Use of conditioning regimen and antiviral central memory cd8 + anti-suppressive cells for haploid identical stem cell transplantation

Abstract

Provided is the use of a conditioning regimen antiviral central memory CD8 + anti-suppressive cell in stem cell transplantation of haploid identity. Accordingly, a method of treating a disease in a subject in need thereof is provided, where the disease includes pathological cells present in the bone marrow of the subject. The method comprises: conditioning the subject under a pre-transplantation conditioning regimen comprising total bone marrow irradiation; immature hematopoietic cells depleted by T cells are transplanted into the body of the subject; administering a cyclophosphamide to the subject; and administering the anti-non-graft versus host disease inducing cell to the subject.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 420,741, filed on October 31, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Background

[0003] The present invention, in some embodiments thereof, involves the use of antiviral central memory CD8 + Use of counter-suppressor cells in haploidentical stem cell transplantation.

[0004] High-dose chemoradiotherapy “conditioning,” a paradigm that is key to achieving durable remissions of hematological malignancies with allogeneic hematopoietic cell transplantation (allo-HCT), has shifted to an emphasis on the immune graft-versus-malignancy effect of the newly formed donor-derived immune system combined with subsequent immunotherapy. However, reduced conditioning, which preserves a more robust host immune system that can protect patients from lethal infections in the early post-transplant period, is also associated with an increased risk of graft rejection. In haploidentical HCT, this can be overcome by using a non-ex vivo T cell-replete transplant, as alloreactive T cells in the graft are able to attack host T cells and pave the way for engraftment of donor stem cells. However, such alloreactive T cells are also associated with life-threatening graft-versus-host (GVH) reactivity, necessitating the use of potent immunosuppressive drugs after transplantation. In recent years, some progress has been made in reducing the severity of graft-versus-host disease (GVHD) using high-dose cyclophosphamide (CY) after transplantation. However, chronic GVHD remains a problem and requires prolonged use of immunosuppressive drugs, which adversely affects the graft-versus-malignancy effect. Furthermore, the development of GVHD in many patients is associated with thymic dysfunction, further limiting adequate antitumor immunity.

[0005] To overcome this obstacle, 'high-dose' T-cell-depleted haploidentical HCT can be used, which does not carry the risk of GVHD even in the absence of post-transplant immunosuppressive therapy. However, achieving graft engraftment after non-myeloablative (NMA) conditioning remains a significant challenge due to the high levels of anti-donor T-cell clones that survive the pre-transplant conditioning regimen. One approach to addressing this challenge is to use high-dose CY shortly after transplantation (discussed in PCT Publication Nos. WO 2013 / 093920 and WO 2013 / 093919).

[0006] In addition, various methods have been considered for generating tolerance-inducing cells (e.g., anti-suppressor cells) that are depleted of GVH reactivity and used as adjuvant therapy for transplantation (see, e.g., PCT Publication Nos. WO 2001 / 49243, WO 2007 / 023491, WO 2010 / 049935, WO 2012 / 032526, WO 2013 / 035099, WO 2018 / 002924, WO 2017 / 009852, WO 2017 / 009853, WO 2018 / 134824). In addition, a strategy combining non-myeloablative conditioning with high-dose HCT and anti-suppressor cells that are depleted of T cells has been described (PCT Publication No.: WO 2021 / 024264).

[0007] Total bone marrow irradiation (TMI) has been introduced as part of a conditioning regimen before hematopoietic cell transplantation with the goal of reducing the toxicity induced by total body irradiation (TBI). TMI concentrates the dose to the entire skeleton without harming other parts of the body, thereby gradually increasing the dose to the bone marrow within an acceptable toxicity range. For example, TMI has been suggested as part of a low-intensity whole-body conditioning regimen where malignant cell reduction is still necessary; TMI has been suggested as a strategy for delivering myeloablative radiation doses to the whole bone marrow in a conditioning regimen; and has been suggested as part of a regimen using strategies to reduce transplant-related mortality [e.g., Jensen et al., (Biol Blood Marrow Transplant, 2018, 24(2): 301-307) and Welliver et al., (International Journal of Radiation Oncology, Biology, Physics, 2018, 102(3): e370-e371)]. Summary of the Invention

[0008] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject, the method comprising:

[0009] (a) conditioning the subject under a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI); and subsequently

[0010] (b) transplanting a therapeutically effective amount of T cell-depleted immature hematopoietic cells into the subject; and subsequently

[0011] (c) administering to the subject a therapeutically effective amount of cyclophosphamide; and subsequently

[0012] (d) administering to the subject a therapeutically effective amount of cells that suppress non-graft-versus-host disease (GVHD) inducing cells,

[0013] thereby treating the disease in the subject.

[0014] According to an aspect of some embodiments of the present invention, there is provided a combination of a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI), a therapeutically effective amount of T cell-depleted immature hematopoietic cells, a therapeutically effective amount of cyclophosphamide, and a therapeutically effective amount of anti-graft-versus-host disease (GVHD)-inducing cells, for use in treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject.

[0015] According to some embodiments of the invention, the disease is cancer.

[0016] According to some embodiments of the invention, the cancer comprises myeloid malignancies or multiple myeloma.

[0017] According to some embodiments of the invention, the myeloid malignancy is selected from the group consisting of AML, CML and MDS.

[0018] According to some embodiments of the invention, the cancer comprises AML or MDS.

[0019] According to some embodiments of the invention, the disease is non-cancerous.

[0020] According to some embodiments of the invention, the disease is selected from the group consisting of sickle cell anemia, aplastic anemia, thalassemia and metabolic genetic diseases.

[0021] According to some embodiments of the invention, the counter-suppression non-GVHD inducing cells and the T cell-depleted immature hematopoietic cells are obtained from the same donor.

[0022] According to some embodiments of the invention, the conditioning further comprises a therapeutically effective amount of spleen irradiation.

[0023] According to some embodiments of the invention, the TMI is administered on day -7 to -1 before transplanting the T cell-depleted immature hematopoietic cells.

[0024] According to some embodiments of the invention, the therapeutically effective amount of the TMI comprises 8-18 Gy in total.

[0025] According to some embodiments of the invention, the therapeutically effective amount of the TMI is administered in at least 2 doses for consecutive days.

[0026] According to some embodiments of the invention, the therapeutically effective amount of the TMI is administered in 4 doses for consecutive days.

[0027] According to some embodiments of the invention, the T cell-depleted immature hematopoietic cells are derived from a donor that is not syngeneic with the subject.

[0028] According to some embodiments of the invention, the non-syngene is allogeneic.

[0029] According to some embodiments of the invention, the allogeneic donor is an HLA-matched sibling, an HLA-matched unrelated donor, an HLA haploidentical related donor, or a donor displaying one or more different HLA determinants.

[0030] According to some embodiments of the invention, the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises less than 5×10 5 CD3 + T cells / kg ideal body weight of the subject.

[0031] According to some embodiments of the invention, the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises less than 2×10 5 CD3 + T cells / kg ideal body weight of the subject.

[0032] According to some embodiments of the present invention, the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises at least 5×10 6 CD34 + cells / kg of the subject's ideal body weight.

[0033] According to some embodiments of the invention, the therapeutically effective amount of the T cell-depleted immature hematopoietic cells depletes CD3 + and / or CD19 + expressing cells.

[0034] According to some embodiments of the invention, the therapeutically effective amount of the cyclophosphamide comprises 25-200 mg cyclophosphamide per kilogram of the subject's ideal body weight.

[0035] According to some embodiments of the invention, the therapeutically effective amount of the cyclophosphamide is administered to the subject in two doses between 2 and 5 days after the transplantation of the T cell-depleted immature hematopoietic cells.

[0036] According to some embodiments of the invention, the therapeutically effective amount of the cyclophosphamide is administered to the subject in two doses 3 and 4 days after the transplantation of the T cell-depleted immature hematopoietic cells.

[0037] According to some embodiments of the invention, the counter-suppression non-GVHD inducing cells comprise a central memory T lymphocyte (Tcm) phenotype.

[0038] According to some embodiments of the present invention, the counter-suppressive non-GVHD inducing cells have antiviral activity.

[0039] According to some embodiments of the present invention, the counter-suppression non-GVHD inducing cells can be obtained by the following method:

[0040] (i) a first population of peripheral blood mononuclear cells (PBMC) from a donor is subjected to a cytokine receptor capable of binding to CD14 + The cells expressing the antibody were contacted and CD14 that could mature into antigen presenting cells were selected. + expressing cells;

[0041] (ii) loading the antigen-presenting cells with viral peptides;

[0042] (iii) using a drug that can deplete CD4 + 、CD56 + and CD45RA + A second population of PBMCs from the same donor as the first population of PBMCs is treated with one or more agents expressing cells to obtain cells enriched for CD45RA expression. - CD8 + Phenotypic memory T cell populations;

[0043] (iv) contacting the cell population comprising the T cells enriched in the memory T cells with the antigen-presenting cells loaded with the viral peptides of step (ii) in the presence of IL-21, so as to allow enrichment of virus-reactive memory T cells; and

[0044] (v) culturing the cells obtained in step (iv) in the presence of IL-21, IL-15 and / or IL-7 to allow proliferation of cells comprising the Tcm phenotype.

[0045] According to some embodiments of the invention, the therapeutically effective amount of the counter-suppressive non-GVHD inducing cells is administered on day 6-9 after the transplantation of the T cell-depleted immature hematopoietic cells.

[0046] According to some embodiments of the present invention, the therapeutically effective amount of the reverse-suppressive non-GVHD inducing cells comprises at least 2.5×10 6 CD8 + cells / kg of the subject's ideal body weight.

[0047] According to some embodiments of the invention, the subject is not receiving long-term treatment with GVHD prophylaxis after the transplant.

[0048] According to some embodiments of the invention, the conditioning further comprises anti-B cell therapy.

[0049] According to some embodiments of the invention, the anti-B cell therapy comprises an anti-B cell antibody.

[0050] According to some embodiments of the invention, the anti-B cell therapy comprises rituximab.

[0051] According to some embodiments of the invention, the conditioning further comprises T cell depletion.

[0052] According to some embodiments of the invention, the T cell depletion is achieved by antibodies, and optionally wherein the antibodies comprise at least one of anti-thymocyte globulin (ATG) antibodies, anti-CD52 antibodies or anti-CD3 antibodies.

[0053] According to some embodiments of the invention, the T cell depletion is achieved by anti-thymocyte globulin (ATG) antibodies.

[0054] According to some embodiments of the invention, the conditioning further comprises a chemotherapeutic agent.

[0055] According to some embodiments of the invention, the chemotherapeutic agent comprises at least one of fludarabine, busulfan, melphalan, thiotepa, and cyclophosphamide.

[0056] According to some embodiments of the invention, the chemotherapeutic agent comprises fludarabine.

[0057] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification, including definitions, shall prevail. In addition, materials, methods, and examples are illustrative only and are not intended to be necessarily restrictive.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Some embodiments of the present invention are described herein by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is emphasized that the details shown are by way of example and for purposes of illustrative discussion of embodiments of the present invention. In this regard, the description taken in conjunction with the drawings will make it apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0060] In the attached figure:

[0061] Figure 1 is a schematic diagram of a treatment regimen according to some embodiments of the present invention. Optionally, the regimen further comprises administering 375 mg / m2 on day -15. 2 Rituximab.

[0062] Figure 2 The present invention provides an overview of antiviral CD8 + Flowchart of the processing and testing of cells depleted of counter-repressor cells. DETAILED DESCRIPTION

[0063] The present invention, in some embodiments thereof, involves conditioning regimens with antiviral central memory CD8 + Use of counter-suppressor cells in haploidentical stem cell transplantation.

[0064] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

[0065] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not necessarily limited to the details set forth in the following description or illustrated by the examples in its application. The present invention is capable of other embodiments or can be practiced or carried out in various ways. In addition, it should be understood that the phrases and terms used herein are for descriptive purposes only and should not be considered as limiting.

[0066] Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are cancerous diseases that result in insufficient hematopoiesis. Chronic fatigue caused by anemia, bleeding caused by thrombocytopenia, and infection caused by neutropenia are typical consequences of these diseases (Weber et al., Front Immunol. 2020;11:627662). Despite the increasing understanding of these diseases and the emergence of new drugs and new treatment options, the overall survival rate of patients diagnosed with these diseases remains low. For example, the 5-year overall survival rate of patients with AML is less than 30% (Weber et al., Front Immunol. 2020;11:627662).

[0067] While putting the present invention into practice, the present inventors developed a regimen for treating cancers such as AML and MDS. The regimen comprises administering total bone marrow irradiation (TMI) and cyclophosphamide to a subject, and simultaneously transplanting a high-dose T cell-depleted allogeneic HSC transplant and antiviral central memory CD8 +Counter-suppressive cells (see Examples 1-3 of the Examples section below). This approach is expected to be a game changer for allogeneic HSC transplantation to treat diseases such as cancer that involve pathological cells present in the subject's bone marrow.

[0068] Thus, according to one aspect of the present invention, there is provided a method of treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject, the method comprising:

[0069] (a) conditioning the subject under a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI); and subsequently

[0070] (b) transplanting a therapeutically effective amount of T cell-depleted immature hematopoietic cells into the subject; and subsequently

[0071] (c) administering to the subject a therapeutically effective amount of cyclophosphamide; and subsequently

[0072] (d) administering to the subject a therapeutically effective amount of cells that suppress non-graft-versus-host disease (GVHD) inducing cells,

[0073] thereby treating the disease in the subject.

[0074] According to an additional or alternative aspect of the present invention, there is provided a combination of a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI), a therapeutically effective amount of T cell-depleted immature hematopoietic cells, a therapeutically effective amount of cyclophosphamide, and a therapeutically effective amount of anti-graft versus host disease (GVHD) inducing cells, for use in treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject.

[0075] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disorder (ie, a disease such as cancer), substantially ameliorating clinical or aesthetic symptoms of a disorder, or substantially preventing the appearance of clinical or aesthetic symptoms of a disorder.

[0076] According to specific embodiments, the treatment regimen described herein is a first line of treatment.

[0077] According to specific embodiments, the treatment regimen described herein is at least a second line of treatment.

[0078] According to specific embodiments, the treatment regimen described herein is at least a third line of treatment.

[0079] As used herein, the term "subject" or "subject in need thereof" refers to a mammal of any sex and any age, preferably a human, diagnosed with a disease as defined herein.

[0080] According to specific embodiments, the subject is a human subject.

[0081] According to specific embodiments, the subject is at least 12, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 years old, each possibility representing a separate embodiment of the invention.

[0082] According to a specific embodiment, the subject is at least 12 years old.

[0083] According to specific embodiments, the subject is not a suitable candidate for a pre-transplant conditioning regimen employing direct total body irradiation (TBI, in which the entire body is directly exposed to ionizing radiation) because the subject may not tolerate the procedure due to its associated toxic side effects.

[0084] According to a specific embodiment, the subject is not a suitable candidate for a pre-transplant conditioning regimen with immediate TBI because the procedure is considered too aggressive for the disease being treated.

[0085] According to specific embodiments, the subject has received at least one treatment cycle (ie, a treatment regimen other than that described herein) for the disease prior to treatment according to the methods and uses described herein.

[0086] The methods and uses described herein are useful for treating diseases involving pathological cells present in the bone marrow.

[0087] Methods for determining the presence of such cells in the bone marrow are known in the art and include, but are not limited to, histology, immunocytochemistry, flow cytometry, Western blot, single cell genomic analysis, PCR, and the like.

[0088] According to specific embodiments, pathological cells comprise less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% or less than 0.01% of BM cells.

[0089] According to a specific embodiment, a bone marrow aspirate or biopsy sample obtained from a subject is tested for the presence of pathological cells by histology, immunocytochemistry, flow cytometry, Western blot, single cell genomic analysis, and / or PCR.

[0090] According to a specific embodiment, the disease is cancer.

[0091] Non-limiting examples of such cancers include myeloid malignancies [eg, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS)] and multiple myeloma.

[0092] According to specific embodiments, the cancer comprises AML or MDS.

[0093] According to a specific embodiment, the cancer is a primary cancer.

[0094] According to a specific embodiment, the cancer is at least a secondary cancer.

[0095] According to a specific embodiment, the cancer is a refractory or relapsed cancer.

[0096] According to a specific embodiment, the disease is non-cancerous.

[0097] Non-limiting examples of such non-cancerous diseases include sickle cell anemia, aplastic anemia, thalassemia, and metabolic genetic diseases.

[0098] Non-limiting examples of such metabolic genetic diseases are described in, e.g., Tan et al., Front Pediatr. 2019; 7:433, the contents of which are incorporated herein by reference in their entirety, and include, e.g., Hurler syndrome, Hunter, Maroteaux-Lamy, Sly, MLD, GLD, Niemann-Pick, Tay Sachs disease, Sandhoff disease, Farber, Alpha-mannosidosis, Fucosidosis, Aspartylglycosminuria, Wolmann syndrome, Pompe disease.

[0099] The methods and uses described herein include pre-transplant conditioning regimens comprising a therapeutically effective amount of total myeloid irradiation (TMI).

