carcinoma carcinoma, transitional cell carcinoma, and adenocarcinoma; prostate cancer, e.g., adenocarcinoma and sarcoma;Testicular cancer, e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenocarcinoma, and lymphoma; liver cancer, e.g., hepatocellular carcinoma; bile duct carcinoma; hepatoblastoma; angiosarcoma; hepatocellular adenoma; and hemangioma; bone cancer, e.g., osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondraginous exostosis), exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor; nervous system cancers, e.g., skull cancer, e.g., osteoma, hemangioma, granuloma, xanthomas, and peptic osteitis; meningeal cancers, e.g., meningioma, meningeal sarcoma, and gliomatosis; brain cancers, e.g., astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; and spinal cancers, e.g., neurofibroma, meningioma, glioma, and sarcoma; gynecological cancers, e.g., uterine cancer, e.g., endometrial carcinoma; cervical cancer, e.g., cervical carcinoma and preneoplastic cervical dysplasia; ovarian cancer, e.g., ovarian carcinoma carcinoma, e.g., serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa cell tumor, Sertoli-Leydig cell tumors, dysgerminoma, and malignant teratoma; carcinoma of the vulva, e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma;Vaginal cancers, such as clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and fallopian tube cancers, such as carcinoma; hematologic cancers, such as cancers of the blood, e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, and myelodysplastic syndromes, Hodgkin's lymphoma, non-Hodgkin's lymphoma (malignant lymphoma), and Waldenstrom's macroglobulinemia; skin cancers, such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, and squamous cell carcinoma. carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal carcinoma and neuroblastoma. In certain embodiments, when the disease is cancer, the disease can be, for example, lung cancer tumor, breast cancer tumor, prostate cancer tumor, brain cancer tumor, or skin cancer tumor.

[0076] The compositions of the present invention may be administered to an individual by a variety of routes (e.g., oral, topical, parenteral, vaginal, systemic, intramuscular, rectal, or intravenous). In certain embodiments, the compositions are formulated with a pharmaceutical carrier. Preferably, the compositions are administered intravenously.

[0077] In some embodiments, the compositions are used in combination with other anti-viral or anti-cancer therapies, such as administering an anti-viral or anti-cancer agent, radiation therapy, phototherapy, or immunotherapy. The anti-viral or anti-cancer agent may be administered together with the compositions of the invention, either in the same formulation or in separate formulations, to enhance the treatment. In these embodiments, the composition and the other therapy may be administered at the same time (concurrently) or at separate times (sequentially), provided that they are administered in such a manner and sufficiently close in time to have the desired effect.

[0078] The compositions of the invention may also be administered in combination with existing methods for treating cancer (e.g., chemotherapy, radiation, or surgery). Accordingly, there is further provided a method for treating cancer (said individual in need of such treatment) comprising administering an effective amount of a composition of the invention to an individual, wherein an effective amount of at least one additional cancer chemotherapeutic agent is also administered to said individual. Examples of suitable chemotherapeutic agents include any of the following: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, intravenous busulfan, oral busulfan, calsterone, capecitabine, and carboplatin. , carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, estramustine, etoposide phosphate , etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leukotriene Vorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab,Pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, and zoledronate.

[0079] In one embodiment, preparing an allogeneic lymphocyte composition for administration to the subject includes: vaccinating a human donor against an antigen present in a recipient, wherein the recipient is the subject; and providing a peripheral blood cell composition derived from the human donor that is allogeneic to the recipient, the peripheral blood cell composition comprising some CD4+ T-cells, some CD8+ T-cells, and some natural killer cells, wherein (i) the donor is allogeneic to the CD4+ T-cells, some CD8+ T-cells, and some natural killer cells, wherein (i) the donor is allogeneic to the antigen present in the recipient. (ii) the donor has T-cell immunity, (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele match to the recipient, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, and (iii) the recipient has no detectable antibodies reactive to the donor's human leukocyte antigens; and producing the allogeneic lymphocyte composition from the peripheral blood cell composition by reducing the number of CD8+ T-cells in the peripheral blood cell composition by at least one order of magnitude, wherein the number of CD4+ T-cells in the allogeneic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%, and wherein the number of natural killer cells in the allogeneic lymphocyte composition is less than or equal to the number of natural killer cells in the peripheral blood cell composition.

[0080] In various embodiments, prior to administering the allogeneic lymphocyte composition to the subject, the method further comprises: prescribing a lymphodepleting, non-lymphocyte-ablative treatment to the subject to induce transient lymphopenia in the subject; prescribing a treatment to reduce or inhibit myeloid-derived suppressor cells; prescribing a treatment to reduce or inhibit tumor-associated macrophage cells, or prescribing a treatment to reduce regulatory T cells; or wherein, following administering the allogeneic lymphocyte composition to the subject, the method further comprises administering an agent to induce a selective reduction in alloreactive T cells.

[0081] In certain embodiments, the lymphocyte-reducing, non-lymphocyte-depleting treatment comprises treating the subject with one or more cytoreductive agents selected from the group consisting of alkylating agents, alkyl sulfonates, nitrosoureas, triazenes, antimetabolites, pyrimidine analogs, purine analogs, vinca alkaloids, epipodophyllotoxins, antibiotics, dibromomannitol, deoxyspergualin, dimethyl myleran, and thiotepa. In certain embodiments, the lymphocyte-reducing, non-lymphocyte-depleting treatment comprises treating the subject with an alkylating agent, and the alkylating agent is cyclophosphamide. In certain embodiments, after administering the first allogeneic lymphocyte composition to the subject, the method further comprises administering to the subject an anti-tumor monoclonal antibody or an anti-tumor monoclonal antibody / drug conjugate.

[0082] "Lymphocyte-depleting therapy," "lymphocyte-reductive therapy," and the like, are meant to refer to drugs or other agents, such as chemotherapeutic agents, that reduce the concentration of lymphocytes in the peripheral blood. "Reduce" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0083] In certain embodiments, the method further comprises administering an anti-tumor monoclonal antibody, and the anti-tumor monoclonal antibody is selected from the group consisting of rituximab, cetuximab, trastuzumab, and pertuzumab. In certain embodiments, the method further comprises administering an anti-tumor monoclonal antibody / drug conjugate, and the anti-tumor monoclonal antibody / drug conjugate is selected from the group consisting of brentuximab vedotin, gemtuzumab ozogamicin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumumab vedotin, lorvotuzumab mertansine, cantuzumab mertansine, and milatuzumab-doxorubicin. In some embodiments, the allogeneic lymphocyte composition is administered first, followed by the administration of a chemotherapeutic agent to the subject. For example, the chemotherapeutic agent is selected from the group consisting of dasatinib, nilotinib, ponatinib, imatinib, lapatinib, and vismodegib.

[0084] In another aspect, prior to administering the allogeneic lymphocyte composition to the subject, the treatment comprises administering a drug selected from the group consisting of dasatinib, 5-fluorouracil, taxotere, clodronate, gemcitabine, cyclophosphamide, fludarabine, denileukin diftitox, and daclizumab.

[0085] In some embodiments, the peripheral blood cell composition is a whole blood product or an apheresis product.

[0086] In various embodiments, the subject has received an injection of a nanoparticle composition into a tumor, wherein the nanoparticle composition comprises nanoparticles comprising an antigen not present in the subject. In many embodiments, the nanoparticles further comprise a cytokine, wherein the cytokine is an interleukin and is selected from the group consisting of IL-2, IL-7, IL-12, and IL-15; or the cytokine is an interferon and is selected from the group consisting of interferon gamma, interferon beta, interferon alpha, interferon tau, interferon omega, and consensus interferon.

[0087] In a further embodiment, the present invention provides a cell bank comprising CD4+ T cells specific for a viral antigen, a tumor neo-antigen, or a combination thereof, wherein the cell bank comprises CD4+ T cells collected from donors of different human leukocyte antigen (HLA) types.

[0088] In various embodiments, the viral antigen is an HPV antigen.

[0089] In one embodiment, the present invention provides an allogeneic lymphocyte composition for administration to a human recipient, obtained by the methods of the present invention described herein.

[0090] If the recipient is seropositive for a CMV antigen, the donor's status is not important. In certain embodiments in which the donor has not been immunized against an antigen present in or to be delivered to the recipient, delivery of CD4+ T-cell help is contingent upon donor CD4+ T-cell recognition of allogeneic HLA class II molecules on the recipient's cells. An example of an "ideal donor" for purposes of illustrating these embodiments of the invention is one that is fully mismatched for HLA class II alleles (particularly HLA-DRB1, HLA-DQB1, and HLA-DPB1) and fully matched for class I alleles (thus maximizing donor cell survival in the recipient and minimizing allogeneic antibody production against class I molecules). Furthermore, the ideal donor is a perfect mismatch with the non-shared HLA of the recipient's first degree relatives, who are potential donors for allogeneic stem cell transplantation.

[0091] In one embodiment, an allogeneic lymphocyte composition derived from a peripheral blood cell composition derived from a human allogeneic donor for administration to a human recipient, the allogeneic lymphocyte composition comprising a number of CD4+ T-cells and a number of natural killer cells derived from the peripheral blood cell composition of the donor, wherein (i) the donor comprises at least one human leukocyte antigen (HLA) class II allele mismatch to the recipient in the donor-to-recipient direction, and the HLA class II allele mismatch is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (ii) the recipient has no detectable antibodies reactive to the donor's human leukocyte antigens, and (iii) the number of CD4+ T-cells in the allogeneic lymphocyte composition is greater than or equal to the number of CD4+ T-cells in the peripheral blood cell composition. The number of T-cells may differ by less than about 50% (for example, but not limited to, less than about 50%, less than about 40%, preferably less than about 20%, more preferably less than about 10%).

[0092] Also included are methods of treating a disease or condition in a human subject, the method comprising administering to the subject a lymphopenic (in some embodiments of this aspect of the invention, it is desirable to provide lymphopenic, non-lymphocyte-ablative treatment to promote homeostatic proliferation and differentiation of the administered lymphocytes; in other embodiments, it is desirable that the treatment is also myelopenic [i.e., inhibiting or reducing suppressive myeloid populations, including myeloid-derived suppressor cells, tumor-associated macrophages, and / or N2 neutrophils]) non-lymphocyte-ablative treatment to transiently induce lymphopenia in the subject; and subsequently administering to the subject a first allogeneic lymphocyte composition derived from a peripheral blood cell composition derived from a human, allogeneic donor, the first allogeneic lymphocyte composition comprising a number of CD4+ T-cells and a number of natural killer cells derived from the peripheral blood cell composition of the donor, wherein (i) (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele mismatch to the subject in the donor-to-subject direction, and the HLA class II allele mismatch is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1; (ii) the subject has no detectable antibodies reactive to the donor's human leukocyte antigens; (iii) the number of CD4+ T-cells in the first allogeneic lymphocyte composition differs by less than about 50% from the number of CD4+ T-cells in the peripheral blood cell composition, and the number of donor CD4+ T-cells based on the subject's ideal body weight in kilograms (kg) is about 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9 (v) the number of natural killer cells in said first allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in said peripheral blood cell composition; and (vi) the first allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T cells compared to said peripheral blood cell composition.

[0093] Without wishing to be bound by any particular theory, the injected CD4+ cells provide signals to other cell types, primarily the subject's CD8+ cells, macrophages, and / or antigen-presenting cells, that enhance the cytotoxic function of these cells in the subject (subject's tolerized CD4+ cells / subject's exhausted CD8+ cells); an example of treating a disease or condition by this method is exemplified by the treatment of at least myelodysplastic syndrome.

[0094] In one embodiment, a kit for use in treating a disease or condition in a subject is provided, the kit comprising: a lymphocyte composition described herein, wherein the subject is a human recipient; and a nanoparticle composition comprising nanoparticles comprising an antigen not present in the human recipient. In one embodiment, the nanoparticles further comprise a cytokine, e.g., an interleukin or interferon. The cytokine may be an interleukin and is selected from the group consisting of IL-2, IL-7, IL-12, and IL-15. The cytokine may be an interferon, e.g., interferon gamma, interferon beta, interferon alpha, interferon tau, interferon omega, and consensus interferon.

[0095] The nanoparticles may further comprise a compound selected from the group consisting of a chemokine, an imaging agent, an optical antenna molecule, a thermal antenna molecule, and a Toll-like receptor ligand, a ligand that promotes the differentiation of CD4+ T-cells into type I (e.g., IFN-γ-producing) CD4+ memory T-cells, or a ligand for a receptor that induces activation of antigen-presenting cells (e.g., an anti-CD40 antibody or an aptamer). Additionally, the nanoparticles may comprise an agent that targets the nanoparticles to tumor cells or antigen-presenting cells.

[0096] In one embodiment, following administering the first allogeneic lymphocyte composition to the subject, the method further comprises administering a monoclonal antibody / CD4+ T-cell epitope conjugate to the subject. In one embodiment, following administering the first allogeneic lymphocyte composition to the subject, the method further comprises administering to the subject a subsequent lymphocyte-reducing, non-lymphocyte-ablative treatment to induce transient lymphopenia in the subject; and subsequently administering to the subject a subsequent allogeneic lymphocyte composition derived from a subsequent peripheral blood cell composition derived from a subsequent human, allogeneic donor, the subsequent allogeneic lymphocyte composition comprising a number of CD4+ T cells and a number of natural killer cells derived from the subsequent peripheral blood cell composition of the subsequent donor, wherein (i) the subsequent donor comprises at least one human leukocyte antigen (HLA) class II allele mismatch to the subject in the direction of the subsequent donor versus the subject, and the HLA class II allele mismatch is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, and (ii) (iii) the number of CD4+ T-cells in the subsequent allogeneic lymphocyte composition differs from the number of CD4+ T-cells in the subsequent peripheral blood cell composition by less than about 50%; and (iv) the number of additional donor CD4+ T-cells based on the subject's ideal body weight in kilograms (kg) is less than about 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9(v) the number of natural killer cells in the subsequent allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the further peripheral blood cell composition; and (vi) the subsequent allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T cells than the further peripheral blood cell composition. Following administering the first allogeneic lymphocyte composition to the subject, the method further comprises administering an agent that blocks negative signaling in T cells. The agent that blocks negative signaling in T cells is selected from the group consisting of an anti-PD-1 antibody, ipilimumab, an anti-PD-L2 antibody, and a PD-1 fusion protein. The disease or condition is selected from the group consisting of cancer, an autoimmune disorder, organ transplant, allograft rejection, and a viral infection. For example, the disease or condition is cancer, and the cancer is a myelodysplastic syndrome.

[0097] In one embodiment, the present invention provides a method for producing an allogeneic lymphocyte composition for administration to a human recipient, the method comprising providing a peripheral blood cell composition derived from a human donor that is allogeneic to the recipient, wherein the peripheral blood cell composition comprises some CD4+ T cells, some CD8+ T cells, and some natural killer cells, wherein (i) the donor has CD4+ T cell immunity to an antigen present in the recipient, (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele match to the recipient, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, and (iii) the recipient has no detectable antibodies reactive to human leukocyte antigens of the donor; and producing the allogeneic lymphocyte composition from the peripheral blood cell composition by reducing the number of T-cells by at least one order of magnitude, wherein (a) the number of CD4+ T-cells in the allogeneic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%, and (b) the number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition.

[0098] In one embodiment, a potential allogeneic donor is selected from a plurality of potential allogeneic donors based on minimizing mismatches between the potential allogeneic donor and the recipient at a plurality of HLA class II alleles, and the potential allogeneic donor is selected as the donor. A potential allogeneic donor is selected from a plurality of potential allogeneic donors based on minimizing mismatches between the potential allogeneic donor and the recipient at a plurality of HLA class I alleles, and the potential allogeneic donor is selected as the donor.

[0099] In one embodiment, the antigen present in the recipient to which the donor is immune is a viral antigen, and the viral antigen is selected from the group consisting of a human papillomavirus antigen, an Epstein-Barr virus antigen, a Kaposi's sarcoma-associated herpesvirus (KSHV) antigen, a hepatitis A virus antigen, a hepatitis B virus antigen, and a hepatitis C virus antigen. For example, the viral antigen is a human papillomavirus antigen, and the human papillomavirus antigen is an E6 or E7 antigenic peptide. In one embodiment, the number of CD4+ T-cells in the allogeneic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 20%.

[0100] In one embodiment, an allogeneic lymphocyte composition is provided for administration to a human recipient, the allogeneic lymphocyte composition being derived from a peripheral blood cell composition derived from a human allogeneic donor, the allogeneic lymphocyte composition comprising some CD4+ T-cells and some natural killer cells derived from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cell immunity to an antigen present in the recipient, (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele match to the recipient, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipient has no detectable antibodies reactive to the donor's human leukocyte antigens, and (iv) the CD4+ T-cells in the allogeneic lymphocyte composition are not present in the recipient. The number of T-cells differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%. The number of donor CD4+ T-cells, based on the recipient's ideal body weight in kilograms (kg), is about 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9 the number of natural killer cells in the allogeneic lymphocyte composition is between 10 and 15 CD4+ T-cells / kg, the number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition, and the allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells compared to the peripheral blood cell composition.

[0101] The present invention provides an allogeneic lymphocyte composition derived from a peripheral blood cell composition derived from a human allogeneic donor for administration to a human recipient, wherein the allogeneic lymphocyte composition comprises some CD4+ T-cells and some natural killer cells derived from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cell immunity to an antigen not present in the recipient, (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele match to the recipient, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipient has no detectable antibodies reactive to the donor's human leukocyte antigens, and (iv) the CD4+ T-cells in the allogeneic lymphocyte composition are not present in the recipient. The number of T-cells differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%. The number of donor CD4+ T-cells, based on the recipient's ideal body weight in kilograms (kg), is about 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9 (vi) the number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition; and (vii) the allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells compared to the peripheral blood cell composition.

[0102] The present invention provides a method for treating a disease or condition in a human subject, the method comprising administering to the subject an allogeneic lymphocyte composition derived from a peripheral blood cell composition derived from a human, allogeneic donor, the allogeneic lymphocyte composition comprising a number of CD4+ T-cells and a number of natural killer cells derived from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cell immunity to an antigen present in the subject, (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele match to the subject, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the subject has no detectable antibodies reactive to human leukocyte antigens of the donor, and (iv) the number of CD4+ T-cells in the allogeneic lymphocyte composition is greater than or equal to the number of CD4+ T-cells in the peripheral blood cell composition. (v) the number of donor CD4+ T-cells based on the subject's ideal body weight in kilograms (kg) is less than about 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9 (vi) the number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition; and (vii) the allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells compared to the peripheral blood cell composition.

[0103] In the methods described herein, optionally, prior to administering the allogeneic lymphocyte composition to the subject, the method further comprises prescribing a treatment to reduce or inhibit myeloid-derived suppressor cells. Treatment to reduce or inhibit myeloid-derived suppressor cells includes, for example, administering a drug selected from the group consisting of dasatinib, 5-fluorouracil, taxotere, clodronate, and gemcitabine. Optionally, prior to administering the allogeneic lymphocyte composition to the subject, the method further comprises prescribing a treatment to reduce or inhibit tumor-associated macrophage cells. Optionally, prior to administering the allogeneic lymphocyte composition to the subject, the method further comprises prescribing a treatment to reduce regulatory T cells. The treatment to reduce regulatory T cells may include administering a drug selected from the group consisting of cyclophosphamide, idelalisib, denileukin diftitox, and daclizumab.

[0104] In another embodiment, the present invention provides a method of treating a disease or condition in a human subject, comprising administering into a tumor a nanoparticle composition (which may be administered by injection or infusion to the subject, wherein the nanoparticles further comprise a targeting agent, and the targeting agent binds to target cells, such as disseminated / delocalized neoplasms (e.g., lymphoma or leukemia) (where direct injection to all possible sites is not practical or feasible), wherein the nanoparticle composition comprises nanoparticles comprising an antigen not present in the subject, thereby introducing the antigen into the subject; administering to the subject an allogeneic lymphocyte composition derived from a peripheral blood cell composition derived from a human allogeneic donor, the allogeneic lymphocyte composition comprising some CD4+ T-cells and some natural killer cells derived from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cells against the antigen. (ii) the donor comprises at least one human leukocyte antigen (HLA) class II allele match to the subject, and the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1; (iii) the subject has no detectable antibodies reactive to the donor's human leukocyte antigens; (iv) the number of CD4+ T-cells in the allogeneic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%; and (v) the number of donor CD4+ T-cells based on the subject's ideal body weight in kilograms (kg) is about 1 x 10 5 CD4+ T-cells / kg and approximately 1 x 10 9(vi) the number of natural killer cells in the allogeneic lymphocyte composition is equal to or less than the number of natural killer cells in the peripheral blood cell composition; and (vii) the allogeneic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells than the peripheral blood cell composition. In one embodiment, the antigen is a non-human animal antigen, and the non-human animal antigen is keyhole limpet hemocyanin antigen. In another embodiment, the antigen is a viral antigen, and the viral antigen is selected from the group consisting of a human papillomavirus antigen, an Epstein-Barr virus antigen, a Kaposi's sarcoma-associated herpesvirus (KSHV) antigen, a hepatitis A virus antigen, a hepatitis B virus antigen, and a hepatitis C virus antigen.

[0105] The following examples are intended to illustrate, but not limit, the present invention. [Example]

[0106] Myelodysplastic syndromes (MDS) are a diverse group of malignant stem cell disorders characterized by dysplasia of blood cells and failure of bone marrow production of blood cells, and a variable risk of transformation to acute leukemia. These disorders may develop de novo or years after exposure to potentially mutagenic chemotherapy.

[0107] Approximately 12,000–20,000 new cases of MDS will be diagnosed in the United States this year, with a median age of onset of 60–72 years. Current treatment outcomes for myelodysplastic syndromes are disappointing. Age, performance status, and disease risk category (usually determined by the International Prognostic Scoring System (IPSS)) determine treatment modality options. Patients younger than 60 years, with good or excellent performance status, and in the IPSS intermediate-2 or high-risk categories are primarily considered for intensive therapy, as these IPSS categories have median survival times of 1.2 years and 0.4 years, respectively. Intensive therapy is defined as treatment requiring hospitalization, including intensive combination chemotherapy and hematopoietic cell transplantation.

[0108] For patients in the low or moderate-1 category, low-intensity therapy is generally considered. These include treatments that can be administered in an outpatient clinic, such as hematopoietic growth factors, differentiation-inducing agents, biological response modifiers, and low-intensity chemotherapy. For patients with poor performance status, supportive care or low-intensity therapy is considered.

[0109] Example 1 - IDE Device The investigational agent used in this study was the CliniMACS® System with CliniMACS® CD8 Reagent, a medical device used to enrich or deplete CD8+ T cells from human blood products. The CliniMACS® System, designed to select CD8+ cells, includes four major components: 1) CliniMACS® CD8 Reagent—colloidal superparamagnetic iron-dextran beads conjugated to a mouse antibody against human CD8; 2) CliniMACSplus Instrument—a software-controlled instrument for processing blood samples (cell preparations); 3) CliniMACS® Tubing Set (standard or LS)—a single-use, sterile, disposable tubing set containing two unique cell sorting columns; and 4) CliniMACS® PBS / EDTA Buffer—a sterile, isotonic, phosphate-buffered, 1 mM EDTA saline solution used as an external wash and transfer fluid for in vitro blood cell preparation. The system utilizes magnetic cell sorting (MACS®), a powerful tool for isolating many cell types, to selectively enrich or deplete cell populations of interest. In this case, CD8+ T cells are labeled with monoclonal antibodies bound to superparamagnetic particles and then depleted from the blood product by passing them through the CliniMACS system, which incorporates a separation column with a strong permanent magnet and ferromagnetic matrix to remove the labeled cells. The therapeutic agent (CD8+ T cell-depleted blood cells) exits the device and is not intended to contain any of the device components.