[0100] Conditioning of T cell-depleted immature hematopoietic cells prior to transplantation can be performed under sublethal, lethal, or supralethal conditions.

[0101] As used herein, the terms "sublethal," "lethal," and "supralethal," when referring to the conditioning of subjects, refer to myelotoxic and / or lymphocytotoxic treatments that, when respectively applied to a representative population of subjects, are typically: not lethal to substantially all members of the population; lethal to some but not all members of the population; or lethal to substantially all members of the population under normal sterile conditions.

[0102] According to a specific embodiment, said conditioning is myeloablative.

[0103] According to a specific embodiment, said conditioning is non-myeloablative.

[0104] "Total bone marrow irradiation (TMI)" refers to irradiation of the entire skeleton, allowing the radiation to penetrate the entire bone marrow while significantly minimizing exposure to the rest of the body. It is worth noting that TMI may involve leakage of radiation to other body areas, accounting for about 25% of the total radiation dose. However, according to an embodiment of the present invention, no active TBI application is envisioned. The realization of TMI has benefited from the development of modulated radiotherapy in recent years, which is a technology that can provide radiation doses to large target areas while reducing the doses to non-target organs and organs at risk. Such methods include, but are not limited to, helical tomotherapy (HT), intensity-modulated radiotherapy (IMRT), volumetric arc-based intensity-modulated radiotherapy (VMAT) and 3D conformal radiotherapy (3D-CRT).

[0105] Methods for achieving TMI are known in the art and are disclosed, for example, in the following literature: Jensen et al. (Biol Blood Marrow Transplant, 2018, 24(2):301-307), Welliver et al. (International Journal of Radiation Oncology, Biology, Physics, 2018, 102(3):e370-e371), Mancosu et al. (Physics and Imaging in Radiation Oncology 11(2019)47–53), Wong et al. (Lancet Oncol 2020; 21:e477–87), Chilukuri1 et al. (Radiat Oncol J 2020; 38(3):207-216), Haraldsson et al. (Physica Medica 60(2019)162–167), the contents of which are incorporated herein by reference in their entirety and are further described herein and in the Examples section that follows.

[0106] According to a specific embodiment, TMI is performed with the aid of computed tomography (CT) scanning to define target organs, delineate organs at risk and calculate doses.

[0107] Avoidance organs (ie, non-target organs) include, for example, the lungs, heart, kidneys, liver, esophagus, oral cavity, parotid gland, thyroid gland, eye, lens, brain, stomach, small intestine, rectum, bladder, testicles, and the like.

[0108] Typically, TMI is performed with the subject in the supine position.

[0109] Depending on the specific implementation, the subject is positioned in a full-body vacuum bag or pad with their arms relaxed at their sides in a non-akimbo position, with their fingers gripping the bag. Other devices may be used as needed to ensure patient comfort and thus position reproducibility, such as a face mask, neck support, or cotton pads.

[0110] According to a specific embodiment, the size of the dose grid should be ≤ 3 mm in all directions.

[0111] Depending on the specific embodiment, TMI is achieved using volumetric modulated arc therapy (VMAT) and / or 3D conformal radiation therapy (3D-CRT).

[0112] According to one specific embodiment, TMI is achieved using volumetric modulated arc therapy (VMAT) for the body and 3D conformal radiation therapy (3D-CRT) for the legs.

[0113] As a non-limiting example, for a VMAT body plan, 6 to 12 overlapping arcs with 3 to 6 different isocenters may be used. Larger patients may require more arcs and isocenters depending on their height and anatomy. The isocenters and field sizes of these arcs are determined based on the patient's anatomy to optimize the travel of the multi-leaf collimator. According to a specific embodiment, each arc has an overlap area of ≥2 cm with the arc above it and an overlap area of ≥2 cm with the arc below it.

[0114] As a non-limiting example, for a 3D leg plan, the multi-leaf collimator is matched to the PTV of the beam view angle.

[0115] According to a specific embodiment, there is an overlap region of ≥ 2 cm between the VMAT body plan and the 3D leg plan.

[0116] A non-limiting example of an irradiator includes 6 megavolt (MV) photons delivered by a TrueBeam (Varian Medical Systems, Palo Alto, CA) linear accelerator equipped with a Millennium 120 multi-leaf collimator.

[0117] A detailed description of an irradiation protocol that can be used in specific embodiments of the present invention is provided in Example 1 of the Examples section that follows, which constitutes an integral part of this specification.

[0118] According to specific embodiments, the therapeutically effective amount of TMI comprises a total dose of 5-25 Gy, 5-20 Gy, 5-18 Gy, 8-18 Gy, 8-15 Gy, 10-15 Gy, or 11-14 Gy.

[0119] According to a specific embodiment, the therapeutically effective amount of TMI comprises a total dose of 8-18 Gy.

[0120] According to one embodiment, the therapeutically effective amount of TMI comprises a total dose of about 12 Gy.

[0121] Conditioning can be achieved with a single radiation dose or with fractionated radiation doses.

[0122] Thus, according to specific embodiments, the subject receives at least 2, at least 3, or at least 4 TMIs.

[0123] According to one embodiment, the subject receives 4 TMIs.

[0124] According to a specific embodiment, the dose is administered over several consecutive days.

[0125] According to a specific embodiment, the therapeutically effective amount of TMI is administered in at least 2 doses for several consecutive days.

[0126] According to a specific embodiment, the therapeutically effective amount of TMI is administered in 4 doses over several consecutive days.

[0127] According to specific embodiments, the first dose of TMI is administered to the subject at least 4 days, at least 5 days, at least 6 days, at least 7 days, or at least 10 days prior to T cell-depleted hematopoietic cell transplantation.

[0128] According to specific embodiments, the last dose of TMI is administered to the subject no later than 1 day, no later than 2 days, no later than 3 days, no later than 4 days before T cell-depleted hematopoietic cell transplantation.

[0129] According to a specific embodiment, the subject is administered TMI on day -14 to -1, day -14 to -3, day -14 to -4, day -10 to -1, day -10 to -3, day -10 to -4, day -7 to -1, day -7 to -3, or day -7 to -4 prior to T cell-depleted hematopoietic cell transplantation.

[0130] According to a specific embodiment, the TMI is administered on day -7 to -1 before the transplantation of T cell-depleted immature hematopoietic cells.

[0131] According to one embodiment, the subject is subjected to four TMIs on days -7, -6, -5, and -4 prior to T cell-depleted hematopoietic cell transplantation.

[0132] According to one embodiment, the subject is administered four TMIs at a dose of approximately 3 Gy each on days -7, -6, -5, and -4 prior to T cell-depleted hematopoietic cell transplantation.

[0133] According to specific embodiments, the conditioning further comprises a therapeutically effective amount of splenic irradiation.

[0134] Splenic irradiation can be performed in the same manner as TMI, using the same methods and machinery to mark the spleen as a target organ (as described in further detail above and in the Examples section that follows).

[0135] According to specific embodiments, the therapeutically effective amount of spleen irradiation comprises a total dose of 5-25 Gy, 5-20 Gy, 5-18 Gy, 8-18 Gy, 8-15 Gy, 10-15 Gy, or 11-14 Gy.

[0136] According to a specific embodiment, the therapeutically effective amount of spleen irradiation comprises a total dose of 8-18 Gy.

[0137] According to a specific embodiment, the therapeutically effective amount of spleen irradiation comprises a total dose of about 12 Gy.

[0138] Conditioning can be achieved with a single splenic irradiation dose or with fractionated irradiation doses.

[0139] According to a specific embodiment, spleen irradiation is performed simultaneously with TMI.

[0140] Thus, according to specific embodiments, the subject receives spleen irradiation at least 2 times, at least 3 times, at least 4 times.

[0141] According to one embodiment, the subject receives splenic irradiation four times.

[0142] According to a specific embodiment, the dose is administered over several consecutive days.

[0143] According to a specific embodiment, a therapeutically effective amount of spleen irradiation is administered in at least 2 doses over several consecutive days.

[0144] According to a specific embodiment, the therapeutically effective amount of spleen irradiation is administered in 4 doses over several consecutive days.

[0145] According to a specific embodiment, the subject is administered a first dose of splenic irradiation at least 4 days, at least 5 days, at least 6 days, at least 7 days, or at least 10 days prior to T cell-depleted hematopoietic cell transplantation.

[0146] According to a specific embodiment, the last dose of splenic irradiation is administered to the subject no later than 1 day, no later than 2 days, no later than 3 days, no later than 4 days prior to T cell-depleted hematopoietic cell transplantation.

[0147] Depending on the specific embodiment, the subject is administered splenic irradiation on days -14 to -1, -14 to -3, -14 to -4, -10 to -1, -10 to -3, -10 to -4, -7 to -1, -7 to -3, or -7 to -4 prior to T cell-depleted hematopoietic cell transplantation.

[0148] According to a specific embodiment, spleen irradiation is administered on day -7 to -1 prior to transplantation of T cell-depleted immature hematopoietic cells.

[0149] According to one embodiment, the subject receives spleen irradiation four times on days -7, -6, -5, and -4 prior to T cell-depleted immature hematopoietic cell transplantation.

[0150] According to one embodiment, the subject receives splenic irradiation four times at a dose of about 3 Gy each time on days -7, -6, -5, and -4 before transplantation of T cell-depleted immature hematopoietic cells.

[0151] According to a specific embodiment, the conditioning further comprises a chemotherapeutic agent.

[0152] Exemplary chemotherapeutic agents include, but are not limited to, busulfan, busulfan, cyclophosphamide, fludarabine, melphalan, busulfan, rapamycin, trisulphan, and thiotepa.

[0153] According to a specific embodiment, the chemotherapeutic agent comprises at least one of fludarabine, busulfan, melphalan, thiotepa, and cyclophosphamide.

[0154] One or more chemotherapeutic agents are administered to the subject prior to transplantation in a single dose or in several doses, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses (eg, daily doses).

[0155] According to a specific embodiment, the subject is administered a chemotherapeutic agent (e.g., fludarabine at a dose of 20-40 mg / m2) for 3, 4, 5, or 6 consecutive days (e.g., 4 consecutive days) prior to transplantation (e.g., days -11 to -8 prior to transplantation of T cell-depleted immature hematopoietic cells). 2 / sky).

[0156] Fludarabine is commercially available, for example, under trade names such as Fludara, from, for example, Sanofi Genzyme, Bayer and Teva.

[0157] According to a specific embodiment, the conditioning further comprises in vivo T cell depletion.

[0158] According to some embodiments, the in vivo T cell depletion is effected by antibodies.

[0159] According to some embodiments of the invention, the antibody comprises an anti-C-D8 antibody, an anti-CD4 antibody, or both.

[0160] According to some embodiments of the invention, the antibody comprises at least one of an anti-thymocyte globulin (ATG) antibody, an anti-CD52 antibody, or an anti-CD3 (OKT3) antibody.

[0161] Such antibodies are commercially available, for example ATG is commercially available from companies such as Gzyme and Pfizer, for example under the trade names such as Thymoglobulin and Atgam.

[0162] According to one embodiment, the antibody is administered to the subject in a single dose or in several doses, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses (e.g., daily doses) prior to transplantation.

[0163] According to a specific embodiment, the subject is administered an antibody therapeutic (e.g., ATG at a dose of 0.5-4 mg / kg ideal body weight, e.g., about 2 mg / kg ideal body weight) for 2, 3, 4, or 5 consecutive days (e.g., 3 consecutive days) prior to transplantation (e.g., day -18 to -12, day -14 to -12, or day -12 prior to T cell-depleted immature hematopoietic cell transplantation).

[0164] According to a specific embodiment, pre-transplant conditioning does not include in vivo T cell depletion.

[0165] According to a specific embodiment, the subject has not been treated with ATG prior to transplantation.

[0166] It will be appreciated that higher radiation doses may be used as part of a conditioning regimen when no ATG is used or lower doses of ATG are used, such as a single dose or two doses (eg, each at about 2 mg / kg ideal body weight).

[0167] As used herein, the term "ideal body weight" refers to a measurement that is used clinically to adjust drug dosages, help assess renal function, and pharmacokinetics (eg, in obese patients).

[0168] The formula used to estimate ideal body weight (kg) is as follows:

[0169] Men: IBW = 50 kg + 2.3 kg for every 5 inches increase.

[0170] Women: IBW = 45.5 kg + 2.3 kg for every 5 kg increase.

[0171] Ideal body weight is discussed in detail in Peterson et al. [Am J Clin Nutr 2016;103:1197–203], which is incorporated herein by reference.

[0172] According to a specific embodiment, the conditioning further comprises in vivo anti-B cell therapy.

[0173] Without wishing to be bound by theory, the present inventors propose that anti-B cell therapy may be used to reduce the amount of B cells and thereby reduce the incidence of post-transplant autoimmunity, which may be affected by insufficient regulation of autoantibodies.

[0174] According to some embodiments, anti-B cell therapy is effected by antibodies.

[0175] Such antibodies include, for example, Rituximab, Ocrelizumab, Ofatumumab, Belimumab, Obinutumumab, Epratuzumab, and Ulituximab.

[0176] According to some embodiments of the invention, the antibody is an anti-CD20 antibody.

[0177] According to a specific embodiment, the anti-B cell therapy comprises rituximab.

[0178] Rituximab is commercially available, for example, from Genentech and Roche under trade names such as Mabthera, Rixathon, Truxima, Rituxan.

[0179] According to one embodiment, the subject is administered anti-B cell therapy in a single dose or several doses, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses (e.g., daily doses), prior to cell transplantation (e.g., on days -20 to -4).

[0180] According to a specific embodiment, a single dose of an anti-B cell therapeutic agent (e.g., rituximab, e.g., at a dose of 100-800 mg / m2) is administered to the subject prior to transplantation (e.g., on days -17 to -1, -17 to -7, or -15 prior to transplantation of T cell-depleted immature hematopoietic cells). 2 Ideal body weight, for example, about 375 mg / m 2 ideal weight).

[0181] According to a specific embodiment, pre-transplant conditioning does not include in vivo anti-B cell therapy.

[0182] According to a specific embodiment, the subject is not treated with rituximab prior to transplantation.

[0183] According to specific embodiments, the conditioning does not include direct total body irradiation (TBI, in which the entire body is directly exposed to ionizing radiation) or total lymphatic irradiation (TLI, in which the lymph nodes, spleen, and thymus are directly exposed to ionizing radiation).

[0184] According to a specific embodiment, the conditioning comprises a T-cytoreductive agent, a chemotherapeutic agent, and a TMI.

[0185] According to a specific embodiment, the conditioning comprises anti-B cell therapy, T cell depleting agents, chemotherapeutic agents and TMI.

[0186] According to a specific embodiment, the conditioning comprises T-cytoreductive agents, chemotherapeutic agents, TMI, and splenic irradiation.

[0187] According to a specific embodiment, the conditioning comprises anti-B cell therapy, T cell depletion agents, chemotherapeutic agents, TMI and spleen irradiation.

[0188] According to a specific embodiment, the immunosuppressive agent administered in the conditioning consists of a T cell depleting agent, a chemotherapeutic agent and a TMI.

[0189] According to a specific embodiment, the immunosuppressive agents administered in said conditioning consist of anti-B cell therapy, T cell depleting agents, chemotherapeutic agents and TMI.

[0190] According to a specific embodiment, the immunosuppressive agents administered in said conditioning consist of T-cytoreductive agents, chemotherapeutic agents, TMI and spleen irradiation.

[0191] According to a specific embodiment, the immunosuppressive agents administered in said conditioning consist of anti-B cell therapy, T cell depleting agents, chemotherapeutic agents, TMI and spleen irradiation.

[0192] According to a specific embodiment, the conditioning agent comprises ATG, fludarabine and TMI.

[0193] According to a specific embodiment, the conditioning comprises rituximab, ATG, fludarabine and TMI.

[0194] According to a specific embodiment, the conditioning comprises ATG, fludarabine, TMI and spleen irradiation.

[0195] According to a specific embodiment, the conditioning comprises rituximab, ATG, fludarabine TMI and spleen irradiation.

[0196] According to a specific embodiment, the immunosuppressive agent administered in said conditioning consists of ATG, fludarabine and TMI.

[0197] According to a specific embodiment, the immunosuppressive agent administered in said conditioning consists of rituximab, ATG, fludarabine and TMI.

[0198] According to a specific embodiment, the immunosuppressive agents administered in said conditioning consist of ATG, fludarabine, TMI and spleen irradiation.

[0199] According to a specific embodiment, the immunosuppressive agents administered in said conditioning consist of rituximab, ATG, fludarabine, TMI and spleen irradiation.

[0200] According to a specific embodiment, chemotherapy, T-cytoreductive agents and / or anti-B cell therapy are administered prior to TMI.

[0201] According to a specific embodiment, T-cytoreductive agents and / or anti-B cell therapy are administered prior to chemotherapy.

[0202] According to a specific embodiment, anti-B cell therapy is administered prior to T cell debulking agents.