[0110] Example 2 - Transiently engrafted donor lymphocytes induce clinical tumor responses To date, only two treatments can extend the survival of patients with MDS. The first is allogeneic BMT, which has achieved some long-term cure as well as delayed disease progression. This treatment is only available to a small proportion of affected individuals due to age, donor availability, and comorbidities. The second is the methyltransferase inhibitor 5-azacytidine. This therapy has been shown to extend median survival by 7 months compared with supportive care alone. Some patients with MDS respond to immunosuppressive regimens, such as cyclosporine, antithymocyte globulin (ATG), or steroids, resulting in persistent increases in blood counts. This finding is similar to that of aplastic anemia, where immunosuppression treats the autoimmune component that leads to cytopenias. The positive results achieved with agents specifically targeting the immune system suggest that MDS is a disease amenable to immunomodulation. One possible explanation for the benefit of ATG, cyclosporine, and steroids is that these drugs selectively inhibit or kill lymphocytes that suppress antitumor immunity, thereby unleashing the activity of endogenous antitumor immune responses. Furthermore, in a study of nonmyeloablative, partially HLA-mismatched (haploidentical) allogeneic bone marrow transplantation, five patients were found to experience disease responses despite graft rejection, potentially providing evidence for the existence of a hidden, endogenous immune response to MDS. All five patients experienced at least a transient decrease in the percentage of bone marrow blasts, and three of the five patients (each of whom was dependent on red blood cell + / - platelet transfusions before transplantation) became transfusion-independent. Table 1 shows that despite the lack of donor cell engraftment 30 days after BMT, at least three of the five patients experienced a decrease in bone marrow blasts that persisted for at least 6 months after BMT. [Table 1]

[0111] Figure 1 shows the hematocrits of the same five patients after bone marrow transplantation, with indentations reflecting the effects of transfusions. Three of the five patients became transfusion-independent, and patient #1 maintained morphologic and hematologic remission for at least three years. More interestingly, patient #2 demonstrated a delayed hematologic response, becoming transfusion-independent four months after BMT and three months after documentation of graft rejection. Given the sensitivity of MDS to immunotherapy, it was hypothesized that immunological perturbations provided by transiently engrafted donor lymphocytes could reawaken endogenous (i.e., host-derived) antitumor immune responses. This hypothesized mechanism (graft rejection followed by the elicitation of endogenous anti-tumor responses) may explain the induction of leukemia remission in patients receiving white blood cell transfusions after no conditioning or only 100 cGy of total body irradiation. The hypothesis that transient engraftment of donor lymphocyte infusions (DLI) induces anti-tumor immune responses from host T cells was tested in a mouse model. BALB / c × C57BL / 6 F1 mice were treated with Cy on day -1 and 10 6 A20 lymphoma cells (BALB / c origin) were administered intravenously with either no treatment, haploidentical DLI alone, autologous tumor cell vaccine alone, or DLI plus vaccine. Compared with animals receiving either no treatment or vaccine alone after Cy conditioning, animals conditioned with Cy and then treated with DLI alone or DLI plus vaccine survived significantly longer, with five and four animals clearly cured, respectively (Figure 2). None of the nine cured animals had any detectable donor chimerism when examined more than 100 days after DLI, suggesting that the donor T cells were rejected.

[0112] These results demonstrated that the combination of Cy and subsequent partial MHC-mismatched DLI induced significant antitumor effects. To characterize the role of donor CD4+ versus CD8+ T cells in the antitumor effect, this experiment was repeated in recipients of Cy + vaccine + 50 million mismatched splenocytes (spleen cells were untreated or depleted of CD4+ T cells, CD8+ T cells, or both). In this experiment, all recipients of whole-spleen DLI died of GVHD before day 20 (Figure 3A). In contrast, mice receiving vaccine plus CD8+ T cell-depleted splenocytes survived significantly longer than mice receiving vaccine plus pan-T cell-depleted splenocytes (p = .04), indicating that CD8+ T cell depletion abolished lethal GVHD without abolishing anti-tumor immunity. To understand why CD8+ T cell depletion from allogeneic DLI abolished GVHD, we investigated donor cell survival in mice conditioned with Cy and then injected with mismatched splenocytes, either untreated or depleted in one or both T cell subsets. Interestingly, CD8+ T cell-depleted splenocytes engrafted only transiently, with donor CD4+ T cell chimerism peaking at day 7 after DLI and declining to undetectable levels by day 21 (Figure 3B). In contrast, persistent engraftment of donor cells was observed in all mice receiving DLI containing CD8+ T cells, and most of these animals ultimately died of GVHD. Taken together, this animal study demonstrated that DLI, which depletes CD8+ T cells after Cy, induces transient engraftment of donor cells and significant antitumor effects without inducing acute GVHD.More recently, it was found that depleting host CD8+ T cells prior to Cy+ DLI significantly reduced the therapeutic effect, strongly suggesting that host CD8+ T cells are important mediators of the anti-tumor effect.

[0113] Example 3 - Clinical Experience with Injection of CD8+ T Cell-Depleted Allogeneic Stem Cells or Lymphocytes There are no reports of patients treated with Cy followed by CD8+ T cell-depleted PBMCs from haploidentical donors. Therefore, preliminary safety data cannot be provided. However, there are reports of patients undergoing allogeneic BMT who received CD8+ T cell-depleted grafts or who relapsed after allogeneic BMT and received injections of CD8+ T cell-depleted PBMCs. The goal of CD8+ T cell depletion was to reduce the incidence of GVHD while maintaining the anti-leukemic effects of the injections. Regarding GVHD, these studies have not provided a definitive answer; some have indicated a possible benefit, while others have shown no benefit. Interestingly, injections of CD8+ T cell-depleted DLI induced activation of endogenous CD8+ T cells, a finding consistent with the hypothesis that CD8+ T cell-depleted DLI can effectively stimulate host CD8+ T cell responses against cancer.

[0114] The results of two other trials are relevant for considering the safety of the proposed clinical trial. In the first study, patients with various hematologic malignancies received bone marrow from unrelated donors who were mismatched for either one HLA-DR allele or one HLA class I (HLA-A or HLA-B) antigen. Patients received CD4+ T cell-depleted grafts containing a titrated dose of CD8+ T cells. The main finding was that despite myeloablative conditioning, graft rejection occurred at rates < 3.1 x 10 6occurred in 6 of 10 patients receiving grafts containing > 3.1 x 10 CD8+ T cells / kg recipient body weight. 6 In 15 patients receiving 100 CD8+ T cells / kg, none developed graft rejection. Thus, depletion of CD8+ T cells, even after myeloablative conditioning, significantly increases the risk of graft rejection, thereby negating the risk of graft-induced hypoplasia and GVHD.

[0115] In the second study, the mean values ​​were 2.7 × 10 4 pcs or 3.5 x 10 4 CD3+ T cells / kg (this corresponds to a CD8+ T cell dose of approximately 1-1.5 x 10 4 Patients received T-cell-depleted, haploidentical peripheral blood stem cells (PBSCs; n = 15) or PBSCs plus bone marrow grafts (n = 28). To promote engraftment in the face of T-cell depletion, patients were heavily conditioned, and the mean T cell counts for PBSCs plus bone marrow versus PBSCs alone recipients were 14.0 × 10, respectively. 6 or 10.6 x 10 6 Patients received "mega-dose" stem cell grafts containing CD34+ cells / kg. Given the low T cell content, no GVHD prophylaxis was administered. Engraftment occurred in all 43 patients, and none experienced acute or chronic GVHD as a result of the transplant procedure. These data suggest that even if sustained engraftment occurs in patients who underwent myeloablative conditioning, it is unlikely that <10 4 These results suggest that haploidentical grafts containing 100 CD8+ T cells / kg are unlikely to cause GVHD. Because a >2 log CD8+ T cell depletion is typically achieved with this device, the starting dose of CD8+ T cell-depleted PBC in this study is likely to be 10 4The dose contains fewer than 100 CD8+ T cells / kg. Therefore, patients receiving this dose are unlikely to engraft or, even if durable engraftment occurs, will likely not develop GVHD.

[0116] Example 4 - CD8 depletion using the CliniMACS system with CliniMACS-CD8 reagent In this study, CD8+ T cell depletion was performed using the CliniMACS system with CliniMACS CD8 reagent (Miltenyi Biotec, Woburn, MA). Three trials were performed, the first using leukapheresis preparations and the final two using phlebotomy specimens. Flow cytometry results from the final depletion are shown in Figure 4 and demonstrate successful depletion of CD8+ T cells (from 8.40% to 0.03% of total cells) and corresponding enrichment of CD4+ T cells and CD16+ or CD56+ NK cells. The table below demonstrates that all three preparations met the protocol criteria of a CD8+ T cell count <3.2% of the CD4+ T cell count. Preparations #2 and #3 were administered to a hypothetical recipient with an ideal body weight of 70 kg for 10 days. 6 If used to deliver a dose of 2.6 x 10 CD4+ T cells / kg, they would be 2.6 x 10 3 and 7.7 x 10 2 CD8+ T cells / kg IBW (a dose well below the threshold for engraftment or GVHD induction). By comparison, the five patients who responded despite graft rejection contained a median of 1.43 x 10 7 CD4+ T cells / kg (range 0.84-3.14 × 10 7 / kg) and 1.86 × 10 7 CD8+ T cells / kg (range 0.43-2.16 × 10 7 1 / kg), thus allowing MDS patients to reject this T cell-rich, haploidentical cell injection. [Table 2]

[0117] Example 5 - Laboratory correlation studies to predict response to treatment Given the potential toxicity of immunosuppressive therapy, such as antithymocyte globulin, in patients with MDS, many investigators have focused efforts on identifying patient characteristics that correlate with response to treatment. Such predictive characteristics include a hypocellular bone marrow, an abnormal T-cell receptor repertoire as determined by spectratyping analysis, the presence of cells with a phenotype characteristic of paroxysmal nocturnal hemoglobinuria (PNH), HLA-DR15 expression, trisomy 8, younger age, and a shorter transfusion history. Therefore, one goal was to determine whether characteristics predictive of response to immunosuppressive therapy also predict response to haploidentical DLI, which depletes Cy+ CD8+ T cells. Patients enrolled in these studies were tested for T-cell receptor beta-chain variable region diversity both before and after therapy. Cytogenetics and HLA typing are routinely performed on all patients.

[0118] Recent studies have demonstrated the role of alloreactive donor natural killer cells in preventing relapse of acute leukemia after haploidentical stem cell transplantation. More recently, alloreactive natural killer cells of donor origin have been found to prevent relapse of AML and MDS after transplantation of HLA-identical stem cells. These results emphasize the need to characterize the expression repertoire of killer immunoglobulin-like receptors (KIRs) on donor NK cells using both molecular and flow cytometric methods to identify donors that express KIRs (their HLA ligands are absent on recipient cells).

[0119] Example 6 - Rationale for proposed study design This is a standard Phase I / II study design, aiming to determine the maximally tolerated dose (MTD) of CD8+ T cell-depleted haploidentical peripheral blood cells (CD8- PBC) when administered by injection after cyclophosphamide (Cy) in the Phase I portion of the study, and then to estimate the efficacy of treatment with the MTD of Cy plus CD8- PBC in the Phase II portion of the study. High doses of Cy (>100 mg / kg) have been widely used as part of transplant conditioning for patients with hematological malignancies, and its safety has been well documented in this population, including elderly patients (55-66 years) with myelodysplastic syndromes. The most serious risks of treatment, due to their potential for mortality, are persistent aplasia and graft-versus-host disease, both of which require sustained engraftment of the donor cells. Selection of the initial cell dose (10 5 CD4+ T cells / kg and <3.2 × 10 3 CD8+ T cells / kg) was for safety reasons only. 4 Grafts containing <10 CD8+ T cells / kg do not cause severe GVHD, even in patients who have undergone lethal conditioning and who do not receive pharmacological immunosuppression after transplantation. Furthermore, partial depletion of CD8+ T cells from standard bone marrow grafts significantly increases the risk of graft rejection. 33 This is the desired outcome of treatment in this study. For these reasons, we recommend a graft containing <10 4 DLI formulations containing CD8+ T cells / kg appear to be less likely to cause serious adverse events.