[0203] According to a specific embodiment, the pre-transplant conditioning regimen includes:

[0204] (i) administering a therapeutically effective amount of a T-cell depleting agent (e.g., ATG, a total dose of about 6 mg / kg) three times a day from day -14 to day -12 before transplantation of T-cell-depleted immature hematopoietic cells;

[0205] (ii) Administer a therapeutically effective amount of chemotherapy (e.g., fludarabine, total dose of about 120 mg / m2) four times daily from days -11 to -8 before transplantation of T cell-depleted immature hematopoietic cells. 2 );and

[0206] (iii) administering a therapeutically effective amount of TMI in a total dose of about 12 Gy in 4 daily doses from day -7 to day -4 before transplantation of T cell-depleted immature hematopoietic cells, and optionally irradiating the spleen.

[0207] According to a specific embodiment, the pre-transplant conditioning regimen comprises:

[0208] (i) Administer a therapeutically effective amount of anti-B cell therapy (e.g., rituximab at a dose of approximately 375 mg / m2) as a single dose on day -15 before transplantation of T cell-depleted immature hematopoietic cells. 2 ideal body weight);

[0209] (ii) administering a therapeutically effective amount of a T-cytoreductive agent (e.g., ATG, a total dose of about 6 mg / kg) three times a day from day -14 to day -12 before transplantation of T-cell-depleted immature hematopoietic cells;

[0210] (iii) Administer a therapeutically effective amount of chemotherapy (e.g., fludarabine, total dose of about 120 mg / m2) four times daily from days -11 to -8 before transplantation of T-cell-depleted immature hematopoietic cells. 2 );and

[0211] (iv) administering a therapeutically effective amount of TMI in a total dose of about 12 Gy in 4 daily doses from day -7 to day -4 before transplantation of T cell-depleted immature hematopoietic cells, and optionally irradiating the spleen.

[0212] The methods and uses described herein comprise transplanting a therapeutically effective amount of T cell-depleted immature hematopoietic cells.

[0213] According to a specific embodiment, the transplantation is performed after conditioning.

[0214] The T cell-depleted immature hematopoietic cells of some embodiments of the invention can be transplanted into a subject using any method known in the art for cell transplantation, such as, but not limited to, cell infusion (eg, intravenous injection) or via the intraperitoneal route.

[0215] After the cells are transplanted into a subject according to the present teachings, it is recommended that the growth function and immunocompatibility of the cells be monitored using various standard techniques according to standard medical practice. For example, the number of immature hematopoietic cells in the subject can be monitored by standard blood and bone marrow tests (e.g., by FACS analysis).

[0216] As used herein, the phrase "immature hematopoietic cells" refers to hematopoietic tissue or cell preparations containing precursor hematopoietic cells (e.g., hematopoietic stem cells). Such tissue / cell preparations include or are derived from biological samples, e.g., bone marrow, mobilized peripheral blood (e.g., mobilized CD34 + cells to increase their concentration), umbilical cord blood (e.g., umbilical cord), fetal liver, yolk sac, and / or placenta. + cells or other hematopoietic stem cells such as CD131 + Cells can be used according to embodiments of the present teachings with or without in vitro expansion.

[0217] According to one embodiment, the immature hematopoietic cells include T cell-depleted immature hematopoietic cells.

[0218] As used herein, the phrase "T cell-depleted immature hematopoietic cells" refers to a population of precursor hematopoietic cells that are depleted of T lymphocytes. T cell-depleted immature hematopoietic cells may include, for example, CD34 + 、CD33 + and / or CD56 + T cell-depleted immature hematopoietic cells can be depleted of CD3 + cells, CD2 + cells, CD8 + cells, CD4 + cells, α / β T cells and / or γ / δ T cells.

[0219] According to one embodiment, immature hematopoietic cells include cells rich in CD34 + T cell depletion of immature hematopoietic cells by mobilized blood cells.

[0220] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises at least about 0.1×10 6 CD34 + cells, 0.5×10 6 CD34 + cells, 1×10 6 CD34 + cells, 2×10 6 CD34 + cells, 3×10 6 CD34 + cells, 4×10 6 CD34 + cells, 5×10 6 CD34 + cells, 6×10 6 CD34 + cells, 7×10 6 CD34 + cells, 8×10 6 CD34 + cells, 9×10 6 CD34 + cells, 10×10 6 CD34 + cells, 15×10 6 CD34 + cells or 20×10 6 CD34 + cells / kg of the subject's ideal body weight.

[0221] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises at least 2.5-20×10 6 CD34 + cells (e.g. 5-10×10 6 CD34 + cells) / kg of the subject's ideal body weight.

[0222] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises at least 5×10 6 CD34 + cells / kg of the subject's ideal body weight.

[0223] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises at least 6×10 6 CD34 + cells / kg of the subject's ideal body weight.

[0224] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises at least 8×10 6 CD34 + cells / kg of the subject's ideal body weight.

[0225] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises at least 10×10 6 CD34 + cells / kg ideal body weight of the subject.

[0226] According to one embodiment, immature hematopoietic cells are depleted of CD3 + and / or CD19 + cell.

[0227] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than about 50×10 5 CD3 + cells, 40×10 5 CD3 + cells, 30×10 5 CD3 + cells, 20×10 5 CD3 + cells, 15×10 5 CD3 + cells, 10×10 5 CD3 + cells, 9×10 5 CD3 + cells, 8×10 5 CD3 + cells, 7×10 5 CD3 + cells, 6×10 5 CD3 + cells, 5×10 5 CD3 + cells, 4×10 5 CD3 + cells, 3×10 5 CD3 + cells, 2×10 5 CD3 + cells, 1×10 5 CD3 + cells, 0.5×10 5 CD3 + cells or 0.1×10 5 CD3 + cells / kg of the subject's ideal body weight.

[0228] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 1-5×10 5 CD3 + cells (e.g. 2-5×10 5 CD3 + cells) / kg of the subject's ideal body weight.

[0229] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 5×10 5 CD3 + cells / kg of the subject's ideal body weight.

[0230] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 4×10 5 CD3 + cells / kg of the subject's ideal body weight.

[0231] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 3×10 5 CD3 + cells / kg of the subject's ideal body weight.

[0232] According to a specific embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 2×10 5 CD3 + cells / kg of the subject's ideal body weight.

[0233] According to one embodiment, immature hematopoietic cells are depleted of CD8 + cell.

[0234] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 1×10 4 -5×10 5 CD8 + cells (e.g. 0.1-4×10 5 CD8 + cells, e.g. 1-3 × 10 5 CD8 + cells) / kg of the subject's ideal body weight.

[0235] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than about 50×10 5 CD8 + cells, 25×10 5 CD8 + cells, 15×10 5 CD8+ cells, 10×10 5 CD8 + cells, 9×10 5 CD8 + cells, 8×10 5 CD8 + cells, 7×10 5 CD8 + cells, 6×10 5 CD8 + cells, 5×10 5 CD8 + cells, 4×10 5 CD8 + cells, 3×10 5 CD8 + cells, 2×10 5 CD8 + cells, 1×10 5 CD8 + cells, 9×10 4 CD8 + cells, 8×10 4 CD8 + cells, 7×10 4 CD8 + cells, 6×10 4 CD8 + cells, 5×10 4 CD8 + cells, 4×10 4 CD8 + cells, 3×10 4 CD8 + cells, 2×10 4 CD8 + cells or 1×10 4 CD8 + cells / kg of the subject's ideal body weight.

[0236] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 5×10 5 CD8 + cells / kg of the subject's ideal body weight.

[0237] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 4×10 5 CD8 + cells / kg of the subject's ideal body weight.

[0238] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 3×10 5 CD8 + cells / kg of the subject's ideal body weight.

[0239] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than about 1×10 6 CD8 + TCRα / β - cells, 0.5×10 6 CD8 + TCRα / β - cells, 1×10 5 CD8 + TCRα / β - cells, 0.5×10 5 CD8 + TCRα / β - cells, 1×10 4 CD8 + TCRα / β - cells, 0.5×10 4 CD8 + TCRα / β - cells, 1×10 3 CD8 + TCRα / β - cells or 0.5×10 3 CD8 + TCRα / β - cells / kg of the subject's ideal body weight.

[0240] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 1×10 5 -1×10 6 CD8 + TCRα / β - cells / kg of the subject's ideal body weight.

[0241] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 1×10 6 CD8 + TCRα / β - cells / kg of the subject's ideal body weight.

[0242] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 5×10 5 CD8 + TCRα / β -cells / kg of the subject's ideal body weight.

[0243] According to one embodiment, the therapeutically effective amount of T cell-depleted immature hematopoietic cells comprises less than 1×10 5 CD8 + TCRα / β - cells / kg of the subject's ideal body weight.

[0244] According to one embodiment, the immature hematopoietic cells are depleted of B cells.

[0245] According to one embodiment, immature hematopoietic cells are depleted of B cells (CD19 + and / or CD20 + B cells).

[0246] According to one embodiment, the therapeutically effective amount of immature hematopoietic cells comprises less than about 50×10 5 CD19 + and / or CD20 + cells, 40×10 5 CD19 + and / or CD20 + cells, 30×10 5 CD19 + and / or CD20 + cells, 20×10 5 CD19 + and / or CD20 + cells, 10×10 5 CD19 + and / or CD20 + cells, 9×10 5 CD19 + and / or CD20 + cells, 8×10 5 CD19 + and / or CD20 + cells, 7×10 5 CD19 + and / or CD20 + cells, 6×10 5 CD19 + and / or CD20 + cells, 5×10 5 CD19 + and / or CD20 + cells, 4×10 5 CD19 + and / or CD20 + cells, 3×10 5 CD19+ and / or CD20 + cells, 2×10 5 CD19 + and / or CD20 + cells or 1×10 5 CD19 + and / or CD20 + cells / kg of the subject's ideal body weight.

[0247] According to one embodiment, the therapeutically effective amount of immature hematopoietic cells comprises less than 1-5×10 5 CD19 + and / or CD20 + Thin (e.g. 3-5×10 5 CD19 + and / or CD20 + cells) / kg of the subject's ideal body weight.

[0248] According to one embodiment, the therapeutically effective amount of immature hematopoietic cells comprises less than 4×10 5 CD19 + and / or CD20 + Ideal body weight in kg / kg.

[0249] According to one embodiment, the therapeutically effective amount of immature hematopoietic cells comprises less than 3×10 5 CD19 + and / or CD20 + cells / kg of the subject's ideal body weight.

[0250] According to one embodiment, the therapeutically effective amount of immature hematopoietic cells comprises less than 2×10 5 CD19 + and / or CD20 + cells / kg of the subject's ideal body weight.

[0251] According to a specific embodiment, the T cell-depleted immature hematopoietic cells comprise two or more cell batches, for example comprising CD34 + The first batch of cells was selected and contained CD3 + / CD19 + A second batch of depleted cells (ie, obtained from the same donor). It will be appreciated that these cells can be used simultaneously or sequentially (eg, on the same day or, for example, within about 1, 2, 3, 4, 5, 6, 7 days, as discussed below).

[0252] T cells (e.g. CD3 + 、CD2 + 、TCRα / β+ 、CD4 + and / or CD8 + cells), or B cells (e.g. CD19 + and / or CD20 + Depletion of the cells) can be performed using any method known in the art, such as by eradication (e.g., killing) with specific antibodies or by affinity-based purification, such as by using magnetic cell separation techniques, FACS sorters, and / or capture ELISA labels.

[0253] Such methods are further described herein and in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Volumes 1 to 4, (DN Weir, ed.) and FLOW CYTOMETRY AND CELL SORTING (A. Radbruch, ed., Springer Verlag, 1992). For example, cells can be sorted by, for example, flow cytometry or FACS. Thus, fluorescence activated cell sorting (FACS) can be used, and can have varying degrees of color channels, low angle and dull light scattering detection channels, and impedance channels. Any ligand-dependent separation technique known in the art can be used in combination with both positive and negative separation techniques, and the separation technique relies on the physical properties of the cells rather than antibody affinity, including but not limited to elution and density gradient centrifugation.

[0254] Other methods for cell sorting include, for example, panning and separation using affinity techniques, including those using solid supports such as plates, beads, and columns. Thus, a biological sample can be separated by "panning" with antibodies attached to a solid matrix such as a plate.

[0255] Alternatively, cells can be sorted / separated by magnetic separation techniques, and some of these methods utilize magnetic beads. Different magnetic beads are available from many sources, including, for example, Dynal (Norway), Advanced Magnetics (Cambridge, Massachusetts, USA), Immuncon (Philadelphia, USA), Immunotec (Marseille, France), Invitrogen, Stem cell Technologies (USA), and Cellpro (USA). Alternatively, the antibody can be biotinylated or conjugated to digoxigenin and used in conjunction with an affinity column coated with avidin or anti-digoxigenin.

[0256] According to one embodiment, different depletion / separation methods can be combined, for example, magnetic cell sorting can be combined with FACS to increase the quality of separation or allow sorting by multiple parameters.

[0257] According to a specific embodiment, the method comprises obtaining T cell-depleted immature hematopoietic cells.

[0258] According to a specific embodiment, T cell-depleted immature hematopoietic cells are obtained by a method comprising collecting mobilized PBMCs from a donor subject.

[0259] The term "peripheral blood mononuclear cells (PBMC)" refers to a portion of a blood sample that contains lymphocytes (including T cells, B cells, NK cells, etc.) and monocytes.

[0260] The term "mobilized PBMCs" refers to PBMCs obtained after administration of induced stem cells mobilized from the bone marrow into the blood. Such agents are well known in the art and include, but are not limited to, G-CSF, GM-SCF, plyrixafor, T140, and T134.

[0261] According to one embodiment, mobilization is effected by G-CSF.

[0262] According to one embodiment, mobilization is effected by G-CSF and plerixafor.

[0263] According to a specific embodiment, the collection of mobilized PBMCs is obtained by a single collection.

[0264] According to a specific embodiment, the collection of mobilized PBMCs is obtained by collecting two, three, four, five or more times a day, such as twice a day (eg, on consecutive days or on alternate days).

[0265] According to one embodiment, the 6 CD34 + cells, for example 2 × 10 6 CD34 + cells, for example 1×10 6 CD34 + A spare fraction of unmodified mobilized PBMCs (eg, obtained from the first collection) at 10 cells / kg ideal body weight is set aside and stored frozen.

[0266] According to one embodiment, PBMCs expressing CD34 can be enriched by incubating them with a CD34 binding agent. + cells.

[0267] According to a specific embodiment, the CD34 binding agent is an antibody.

[0268] According to a specific embodiment, the CD34 antibody is a monoclonal antibody.

[0269] According to one embodiment, the CD34 monoclonal antibody is conjugated to magnetic particles.

[0270] According to a specific embodiment, the CD34 monoclonal antibody is conjugated to superparamagnetic particles.

[0271] According to one embodiment, CD34 + Labeled cells are selected by magnetic separation.

[0272] According to a specific embodiment, CD34 magnetically labeled cells (i.e., CD34 + expressing cells) are retained by the separation column (i.e., positive selection), and CD34 - The cells were then released from the column and collected + According to one embodiment, samples are taken from each fraction for cell counting, viability, and / or immunophenotyping.

[0273] According to one embodiment, mobilized PBMCs are depleted of platelets using, for example, COBE 2991.

[0274] According to one embodiment, the platelet-depleted PBMC preparation of one embodiment is incubated with IVIg for 5-30 minutes, such as 10-15 minutes. + and CD19 + The binding agent is added to the cell preparation and incubated on an orbital rotator for, eg, 10-60 minutes, eg, 30 minutes.

[0275] According to a specific embodiment, the CD3 and / or CD19 binding agent is an antibody.

[0276] According to a specific embodiment, the CD3 and / or CD19 antibody is a monoclonal antibody.

[0277] According to a specific embodiment, CD3 and / or CD19 monoclonal antibodies are conjugated to magnetic particles.

[0278] According to a specific embodiment, CD3 and / or CD19 monoclonal antibodies are conjugated to superparamagnetic particles.

[0279] According to one embodiment, after the incubation is complete, the cells are washed by centrifugation and the cell pellet is resuspended in a buffer (eg COBE 2991) to remove excess reagents.

[0280] According to one embodiment, CD3 + / CD19 + Labeled cells are selected by magnetic separation.

[0281] According to a specific embodiment, CD3 + / CD19 + Labeled cells in Processed on column.

[0282] According to a specific embodiment, CD3 + / CD19 + Magnetic labeled cells (i.e., CD3 + / CD19 + expressing cells) are retained by the separation column (i.e., negative selection), and CD4 - / CD56 - / CD45RA - According to one embodiment, the collected cells (CD3 - / CD19 - According to one embodiment, samples from each fraction are taken out for cell counting, viability and / or immunophenotyping.

[0283] According to one embodiment, wherein more than about 1×10 5 CD3 to 5 × 10 5 CD3, for example 1 × 10 5 CD3 to 3 × 10 5 CD3, for example 2.5 × 10 5 CD3, for example 2 × 10 5 In the case where / kg ideal body weight is present in the collected cell fraction, a second CD3 + Depletion step.