[0120] Experience with haploidentical DLI without CD8+ T-cell depletion has recently been published. In a phase I / II trial, 41 patients with relapsed / refractory malignancies underwent non-ablative conditioning with 100 cGy of total body irradiation followed by 1 × 10 6 -2×10 8Twenty-nine patients received the highest dose of haploidentical CD3+ cells / kg injected. At higher dose levels, the desired response was achieved. In particular, 1 × 10 8 CD3+ cells / dose was the lowest dose associated with a response (25% response rate, or 2 of 8 patients), and 2 × 10 8 CD3+ cells / dose (the highest rating) was associated with the greatest response rate (nearly 50%, 10 of 21 patients). As proof of principle, all responses occurred in the absence of sustained donor chimerism. Transient donor chimerism was observed in the highest-dose cohort, but it resolved within 2 weeks in most patients. Of the two patients who converted to complete donor chimerism, one developed severe acute GVHD (steroid-responsive, followed by fatal sepsis). At higher doses, an acute clinical syndrome termed a "haploimmune storm" (characterized by one or more of the following: fever, fatigue, LFT abnormalities, rash, and diarrhea), likely due to cytokine flux, was common and responded well to steroids. This study demonstrated the biological activity and manageable safety profile of this approach. The minimum CD3+ T-cell dose (without CD8+ depletion) required for response in this study was 1 x 10 8 The cell count was 100 cells / kg.

[0121] Example 7 - Patent selection Patients must have pathologically confirmed myelodysplastic syndrome (MDS), Int-2, or a high IPSS score (using the IPSS scoring system). Patients must have failed treatment with, or be ineligible or intolerant to, 5-azacytidine.

[0122] Example 8 - Treatment Regimen All patients will require a detailed medical history and physical examination, as well as standard assessments of cardiac, liver, and renal function. All patients will undergo bone marrow aspiration and biopsy for morphologic, cytogenetic (if applicable), and flow cytometric (if applicable) evaluation, along with standard disease assessment (e.g., CT of the chest, abdomen, and pelvis), within one month prior to protocol enrollment.

[0123] Pretreatment evaluation

[0124] Cyclophosphamide is administered as an IV infusion over 1-2 hours (depending on volume) on days -2 and -1. The cyclophosphamide dose is 50 mg / kg / day. Dose is calculated based on adjusted ideal body weight or actual body weight, whichever is less. Weight and height are measured directly. Approximate weight-for-height may be calculated from reference tables or equations that reflect ideal "values."

[0125] Cyclophosphamide and pre-DLI regimen

[0126] Prior to cyclophosphamide administration, patients are instructed to increase fluid volume overnight. Hydration with normal saline at 3 cc / kg / hr iv is initiated 2 hours before cyclophosphamide, then the rate is reduced to 2 cc / kg / hr 1 hour before cyclophosphamide and continued until 8 hours after cyclophosphamide. Mesna is given iv in divided doses 30 minutes before cyclophosphamide and 3, 6, and 8 hours after.

[0127] The mesna dose is based on the cyclophosphamide dose administered. The total daily dose of mesna is equal to 80% of the total daily dose of cyclophosphamide.

[0128] Prophylactic antibacterial therapy will begin on day 0 and will be in accordance with institutional practice.

[0129] Antifungal prophylaxis is administered as follows: fluconazole 400 mg orally or intravenously once daily, starting on day 0 and continuing until ANC is >500 for 3 consecutive days (or on 2 consecutive measurements over 3 days). Other appropriate antifungal prophylaxis may be substituted. Pneumocystis carinii pneumonia (PCP) prophylaxis should be initiated on day 0 and continued through day 60. Patients intolerant to trimethoprim / sulfamethoxazole (Bactrim) receive either dapsone or pentamidine for PCP prophylaxis. Viral prophylaxis consists of valacyclovir or acyclovir from days 0 through 60. Oral quinolone (eg, moxifloxacin or norfloxacin) is administered according to institutional preference after DLI until ANC is >500 for 3 consecutive days (or 2 consecutive measurements over 3 days).

[0130] All patients will receive an injection of CD8+ T cell-depleted haploidentical PBCs using the CliniMACS® System with the CliniMACS® CD8 Reagent. Dose levels are numbered from lowest to highest cell dose. Patients in the first cohort (dose level 1) will receive Cy+ CD8+ T cell-depleted haploidentical PBCs (CD8- PBCs) (1 x 10 5 If the criteria for dose escalation are met, patients at dose levels 2, 2b, 3, or 4 will receive 1 x 10 CD4+ T cells / kg recipient IBW, intended to include a dose of 1 x 10 6 , 3×10 6 , 1×10 7 , or 5 x 10 7 CD8- PBCs containing CD4+ T cells / kg, intended to be administered at a dose of 100-1500 mg / kg.

[0131] DLI dose calculation

[0132] The formula for calculating the volume of the final (CD8-depleted) product that delivers the intended dose of CD4+ T cells is as follows: Intended Volume (ml) = Intended CD4+ T Cell Dose (cells / kg) × Recipient IBW* (kg) / CD4+ T Cell Concentration (cells / ml). *Note - If actual weight < ideal weight, use actual weight.

[0133] However, the total number of CD8+ T cells administered should not exceed 3.2% of the intended number of CD4+ T cells to be administered (the numerator in the above formula). If the CD4+ / CD8+ cell ratio in the depleted product is less than 31.25 (= 1 / 0.032), the volume of the product to be administered is determined by the following formula: if the CD4 / CD8 ratio of the final product is less than 31.25, then the volume to be administered (ml) = the intended volume x (CD4 / CD8 ratio) / 31.25; if the CD4 / CD8+ cell ratio in the depleted product is 31.25 or greater, then the volume of the product to be administered is the intended volume (Formula 1): if the CD4 / CD8 ratio of the final product is greater than 31.25, then the volume to be administered (ml) = the intended volume.

[0134] Transfusion Support

[0135] Platelet and packed red cell transfusions will be performed according to current institutional recommendations.

[0136] Example 9 - Duration of Treatment Patients are eligible for only one lymphocyte injection. This restriction is appropriate because rejection of the injected lymphocytes is expected to induce anamnestic immunity against the donor's cells or other close relatives. On day 60, patient peripheral blood is obtained and tested for the presence of human anti-mouse antibodies (HAMA) and for cytotoxic antibodies against the donor cells.

[0137] Follow-up period

[0138] Patients will be followed for at least 60 days after DLI and thereafter until death or disease progression, whichever occurs first. Patients who are removed from the study due to intolerable adverse events or who experience treatment-related adverse events will be followed until the adverse events resolve or stabilize.

[0139] Post-DLI monitoring:

[0140] Patients remaining on the study will have blood drawn at 14, 28, and 60 days after DLI, as well as at 6 months. At these blood draws, a CBC with manual differential will be obtained. Lymphocyte subsets, including the percentage of cells expressing CD4 or CD8, will be analyzed by flow cytometry. From day 60 onward, patients will have a complete blood count with differential monthly until 6 months after DLI, unless disease progression is confirmed.

[0141] Disease evaluation

[0142] In addition to the disease assessments detailed above, the results of additional disease assessments performed as standard of care will be collected for study purposes until death or disease progression, whichever occurs first.

[0143] Example 10 - Dosing Delay / Dose Modification The cyclophosphamide dose will not be changed. If the CD8+ T cell content is excessive, the DLI dose will be changed.

[0144] Adverse Events: Listing and Reporting Requirements

[0145] For all patients with acute GVHD, collect the following information:

[0146] Date of onset (defined as the date of the first biopsy confirming GVHD), GVHD Assessment Form at onset, weekly until resolution of GVHD, and day 60. Initial overall clinical grade, maximum overall clinical grade, date of onset of grade III-IV acute GVHD, if any.

[0147] The occurrence and severity of acute and chronic GVHD from day 60 onwards will be captured at the patient's 6-month evaluation.

[0148] All cases of grade II-IV acute GVHD will be captured as adverse events. Grade III-IV GVHD will be reported as serious adverse events.

[0149] DLI-induced hypoplasia was defined as neutropenia (absolute neutrophil count <500 / ml) and any evidence of donor chimerism after day 60. All cases of DLI-induced hypoplasia were reported as serious adverse events.

[0150] Example 11 - Drug Information Cyclophosphamide (Cytoxan 登録商標 )

[0151] Cyclophosphamide is commercially available. Cyclophosphamide is an alkylating agent that primarily blocks cell division by cross-linking DNA strands. Cyclophosphamide is non-specific for the cell cycle. Injectable cyclophosphamide is available in 2000 mg vials, which are reconstituted with 100 ml of sterile water for injection. The concentration of the reconstituted product is 20 mg / ml. The calculated dose is further diluted in 250-500 ml of 5% dextrose in water. Each dose is administered by infusion over 1-2 hours (depending on the total volume).

[0152] Clinical toxicities of cyclophosphamide include alopecia, nausea and vomiting, headache and dizziness, hemorrhagic cystitis, cardiotoxicity, immunosuppression, bone marrow suppression, pulmonary fibrosis, elevated liver enzymes, and syndrome of inappropriate antidiuretic hormone (SIADH). Cyclophosphamide is dispensed by Oncology Pharmacy and manufactured by Mead Johnson Pharmaceuticals.

[0153] Mesna (sodium 2-mercaptoethane sulfonate)

[0154] Mesna is a prophylactic drug used to prevent hemorrhagic cystitis induced by oxazophosphorine (cyclophosphamide and ifosfamide). It has no inherent toxicity and does not antagonize chemotherapy. Mesna binds with acrolein, a uremic metabolite produced by oxazophosphorine, to produce a non-toxic thioether, and slows the rate at which acrolein is produced by binding with the 4-hydroxy metabolite of oxazophosphorine.

[0155] Mesna is available in 200 mg, 400 mg, and 1000 mg vials containing a 100 mg / ml solution. Each dose of mesna is further diluted with 50 ml of saline and administered intravenously over 15 minutes. The dose of mesna is based on the dose of cyclophosphamide administered. The total daily dose of mesna is equal to 80% of the total daily dose of cyclophosphamide. At doses used for urinary tract protection, mesna is substantially non-toxic. However, adverse effects that can be attributed to mesna include nausea and vomiting, diarrhea, abdominal pain, altered taste, rash, hives, headache, joint or limb pain, low blood pressure, and fatigue.

[0156] CBER IDE Device

[0157] Donors will collect their blood via peripheral whole blood collection (450 ml into CPDA-1) or leukapheresis to collect peripheral leukocytes at steady state (without mobilization). Each leukapheresis collection will be performed with a continuous flow cell separator (COBE Spectra, Gambro) using the institution's standard operating procedures for lymphocyte collection. The method of donation (phlebotomy vs. leukapheresis) will be determined by obtaining an absolute peripheral blood CD4+ T cell count 30 days prior to donation and estimating the volume of blood required to obtain the targeted CD4+ T cell dose. The normal range for peripheral blood CD4+ T cell counts is 0.5-1.5 × 10 6 / ml, so for dose levels 1-2 simple phlebotomy is sufficient, for level 2b apheresis is required, but for dose levels 3 and 4 leukapheresis is required.