[0284] Alternatively, utilize only a portion of the T cell depleted fraction for infusion in order to avoid infusion of more than 5 × 10 5 CD3, for example 3 × 10 5 CD3, for example 2 × 10 5 Infusion of CD3 cells / kg ideal body weight.

[0285] T cell-depleted immature hematopoietic cells can be used as fresh cells (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, such as within about 3 days).

[0286] Alternatively, cells can be cryopreserved until needed (e.g., 1 day, 2-6 days, 1 week, 2 weeks, 1 month, 2 months, 4 months, 6 months, 1 year, or longer).

[0287] The protocols of some embodiments of the present invention are generally used for non-syngeneic applications, and therefore, the T cell-depleted immature hematopoietic cells (and / or counter-suppressive non-GVHD inducing cells and / or PBMCs, as further described below) are generally allogeneic with respect to the recipient subject (e.g., from an allogeneic donor). Similarly, in cases where xenogeneic applications may be beneficial, the cells used may be of xenogeneic origin as described below. However, in cases where syngeneic applications may be beneficial, the cells used may be autologous with respect to the recipient subject (e.g., from a subject). Such determinations are well within the capabilities of those skilled in the art, especially in light of the disclosure provided.

[0288] Thus, the T cell-depleted immature hematopoietic cells can be syngeneic or non-syngeneic with respect to the subject.

[0289] As used herein, the term "syngeneic" cell refers to a cell that is substantially genetically identical to a subject or substantially all of a subject's lymphocytes. Examples of syngeneic cells include cells derived from the subject (also referred to in the art as "autologous"), derived from a clone of the subject, or derived from an identical twin of the subject.

[0290] As used herein, the term "non-syngeneic" cell refers to a cell that is not substantially genetically identical to the subject or substantially all of the subject's lymphocytes (eg, an allogeneic cell or a xenogeneic cell).

[0291] According to a specific embodiment, the T cell-depleted immature hematopoietic cells are derived from a donor that is non-syngeneic to the subject.

[0292] According to a specific embodiment, non-syngeneic is allogeneic.

[0293] As used herein, the term "allogeneic" refers to cells derived from a donor subject that is of the same species as the recipient subject but is substantially non-clonal to the recipient subject. Typically, non-zygotic twin mammals of the same species are allogeneic to each other. It should be understood that allogeneic cells can be HLA-identical, partially HLA-identical, or HLA-incompatible (i.e., display one or more different HLA determinants) relative to the recipient subject. According to specific embodiments, the allogeneic donor is an HLA-matched sibling, an HLA-matched unrelated donor, a related donor with identical HLA units, or a donor displaying one or more different HLA determinants.

[0294] According to one embodiment, the donor is a human.

[0295] As used herein, the term "xenogeneic" refers to cells that express substantially different kinds of antigens relative to the basic proportions of the subject's lymphocytes. Typically, outbred mammals of different species are xenogeneic to each other.

[0296] The present invention contemplates that xenogeneic cells are derived from various species. Therefore, according to one embodiment, cells can be derived from any mammal. The suitable species origin of the cell comprises main domesticated animals or livestock animals and primates. Such animals include but are not limited to porcines (e.g., pigs), bovines (e.g., cattle), equines (e.g., horses), ovines (e.g., goats, sheep), felines (e.g., house cats (Felis domestica)), canines (e.g., Canis domestica), rodents (e.g., mice, rats, rabbits, guinea pigs, gerbils, hamsters) and primates (e.g., chimpanzees, rhesus monkeys, macaques, marmosets). Cells of xenogeneic origin (e.g., porcines origin) are preferably derived from sources without known zoonotic diseases, such as porcine endogenous retroviruses. Similarly, cells or tissues derived from humans are preferably obtained from sources that are substantially free of pathogens.

[0297] The methods and uses described herein comprise administering a therapeutically effective amount of cyclophosphamide.

[0298] According to a specific embodiment, the administration of cyclophosphamide is performed after transplantation of T cell-depleted immature hematopoietic cells.

[0299] According to one embodiment, in addition to the post-transplantation administration described herein, the present invention further contemplates administration of cyclophosphamide prior to transplantation (eg, on days 6, 5, 4, or 3 prior to transplantation, ie, D-6 to -3).

[0300] According to specific embodiments, the therapeutically effective amount of cyclophosphamide comprises about 1-25 mg, 1-50 mg, 1-75 mg, 1-100 mg, 1-250 mg, 1-500 mg, 1-750 mg, 1-1000 mg, 5-50 mg, 5-75 mg, 5-100 mg, 5-250 mg, 5-500 mg, 5-750 mg, 5-1000 mg, 10-50 mg, 10-75 mg, 10-100 mg, 10-2 50mg, 10-500mg, 10-750mg, 10-1000mg, 25-50mg, 25-75mg, 25-100mg, 25-125mg, 25-200mg, 25-300mg , 25-400mg, 25-500mg, 25-750mg, 25-1000mg, 50-75mg, 50-100mg, 50-125mg, 50-150mg, 50-175mg, 50 -200mg, 50-250mg, 50-500mg, 50-1000mg, 75-100mg, 75-125mg, 75-150mg, 75-250mg, 75-500mg, 75-1 000mg, 100-125mg, 100-150mg, 100-200mg, 100-300mg, 100-400mg, 100-500mg, 100-1000mg, 125-150 mg, 125-250mg, 125-500mg, 125-1000mg, 150-200mg, 150-300mg, 150-500mg, 150-1000mg, 200-300mg, 200-400mg, 200-500mg, 200-750mg, 200-1000mg, 250-500mg, 250-750mg, 250-1000mg / kg of the subject's ideal body weight.

[0301] According to one embodiment, the therapeutically effective amount of cyclophosphamide is about 25-200 mg / kg of the subject's ideal body weight.

[0302] According to one embodiment, cyclophosphamide is administered in a single dose.

[0303] According to one embodiment, cyclophosphamide is administered in multiple doses, for example in 2, 3, 4, 5 or more doses.

[0304] According to a specific embodiment, cyclophosphamide is administered in two doses.

[0305] The dose of each cyclophosphamide administration can comprise about 5 mg, 7.5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg or 500 mg per kilogram of the subject's ideal body weight.

[0306] According to a specific embodiment, each dose of cyclophosphamide is 50 mg / kg of the subject's ideal body weight.

[0307] According to a specific embodiment, cyclophosphamide may be administered to the subject between day 2 and day 5 following transplantation of the T cell-depleted immature hematopoietic cells.

[0308] According to a specific embodiment, cyclophosphamide is administered to the subject in two doses 3 days and 4 days after the transplantation of T cell-depleted immature hematopoietic cells.

[0309] Cyclophosphamide is commercially available under the trade names Endoxan, Cytoxan, Neosar, Procytox and Revimmune from, for example, Zydus (German Remedies), Roxane Laboratories Inc - Boehringer Ingelheim, Bristol-Myers Squibb Co - Mead Johnson and Co and Pfizer - Pharmacia & Upjohn.

[0310] According to one embodiment, the subject is treated with additional supportive medications such as adjuncts to chemotherapy.

[0311] According to one embodiment, the subject is treated with a dose of mesna (e.g., 10 mg / kg via intravenous piggy back (IVPB)) immediately prior to the first dose of cyclophosphamide (e.g., 2 hours, 1 hour, 30 minutes, 15 minutes prior to the first dose of cyclophosphamide). According to one embodiment, the administration of mesna is repeated every 4 hours for a total of 10 doses.

[0312] Mesna is commercially available under the trade names Uromitexan and Mesnex from, for example, Baxter.

[0313] According to one embodiment, the subject is treated with ondansetron (or another antiemetic) prior to each dose of cyclophosphamide (Cy).

[0314] According to one embodiment, the subject is not being treated with an immunosuppressive agent (eg, other than CY and counter-suppressive cells as discussed herein).

[0315] According to one embodiment, the subject is treated with an immunosuppressant.

[0316] Examples of immunosuppressants include, but are not limited to, tacrolimus (also known as FK-506 or tacrolimus, trade names: Prograf, Advagraf, Protopic), mycophenolate mofetil, mycophenolate sodium, prednisone, methotrexate, cyclophosphamide, cyclosporine, cyclosporine A, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNFα blockers, biologics targeting inflammatory cytokines, and nonsteroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenac, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus) and rapamycin analogs (e.g., CCI-779, RAD001, AP23573). These agents may be administered alone or in combination.

[0317] According to one embodiment, no corticosteroids are administered as pre-treatment to the counter-suppressed cells.

[0318] According to a specific embodiment, the subject is not treated long-term (e.g., for an extended period of time, e.g., greater than 8, 9, 10, 12, 14, 21, 25, 30, 45, 60, or 90 days, e.g., 10-21 days, e.g., 10 or 14 days) with GVHD prophylaxis (e.g., with corticosteroids and / or immunosuppressants) after transplantation.

[0319] According to one embodiment, in the case of recurrence after hematopoietic stem cell transplantation, the subject can be further treated by donor lymphocyte infusion (DLI).For example, the subject can be as previously described by the people such as Dazzi [Dazzi, Szydlo et al., Blood, (2000) 96:2712-6], with the T cell of graded dose being applied, and described reference is incorporated herein by reference in its entirety.

[0320] The methods and uses disclosed herein comprise administering a therapeutically effective amount of cells that counter-suppress non-graft versus host disease (GVHD) inducing cells.

[0321] According to a specific embodiment, the counter-suppressive non-GVHD inducing cells are administered after transplantation of T cell-depleted immature hematopoietic cells.

[0322] According to a specific embodiment, the counter-suppressive non-GVHD inducing cells are administered after the administration of cyclophosphamide.

[0323] The counter-suppressive non-GVHD inducing cells of some embodiments of the present invention can be transplanted into a subject using any method known in the art for cell transplantation, such as, but not limited to, cell infusion (eg, intravenous injection) or via the intraperitoneal route.

[0324] The term "anti-suppressor cell" relates to an immune cell (e.g., a donor-derived T cell) that, upon recognition and binding to the anti-suppressor cell, results in the inactivation of anti-donor recipient T cells. According to one embodiment, the inactivation results in apoptosis of the anti-donor recipient T cells. According to a specific embodiment, the anti-suppressor cell is a T cell.

[0325] In general, a particular feature of countersuppressor cell biology is that the specificity of the countersuppressor activity is achieved by unidirectional recognition of the countersuppressor cell by a responder T cell (i.e., an anti-donor recipient T cell directed against the countersuppressor cell), which is killed upon binding to its countersuppressor target due to the exchange of signals following this interaction. Thus, for example, when the countersuppressor cell is a T cell, although the countersuppressor cell contains its own specific TCR, the specificity of the countersuppressor activity is not determined by the TCR of the countersuppressor cell, but rather by the TCR of the anti-donor recipient T cell in a TCR-independent manner.

[0326] Anti-suppressive cells are also known as tolerance-inducing cells.

[0327] As used herein, the phrase "tolerance-inducing cells" refers to cells that, when contacted with donor cells, induce reduced responsiveness of recipient cells (e.g., recipient's T cells) compared to the responsiveness of the recipient cells in the absence of the administered tolerance-inducing cells.

[0328] The counter-suppressor cells disclosed herein are non-graft versus host disease (GVHD) inducing cells.

[0329] As used herein, the term "non-graft versus host disease" or "non-GVHD" refers to cells that have substantially reduced or no graft versus host (GVH) induction reactivity. Thus, the cells of some embodiments of the present invention do not significantly cause graft versus host disease (GVHD), as demonstrated by survival, weight, and overall appearance of the transplanted subject within 30-120 days after transplantation. Methods for assessing subjects for reduced GVHD are well known to those skilled in the art.

[0330] A large number of GVH-free suppressor cells have been described in the art, any of which can be used with embodiments of the present invention. Such suppressor cells include, for example, those described in PCT Publication Nos. WO2001 / 049243, WO2002 / 102971, WO2007 / 023491, WO2010 / 049935, WO2012 / 032526, WO2013 / 035099, WO2018 / 002924, WO2017 / 009852, WO2017 / 009853, WO2018 / 134824, and WO2021 / 024264, the contents of which are incorporated herein by reference in their entirety.

[0331] According to one embodiment, the counter-suppressive non-GVHD inducing cells are not naturally occurring and are not natural products. These cells are typically generated by ex vivo manipulation (e.g., exposure to one or more antigens in the presence of specific cytokines).

[0332] According to a specific embodiment, the counter-suppression non-GVHD inducing cells comprise a central memory T lymphocyte (Tcm) phenotype.

[0333] As used herein, the phrase "central memory T lymphocyte (Tcm) phenotype" or "Tcm cells" refers to a subset of T cells that home to lymph nodes. In humans, cells with the Tcm phenotype typically contain CD3 + / CD8 + / CD62L + / CD45RO + / CD45RA - Characteristics. It should be understood that Tcm cells may express all characteristic markers on a single cell, or may express only some characteristic markers on a single cell. Cell phenotypes can be determined using any method known to those skilled in the art, such as by fluorescence activated cell sorting (FACS) or capture ELISA labeling.

[0334] According to one embodiment, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% of the counter-suppression non-GVHD inducing cells have Tcm cell characteristics.

[0335] According to a specific embodiment, the cells having the Tcm phenotype comprise about 20%, about 30%, about 40%, about 50%, about 60% or about 70% of counter-suppressive non-GVHD inducing cells.

[0336] According to a specific embodiment, about 10-20%, about 10-30%, about 10-40%, about 10-50%, about 20-30%, about 20-40%, about 30-50%, about 40-60%, about 50-70%, about 60-80%, about 70-90%, about 80-100%, or about 90-100% of the counter-suppressive non-GVHD inducing cells have Tcm cell characteristics.

[0337] As mentioned, Tcm cells typically home to lymph nodes after transplantation. According to some embodiments, the counter-suppressive non-GVHD-inducing cells of some embodiments of the present invention can home to any lymph node after transplantation, such as, for example, peripheral lymph nodes and mesenteric lymph nodes. The homing properties of these cells allow them to exert their counter-suppressive effect in a rapid and efficient manner.

[0338] Additionally or alternatively, the counter-suppression non-GVHD inducing cells of some embodiments of the invention comprise anti-disease activity.

[0339] The term "anti-disease activity" refers to the function of a cell against a diseased cell. The anti-disease activity may be directly directed against the diseased cell, e.g., the ability to kill the diseased cell. When the cell is a T cell, this activity may be due to TCR-dependent or TCR-independent killing [e.g., mediated by LFA1-I / CAM1 binding [Arditti et al., Blood (2005) 105(8):3365-71. Epub 2004 Jul 6]. Additionally or alternatively, the anti-disease activity may be indirect, e.g., by activating other types of cells (e.g., CD4 + T cells, B cells, monocytes, macrophages, NK cells), which cause the death of the diseased cells (e.g., by killing, apoptosis, or by secretion of other factors such as antibodies, cytokines, etc.).

[0340] Diseased cells can include, for example, virus-infected cells, bacteria-infected cells, cancer cells [such as cells of a solid tumor or leukemia / lymphoma cells, also referred to herein as cells with graft-versus-leukemia (GVL) activity], cells associated with an autoimmune disease, cells associated with an allergy, or cells altered due to stress, radiation, or age.

[0341] Thus, according to specific embodiments, the counter-suppressive non-GVHD inducing cells have antiviral, anti-bacterial, anti-fungal and / or anti-cancer activity.

[0342] According to one embodiment, the counter-suppressing non-GVHD inducing cells have antiviral activity.

[0343] The term "antiviral activity" refers to the function of a cell against virus-infected cells (e.g., expression of one or more viral antigens in the context of an MHC-peptide complex on the cell surface). Typically, antiviral activity results in the killing of virus-infected cells.

[0344] According to a specific embodiment, the counter-suppressing non-GVHD inducing cells have anti-cancer activity.

[0345] The term "anti-cancer activity" refers to the function of a cell against cancer cells. Typically, anti-cancer activity results in the killing of cancer cells. According to one embodiment, the anti-cancer activity comprises graft versus leukemia / lymphoma (GVL) activity.

[0346] Thus, according to specific embodiments, the counter-suppressing non-GVHD inducing cells specifically recognize a third party antigen (eg, a T cell comprising a TCR specific for the third party antigen).

[0347] "Third party antigen" refers to an antigen that is not present in healthy cells of the donor or recipient.

[0348] Such antigens may be, for example, viral peptides, bacterial peptides, fungal peptides and / or cancer / tumor peptides.

[0349] According to some embodiments, depending on the desired application (e.g., the disease to be treated), the counter-suppression non-GVHD-inducing cells can be non-genetically modified cells or genetically modified cells (e.g., cells that have been genetically engineered to express or not express a specific gene, marker, or peptide, or to secrete or not secrete a specific cytokine). Such determination is well within the capabilities of those of ordinary skill in the art.

[0350] According to one embodiment, the cell expresses a chimeric antigen receptor (CAR) or a modified T cell receptor (TCR).

[0351] Thus, cells of some embodiments of the invention can be transduced to express a TCR or a CAR.