[0158] Based on extensive past experience, a 4-hour leukapheresis procedure can deliver 5 x 10 7This will be sufficient to obtain 100 CD4+ T cells / kg recipient IBW. Aim to collect at least 30% more than the desired dose to accommodate for cell loss during the depletion process.

[0159] The product is subjected to CD8 depletion in the Graft Engineering Laboratory. Standard operating procedures are as follows: The product is analyzed for nucleated cell counts, CD3, CD4, CD8, CD16, and CD56. The product is stored overnight, and CD8 depletion is performed using the CliniMACS® Selection System (Miltenyi Biotec, Auburn, CA). Prior to CD8 depletion, the whole blood product is first processed to prepare a buffy coat concentrate, and for major ABO-incompatible donor / recipient pairs, the buffy coat concentrate is further processed using lymphocyte separation medium to remove contaminating red blood cells. The processed whole blood product or apheresis product is then concentrated and resuspended in PBS / EDTA supplemented with 0.5% human serum albumin.

[0160] Add mouse monoclonal CD8 antibody conjugated to iron-dextran superparamagnetic particles and incubate at room temperature for 30 minutes. Use one vial of antibody to measure 40 x 10 total white blood cells. 9 and CD8+ cells were 4 × 10 9The column is processed until the total volume reaches 100 ml. Excess antibody is removed by washing once, and the volume of the product is adjusted to 100 ml with PBS / EDTA supplemented with albumin. It is then connected to the CliniMACS Selection System using a sterile disposable tubing set. The run is initiated by a pre-programmed computer program that controls: (i) flow of the antibody-treated cells; (ii) washing to remove remaining unbound cells; (iii) removal of the magnetic field around the column to release the selected cells; and (iv) final collection of the CD8-depleted cells into a pouch. The entire process takes approximately 2-6 hours after the initial product enrichment. The product is then analyzed for cell count, viability, and CD3, CD4, CD8, CD16, and CD56 content. The CD4 concentration is used to calculate the patient dose. The calculated volume is taken and prepared for injection according to the institution's standard operating procedures.

[0161] Correlation / Special Tests

[0162] Phenotypic immune reconstitution

[0163] Peripheral blood concentrations of lymphocyte subsets, such as CD4+ and CD8+ T cells, will be determined using absolute lymphocyte counts and flow cytometry at 14, 28, 60 days, and 6 months after DLI.

[0164] Analysis of Host CD8+ T Cell Repertoire Diversity by Spectratype Analysis. Recent studies have demonstrated that the diversity of the T cell repertoire can be assessed by T cell receptor V region spectratyping, which evaluates the CDR3 / diversity / combining region (Vf3D- -DJ--Cf3) of cells expressing a given V gene. This region confers T cell receptor specificity. Immunoscoping, or V spectratyping, is very useful for assessing antitumor immune responses after therapy and immune reconstitution after bone marrow transplantation. 49 Furthermore, spectratype analysis of MDS patients before and after immunosuppressive therapy revealed deviations in the T cell repertoire that normalized with response to therapy. Therefore, we hypothesize that patients with MDS and probable CMML have a biased T cell repertoire before treatment, that the DLI initially induces a population of alloreactive T cells, and that responders eventually acquire a normal T cell repertoire as revealed by spectratyping. Pretreatment CD8+ T cells are obtained from the patient's peripheral blood mononuclear cells (PBMCs). To identify the patient's anti-donor-responsive T cells, the patient's pretreatment PBMCs are cultured with irradiated donor PBMCs for 7 days before cell sorting. This culture period allows the patient's anti-donor T cells to undergo clonal expansion. PBMCs are harvested, and CD8+ T cells are purified at days 14, 28, 60, and 6 months.

[0165] Example 12 - NEDLI model for enhancing anti-tumor effects neoAg-specific CD8 +T cell exhaustion is a major barrier to the success of cancer immunotherapy in humans. Current efforts to overcome this barrier have focused on the use of immune checkpoint inhibitors (CIs), either alone or in combination with drugs designed to alter the tumor immune microenvironment. To date, these efforts have met with limited success, and most cancer patients fail to benefit from immunotherapeutic strategies. CD4 + T cells are CD8 + Not only can it prevent T cell exhaustion, but it can also reverse established exhaustion, thereby restoring anti-cancer immune responses. + T cells can induce CD8 T cells by licensing antigen-presenting cells (APCs) via the CD40 molecule on the APCs. + Provides help to T cells. CD4 + T cell help, CD8 + To generate T cell memory, and CD8 + Importantly, for help to be delivered, the CD4 + and CD8 + T cells do not have to see the same antigen, but they must see antigens presented on the same APC. CD4 help can reverse established exhaustion, but growing cancers require tumor-specific CD4 + Evidence is emerging that CD8 induces tolerance in T cells, and to date, + To restore T cell helper function, CD4 + There are no known strategies for restoring tolerance in T cells. Agonistic antibodies against the CD27 molecule inhibit CD4 + They can mimic some aspects of T cell help and thus stimulate anti-tumor immunity, especially in combination with CI, but are unlikely to restore T cells from established exhaustion in well-advanced cancers.

[0166] In cancer, MHC-alloreactive CD4 T cells derived from healthy donors are used to restore T cell exhaustion. + When T cells are injected, the injected cells are eventually rejected, but the recipient CD8 + Mechanisms requiring T cells can promote the regression of established cancers. Vaccination of healthy allogeneic donors with viral antigens enhances the therapeutic efficacy of non-engrafting donor lymphocyte infusion (NEDLI) against tumors expressing the antigens, thus enhancing the therapeutic efficacy of CD4 T cells derived from healthy donors vaccinated against tumor neoantigens. + T cells can be used to treat sporadic human cancers.

[0167] CD4 + T cells can also activate CD8 by licensing APCs. + Recovers T cells from exhaustion. CD4 + T cells can also activate CD8 by licensing APCs. + Provides help to T cells, and CD4 + T cell help restores CD8 T cells from exhaustion, but not CD4 + It is unclear whether T cells can recover from exhaustion by licensing APCs. Vaccination of donors against CD4 neoepitopes could enhance the antitumor effects of NEDLI. Vaccination of donors can enhance the antitumor effects of allogeneic BMT, and healthy donor CD8 +T cells respond vigorously to neoantigens, but identifying CD4 neoepitopes has been challenging. We used a previously validated CD4 neoepitope, M30, to demonstrate proof of principle while technology for identifying CD4 neoepitopes advances.

[0168] Currently available cell therapies for cancer (allogeneic blood or bone marrow transplants, CAR T cells or T cell receptor-modified T cells, tumor-infiltrating lymphocytes) use injectable lymphocytes that directly attack cancer cells. In contrast, the goal of NEDLI is to restore endogenous anti-tumor immunity by providing helper signals via the recipient's antigen-presenting cells (APCs). + Transient engraftment of T cells resulted in neoAg-specific CD8 + The hypothesis that T cells can be restored from exhaustion is quite revolutionary. U.S. Patent No. 9,931,359 B2 describes a method for selecting HLA class II mismatched donors for injection of CD8-depleted non-engrafted donor lymphocytes to restore endogenous anti-tumor immunity. In this application, the donor has at least one HLA class II allele match with the recipient, the donor is vaccinated against tumor or viral neoantigens, and then virus- or tumor-specific T cells are further expanded ex vivo.

[0169] The anti-tumor effect of this therapy is enhanced by MHC class II mismatch in the graft-versus-host direction, and alloreactive donor memory CD4 + Ensure high frequencies of T cells (not naive but memory CD4 T cells via heterologous immunization) + T cells may overcome tumor-induced immunosuppression and deliver appropriate helper signals. +T cell help can be achieved by directing CD8 T cells to multiple neoepitopes via epitope spreading (see Figure 12), so that the targeted antigen does not need to be expressed on all cancer cells. + It could revive T cells.

[0170] The disease response induced by transient lymphocyte engraftment may be best explained by an indirect process initiated by short-lived donor cells but carried to completion by host cells. The present invention's findings support the discovery that cyclophosphamide, followed by CD8 + Injection of cell-depleted MHC-mismatched donor lymphocytes (Cy + CD8 - Treatment of mice with donor cells (DLI) induces regression of established tumors with minimal toxicity in both hematologic and solid cancer models, even though the donor cells are ultimately rejected by the host's immune system. - The optimal antitumor effect of DLI is due to the donor CD4 + Presence of T cells, host CD8 + T cells and expression of alloantigens by normal host but not tumor tissue. Importantly, the DLI-derived CD8 + Cell depletion eliminated the risk of persistent donor cell engraftment and the risk of lethal GVHD, but did not compromise antitumor efficacy. Based on these results, alloreactive donor CD4 + T cells deliver signals via APCs that cross-present tumor antigens, thereby targeting tumor-specific recipient CD8 + We proposed a model for recovering T cells from exhaustion (Figure 5). As shown in Figure 5, alloreactive type 1 (T h 1) CD4 +Proposed mechanism for T cell recovery from exhaustion. Recognition of major histocompatibility complex (MHC) class II alloantigens on the surface of recipient antigen-presenting cells (APCs) leads to activation of alloreactive donor CD4+ T cells, followed by upregulation of activating ligands on the APCs, including CD154, the ligand for CD40. Ligation of CD40 induces upregulation of molecules on the APC surface, including CD70, the ligand for CD27 on CD8+ T cells. This signaling cascade has been shown to be necessary for the induction of CD8+ T cell memory; the same interaction has been proposed to be involved in the recovery of CD8+ T cells from exhaustion and their reversion to CD8+ memory T cells. Interferon-gamma and / or interleukin-21, secreted by CD4+ T cells, may also be involved in the recovery of T cells from exhaustion.

[0171] NEDLI can induce potent antitumor effects against hematological malignancies, but its effects against solid tumors are generally weaker and non-curative. Priming donors against tumor-specific antigens may enhance the antitumor effects of NEDLI. To test this hypothesis, healthy syngeneic or MHC-haploidentical donors were vaccinated against the human papillomavirus E7 antigen, and splenocytes from the primed donors were injected into mice that had been inoculated 14 days prior with TC1 (an E7-expressing lung cancer) with either undepleted or CD8-depleted spleen cells (Figure 6A).

[0172] Tumor-bearing recipients were cured by unreduced DLI from E7-primed syngeneic donors (Fig. 6B, left), and the therapeutic effect was abolished by CD8-depletion.

[0173] Interestingly, when haploidentical donors were used and the donor cells were rejected, a beneficial effect was achieved by CD8-depleted (but not non-depleted) DLI from the primed donor (Figure 6B, right). Because CD8-depleted syngeneic DLI did not prolong survival, we hypothesized that alloreactive and E7-specific CD4 + T cells cooperate to induce anti-tumor immunity in non-engrafted patients, as well as alloreactive CD8 + T cells likely express endogenous, tumor-specific CD8 + We conclude that damage to (recipient) APCs, which provide signals that allow T cells to recover from exhaustion, impairs anti-tumor immunity.

[0174] We next tested whether the antitumor efficacy of NEDLI could be enhanced by stimulating primed T cells ex vivo with E7-peptide-pulsed dendritic cells. Spleen cells from an E7-primed CB6 F1 donor were either undepleted or CD8-depleted, and then cocultured ex vivo for one week with syngeneic CB6 F1 or haploidentical B6C3 F1 dendritic cells pulsed with overlapping pentadecamers covering the full-length HPV16 E7 protein. After culture, cells were harvested and injected into Cy-treated B6C3 F1 mice bearing TC1. Figure 7 shows that ex vivo culturing of E7-primed donor cells with E7 peptide enhanced the antitumor efficacy of NEDLI. Furthermore, CD8-depletion of ex vivo cultured donor cells was associated with increased tumor cell proliferation. + The T cells did not cause fatal GVHD.

[0175] The cured mice in Figure 7 were treated with donor (H-2 d+ ) had no evidence of chimerism, but the immunodominant Kb -Fluorescent dye-conjugated H-2K pulsed with binding peptide b E7-specific CD8 as demonstrated by staining with tetramers of MHC molecules + The expanded population of E7-specific CD8+ T cells included memory (CD127 + PD-1 - ) phenotype (Figure 8F). Cured animals were re-challenged with TC1 (which expresses E7-specific CD8 + T cells showed resistance to further clonal expansion [Fig. 8C, D], and they upregulated PD-1 expression [Fig. 8G, H].

[0176] As shown in Figure 8, the percentage of recipient CD8+ T cells responsive to the immunodominant H-2Kb-restricted peptide of E7 was determined by staining with H-2Kb-tetramers pulsed with the peptide (top row). The bottom row shows the cell surface expression of CD127 and PD-1 on gated E7-specific CD8+ T cells. Memory CD8+ T cells express CD127+ and PD-1. low activated T cells express CD127+PD-1 high and exhausted T cells express CD127 - and PD-1 high is.

[0177] When CD8-depleted lymphocytes (open triangles in Figure 7) from cured animals were cultured with E7 peptide, IFN-γ-secreting CD4 + IFN-γ also stimulated proliferation of naive CD4 T cells (Fig. 9A and 9B). +NeoAg-specific Th1 cells were also generated by culturing T cells with E7 peptide (Figures 9A and 9B), raising the possibility that neoAg-specific Th1 cells for adoptive cancer immunotherapy could be expanded ex vivo from healthy, unvaccinated donors, thereby ameliorating donor safety concerns.

[0178] As shown in Figure 9, CD4 T cells from untreated B6 x C3H F1 mice (naive; a, b) or from mice cured by injection of non-engrafted donor lymphocytes (c) were detected. + Staining of T cells for intracellular interferon-gamma (IFNγ) and tumor necrosis factor-α was measured 5 days after stimulation with unpulsed B6x C3H F1 dendritic cells (a) or DCs pulsed with E7 peptide (b, c). Panel d shows IFNγ secretion from the same cells by ELISPOT assay.

[0179] As shown in Figure 10, TC1-bearing B6 x C3H F1 mice were cured by cyclophosphamide plus CD8-depleted lymphocytes (harvested from E7-vaccinated donors and expanded ex vivo with E7 peptide). 300 days after lymphocyte injection, spleen cells from the cured mice were CD8 + The cells were depleted, cultured with E7 peptide for 1 week, and transplanted into TC1-bearing BALB / c x B6 F1 mice (20 million cells per recipient) that had been treated with cyclophosphamide 1 day prior. Alternatively, TC1-bearing BALB / c x B6 F1 mice were transfected with CD8 + Spleen cells from a naive B6 x C3H F1 donor were administered, depleted and cultured with E7 peptide one week prior to adoptive transfer. Survival is shown in Figure 10B.

[0180] Ex vivo-cultured CD8-depleted splenocytes from NEDLI-cured B6C3 mice were again able to prolong mouse survival despite being rejected by tumor-bearing CB6 F1 recipients (Figure 10B). Thus, mice cured by injection of non-engrafting donor lymphocytes contain CD4+ T cells that can continuously transfer anti-tumor immunity when administered as NEDLI.

[0181] Human papillomavirus (HPV) is associated with several human cancers, including oropharyngeal, cervical, penile, and anal cancers. It has been found that the virus can transform epithelial cells by expressing two viral antigens (E6 and E7) intracellularly. E6 inactivates the p53 tumor suppressor protein, and E7 inactivates the retinoblastoma protein, both proteins closely involved in cell cycle regulation. Transformed cells are no longer subject to growth control, and the E6 and E7 proteins are essential for progression to malignant tumors.

[0182] The immune system is able to respond to and eliminate HPV. A key determinant of whether an individual exposed to a carcinogenic strain (usually HPV16 or HPV18) will clear the virus or become chronically infected is the T cell response to HPV, or more precisely, the CD4 T cell response to HPV. + HPV infection is a T cell response. Individuals who mount a type I CD4+ T cell response, including the production of IFN-γ, typically clear the virus, but those who fail to mount such a type I response may become chronically infected and susceptible to malignant transformation. Investigational vaccines are currently available to generate responses against the E6 and E7 antigens of HPV16 and HPV18.

[0183] In animal models, when donors and recipients are matched for all major histocompatibility complex (MHC) genes, vaccination of the donor against HPV E7 significantly increases the CD4 count of the vaccinated donor. + T cells and CD8 + We found that anti-tumor immunity could be transferred by transferring T cells to the recipient (Figure 6B, left panel). On the other hand, when the donor and recipient were MHC-haploidentical, the donor was vaccinated against HPV E7, and the vaccinated CD8 + Vaccinated CD4, but not T cells + It was found that anti-tumor immunity can be transferred by transferring T cells into recipients (Fig. 6B, right panel). The anti-tumor efficacy of NEDLI derived from donors vaccinated against tumor-specific antigens can be enhanced by culturing the vaccinated T cells in vitro with tumor peptides before injecting them into the recipients (Fig. 7B). CD4 T cells from vaccinated and ex vivo stimulated donors + Injection of T cells restores the recipient's immune response, and cured animals now express CD8 + T cells (Figure 8B-D) and CD4 + The recipients had T cells (Fig. 9C, D) that responded to tumor antigens and rejected tumor cells despite the elimination of donor cells, indicating that immunity against HPV was effectively transferred from the donor to the recipient.

[0184] The present invention contemplates the injection of CD8-depleted cells from donors vaccinated against tumor-specific antigens (viral antigens or tumor neoantigens) and cultured with virus-derived peptides or neoantigens. Alternatively, the present invention contemplates the injection of CD8-depleted cells from unimmunized donors stimulated ex vivo with peptide-pulsed dendritic cells to stimulate virus-specific or neoantigen-specific CD4+ T cells. The present invention also contemplates the creation of cell banks containing HPV-primed CD4+ T cells from donors of various human leukocyte antigen (HLA) types. For example, the following table shows that a bank of 10 cell lines (each cell line uniquely expressing one of the 10 most common HLA class II alleles) is sufficient to provide therapeutic treatment to 95.7% of the US Caucasian population. Examples of HLA class II molecules include HLA-DRB1, HLA-DPB1, and HLA-DQB1. [Table 3]

[0185] The anti-tumor efficacy of NEDLI was evaluated by neoAg-specific CD4 + A key point of this study is whether increasing the frequency of T cells can enhance alloreactive CD4 T cells. Preliminary data suggest that alloreactive CD4 T cells play a key role in mediating antitumor immunity after NEDLI. + T cells and tumor-specific CD4 + The effect of MHC mismatch in a model of donor neoAg vaccination will also be examined, as a clear synergistic effect between the two was demonstrated. By carefully selecting donor and recipient strains, it should be possible to readily analyze the relative roles of alloresponsiveness versus neoAg priming on antitumor efficacy.

[0186] Example 13 - Characterization of the effect of neoAg vaccination of healthy donors on the anti-tumor efficacy of NEDLI Comparison of the benefits of in vivo donor vaccination versus ex vivo T cell expansion, and the interaction between alloreactive and neoAg-specific T cells.

[0187] The purpose of this experiment was to identify CD4 + Boosting T cell help and CD8 against 'sporadic' tumors - The objective of this study was to test two methods to increase the anti-tumor efficacy of NEDLI: 1) CD4 + 1) in vivo vaccination with T cell neoepitopes (with or without subsequent ex vivo neoepitope stimulation); or 2) CD4 + In vitro "priming" of T cells using sequential stimulation. The experimental design is shown in Table 3 below. C57BL / 6 (B6; H-2 b ) The original B16-F10 melanoma grows in F1 hybrids; immunogenic CD4 + The neo-epitope has been identified.

[0188] 1) B6 × C3H(B6C3; H-2 bxk ) F1 or MHC-haploidentical BALB / C × B6 (CB6; H-2 bxd )F1 mice were vaccinated with either the mutant neo-epitope M30 (groups 2 and 6) encoded by the kinesin family member 18b gene (Kif18b) or the corresponding wild-type peptide (groups 1 and 5). The vaccines contained 100 jtg of synthetic peptide and 50 jtg of poly(I:C) in a volume of 200 jtl of phosphate-buffered saline and were injected into the flank. Vaccination efficacy and responder cell phenotype (CD4 vs. CD8) were tested by flow cytometry and intracellular cytokine staining (ICS) for interferon gamma (IFNγ) or tumor necrosis factor alpha (TNFα). Two weeks after vaccination, donor mice were euthanized, and spleen cells were purified to identify CD8 +The cells were depleted and injected into B16-F10-bearing B6C3 mice that had been treated with cyclophosphamide (Cy) the day before injection. Alternatively, CD8-depleted cells from immunized donors were cultured with M30-pulsed donor DCs for 5 days before injection (groups 3 and 7);

[0189] 2) Spleen cells from naive B6C3 or CB6 F1 mice were stimulated twice weekly with M30-pulsed autologous dendritic cells plus 20 U / ml IL-2 (groups 4 and 8). + CD4 + The frequency of T cells is measured by ICS before and after ex vivo stimulation. + T cells may be purified using an IFN'y capture assay (Miltenyi Biotec) and further expanded using anti-CD3 and anti-CD28 coated beads. [Table 4] [ka]

[0190] Based on historical data on similar treatment groups, a non-parametric Mann-Whitney test for comparing two treatment groups was simulated. A sample size of 14 per treatment group was estimated to be sufficient with 80% power and a two-sided type-I error of 0.05.

[0191] The benchmark for success was expanded (by vaccination or ex vivo culture) M30-specific CD4 + In recipients of CD8-depleted cells containing T cell populations, survival was significantly prolonged (groups 6, 7, or 8 compared with 5), suggesting the contribution of CD4 to the tumor microenvironment. +Reflecting the benefit of receiving T cell help, myeloid cells are reprogrammed to become more immunostimulatory and express CD8 + T cell help. The anti-tumor efficacy of primed NEDLI is likely to be enhanced by the allogeneic responsiveness of the donor population (e.g., compare groups 6 with 2, 7 with 3, and 8 with 4). A single neoAg, single dose of vaccine, or vaccine formulation may be insufficient to enhance anti-tumor immunity. If survival is not extended, vaccinate the donor with an mRNA pentatope vaccine. Donor vaccination may exacerbate GVHD or cytokine release syndrome; this may be mitigated by reducing the donor cell dose.

[0192] To compare the anti-tumor effects of neoAg-primed NEDLI and immune checkpoint blockade (ICB), alone or in combination. + Intratumoral delivery of recombinant adenovirus encoding CD40 ligand, a key molecule involved in delivering T cell help (Figure 5), was shown to be superior to and synergistic with the combined anti-PD-1 and anti-CTLA4 antibody ICB. We predict that NEDLI plus neoAg-priming will be superior to and synergistic with ICB. Sixty-five B6C3 F1 mice were each administered 5 × 10 5B16-F10 melanoma cells are inoculated subcutaneously in the flank on day -8, and Cy 200 mg / kg is administered IP on day 0. On day 1, groups of 13 mice each received either: 1) no treatment; 2) treatment with 20 million CD8-depleted splenocytes from unprimed CB6 F1 donors (which confers a modest but significant survival benefit); 3) treatment with 20 million CD8-depleted splenocytes from M30 peptide-vaccinated (see Experiment 1) CB6 F1 donors; 4) 200 jtg of ICB bearing monoclonal antibodies against mouse PD-1 (clone RMP1-14) and CTLA-4 (clone 9H10, both from Bio X Cell), respectively, administered intraperitoneally every 3 days for four doses; or 5) treatment with M30-primed CD8-depleted CB6 F1 splenocytes plus ICB. Tumor growth is measured three times a week in seven mice per group. + To examine the effect of NEDLI on T cells, we used H-2K IgG1, a gene specific for the p15E peptide (KSPWFTTL) of retroviral protein gp70, which is expressed in B16-F10 but not in normal tissues of C57BL / 6 mice. b -Restricted CD8 + T cell phenotype and function were characterized. On days 7 and 14 of each treatment, three mice per group were sacrificed, and single cell suspensions from spleens, tumor-draining lymph nodes, and disaggregated tumors were analyzed with p15E peptide (MBL International, Woburn, MA) and CD62L, Tbet Fluorescent dye-conjugated H-2K loaded with antibodies against CD127, CD27, and KLRG-1 b The tumor environment is stained with CD80, CD86, and PD-L1 tetramers, and CD11c + Characterized by assessing expression in bone marrow cells and total CD4 +Foxp3 on T cells + The Treg ratio and CD8 / Treg ratio were measured. TILs and lymph node and spleen cells were stimulated with p15E peptide, IFN-γ, and TNF-α, and intracellular cytokine-induced granzyme B production was measured by staining and flow cytometry. NeoAg priming resulted in slower sc tumor growth and tumor-specific CD8 + It is expected to enhance the anti-tumor efficacy of NEDLI, as demonstrated by enhanced T cell activation. + T cell help, CD8 + Given the evidence that NEDLI but not ICI reverses T cell exhaustion, we predict that neoAg-primed NEDLI will have superior antitumor efficacy than ICI.