[0352] As used herein, "transduction with TCR" refers to the cloning of two chains (i.e., polypeptide chains), such as the alpha chain of a T cell receptor (TCR), the beta chain of TCR, the gamma chain of TCR, the delta chain of TCR, or a combination thereof (e.g., an alpha beta chain or a gamma delta chain). According to one embodiment, the TCR comprises the variable region of the TCR (e.g., an alpha and beta chain or a gamma and delta chain). Methods for transducing cells (e.g., T cells) with TCR (e.g., to generate TCR-T cells) are known in the art and are disclosed in, for example, Nicholson et al. Adv Hematol. 2012; 2012: 404081; Wang and Rivière Cancer Gene Ther. March 2015; 22 (2): 85-94); and Lamers et al., Cancer Gene Therapy (2002) 9, 613–623.

[0353] As used herein, "transduction with CAR" refers to the cloning of a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR includes an antigen recognition portion and a T cell activation portion. Chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen binding domain (e.g., single-chain variable fragment (scFv)) of an antibody connected to a T cell signaling or T cell activation domain. Methods for transducing cells (e.g., T cells) with CAR (e.g., to generate CAR-T) are known in the art and are disclosed in, for example, Davila et al. Oncoimmunology. December 1, 2012; 1(9): 1577-1583; Wang and Rivière Cancer Gene Ther. March 2015; 22(2): 85-94); and Maus et al. Blood. April 24, 2014; 123(17): 2625-35.

[0354] According to a specific embodiment, the method comprises obtaining counter-suppressive non-GVHD inducing cells.

[0355] Methods for obtaining counter-suppressive non-GVHD cells that can be used with specific embodiments of the present invention are known in the art and are disclosed, for example, in PCT Publication Nos. WO2001 / 049243, WO2002 / 102971, WO2007 / 023491, WO2010 / 049935, WO2012 / 032526, WO2013 / 035099, WO2018 / 002924, WO2017 / 009852, WO2017 / 009853, WO2018 / 134824, and WO2021 / 024264, the contents of which are incorporated herein by reference in their entirety.

[0356] According to one embodiment, the counter-suppressive non-GVHD cells are obtained or obtainable according to the method described in PCT Publication No. WO 2021 / 024264.

[0357] According to a specific embodiment, the counter-suppressed non-GVHD cells are obtained or obtainable according to the method described in Examples 1-2 of the following Examples section, which serves as an integral part of the present description.

[0358] According to a specific embodiment, the counter-suppression non-GVHD inducing cells are obtained or obtainable by a method comprising the following steps:

[0359] (i) a first population of peripheral blood mononuclear cells (PBMC) from a donor is subjected to a cytokine receptor capable of binding to CD14 + The cells expressing the antibody were contacted and CD14 that could mature into antigen presenting cells were selected. + expressing cells;

[0360] (ii) loading the antigen-presenting cells with viral peptides, bacterial peptides, fungal peptides and / or cancer / tumor peptides;

[0361] (iii) using a drug that can deplete CD4 + 、CD56 + and CD45RA + A second population of PBMCs from the same donor as the first population of PBMCs is treated with one or more agents expressing cells to obtain cells enriched in (e.g., containing at least 40%) expressing CD45RA - CD8 + Phenotypic memory T cell populations;

[0362] (iv) contacting the cell population comprising the T cells enriched in the memory T cells with the antigen-presenting cells loaded with the viral peptides of step (ii) in the presence of IL-21, so as to allow enrichment of virus-reactive memory T cells; and

[0363] (v) culturing the cells obtained in step (iv) in the presence of IL-21, IL-15 and / or IL-7 to allow proliferation of cells comprising said Tcm phenotype, as further described below.

[0364] According to one embodiment, a cell population refers to a heterogeneous mixture of cells.

[0365] As described above for T cell-depleted immature hematopoietic cells, counter-suppressive non-GVHD-inducing cells or PBMCs can be obtained from an allogeneic, xenogeneic, or syngeneic donor with respect to the recipient subject.

[0366] According to a specific embodiment, the counter-suppressive non-GVHD inducing cells or PBMCs are non-syngeneic to the subject, as discussed in detail above.

[0367] According to a specific embodiment, the counter-suppressive non-GVHD inducing cells or PBMCs are obtained from the same donor as the T cell-depleted immature hematopoietic cells.

[0368] According to one embodiment, the PBMCs used to generate counter-suppressive non-GVHD inducing cells are non-mobilized (i.e., antigen-naive) PBMCs, i.e., cells obtained without the use of drugs to induce the mobilization of hematopoietic precursors (e.g., stem cells) from the bone marrow into the peripheral blood circulation.

[0369] According to one embodiment, PBMCs for generating reverse suppression non-GVHD inducing cells are collected 5-15 days (e.g., 7-10 days, e.g., 7 days, e.g., 8 days) before the planned transplant date (i.e., day 0) of immature hematopoietic cells, as discussed in detail below. However, it should be understood that PBMCs for generating reverse suppression non-GVHD inducing cells can be collected at any time before the planned transplant date. Such PBMCs can be stored as is, or can be processed as discussed below and then stored for future use (e.g., cryopreservation).

[0370] According to one embodiment, the PBMCs are collected from a donor subject using standard techniques. According to a specific embodiment, the PBMCs are collected using leukapheresis (i.e., a process in which the PBMCs are substantially removed from a donor subject and the remaining blood components are returned to the donor subject).

[0371] According to one embodiment, the PBMC collection procedure (e.g., leukapheresis) yields, for example, 0.01-1000×10 10 Mononuclear cells (MNC), for example 0.1-100×10 10 , for example 1×10 10 MNCs.

[0372] According to one embodiment, the PBMC collection procedure (e.g., leukapheresis) yields a minimum of 500×10 6 mononuclear cells (MNCs), for example 500×10 10 Up to 1000×10 10 MNCs, for example, 1×10 10 MNCs.

[0373] According to one embodiment, PBMCs are collected in a single collection procedure.

[0374] According to one embodiment, PBMCs are collected in two, three, four, five or more collection procedures (eg, to obtain a desired number of MNCs).

[0375] According to one embodiment, if PBMCs are collected in two or more collection procedures (eg, from the same donor subject), the PBMCs can be pooled together (eg, for further processing) or used individually.

[0376] According to one embodiment, any of the aforementioned collections can be referred to as batches. Alternatively, any group of collections (e.g., over a few days, such as 1, 2, 3, 4, 5 days, such as 3 days, in the context of collection, from the same donor subject) can be referred to as batches.

[0377] According to one embodiment, any of the aforementioned PBMC collections can be maintained in the collection tube for one or more days (e.g., 1, 2, 3, 4, 5 days, such as 1-2 days) prior to further processing (e.g., MNC isolation as discussed below).

[0378] According to one embodiment, mononuclear cells (MNCs) are isolated from PBMCs. Any standard technique known in the art can be used to isolate MNCs from PBMCs. For example, according to one embodiment, the collected PBMCs are diluted (e.g., 1:2) with Dulbecco's Phosphate-Buffered Saline (DPBS), e.g., without calcium and magnesium and supplemented with, e.g., 0.6% ACD-A and 0.5% 25% HSA, and the MNCs are separated by ficoll density gradient separation. After ficoll density gradient separation, the MNC preparation of one embodiment is platelet washed (i.e., thrombowashed) by manual centrifugation, e.g., 1-5 times, e.g., 1-3 times, e.g., 2 times, and resuspended with wash buffer (e.g., PBS containing ACD-A and 0.5% 25% HSA).

[0379] The PBMC preparation or MNC preparation of some embodiments of the present invention is divided into two fractions (e.g., equal fractions). One PBMC or MNC fraction is further processed into antigen presenting cells (i.e., referred to herein as the first population of PBMCs), and the second PBMC or MNC fraction (i.e., referred to herein as the second population of PBMCs) is enriched for CD8 + Memory T cells.

[0380] According to one embodiment, the first population of PBMCs and the second population of PBMCs are from the same batch.

[0381] Alternatively, two PBMC preparations from different PBMC collection procedures may be used.Thus, according to one embodiment, the first population of PBMCs and the second population of PBMCs are from different batches.

[0382] According to one embodiment, antigen presenting cells (eg, dendritic cells) are generated by firstly incubating a first population of PBMCs with a dendritic cell capable of binding to CD14 + Cells expressing CD14 were contacted with antibodies and selected + expressing cells.

[0383] According to one embodiment, the antibody capable of binding to CD14+ expressing cells is a CD14 monoclonal antibody. Such antibodies can be obtained commercially, for example, from BD Biosciences, Santa Cruz Biotechnology, and R&D Systems.

[0384] CD14 selection using CD14 monoclonal antibody + Expressing cells can be performed using any method known in the art, such as by using magnetic activated cell sorting (MACS™), a FACS sorter, and / or capture ELISA labels, available from, for example, Miltenyi Biotec.

[0385] According to one embodiment, different depletion / separation methods can be combined, for example, magnetic cell sorting can be combined with FACS to increase the quality of separation or to allow sorting by multiple parameters.

[0386] According to one embodiment, CD14 + The selection of expressing cells is not affected by plastic adherence.

[0387] According to one embodiment, CD14 is selected by magnetic separation technique. + Expressing cells. Different magnetic beads are available from many sources, including, for example, Dynal (Norway), Advanced Magnetics (Cambridge, Massachusetts, USA), Immuncon (Philadelphia, USA), Immunotec (Marseille, France), Invitrogen, Stemcell Technologies (USA), Cellpro (USA), and Miltenyi Biotec GmbH (Germany). Alternatively, the antibody can be biotinylated or conjugated with digoxigenin and used in conjunction with an avidin or anti-digoxigenin-coated affinity column.

[0388] According to one embodiment, the CD14 monoclonal antibody is conjugated to magnetic particles.

[0389] According to one embodiment, the magnetic particles comprise superparamagnetic iron dextran particles.

[0390] According to a specific embodiment, the cells marked by CD14 are Processed on column.

[0391] According to a specific embodiment, CD14-labeled cells are selected on SuperMACSTMII using an XS separation column.

[0392] According to one embodiment, CD14 magnetically labeled cells (i.e., CD14 + expressing cells) are retained by the separation column (i.e., positive selection), and CD14 - cells. Then CD14 + The cells are released from the column and collected. According to one embodiment, a sample from each fraction is removed for cell counting, viability and / or immunophenotyping.

[0393] According to one embodiment, viability is assessed by positive expression of 7AAD, i.e., 7AAD + cell.

[0394] According to one embodiment, CD14 + Enriched cell preparations are for example 1-10×10 6 cells / ml, for example 3×10 6 In one embodiment, the cell culture medium is supplemented with cytokines and growth factors. The determination of cytokines and growth factors to be used is within the technology of those skilled in the art. For example, the cell culture medium is supplemented with IL-4 (e.g., 200-2000 IU / mL, e.g., 1000 IU / mL) and GM-CSF (e.g., 1000-4000 IU / mL, e.g., 2000 IU / mL). The cell suspension is then inoculated (e.g., in a cell culture plate, e.g., a CellFactory plate) and incubated at 37°C, 5% CO2 for 12-36 hours, e.g., 16-24 hours, e.g., 24 hours.

[0395] According to one embodiment, in order to induce CD14 + CD14 expressing cells mature into antigen presenting cells (e.g. dendritic cells) + The enriched cell preparation is cultured in the presence of a maturation factor (e.g., a dendritic cell maturation factor). The determination of the maturation factor to be used is within the skill of those skilled in the art. Thus, according to one embodiment, the seeded (e.g., in a cell culture plate, e.g., a CellFactory plate) CD14+ The enriched cells are cultured in the presence of IL-4 (e.g., 200-2000 IU / mL, e.g., 1000 IU / mL), GM-CSF (e.g., 1000-4000 IU / mL, e.g., 2000 IU / mL), LPS (e.g., 10-100 ng / mL, e.g., 40 ng / mL), and IFN-γ (e.g., 50-500 IU / mL, e.g., 200 IU / mL) at 37° C., 5% CO 2 for 10-24 hours, e.g., 14-18 hours, e.g., 16 hours.

[0396] After the incubation period, antigen-presenting cells (e.g., mature dendritic cells, i.e., mDCs) are obtained from the cell culture. According to one embodiment, non-adherent cells are removed and antigen-presenting cells (i.e., adherent cells) are detached from the culture plate using any method known in the art (e.g., by adding ice-cold buffer, e.g., ACD-A and DPBS buffer containing 0.5% HSA, and incubated on ice or frozen gel packs for 10-60 minutes, e.g., 30 minutes). The antigen-presenting cells harvested are then centrifuged, washed, and resuspended in culture medium.

[0397] According to one embodiment, antigen presenting cells (eg, mDCs) are loaded with one or more antigens.

[0398] As used herein, the phrase "loading" refers to the attachment of one or more antigens (eg, peptides) to MHC peptides (eg, MHC class I or class II) presented in a peptide-MHC complex on the surface of an antigen presenting cell (APC).

[0399] As used herein, "antigen(s)" refers to soluble or insoluble (eg, membrane associated) molecules that are capable of inducing an immune response.

[0400] For example, the one or more antigens can be whole cells (eg, live or dead cells), cell fractions (eg, lysed cells), cellular antigens (eg, cell surface antigens), protein extracts, purified proteins, or synthetic peptides.

[0401] According to one embodiment, the one or more antigens comprise viral antigens.

[0402] According to one embodiment, the viral antigens are derived from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more different types of viruses.

[0403] According to one embodiment, the viral antigens are derived from 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-10, 2-8, 2-6, 2-4, 3-50, 3-40, 3-30, 3-20, 3-10, 3-9, 3-7, 3-5, 3-4, 4-50, 4-40, 4-30, 4-20, 4-10, 4-8 or 4-6 types of viruses.

[0404] According to a specific embodiment, the viral antigens are derived from 1-20 types of viruses.

[0405] According to a specific embodiment, the viral antigens are derived from 1-10 types of viruses.

[0406] According to a specific embodiment, the viral antigens are derived from 1-4 types of viruses.

[0407] According to a specific embodiment, the viral antigens are derived from 2-10 types of viruses.

[0408] According to a specific embodiment, the viral antigens are derived from 2-4 types of viruses.

[0409] According to a specific embodiment, the viral antigens are derived from 4-20 types of viruses.

[0410] According to a specific embodiment, the viral antigens are derived from 4-10 types of viruses.

[0411] According to a specific embodiment, the viral antigens are derived from 4-8 types of viruses.

[0412] According to a specific embodiment, the viral antigens are derived from 4-6 types of viruses.

[0413] Exemplary viruses from which the antigen can be derived (i.e., originate) include, but are not limited to, Epstein-Barr virus (EBV), adenovirus (Adv), cytomegalovirus (CMV), cold viruses, influenza viruses, hepatitis A, B, and C viruses, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, respiratory syncytial virus, rubella, smallpox, varicella-zoster, rotavirus, West Nile virus, polyomavirus (e.g., BK virus), Zika virus, parvovirus (e.g., parvovirus B19), varicella-zoster virus (VZV), herpes simplex virus (HSV), severe acute respiratory syndrome (SARS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0414] Specific examples of viruses and their respective antigens include, but are not limited to, BK virus antigens, including but not limited to BKV LT; BKV (capsid VP1), BKV (capsid protein VP2), BKV (capsid protein VP2, isotype VP3, BKV (small T antigen); adenovirus antigens include but are not limited to Adv penton or Adv hexon; CMV antigens include but are not limited to envelope glycoprotein B, CMV IE-1 and CMV pp65, UL28, UL32, UL36, UL40, UL48, UL55, UL84, UL94, UL99 UL103, UL151, UL153, US29, US 32; EBV antigens include but are not limited to EBV LMP2, EBV BZLF1, EBV EBNA1, EBV P18 and EBV P23; hepatitis antigens include but are not limited to S, M and L proteins of hepatitis B virus, pre-S antigen of hepatitis B virus, HBCAG DELTA, HBVHBE, hepatitis C virus RNA, HCV NS3 and HCV NS4; Herpes simplex virus antigens include but are not limited to immediate early protein and glycoprotein D; HIV antigens include but are not limited to gene products of gag, pol and env genes, such as HIV gp32, HIVgp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66POL, HIV TAT, HIV GP36, Nef protein and reverse transcriptase; influenza antigens include but are not limited to hemagglutinin and neuraminidase; Japanese encephalitis virus antigens include but are not limited to proteins E, ME, ME-NS1, NS1, NS1-NS2A and 80%E; measles antigens include but are not limited to measles virus fusion protein; rabies antigens include but are not limited to rabies glycoprotein and rabies nucleoprotein; respiratory syncytial virus antigens include but are not limited to RSV fusion protein and M2 protein; rotavirus antigens include but are not limited to VP7sc; rubella antigens include but are not limited to proteins E1 and E2; severe acute respiratory syndrome (SARS-CoV) antigens include but are not limited to S1, RBD, nucleocapsid and Plpro; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens include but are not limited to S1, S2, S1+S2 ECD, RBD, N antigen, S antigen and nucleocapsid; and varicella-zoster virus antigens include but are not limited to gpl and gpl1.

[0415] According to one embodiment, the one or more antigens comprise a viral peptide (or fragment thereof).