[0193] Mixed-effects model analysis was performed using the MIXED procedure in SAS (Cary, NC) and the Westfall and Young parametric residual resampling method using the Glinmix procedure, adjusting for post hoc tests and limiting to only interesting comparisons. Flow cytometry data are expressed as the mean % of positive cells or mean fluorescence intensity (MFI), with confidence intervals. Group means are compared using the Mann-Whitney U test.

[0194] Example 14 - Characterization of how virus-antigen or neoAg-primed donor-derived NEDLI induces tumor immunity Tumor-specific recipient CD8 receptors are expressed via recipient APCs. + By providing signals to T cells, alloreactive donor CD4 + Whether the T cells enhance anti-tumor immunity will be determined.

[0195] Previous studies have shown that the therapeutic effect of injecting MHC-mismatched CD8-depleted donor lymphocytes is significantly greater than that of donor CD4 lymphocytes. + T cells, recipient CD8 +Based on these results, alloreactive CD4 T cells and the expression of alloantigens on normal host tissue cells were shown to be required. + T cells express tumor-specific CD8 + It is hypothesized that CD4 + T cells and CD8 + The antigens recognized by the T cells must be presented on the same APC.

[0196] The key to this experiment is CD4 + T cells and CD8 + The goal is to separate the presentation of alloantigens and tumor antigens on different APCs so that they do not communicate with T cells. This is done by using the parental B6 (H-2 b ) but tumor-specific H-2b-restricted CD8 + The other line, C3H, is able to present tumor antigens to T cells, while the other line, C3H, presents MHC class II alloantigens (H-2 IA) to non-engrafted donor CD4+ T cells. k or IE k This was accomplished by generating biparental bone marrow chimeras (Figure 11A; and groups 3 and 4 in Table 4 below) that display CD4+ T cells and neoantigen-specific CD8 T cells. The positive control for this experiment was generating F1-parental chimeras (Figure 11B; and groups 5 and 6 below), in which the APCs were CD4+ T cells and neoantigen-specific CD8 T cells. + It can present both alloantigens to T cells, thereby acting as a bridge for these T cells to communicate.

[0197] Methods: Irradiated (950 cGy) B6 mice were treated with 10 7 , reduced T cells (T - ) B6C3 F1 bone marrow (BM) cells (groups 3 and 4), or 5 × 10 6 T's - B6 and 5x10 6 T's - C3H(H-2 kAfter 2 months, chimeras were transplanted with 5 × 10 4 E7-expressing TC1 (H-2 b ) lung cancer cells were administered IV. Two weeks later, tumor-bearing recipients were treated with Cy 200 mg / kg IP, and the next day, either untreated or with ex vivo E7-peptide-stimulated CD8 - Treatment is with spleen + LN cells (open triangles in Figure 7). Survival is compared in the different groups by the Log Rank test.

[0198] B6 APCs cross-present tumor antigens but do not express donor CD4 + Groups 1 and 2 are negative controls for this study because there are no alloantigens to stimulate T cell help. MHC class II alloantigens and tumor antigens are presented on different APCs in groups 3 and 4, but on the same APC in groups 5 and 6. If alloresponding donor CD4 + If T cells enhance endogenous anti-tumor immunity by licensing APCs, mice in group 6 will survive significantly longer than mice in group 4. If alloreactive donor CD4 + If T cells prolong survival solely by secreting cytokines after allogeneic recognition, then survival of mice in groups 4 and 6 should be similar to, but superior to, survival of mice in group 2. [Table 5]

[0199] When exogenous help restores exhaustion through APC licensing, group 4 loses syngeneic CD8+ cells to DLI, which had no effect (Figure 6, left panel, open red squares). Cy+ cells alone (Figure 7, closed diamonds) lose CD8+ cells stimulated ex vivo with peptide-pulsed haplo-DCs. - Comparing with the DLI (open red triangle; similar to group 6) yields a risk ratio of 15. With a two-sided type I error of 0.05 and a sample size of 10 in each group, power = 0.999, and therefore 10 / group is sufficient to test the primary hypothesis.

[0200] Evaluation of the ability of NEDLI to induce epitope spreading.

[0201] Intratumoral clonal heterogeneity and HLA or neoAg loss limit the effectiveness of immunotherapies directed against single tumor antigens (e.g., CAR T cells, anti-tumor monoclonal antibodies, tumor vaccines). Epitope spreading counters tumor evasion by broadening responses to antigens not directly targeted by the therapy. CD4 + T cell help is provided by neoAg-specific CD8 + It is hypothesized that reversing T cell exhaustion promotes epitope spreading (Figure 12). Figure 12 shows transiently engrafting, tumor-specific CD4 + This figure shows a hypothesized mechanism for epitope spreading after T cell injection. Tumor cell killing by CD8+ cytotoxic T cells (CTLs) specific for antigen "A" triggers antigen-presenting cells in the tumor microenvironment to release and cross-present antigen AE. Injected type I (Th1) CD4+ T cells provide a signal to rescue exhausted CD8+ T cells (eCTLs) that recognize antigen A presented by MHC class II molecules and antigens B, C, and E presented by MHC class I molecules on the same antigen-presenting cells.

[0202] To test this hypothesis, the following experiment is designed.

[0203] OT-I transgenic mice (Jackson Labs) express H-2K b A CD8 transgenic mouse model encoding a transgenic T cell receptor for chicken ovalbumin (OVA) peptide 257-264 (SIINFEKL, SEQ ID NO: 3), which is presented by MHC class I molecules. + T cells, whereas OT-II mice (Jackson Labs) contain H-2 IA b CD4 specific for OVA peptide 323-339 (ISQAVHAAHAEINEAGR, SEQ ID NO: 4) presented by + B16-OVA is an MHC class II-negative B16-F10 melanoma line engineered to express chicken ovalbumin, which is recognized by OT-I T cells. By breeding the corresponding inbred strains, B6.SJL x C3H (H-2 kxb , CD45.1 + ), OT-I x C3H (H-2 kxb , CD45.1 - ), and OT-II x BALB / c (H-2 dxb ) F1 mice were generated. 81 B6.SJL x C3H F1 mice were each injected with 2 million CD8 cells derived from OT-I x C3H F1 mice. + T cells were administered IV, and after 2 days, 5 x 10 5 5 x 10 B16-F10 melanoma cells were inoculated into one flank. 5 B16-OVA cells were inoculated subcutaneously into the opposite flank. When tumors on each side grew to approximately 5 mm, 3 mice were euthanized and 13 mice per group were given 200 mg / kg Cy IP, followed one day later by one of the following: 1) Nothing; 2) 2 weeks prior to the administration of Kif18-encoded wild-type peptide [ka] (See Experiment 1 for vaccination details) Primed with CB6 F1; H-2 dxb ) 20 million CD8-depleted splenocytes from mice; 3) 2 weeks prior to the M30 neoepitope [ka] 20 million CD8-depleted splenocytes from CB6 F1 mice primed with; 4) 1 million CD4 cells from unimmunized OT-II x BALB / c F1 mice + CD8-depleted spleen cells containing T cells; 5) CD8-depleted splenocytes obtained from OT-II × BALB / c mice and cultured for 5 days with OVA323-339-pulsed dendritic cells supplemented with IL-2, GM-CSF, and 1 tM ibrutinib (to promote Th1 differentiation); and 6) Selected CD4 from Group 5 + T cells. Tumor size in both flanks is measured three times a week for all surviving mice. Three mice per group are sacrificed on days 3 and 7 after NEDLI (or earlier if tumors begin to regress). Spleens, draining LNs, and tumor-derived cells are analyzed for: 1) Tumor-specific CD8 + T cells: p15E-specific CD8 T cells and OVA-specific T cells (H-2 k+ ) expression of CD27, CD127, PD-1, and KLRG1; 2) Functional: Cells were stimulated with M30, p15E peptide, or class I and II OVA peptides separately for 5 hours. The frequencies of IFN-, TNF-, granzyme B-, IL-4-, and IL-2-producing cells were analyzed by ICS and flow cytometry; 3) Immunohistochemistry: Tumor fragments are snap frozen in liquid nitrogen. 7 tm tumor sections are stained with CD4, CD8, p15E tetramer, or OVA tetramer, along with KLRG-1, PD-1, CD127, or IFN'y; 4) DC activation: Cells from tumor, draining lymph nodes, and spleen are stained with CD11b, CD11c, class II, CD70, CD80, and CD86.

[0204] Example 15 - Materials and Methods All experiments in Examples 13-15 used inbred or transgenic mice purchased from Jackson Laboratories, or F1 hybrid mice purchased commercially or generated by in-house breeding. Mice were purchased at 6-8 weeks of age and were approximately 8-10 weeks old at the start of the experiments. In most experiments, cells from the donor strain were injected into tumor-bearing recipients. In each group of treated mice, equal numbers of males and females were used as recipients. The following procedures were performed: a) Adoptive cell therapy. Recipient mice were conditioned with either 200 mg / kg cyclophosphamide intraperitoneally or 950 cGy total body irradiation (TBI), and 1 day later, lymphocytes were administered intravenously (IV) via the tail vein. TBI-conditioned animals also received T cell-depleted bone marrow cells intravenously to rescue hematopoiesis. b) Mouse tumor model—Recipients received 1x10 B16-F10 melanoma cells. 5 IV or 5x10 pieces 5 5 x 10 5 5 x 10 B16-OVA (B16-F10 transfected with chicken ovalbumin gene) sc or 5 x 10 4 TC1 lung cancer cells were administered intravenously, and treatment began two weeks later. c) Vaccination - Prior to immunization, mice were anesthetized with isoflurane by instillation. Mice were immunized with synthetic peptide and 50 μg of poly(I:C) in the flank, approximately 200 μl / mouse of emulsion. For the E7 cDNA vaccine, mice were administered cDNA (25 μg) by intramuscular electroporation (106 V, eight 20-ms pulses at 200-ms intervals). Mice were checked daily after immunization for pain and were dosed with buprenorphine SR at 0.05–0.1 mg / kg sc or ip q for 48–72 hours, as indicated.

[0205] Vaccination. C57BL / 6 x C3H (B6C3) F1 or BALB / c x C57BL / 6 (CB6) F1 mice were vaccinated weekly for a total of three doses with 25 μg of pcDNA-3-CRT / E71 (a DNA vaccine encoding the E7 antigen of HPV16 serotype). The vaccine was delivered intramuscularly by electroporation using eight pulses of 106 V current, each pulse lasting 20 ms with a 200 ms interpulse interval.