[0416] According to one embodiment, the viral peptides comprise 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-10, 2-8, 2-6, 2-4, 3-50, 3-40, 3-30, 3-20, 3-10, 3-9, 3-7, 3-5, 3-4, 4-50, 4-40, 4-30, 4-20, 4-10, 4-8 or 4-6 viral peptides.

[0417] According to a specific embodiment, the viral peptides comprise 4-50 viral peptides (eg, in a single preparation or in several preparations).

[0418] According to a specific embodiment, the viral peptides comprise 4-40 viral peptides (eg, in a single preparation or in several preparations).

[0419] According to a specific embodiment, the viral peptides comprise 4-30 viral peptides (eg, in a single preparation or in several preparations).

[0420] According to a specific embodiment, the viral peptides comprise 4-20 viral peptides (eg, in a single formulation or in several formulations).

[0421] According to a specific embodiment, the viral peptides comprise 4-10 viral peptides (eg, in a single formulation or in several formulations).

[0422] According to a specific embodiment, the viral peptides comprise 4-8 viral peptides (eg, in a single formulation or in several formulations).

[0423] According to a specific embodiment, the viral peptides comprise 4-6 viral peptides (eg, in a single formulation or in several formulations).

[0424] According to one embodiment, the viral peptides comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 viral peptides (e.g., in a single preparation or several preparations).

[0425] According to one embodiment, the viral peptides comprise four viral peptides (eg, in a single formulation or in several formulations).

[0426] According to one embodiment, the viral peptides comprise five viral peptides (eg, in a single formulation or in several formulations).

[0427] According to one embodiment, the viral peptides comprise six viral peptides (eg, in a single formulation or in several formulations).

[0428] According to one embodiment, the viral peptides comprise 8 viral peptides (eg, in a single formulation or in several formulations).

[0429] According to one embodiment, the viral peptides comprise 10 viral peptides (eg, in a single formulation or in several formulations).

[0430] According to one embodiment, the viral peptides comprise 15 viral peptides (eg, in a single formulation or in several formulations).

[0431] According to one embodiment, the viral peptides comprise 20 viral peptides (eg, in a single formulation or in several formulations).

[0432] According to one embodiment, the viral peptides comprise 30 viral peptides (eg, in a single formulation or in several formulations).

[0433] According to one embodiment, the viral peptides comprise 40 viral peptides (eg, in a single formulation or in several formulations).

[0434] According to one embodiment, the viral peptides comprise 50 viral peptides (eg, in a single formulation or in several formulations).

[0435] According to one embodiment, the viral peptides comprise peptides from a single organism (ie from one type of virus).

[0436] According to one embodiment, the viral peptides comprise peptides from two or more organisms (ie, a mixture from 2, 3, 4, 5 or more virus types).

[0437] According to one embodiment, the viral peptide comprises a BK viral peptide.

[0438] According to a specific embodiment, the viral peptide includes at least one of an Epstein-Barr virus (EBV) peptide, a cytomegalovirus (CMV) peptide, a BK virus peptide, and an adenovirus (Adv) peptide.

[0439] According to a specific embodiment, the viral peptides include Epstein-Barr virus (EBV) peptides, cytomegalovirus (CMV) peptides, BK virus peptides, and adenovirus (Adv) peptides.

[0440] According to one specific embodiment, the viral peptides include at least one of EBV-LMP2, EBV-BZLF1, EBV-EBNA1, EBV-BRAF1, EBV-BMLF1, EBV-GP340 / 350EBNA2, EBV-EBNA3a, EBV-EBNA3b, EBV-EBNA3c, CMV-pp65, CMV-IE-1, Adv penton, Adv hexon, BKV LT, BKV (capsid VP1), BKV (capsid protein VP2), BKV (capsid protein VP2, isotype VP3) and BKV (small T antigen).

[0441] According to a specific embodiment, the viral peptide comprises at least one of AdV5 hexon, hCMV pp65, EBV select (discussed below), and BKV LT.

[0442] Dedicated software can be used to analyze antigen sequences to identify short immunogenic peptides, ie, peptides that are presentable in the context of major histocompatibility complex (MHC) class I or MHC class II.

[0443] According to one embodiment, the one or more antigens comprise a mixture of pepmix, which is a library of overlapping peptides (e.g., 15-mers overlapping by 11 amino acids) spanning the entire protein sequence of three viruses: CMV, EBV, and Adeno (such pepmix can be purchased commercially, e.g., from JPT Technologies, Berlin, Germany).

[0444] According to one embodiment, the viral peptides comprise "EBV Select", a commercial product from Miltenyi Biotec, which contains 43 MHC class 1 and class 2 restricted peptides from 13 different proteins of EBV (e.g. MACS GMPPep EBV Select, e.g., catalog number 170-076-143). Additionally or alternatively, the viral peptides comprise "Collective EBV," a commercial product from JPT comprising a pepmix that includes peptides from 14 different EBV antigens. Additionally or alternatively, the viral peptides comprise PepMix commercially available from JPT. TM BKV (capsid protein VP1), PepMix TM BKV (capsid protein VP2), PepMix TM BKV (capsid protein VP2, isotype VP3), PepMix TM BKV (large T antigen), PepMix TM BKV (small T antigen).

[0445] According to another specific embodiment, the one or more antigens comprise a mixture of seven pepmix spanning EBV-LMP2, EBV-BZLF1, EBV-EBNA1, CMV-pp65, CMV-IE-1, Adv penton and Adv hexon, with a concentration of, for example, 100 ng / peptide or 700 ng / mixture of seven peptides.

[0446] According to one embodiment, the one or more antigens comprise one or more antigens of infectious organisms (eg, bacteria, fungal organisms) that commonly affect immunocompromised subjects, such as transplant patients.

[0447] According to one embodiment, the antigen is a bacterial antigen, such as, but not limited to, anthrax; antigens of gram-negative bacilli, chlamydia, diphtheria, haemophilus influenza, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsia, staphylococcus, streptococcus, and tetanus.

[0448] As further specific examples of bacterial antigens, anthrax antigens include, but are not limited to, anthrax protective antigens; Gram-negative bacillus antigens include, but are not limited to, lipopolysaccharide; Haemophilus influenzae antigens include, but are not limited to, capsular polysaccharide; diphtheria antigens include, but are not limited to, diphtheria toxin; Mycobacterium tuberculosis antigens include, but are not limited to, mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secretory protein, and antigen 85A; pertussis toxin antigens include, but are not limited to, hemagglutinin, pertactin, FIM2, FIM3, and adenylate cyclase; pneumococcal antigens include, but are not limited to, pneumolysin and pneumococcal capsular polysaccharide; rickettsial antigens include, but are not limited to, rompA; streptococcal antigens include, but are not limited to, M protein; and tetanus antigens include, but are not limited to, tetanus toxin.

[0449] According to one embodiment, the antigen is a superbug antigen (e.g., a multidrug resistant bacteria). Examples of superbugs include, but are not limited to, Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae, Enterobacter).

[0450] According to one embodiment, the antigen is a fungal antigen. Examples of fungi include, but are not limited to, Candida, Coccidiodes, Cryptococcus, Histoplasma, Leishmania, Plasmodium, protozoa, parasites, Schistosoma, Tinea, Toxoplasma, and Trypanosoma cruzi.

[0451] As further specific examples of fungal antigens, Coccidioides antigens include, but are not limited to, spherule antigens; Cryptococcus antigens include, but are not limited to, capsular polysaccharides; Histoplasma antigens include, but are not limited to, heat shock protein 60 (HSP60); Leishmania antigens include, but are not limited to, gp63 and lipophosphoglycosides; Plasmodium falciparum antigens include, but are not limited to, merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gametocyte surface antigens, and protozoan and other parasite antigens include the blood stage antigen pf 155 / RESA; Schistosoma antigens include, but are not limited to, glutathione-S-transferase and paramyosin; tinea fungal antigens include, but are not limited to, trichophyton; Toxoplasma antigens include, but are not limited to, SAG-1 and p30; and Trypanosoma cruzi antigens include, but are not limited to, a 75-77 kDa antigen and a 56 kDa antigen.

[0452] According to one embodiment, the one or more antigens comprise an antigen associated with a malignant disease (eg, a cancer or tumor antigen).

[0453] According to one embodiment, the antigen is an antigen (or portion thereof, such as an antigenic epitope) expressed by a cancer cell. According to one embodiment, the antigen (or portion thereof) is derived from a protein expressed in hematopoietic tissue (e.g., a hematopoietic malignancy, such as a leukemia antigen) or a protein expressed in a solid tumor (e.g., melanoma, pancreatic cancer, liver cancer, gastrointestinal cancer, etc.).

[0454] Examples of cancer / tumor antigens include, but are not limited to, A33, BAGE, Bcl-2, B cell maturation antigen (BCMA), BCR-ABL, β-catenin, testicular cancer antigens (CTAs such as MAGE-1, MAGE-A2 / A3, and NY-ESO-1), CA125, CA19-9, CA 50, CA 27.29 (BR 27.29), CA 15-3, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEA, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ELA2, ephrin B2, estrogen receptor, FAP, ferritin, folate binding protein, GAGE, G250 / CAIX, GD-2, GM2, gp75, gp100 (Pmel 17), HA-1, HA-2, HER-2 / neu, HM1.24, HPV E6, HPV E7, hTERT, Ki-67, LRP, mesothelin, mucin-like cancer-associated antigen (MCA), MUC1, p53, PR1, PRAME, PRTN3, RHAMM (CD168), and WT-1. For more tumor antigens, see: Molldrem J. Biology of Blood and Marrow Transplantation (2006) 12: 13-18; Alatrash G. and Molldrem J., Expert Rev Hematol. (2011) 4 (1): 37–50; Renkvist et al., Cancer lmmunol lmmunother (2001) 50: 3-15; van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun (2013), www (.) cancerimmunity (.) org / peptide / ; Rittenhouse, Manderino and Hass, Laboratory Medicine (1985) 16 (9) 556-560; all of these references are incorporated herein by reference.

[0455] According to one embodiment, the one or more antigens comprise a mixture of antigens (e.g., a mixture of antigens from one group of antigens as discussed, such as viral antigens; or a mixture of antigens from different groups of antigens, such as viral and bacterial antigens, viral antigens and cancer / tumor antigens).

[0456] According to one embodiment, the one or more antigens comprise a mixture of viral peptides and cancer / tumor peptides (eg, in a single formulation or in several formulations).

[0457] According to one embodiment, the one or more antigens comprise a mixture of viral and bacterial peptides (eg, in a single formulation or in several formulations).

[0458] According to one embodiment, the one or more antigens comprise a mixture of viral peptides and fungal peptides (eg in a single formulation or in several formulations).

[0459] According to one embodiment, loading antigen-presenting cells (eg, mDCs) with one or more antigens can be performed using any method known in the art.

[0460] According to one embodiment, in order to load (e.g., present) viral peptides on antigen-presenting cells (e.g., mDCs), the viral peptides are co-cultured with antigen-presenting cells (e.g., mDCs) at 37° C. and 5% CO 2 for 30 minutes to 3 hours (e.g., 1 hour). For example, antigen-presenting cells (e.g., mDCs) can be loaded with peptivators (e.g., AdV5 hexon, HCMV pp65, EBV selection, and BKV LT) by incubating at 37° C. and 5% CO 2 for 30 minutes to 3 hours (e.g., 1 hour).

[0461] After incubation, the viral peptide-loaded antigen presenting cells (eg, mDCs) are washed and centrifuged with, for example, 25% HSA in ACD-A and DPBS buffer with 0.5%, and resuspended in cell growth medium (eg, T cell growth medium).

[0462] According to one embodiment, antigen presenting cells (e.g., mDCs) loaded with one or more antigens (e.g., viral peptides) are irradiated via an X-ray source. Thus, according to one embodiment, antigen presenting cells (e.g., mDCs) loaded are irradiated with about 1-5 Gy, about 5-10 Gy, about 10-20 Gy, about 10-30 Gy, about 10-40 Gy, about 10-50 Gy, about 20-30 Gy, about 20-40 Gy, or about 20-50 Gy. According to a specific embodiment, DCs are irradiated with about 10-40 Gy (e.g., 25-30 Gy, e.g., 30 Gy).

[0463] Once irradiation is complete, the loaded antigen presenting cells (eg, viral peptide-loaded mDCs) are washed, centrifuged, and resuspended in cell growth medium (eg, T cell growth medium).

[0464] The antigen-loaded antigen-presenting cells (eg, mDCs) are then ready for generating Tcm cells from a cell population comprising memory CD8 T cells according to some embodiments of the present invention.

[0465] According to a specific embodiment, the antigen presenting cells include dendritic cells (DC).

[0466] According to a specific embodiment, the antigen presenting cells comprise mature dendritic cells (mDCs).

[0467] According to a specific embodiment, at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the antigen presenting cells comprise mature dendritic cells (mDCs).

[0468] According to one embodiment, the antigen presenting cells comprise irradiated dendritic cells.

[0469] According to one embodiment, the antigen presenting cells and the counter-suppression non-GVHD inducing cells and / or immature hematopoietic cells belong to the same donor subject.

[0470] It should be understood that antigen-presenting cells can express all antigens on a single cell, or can express only part of the antigens on a single cell. In addition, different antigen-presenting cells (e.g., in the same preparation) can express different antigens. Accordingly, antigen-presenting cells (e.g., mDCs) comprise a heterogeneous cell mixture.

[0471] According to some embodiments of the present invention, one or more antigens (e.g., viral peptides) can be presented by genetically modified antigen-presenting cells or artificial antigen-presenting cells that display MHC antigens (also referred to as human leukocyte antigens (HLA)) that can be recognized by T cells (e.g., cell lines transfected with one or more antigens). Additionally or alternatively, one or more antigens (e.g., viral peptides) of some embodiments of the present invention can be displayed on artificial vehicles (e.g., liposomes).

[0472] Thus, antigen presenting cells (as discussed above), cell lines, artificial vehicles (e.g., liposomes), or artificial antigen presenting cells (e.g., leukemia or fibroblast cell lines transfected with one or more antigens) can be used to present short synthetic peptides fused thereto or loaded thereto, or to present protein extracts or purified proteins. Such short peptides, protein extracts, or purified proteins can be peptides derived from viral, bacterial, fungal, or cancer / tumor antigens, or peptides representing any other antigen.

[0473] As mentioned above, the method of some embodiments of the present invention is achieved by providing a cell population comprising T cells, wherein the T cells in the cell population are enriched for expressing CD45RA- CD8 + phenotype and depletion of CD4 + 、CD56 + and CD45RA + Memory T cells expressing cells.

[0474] The term "cell population comprising T cells" refers to a heterogeneous mixture of PBMCs comprising T cells, B cells, and myeloid cells. A cell population comprising T cells typically comprises T cells (e.g., cytotoxic T cells, memory T cells, effector T cells, etc.) having numerous characteristics and functions and capable of binding to various antigens.

[0475] According to one embodiment, the cell population comprising T cells does not comprise erythrocytes and granulocytes.

[0476] As used herein, the term "memory T cells" refers to a subpopulation of T lymphocytes that have previously encountered an antigen and responded, also known as antigen-experienced T cells.

[0477] According to one embodiment, memory T cells constitute at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or even 100% of the T cells in a cell population.

[0478] It will be appreciated that under normal conditions (ie as determined in healthy subjects), levels of memory T cells constitute less than 20% of the total number of cells in a population of T cell-containing cells.

[0479] According to one embodiment, memory T cells comprise T cells expressing the CD8 marker (i.e., CD8 + T cells).

[0480] According to another embodiment, the memory T cells comprise CD8 + CD45RO + Phenotype.

[0481] According to another embodiment, the memory T cells comprise CD8 + CD45RA - Phenotype.

[0482] According to another embodiment, the memory T cells comprise CD8 + CD45RO + CD45RA - Phenotype.

[0483] According to one embodiment, memory T cells lack CD45RA + cell.

[0484] According to one embodiment, memory T cells lack CD4 + and / or CD56 + cell.

[0485] Memory CD8 + T cells can be selected by selecting cells that co-express CD8 + and CD45RA - cells and / or co-expressing CD8 + and CD45RO + This can be achieved by purifying cells that have been purified and can be performed using any method known in the art, such as by affinity-based purification (e.g., such as by using MACS beads, a FACS sorter, and / or capture ELISA labels).

[0486] Memory CD8 + T cell selection can further be achieved by selecting effector T cells and central memory T cells, the latter expressing, for example, CD62L, CCR7, CD27 and / or CD28.

[0487] To obtain a cell population in which the T cell population comprises highly pure memory T cells (e.g., at least about 30-50% memory T cells, e.g., 40% memory T cells), or to increase the number of memory T cells, PBMCs can be depleted of naive cells such as CD45RA + cells, CD4 + cells (e.g., T helper cells), CD56 + cells (e.g., NK cells) or any other cells that do not comprise a memory T cell phenotype.

[0488] Naive T cells (e.g., expressing CD45RA + cells), CD4 + and / or CD56 + Depletion of cells can be performed using any method known in the art, for example, by affinity-based purification (eg, such as by using MACS beads, a FACS sorter, and / or capture ELISA tags).

[0489] According to one embodiment, memory T cells are obtained from peripheral blood mononuclear cells (PBMC).

[0490] According to one embodiment, memory T cells are obtained by a method comprising: using a cell culture medium capable of depleting CD4 + 、CD56 + and CD45RA + A second population of PBMCs from the same donor subject as the first population of PBMCs is treated with one or more agents expressing cells to obtain cells comprising cells expressing CD45RA - CD8+ A cell population of at least 40% memory T cells of the phenotype.

[0491] According to one embodiment, the cell population further comprises B cells and myeloid cells.

[0492] According to one embodiment, prior to memory T cell enrichment, the CD14 - The cells are combined with a second population of cells.

[0493] According to one embodiment, the CD14 T cells obtained from the first population of cells are removed prior to the enrichment of memory T cells. - The cells and / or PBMCs obtained from the second population of cells are centrifuged and eluted at, for example, 10-50×10 6 cells / ml, for example 30×10 6 The cells are resuspended in a cell growth medium, such as a T cell growth medium (e.g., Click medium with advanced RPMI 1640 supplemented with 1:100 Glutamaxe and 5% human AB serum) at a concentration of 10 cells / ml. According to one embodiment, the cell growth medium is supplemented with IL-7 (e.g., at a concentration of, for example, 1-100 IU / mL, such as 30 IU / mL). The cell suspension is then inoculated (e.g., in a tissue culture flask) and incubated at 37°C, 5% CO2 for 12-36 hours, such as 16-24 hours, such as 24 hours.

[0494] According to one embodiment, the second population of PBMCs is centrifuged and resuspended in a buffer (e.g. / 0.5% HSA buffer) to a minimum ratio of 1:2.

[0495] According to one embodiment, the second population of PBMCs is subjected to a platelet wash (e.g., thrombowash), centrifuged, and resuspended in a buffer (e.g., / 0.5% HSA buffer).

[0496] According to one embodiment, the platelet-depleted cell preparation of one embodiment is incubated with IVIg for 5-30 minutes, such as 10-15 minutes. + 、CD56 + and CD45RA + The binding agent is added to the cell preparation and incubated on an orbital rotator for, eg, 10-60 minutes, eg, 30 minutes.

[0497] According to one embodiment, CD4 + 、CD56 +and CD45RA + The binding agent is an antibody.

[0498] According to a specific embodiment, CD4 + 、CD56 + and CD45RA + The binding agent is a monoclonal antibody.

[0499] According to a specific embodiment, CD4 + 、CD56 + and CD45RA + Monoclonal antibodies are conjugated to magnetic particles.

[0500] According to a specific embodiment, CD4 + 、CD56 + and CD45RA + Monoclonal antibodies are conjugated to superparamagnetic particles.

[0501] According to one embodiment, after the incubation is complete, the cells are washed by centrifugation and the cell pellet is resuspended in a buffer (e.g. / 0.5% HSA buffer) to remove excess reagent.

[0502] According to one embodiment, CD4 + / CD56 + / CD45RA + Labeled cells are selected by magnetic separation.

[0503] According to a specific embodiment, CD4 + / CD56 + / CD45RA + Labeled cells in Processed on column.

[0504] According to one embodiment, CD4 + / CD56 + / CD45RA + Magnetic labeled cells (i.e., CD4 + / CD56 + / CD45RA + expressing cells) are retained by the separation column (i.e., negative selection), and CD4 - / CD56 - / CD45RA - Cells. According to one embodiment, the collected cells are washed and resuspended in T cell growth medium. According to one embodiment, samples are taken from each fraction for cell counting, viability and / or immunophenotyping.

[0505] According to one embodiment, the collected CD4 - / CD56 - / CD45RA - The cell fraction was adjusted to 0.01-10 × 10 in T cell growth medium supplemented with cytokines and growth factors. 6 cells / ml, for example 2×10 6 Cells / ml. Those skilled in the art will be able to determine the cytokines and growth factors to be used. For example, T cell growth medium is supplemented with IL-7 (e.g., at a concentration of 1-100 IU / mL, such as 30 IU / mL). The cell suspension is then inoculated (e.g., in 100), and incubated at 37° C., 5% CO 2 for 12-36 hours, such as 16-24 hours, such as 24 hours.

[0506] To deplete alloreactive clones from the memory T cell pool by activation with an antigen (eg, a viral antigen), cells comprising the memory T cells of some embodiments of the invention are contacted with an antigenic peptide (eg, a viral antigen).

[0507] According to a specific embodiment, the counter-suppression non-GVHD inducing cells of some embodiments of the present invention are generated by contacting a cell population comprising memory T cells with antigen-presenting cells loaded with antigenic peptides, such as viral peptides (e.g., as described above), in a culture supplemented with IL-21 (e.g., in a culture without other cytokines, i.e., without adding any additional cytokines). This step typically occurs for about 12-24 hours, about 12-36 hours, about 12-72 hours, 12-96 hours, 12-120 hours, about 24-36 hours, about 24-48 hours, about 24-72 hours, about 36-48 hours, about 36-72 hours, about 48-72 hours, about 48-96 hours, about 48-120 hours, 0.5-1 day, 0.5-2 days, 0.5-3 days, 0.5-5 days, 1-2 days, 1-3 days, 1-5 days, 1-7 days, 1-10 days, 2-3 days, 2-4 days, 2-5 days, 2-6 days, 2-8 days, 3-4 days, 3-5 days, 3-7 days, 4-5 days, 4-8 days, 5-7 days, 6-8 days, or 8-10 days, and allows for the enrichment of antigen (e.g., viral antigen) reactive cells.

[0508] According to one embodiment, cells depleted of CD4 + 、CD56 + and CD45RA + cells and contain memory CD8 +A PBMC population of T cells (e.g., a cell population comprising T cells, wherein the T cells in the cell population comprise at least 40% memory T cells) is contacted with one or more antigens (e.g., as described above) for 12 hours to 6 days (e.g., 3 days).

[0509] In cultures supplemented with IL-21, cells containing memory CD8 + Contacting a population of T cells with one or more antigens (e.g., as described above) is typically performed in the presence of 0.001-3000 IU / ml, 0.01-3000 IU / ml, 0.1-3000 IU / ml, 1-3000 IU / ml, 10-3000 IU / ml, 100-3000 IU / ml, 1000-3000 IU / ml, 0.001-1000 IU / ml, 0.01-1000 IU / ml, 0.1-1000 IU / ml, 1-1000 IU / ml, 10-1000 IU / ml, 100-1000 IU / ml, 250-1000 IU / ml, 50 ... ml, 750-1000IU / ml, 10-500IU / ml, 50-500IU / ml, 100-500IU / ml, 250-500IU / ml, 100-250IU / ml, 0.1-100IU / ml, 1-100IU / ml, 10-100IU / ml, 30-100I U / ml, 50-100IU / ml, 1-50IU / ml, 10-50IU / ml, 20-50IU / ml, 30-50IU / ml, 1-30IU / ml, 10-30IU / ml, 20-30IU / ml, 10-20IU / ml, 0.1-10IU / ml or 1-10IU / ml IL-21. According to a specific embodiment, the concentration of IL-21 is 50-500 IU / ml (eg, 100 IU / ml).

[0510] According to one embodiment, in the absence of exogenous cytokines other than IL-21, cells containing memory CD8 + Such culture conditions allow the survival and enrichment of only those cells that have undergone stimulation and activation by the antigen(s) (i.e., antigen-reactive cells, such as virus-reactive memory T cells) because these cells secrete cytokines (e.g., IL-2) that enable them to survive (all remaining cells die under these culture conditions).

[0511] According to one embodiment, the memory CD8 + T cells (i.e. CD4 - CD56 -CD45RA - In some embodiments, the ratio of the cell population (e.g., mDCs) to the antigen-presenting cells (e.g., mDCs) loaded with antigen (e.g., viral) peptides is about 2:1 to about 10:1, e.g., about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, or about 10:1.

[0512] According to one embodiment, the memory CD8 + T cells (i.e. CD4 - CD56 - CD45RA - The ratio of the cell population (eg, cells) / antigen-presenting cells (eg, mDCs) loaded with antigen (eg, viral) peptides is about 2:1 to 8:1, for example, about 5:1.

[0513] According to one embodiment, the memory CD8 + T cells (i.e. CD4 - CD56 - CD45RA - cells) of the cell population, with a concentration of 0.01-10×10 6 cells / ml, for example 1×10 6 cells / ml, together with antigen presenting cells (e.g., mDCs) loaded with viral peptides, at a ratio of about 2:1 to about 8:1, for example, about 5:1 (memory CD8 + The cells were plated at a T cell growth medium with a T cell:antigen presenting cell (e.g., mDC) ratio, along with IL-21 (e.g., at a concentration of 50-500 IU / ml, e.g., 100 IU / ml), at 37° C., 5% CO 2 (e.g., at 100100) 1-5 days (e.g., 3 days).

[0514] According to a specific embodiment, after culturing with IL-21, the resulting cells containing memory CD8 +The cell population of T cells is prepared to allow the obtained counter-suppression non-GVHD inducing cells to proliferate. This step is typically performed for about 12-24 hours, about 12-36 hours, about 12-72 hours, about 12-96 hours, about 12-120 hours, about 12-240 hours, 24-36 hours, 24-48 hours, about 24-72 hours, 24-96 hours, 24-120 hours, 24-240 hours, about 48-72 hours, about 48-120 hours, about 48-240 hours, about 96-240 hours, about 120-144 hours, about 120-240 hours, about 144-240 hours, 0.5-1 day, 0.5-2 days, 0.5-3 days, 0.5-5 days, 0.5-10 days, 1-2 days, 1-3 days, 1-4 days, 1-6 days, 1-8 days, 1-9 days, 1-10 days, 2-3 days, 2-4 days, 2-5 days, 2-6 days, 2-8 days, 2-9 days, 2-10 days, 4-5 days, 4-6 days, 4-8 days, 4-9 days, 4-10 days, 5-6 days, 5-7 days, 5-8 days, 5-9 days, 5-10 days, 5-15 days, 6-7 days, 6-8 days, 6-9 days, 6-10 days, 6-12 days, 7-8 days, 7-9 days, 7-11 days, 7-13 days, 7-15 days, 8-9 days, 8-10 days, 9-10 days, 9-12 days, 9-15 days, 10-12 days, 10-15 days, 12-15 days, 14-16 days, 14-18 days, 16-18 days or 18-20 days. According to one embodiment, the obtained cells containing memory CD8 + The cell population of T cells (ie, after culturing with IL-21) is cultured for about 6-12 days (eg, 9 days).

[0515] This step is typically performed in the presence of IL-21 at a concentration of about 0.001-3000 IU / ml, 0.01-3000 IU / ml, 0.1-3000 IU / ml, 1-3000 IU / ml, 10-3000 IU / ml, 100-3000 IU / ml, 1000-3000 IU / ml, 0.001-1000 IU / ml, 0.01-1000 IU / ml, 0.1-1000 IU / ml, 1-1000 IU / ml, 10-1000 IU / ml, 100-1000 IU / ml, 250-1000 IU / ml, 500-1000 IU / ml, 750-1 000IU / ml, 10-500IU / ml, 50-500IU / ml, 100-500IU / ml, 250-500IU / ml, 100-250IU / ml, 0.1-100IU / ml, 1-100IU / ml, 10-100IU / ml, 30-100IU / ml, 50-100IU / ml, 1-50IU / ml, 10-50IU / ml, 20-50IU / ml, 30-50IU / ml, 1-30IU / ml, 10-30IU / ml, 20-30IU / ml, 10-20IU / ml, 0.1-10IU / ml, or 1-10IU / ml IL-21. According to a specific embodiment, the concentration of IL-21 is 50-500 IU / ml (eg, 100 IU / ml).

[0516] This step is further performed in the presence of IL-15 at a concentration of about 0.001-3000 IU / ml, 0.01-3000 IU / ml, 0.1-3000 IU / ml, 1-3000 IU / ml, 10-3000 IU / ml, 100-3000 IU / ml, 125-3000 IU / ml, 1000-3000 IU / ml , 0.001-1000IU / ml, 0.01-1000IU / ml, 0.1-1000IU / ml, 1-1000IU / ml, 10-1000IU / ml, 100-1000IU / ml, 125-1000IU / ml, 250-1000IU / ml, 500-1000IU / ml, 750-1000IU / ml、10-500IU / ml、50-500IU / ml、100-500IU / ml、125-500IU / ml、250-500IU / ml、250-500IU / ml、125-250IU / ml、100-250IU / ml、0.1-100IU / ml、1-100IU / ml、10-100I In one embodiment, the concentration of IL-15 is 50-500 IU / ml (e.g., 125 IU / ml).

[0517] This step is further performed in the presence of IL-7 at a concentration of about 0.001-3000 IU / ml, 0.01-3000 IU / ml, 0.1-3000 IU / ml, 1-3000 IU / ml, 10-3000 IU / ml, 30-3000 IU / ml, 100-3000 IU / ml, 1000-3000 IU / ml, 0.001-1000 IU / ml, 0.01-1000 IU / ml, 0.1-1000 IU / ml, 1-1000 IU / ml, 10-1000 IU / ml, 30-1000 IU / ml, 100-1000 IU / ml, 250-1000 IU / ml, 500-1000 IU / ml. l, 750-1000IU / ml, 10-500IU / ml, 30-500IU / ml, 50-500IU / ml, 100-500IU / ml , 250-500IU / ml, 100-250IU / ml, 0.1-100IU / ml, 1-100IU / ml, 10-100IU / ml, 30 -100IU / ml, 50-100IU / ml, 1-50IU / ml, 10-50IU / ml, 20-50IU / ml, 30-50IU / ml , 1-30IU / ml, 10-30IU / ml, 20-30IU / ml, 10-20IU / ml, 0.1-10IU / ml or 1-10IU / ml IL-7. According to a specific embodiment, the concentration of IL-7 is 1-100 IU / ml (30 IU / ml).

[0518] According to a specific embodiment, after the step of culturing with IL-21, the culture is supplemented with IL-7 (e.g., at a concentration of, for example, 1-100 IU / ml, for example, 30 IU / ml), IL-15 (e.g., at a concentration of, for example, 50-500 IU / ml, for example, 125 IU / mL) and IL-21 (e.g., at a concentration of, for example, 50-500 IU / ml, for example, 100 IU / mL) at 50% of the culture volume and cultured for about 6-12 days (e.g., 9 days), while supplementing IL-7, IL-15, IL-21 every about 48-96 hours, for example, 48 hours, for example, 72 hours.

[0519] According to one embodiment, the total length of culture time used to generate the cells is about 9, 10, 11, 12, 13, 14, 15, 17, 19 or 21 days (e.g., 12 days).

[0520] According to one embodiment, the cell culture is monitored for glucose levels.

[0521] According to one embodiment, the glucose is at a level comprised between 10-500 mg / dl, such as between 50-170 mg / dl.

[0522] According to one embodiment, the cytokines IL-7, IL-15, IL-21 are added to the culture (as detailed above) when glucose levels are between 170 mg / dL and 130 mg / dL.

[0523] According to one embodiment, when the glucose level is between 129 mg / dL and 100 mg / dL, fresh T cell growth medium, for example 25% volume, for example 100 ml (e.g. 400 ml of 25% volume) is added. 100 volumes) plus cytokines IL-7, IL-15, and IL-21 were added to the culture.

[0524] According to one embodiment, when the glucose level is between 99 mg / dL and 50 mg / dL, fresh T cell growth medium, for example 50% volume, for example 200 ml (e.g. 50% of 400 ml) is added. 100 volumes) plus cytokines IL-7, IL-15, and IL-21 were added to the culture.

[0525] According to one embodiment, the culturing further comprises adding glucose to a concentration of at least about 20 mg / dl, at least about 30 mg / dl, at least about 40 mg / dl, at least about 50 mg / dl, at least about 60 mg / dl, at least about 70 mg / dl, at least about 80 mg / dl, at least about 90 mg / dl, at least about 100 mg / dl.

[0526] According to a specific embodiment, the culturing further comprises adding glucose to a concentration of at least about 50 mg / dl.

[0527] According to one embodiment, the cell culture is monitored for pH levels.

[0528] According to one embodiment, the pH is in the physiological range (eg pH 7.2-7.6).

[0529] In cases where the pH level is not within the physiological range, the pH level can be adjusted using any method known in the art.

[0530] According to one embodiment, in the presence of IL-21, IL-15 and / or IL-7, cultivation is achieved in an antigen-free environment (i.e., without adding one or more antigens, such as viral peptides). However, it should be understood that residual one or more antigens (e.g., viral peptides) may be present in the cell culture, and therefore, an antigen-free environment relates to a cell culture without the addition of supplementary antigen-presenting cells presenting one or more antigens (e.g., viral peptides).