[0206] Preparation of cells for adoptive immunotherapy. One week after the last vaccination, spleen cells from vaccinated or unvaccinated donors were left untreated or depleted of CD8+ cells by magnetic cell sorting (MACS; Miltenyi Biotec). In experiments using uncultured spleen cells, animals were administered either 20 million undepleted cells or CD8-depleted cells containing CD4+ T cells present in 20 million undepleted cells. In some experiments, whole or CD8-depleted spleen cells from vaccinated CB6 F1 donors were incubated with a 500-kDa antibody containing the amino acid sequence of the HPV16 E7 protein. (Peptide sequence: MHGDTPTLHE YMLDLQPETT DLYCYEQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP; SEQ ID NO: 7) (JPT Technologies;) The 22 peptides were cultured with syngeneic CB6 F1 or MHC-haploidentical B6C3 F1 dendritic cells pulsed with 1 μg / ml of each of 22 overlapping 15mers from the E7 sequence. The adjacent pentadecapeptides in the E7 sequence overlap by 11 amino acids at their C-terminus. The first four of the 22 peptides are as follows; the rest can be deduced from the above sequences:

[0207] MHGDTPTLHEYMLDL (SEQ ID NO: 8)

[0208] TPTLHEYMLDLQPET (SEQ ID NO: 9)

[0209] HEYMLDLQPETTDLY (SEQ ID NO: 10)

[0210] LDLQPETTDLYCYEQ (SEQ ID NO: 11)

[0211] Dendritic cells were prepared by pan-DC magnetic cell sorting (MACS; Miltenyi Biotec). 20 million donor spleen cells were cultured with 2,000 peptide-pulsed dendritic cells in 10 ml of RPMI medium supplemented with 10% fetal bovine serum, 2-ME, 25 U / ml recombinant mouse IL-2, and 20 ng / ml recombinant mouse GM-CSF for 5 days. At the end of the 5-day period, cells were washed and resuspended in phosphate-buffered saline. Mice were administered a dose of ex vivo cultured cells corresponding to the CD4+ T cell input contained in 20 million undepleted spleen cells.

[0212] Treatment Protocol. In experiments evaluating the antitumor efficacy of adoptive cell therapy, recipient mice received 5 x 104 TC1 tumor cells intravenously (IV) via the tail vein on day 0, followed by 200 mg / kg cyclophosphamide intraperitoneally on day 13 and donor cells on day 14. The dose of unstimulated, undepleted donor cells was 20 million splenocytes per recipient. The dose of uncultured, CD8-depleted splenocytes was normalized to contain the same number of CD4+ cells as 20 million undepleted splenocytes. The dose of ex vivo cultured cells was normalized to the number of CD4+ cells placed in culture. For example, if a mouse spleen contains 100 million total cells and 20 million CD4+ cells, and the CD8-depleted population contains 88 million cells and 18 million CD4+ cells, and if placing 88 million CD8-depleted spleens in culture for peptide stimulation results in a stimulated population containing 10 million cells and 4 million CD4+ cells, then the DLI dose would be 20 million whole spleens (containing 4 million CD4+ cells), 19.56 million CD8-depleted spleens (containing 4 million CD4+ cells), or 2.2 million peptide-stimulated cells.

[0213] Survival of mice in the various treatment groups was compared by Log-Rank test; a p-value of less than 0.05 was considered significant.

[0214] The proposed experiments involve multiple simultaneous cellular interactions that result in phenomena such as graft-versus-host disease (GVHD), antitumor effects, and lymphocyte responses to therapeutic vaccination. These complex biological phenomena cannot be simply modeled or recapitulated by studying lymphocytes in vitro. The widespread availability of inbred, transgenic, and F1 hybrid strains will enable us to model GVHD and adoptive immunotherapy of cancer in a clinically relevant manner. Specific strains were selected to model the human condition through the injection of HLA-matched sibling or HLA-haploidentical non-engrafting donor lymphocytes (NEDLI). T cell receptor transgenic mice facilitate tracking endogenous, tumor-specific T cells in tumor-bearing animals. Mouse tumors with defined genetic alterations that confer chemotherapy resistance or express defined "tumor antigens" are also widely available. These experiments can only be performed in species such as mice or rats where inbred strains and F1 lines are widely available.

[0215] After NEDLI and tumor implantation via intravenous injection via the tail vein, mice are closely monitored for any tumor-related morbidity or toxicity of the cell injection, such as GVHD (weight loss, hunched posture, ruffled fur, diarrhea, erythema, and reduced mobility) or cytokine release syndrome (fever, diarrhea, weight loss, and immobility). Intravenous administration is only transiently painful and can be performed without anesthesia. The study is terminated when the tumor-free survival of each group remains unchanged over a two-week observation period. For this purpose, 100 days of observation after tumor inoculation is usually sufficient. This is in accordance with the recommendations of the American Veterinary Medical Association (AVMA). Animals are monitored at least three times a week after tumor and / or allogeneic lymphocyte injection for any signs of allogeneic GVHD or progressive cancer morbidity. As soon as tumor-related morbidity is diagnosed, tumor-bearing animals are euthanized to prevent undue suffering. Animals suffering from GVHD are also euthanized.

[0216] In experiments where mice received subcutaneous injections of tumors, the maximum two-dimensional measurement of the tumor was 100 mm 2 Mice are generally euthanized when the tumor size exceeds 100 mg / kg or when the tumor develops extensive ulceration, whichever occurs first. In all experiments, animals that appear to be suffering are euthanized. Experimental conditions that may result in suffering include extensive lung metastases resulting in labor breathing, graft-versus-host disease with significant weight loss, diarrhea, and immobility, or tumor-induced paralysis.

[0217] Cell lines: B16-F10 melanoma (ATCC), B16-OVA melanoma, and TC-1 lung carcinoma (kindly provided by TC Wu, Johns Hopkins University)

[0218] Mouse cell lines are authenticated by short tandem repeat profiling (National Institute of Standards and Technology Mouse Cell Line Authentication Consortium / American Type Culture Collaboration). Cells are also tested for mycoplasma contamination (Genetica Cell Line Testing, Burlington, NC).

[0219] In TC-1, constitutive expression of the E7 oncogene was confirmed by PCR using primers directed against the HPV16 E7 open reading frame.

[0220] Upon obtaining B16-F10 from ATCC, the cells are expanded and then cryopreserved in 100 aliquots of 1 million cells each. Rather than maintaining the cell line through continuous passaging, one vial of frozen cells is thawed for each new experiment.

[0221] All antibodies for flow cytometry were purchased from commercial vendors (e.g., BD Biosciences). Antibodies against PD-1 and CTLA-4 for administration to mice were provided by Bio X Cell. Antibody specificity was confirmed by immunoblot analysis of cell extracts overexpressing the target proteins.

[0222] Ibrutinib was provided by Pharmacyclics. Identity was confirmed by liquid chromatography-tandem mass spectrometry performed by the Johns Hopkins Drug Discovery Core.

[0223] Vertebrates: C57BL / 6, B6.SJL, BALB / c, C3H, B6xC3H F1, BALB / cx B6 F1, OT-I, and OT-II mouse strains are all purchased from Jackson Laboratories (Bar Harbor, ME). The vendor provides certificates of authenticity.

[0224] Those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments disclosed and still obtain similar results without departing from the spirit and scope of the invention. The present invention is not limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Accordingly, the present invention is limited only by the scope of the following claims.

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Claims

1. 1. A method for producing a lymphocyte composition for administration to a recipient, said method comprising: a) providing a peripheral blood cell composition obtained from a donor, wherein the donor has been vaccinated against an antigen present in the recipient, and wherein the peripheral blood cell composition comprises CD8+ T-cells, CD4+ T-cells, and natural killer cells; b) depleting the CD8+ T-cells of the peripheral blood cell composition, wherein depleting the CD8+ T-cells of the peripheral blood cell composition reduces the number of CD8+ T-cells in the peripheral blood cell composition by at least one order of magnitude; and c) expanding CD4+ T-cells specific for the antigen by culturing the CD4+ T-cells with the antigen present in the recipient, wherein the donor has at least one human leukocyte antigen (HLA) class II allele match and at least one HLA class II allele mismatch to the recipient in the donor-versus-recipient direction (graft-versus-host direction), thereby producing a lymphocyte composition.

2. 10. The method of claim 1, wherein the antigen is selected from the group consisting of a neoplasm antigen, a neoplasm idiotype, a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a non-human animal antigen, a tumor neoantigen, and combinations thereof.

3. 3. The method of claim 2, wherein the viral antigen is selected from the group consisting of human papillomavirus (HPV) E6 antigen, HPV E7 antigen, and combinations thereof.

4. 3. The method of claim 2, wherein the viral antigen is selected from the group consisting of Epstein-Barr virus latent membrane protein 1 (LMP1), latent membrane protein 2a (LMP2a), and combinations thereof.

5. 3. The method of claim 2, wherein the antigen is a neoplastic antigen or a tumor neoantigen.

6. 6. The method of claim 5, wherein the tumor neoantigen is an antigen derived from a recipient tumor.

7. 10. The method of claim 1, wherein the donor is a cancer-free donor.

8. 10. The method of claim 1, wherein the HLA class II allele match is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1.

9. 2. The method of claim 1, wherein the HLA class II allele mismatch is in a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1.

10. 2. The method of claim 1, wherein the donor vaccinated against an antigen present in the recipient is a donor with CD4+ T-cell immunity against the antigen present in the recipient.

11. 10. The method of claim 1, wherein the recipient has no detectable antibodies reactive to the donor's human leukocyte antigens.

12. 10. The method of claim 1, wherein depleting CD8+ T-cells in the peripheral blood cell composition comprises using an anti-CD8+ antibody bound to magnetic particles, or an anti-CD8+ antibody plus complement.

13. 10. The method of claim 1, wherein subjects selected from the group consisting of the recipient, the donor, and potential allogeneic donors are screened for serological responsiveness to an antigen of an infectious agent selected from the group consisting of a human immunodeficiency virus (HIV) antigen, a hepatitis virus antigen, an Epstein-Barr virus antigen, and a cytomegalovirus antigen.

14. 10. The method of claim 1, wherein the number of CD4+ T-cells in said lymphocyte composition differs from the number of CD4+ T-cells in said peripheral blood cell composition by less than about 50%.

15. 10. The method of claim 1, wherein the number of donor CD4+ T-cells is between about 1 x 105 CD4+ T-cells / kg and about 1 x 109 CD4+ T-cells / kg, based on the recipient's ideal body weight in kilograms (kg).

16. 1. A lymphocyte cell composition for use in treating cancer in a subject, wherein the lymphocyte cell composition is obtained from a peripheral blood cell composition of a donor having at least one human leukocyte antigen (HLA) class II allele match and at least one HLA class II allele mismatch to the recipient in the donor-versus-recipient direction (graft-versus-host direction), wherein the donor has been vaccinated against a viral antigen and / or a tumor neo-antigen present in the subject. wherein the composition is CD8+ T-cell depleted; and wherein the composition comprises an expanded population of CD4+ T-cells specific for the viral antigen and / or the tumor neo-antigen present in the subject, and lymphocyte-depleting chemotherapy is also prescribed to the subject.

17. 17. The lymphocyte cell composition of claim 16, wherein the lymphocyte-depleting chemotherapy is a lymphocyte-reducing, non-lymphocyte-ablative treatment; a treatment that induces transient lymphopenia; a treatment that reduces or inhibits myeloid-derived suppressor cells; a treatment that reduces or inhibits tumor-associated macrophage cells, or a treatment that reduces regulatory T-cells.

18. 17. The lymphocyte cell composition of claim 16, wherein the lymphocyte-depleting chemotherapy is selected from the group consisting of dasatinib, 5-fluorouracil, taxotere, clodronate, gemcitabine, cyclophosphamide, fludarabine, denileukin diftitox, and daclizumab.

19. 10. The method of claim 1, wherein the peripheral blood cell composition is a whole blood product or an apheresis product.

20. 17. The lymphocyte cell composition of claim 16, wherein the peripheral blood cell composition is a whole blood product or an apheresis product.

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