[0531] According to one embodiment, in order to obtain memory CD8 specific for one or more antigens + T cells, which acquire memory CD8 + T cells before (e.g., after providing a cell population comprising at least 40% memory CD8 + Before the T cell population of T cells is generated, the antigen (e.g., tumor antigen, viral antigen) is administered to the donor subject. Any method that immunizes the cell donor against the antigen in order to elicit an immunogenic response (e.g., memory CD8 + T cell generation).

[0532] The antigen can be administered as is, or as part of a composition comprising an adjuvant (e.g., complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA)). According to one embodiment, the antigen is administered to the donor subject once. According to one embodiment, the donor subject receives at least one additional (e.g., booster) administration (e.g., 2, 3, 4 or more administrations) of the antigen. Such additional administration can be achieved 1, 3, 5, 7, 10, 12, 14, 21, 30 days or longer after the first administration of the antigen.

[0533] To further enrich for memory CD8 against specific antigens + T cells and deplete alloreactive clones from the memory T cell pool, including memory CD8 + The population of T cells can be further contacted with the same antigen(s) (eg, the same antigen(s) as administered to the cell donor), as described above.

[0534] It will be appreciated that cell samples and culture medium samples may be obtained at any stage during the process of generating counter-suppressive non-GVHD inducing cells. These can be used to assess cell number, cell viability, sterility, immunophenotyping, glucose and pH levels, and the like. Any method known in the art may be used to implement such procedures. Non-limiting examples of such methods are described in the Examples section below.

[0535] According to one embodiment, the anti-suppressive non-GVHD inducing cells of some embodiments of the present invention can be used in combination with any CAR-T or TCR-T cells (e.g., CAR-T or TCR-T cells generated from cells of an anti-suppressive cell donor), such as tumor-specific CAR-T or TCR-T cells targeting various tumor antigens. It should be understood that anti-suppressive cells and CAR-T / TCR-T cells can be used simultaneously or sequentially (e.g., on the same day, or for example, within about 1, 2, 3, 4, 5, 6, 7 days).

[0536] Counter-suppressed non-GVHD inducing cells can be used as fresh cells (eg, within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, such as within about 3 days).

[0537] Alternatively, cells can be cryopreserved until needed (eg, 1 week, 2 weeks, 1 month, 2 months, 4 months, 6 months, 1 year, or longer).

[0538] According to a specific embodiment, the counter-suppressing non-GVHD inducing cells are administered in a single administration.

[0539] According to a specific embodiment, the counter-suppressing non-GVHD inducing cells are administered in two or more administrations.

[0540] According to one embodiment, the counter-suppressive non-GVHD inducing cells are administered in single or multiple administrations on days 3-21 after transplantation of T cell-depleted immature hematopoietic cells.

[0541] According to one embodiment, the counter-suppressive non-GVHD inducing cells are administered in single or multiple administrations on day 6-9 after transplantation of T cell-depleted immature hematopoietic cells.

[0542] According to one embodiment, the counter-suppressive non-GVHD inducing cells are administered as a single administration on day 6-9 after transplantation of T cell-depleted immature hematopoietic cells.

[0543] According to one embodiment, the counter-suppressive non-GVHD inducing cells are administered on day 7 after the transplantation of T cell-depleted immature hematopoietic cells.

[0544] According to a specific embodiment, the therapeutically effective amount of cells that suppress non-GVHD induction comprises more than 1×10 6 cells / kg of the subject's ideal body weight.

[0545] According to a specific embodiment, the therapeutically effective amount of cells that suppress non-GVHD induction is in the range of: 0.01×10 6 to 20×10 6 cells / kg ideal body weight, 0.01×106 to 0.5×10 6 cells / kg ideal body weight, 0.01×10 6 to 1×10 6 cells / kg ideal body weight, 0.01×10 6 to 5×10 6 cells / kg ideal body weight, 0.1×10 6 to 0.5×10 6 cells / kg ideal body weight, 0.1×10 6 to 1×10 6 cells / kg ideal body weight, 0.1×10 6 to 5×10 6 cells / kg ideal body weight, 0.5×10 6 to 1×10 6 cells / kg ideal body weight, 0.5×10 6 to 1×10 6 cells / kg ideal body weight, 0.5×10 6 to 5×10 6 cells / kg ideal body weight, 1×10 6 to 5×10 6 cells / kg ideal body weight, 1×10 6 to 20×10 6 cells / kg ideal body weight, 5×10 6 to 20×10 6 cells / kg ideal body weight, 10×10 6 to 15×10 6 cells / kg ideal body weight, 10×10 6 to 20×10 6 cells / kg ideal body weight or 15×10 6 to 20×106×10 6 cells / kg of the subject's ideal body weight.

[0546] According to one embodiment, the therapeutically effective amount of cells to suppress non-GVHD induction comprises at least 2.5×10 6 CD8 + cells / kg of the subject's ideal body weight.

[0547] According to one embodiment, the therapeutically effective amount of cells to suppress non-GVHD induction comprises at least 3×10 6 CD8 + cells, at least 3.5×10 6 CD8 + cells, at least 4×10 6 CD8+ cells, at least 4.5×10 6 CD8 + cells, at least 5×10 6 CD8 + cells or at least 10 × 10 6 CD8 + cells / kg of the subject's ideal body weight.

[0548] According to one embodiment, the therapeutically effective amount of cells to suppress non-GVHD inducing cells comprises at least about 2.5×10 6 CD8 + cells / kg of the subject's ideal body weight.

[0549] According to one embodiment, the therapeutically effective amount of cells to suppress non-GVHD induction comprises about 5×10 6 CD8 + cells / kg of the subject's ideal body weight.

[0550] According to one embodiment, the therapeutically effective amount of cells that suppress non-GVHD induction comprises about 10×10 6 CD8 + cells / kg of the subject's ideal body weight.

[0551] Non-limiting examples of treatment regimens that may be used with embodiments of the present invention are schematically shown in Figure 1 and are also described in detail in Examples 1-2 of the Examples section that follows.

[0552] Thus, according to a further or alternative aspect of the present invention, there is provided a method of treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject, the method comprising:

[0553] (a) conditioning the subject under a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI) and splenic irradiation, wherein the TMI is administered at a total dose of 12 Gy in 4 divided doses from day -7 to day -1 prior to transplantation of T cell-depleted immature hematopoietic cells from a non-syngeneic donor into the subject; and subsequently

[0554] (b) after (a), transplanting a therapeutically effective amount of the T cell-depleted immature hematopoietic cells obtained from the donor non-syngeneic to the subject, wherein the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises less than 5×10 6 CD3 + T cells / kg of the subject's ideal body weight and at least 5×10 6 CD34+ cells / kg of the subject's ideal body weight; and subsequently

[0555] (c) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount of cyclophosphamide comprises 25-200 mg of cyclophosphamide per kilogram of the subject's ideal body weight, and wherein the therapeutically effective amount of cyclophosphamide is administered to the subject in two doses, 3 days and 4 days after the transplantation of the T cell-depleted immature hematopoietic cells; and subsequently

[0556] (d) administering to the subject a therapeutically effective amount of reverse-suppressive non-graft-versus-host disease (GVHD)-inducing cells, said reverse-suppressive non-graft-versus-host disease (GVHD)-inducing cells comprising a central memory T lymphocyte (Tcm) phenotype and having antiviral activity obtained from the same donor as the T cell-depleted immature hematopoietic cells, wherein said reverse-suppressive non-GVHD-inducing cells can be obtained by:

[0557] (i) a first population of peripheral blood mononuclear cells (PBMC) from a donor is subjected to a cytokine receptor capable of binding to CD14 + The cells expressing the antibody were contacted and CD14 that could mature into antigen presenting cells were selected. + expressing cells;

[0558] (ii) loading the antigen-presenting cells with viral peptides;

[0559] (iii) using a drug that can deplete CD4 + 、CD56 + and CD45RA + A second population of PBMCs from the same donor as the first population of PBMCs is treated with one or more agents expressing cells to obtain cells enriched for CD45RA expression. - CD8 + Phenotypic memory T cell populations of T cells;

[0560] (iv) contacting the cell population comprising the T cells enriched in memory T cells with the antigen-presenting cells loaded with the viral peptides of step (ii) in the presence of IL-21, so as to allow enrichment of virus-reactive memory T cells; and

[0561] (v) culturing the cells obtained in step (iv) in the presence of IL-21, IL-15 and / or IL-7 to allow proliferation of cells comprising the Tcm phenotype,

[0562] The subject's disease is thereby treated.

[0563] According to an additional or alternative aspect of the present invention, there is provided a combination of a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI) and spleen irradiation, a therapeutically effective amount of T cell-depleted immature hematopoietic cells obtained from a non-syngeneic donor of the subject, a therapeutically effective amount of cyclophosphamide, and a therapeutically effective amount of cells that counter-suppress non-graft-versus-host disease (GVHD) inducing, for use in treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject,

[0564] wherein the TMI is administered in 4 doses at a total dose of 12 Gy on day -7 to day -1 before the T cell-depleted immature hematopoietic cells,

[0565] wherein the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises less than 5×10 6 CD3 + T cells / kg of the subject's ideal body weight and at least 5×10 6 CD34 + cells / kg of the subject's ideal body weight,

[0566] wherein the therapeutically effective amount of cyclophosphamide comprises 25-200 mg cyclophosphamide per kilogram of the subject's ideal body weight, administered in two doses 3 days and 4 days after the T cell-depleted immature hematopoietic cells,

[0567] wherein the anti-GVHD inducing cells comprise a central memory T lymphocyte (Tcm) phenotype, have antiviral activity, and are obtained from the same donor as the T cell-depleted immature hematopoietic cells, and

[0568] The reverse suppression non-GVHD inducing cells can be obtained by:

[0569] (i) a first population of peripheral blood mononuclear cells (PBMC) from a donor is subjected to a cytokine receptor capable of binding to CD14 + The cells expressing the antibody were contacted and CD14 that could mature into antigen presenting cells were selected. + expressing cells;

[0570] (ii) loading the antigen-presenting cells with viral peptides;

[0571] (iii) using a drug that can deplete CD4 + 、CD56 + and CD45RA + A second population of PBMCs from the same donor as the first population of PBMCs is treated with one or more agents expressing cells to obtain cells enriched for CD45RA expression. - CD8+ Phenotypic memory T cell populations of T cells;

[0572] (iv) contacting the cell population comprising the T cells enriched in the memory T cells with the antigen-presenting cells loaded with the viral peptides of step (ii) in the presence of IL-21, so as to allow enrichment of virus-reactive memory T cells; and

[0573] (v) culturing the cells obtained in step (iv) in the presence of IL-21, IL-15 and / or IL-7 to allow proliferation of cells comprising the Tcm phenotype.

[0574] The number of administrations and therapeutically effective amounts of cells, immunosuppressive drugs, and / or immunosuppressive irradiation before and after transplantation can be adjusted based on the subject's response to the treatment regimen. Determination of the number of administrations and therapeutically effective amounts is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0575] The cells and drugs of some embodiments of the present invention can be administered to an organism per se, or in a pharmaceutical composition in which it is mixed with a suitable carrier or excipient.

[0576] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0577] As used herein, "active ingredient" refers to the cells or drugs described herein that are responsible for a biological effect.

[0578] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are also encompassed by these terms.

[0579] As used herein, "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various types of sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0580] Techniques for drug formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which reference is incorporated herein by reference.

[0581] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially nasal, enteral or parenteral delivery, including intramuscular, subcutaneous and intramedullary injection, as well as intrathecal, direct intraventricular, intracardial, for example, in the right or left ventricular cavity, in the common coronary artery, intravenous, intraperitoneal, intranasal or intraocular injection.

[0582] Conventional methods for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of agents (e.g., generating chimeric fusion proteins comprising a transport peptide with affinity for endothelial cell surface molecules in combination with an agent that itself cannot cross the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of agents (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and temporary disruption of the integrity of the BBB by hypertonic disruption (resulting from infusion of mannitol solutions into the carotid artery or the use of bioactive agents such as angiotensin peptides). However, each of these strategies has limitations, such as inherent risks associated with invasive surgical procedures, size restrictions imposed by inherent limitations of endogenous transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules comprising carrier motifs that may be active outside the CNS, and the risk of possible brain damage in brain regions where the BBB is disrupted, rendering them suboptimal delivery methods.

[0583] Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example, via injection of the pharmaceutical composition directly into a tissue area of the patient.

[0584] Pharmaceutical compositions of some embodiments of the present invention can be manufactured by processes well known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0585] Therefore, the pharmaceutical composition for use according to some embodiments of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the active ingredient into a pharmaceutically acceptable formulation. The appropriate formulation depends on the route of administration selected.

[0586] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0587] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compound with a pharmaceutically acceptable carrier well known in the art. Such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by patients. Pharmaceutical preparations for oral use can be prepared by using solid excipients, optionally grinding the resulting mixture, and, when necessary, adding suitable adjuvants, processing the mixture of particles to obtain tablets or dragee cores. Suitable excipients are particularly fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers, such as polyvinyl pyrrolidone (PVP). When necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or its salts such as sodium alginate can be added.

[0588] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identification or characterization of different combinations of active compound doses.

[0589] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In the soft capsule, the active ingredient can be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer can be added. All formulations for oral administration should be in a dosage suitable for the selected route of administration.

[0590] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0591] For administration by nasal inhalation, for the active component used according to some embodiments of the present invention, the form presented with the aerosol spray from pressurized packaging or sprayer is easily delivered, wherein using suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide.In the case of pressurized aerosol, dosage unit can be determined by providing valve to deliver metered amount. Capsules such as gelatin and liquid cartridges for use in dispensers can be formulated, which contain a powder mix of compound and suitable powder base such as lactose or starch.

[0592] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or multidose containers, with an optional addition of a preservative. The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles and can contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0593] The pharmaceutical composition for parenteral administration includes an aqueous solution of the active agent in a water-soluble form. In addition, the suspension of the active ingredient can be prepared as an injection suspension suitably based on oil or water. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions can contain materials that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension can also contain a suitable stabilizer or an agent that increases the solubility of the active ingredient to allow the preparation of a highly concentrated solution.

[0594] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, a sterile, pyrogen-free, water-based solution, before use.

[0595] Pharmaceutical compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0596] According to one embodiment, the cells are formulated for administration as fresh cells.

[0597] According to one embodiment, the cells are formulated for administration as cryopreserved cells.

[0598] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, it refers to an amount of the active ingredient that is effective to prevent, alleviate or ameliorate the symptoms of a condition (e.g., cancer as defined herein) or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0599] For any formulation used in the methods of the present invention, a therapeutically effective amount or dosage can be initially estimated from in vitro and cell culture assays. For example, a dosage can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful dosages in humans.

[0600] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard in vitro pharmaceutical procedures, in cell culture or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage can vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician taking into account the patient's condition. (See, for example, Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Chapter 1, page 1).

[0601] The amount and interval of dosage can be adjusted individually to provide sufficient levels of active ingredient sufficient to induce or inhibit a biological effect (minimum effective concentration, MEC). The MEC is different for each formulation but can be estimated from in vitro data. The dosage required to reach the MEC depends on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0602] Depending on the severity and responsiveness of the condition to be treated, dosing can be a single administration or multiple administrations, with the course of treatment continuing for several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved.

[0603] The amount of composition to be administered will, of course, depend on the subject to be treated, the severity of the affliction, the mode of administration, the judgment of the prescribing physician, and the like.

[0604] When needed, the compositions of some embodiments of the present invention may be present in a packaging or dispenser device, such as a kit approved by the FDA, which may include one or more unit dosage forms containing an active ingredient. Packaging may, for example, include metal foil or plastic foil, such as a blister pack. Packaging or dispenser devices may be accompanied by instructions for use. Packaging or dispensers may also be accommodated by a notice accompanying the container, the notice being in the form prescribed by a government agency that manages the manufacture, use, or sale of the drug, the notice reflecting the form of the composition approved by the agency or human or veterinary administration. For example, such notices may be labels approved for prescription drugs by the U.S. Food and Drug Administration (USFood and Drug Administration), or product inserts approved. As further described above, compositions of the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for the treatment of the indicated condition.

[0605] As used herein, the term "about" refers to ±10%.

[0606] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0607] The term "consisting of" means "including and limited to."

[0608] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0609] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0610] Throughout this application, various embodiments of the present invention may be presented in the form of ranges. It should be understood that the description in the form of ranges is only for the sake of convenience and brevity, and should not be interpreted as a hard limit to the scope of the present invention. Accordingly, the description of a range should be deemed to have specifically disclosed all possible subranges and each numerical value within the range. For example, a description of a range such as from 1 to 6 should be deemed to have specifically disclosed subranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each number within the range, such as 1, 2, 3, 4, 5 and 6. This applies regardless of the breadth of the scope.

[0611] Whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The phrases "ranging from a first indicated number to a second indicated number / ranging between a first indicated number and a second indicated number" and "ranging from a first indicated number to a second indicated number / ranging from a first indicated number to a second indicated number" are used interchangeably herein and are intended to include the first indicated number and the second indicated number, and all fractions and integers therebetween.

[0612] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to those manners, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or readily developable from known manners, means, techniques and procedures.

[0613] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable subcombination or, where appropriate, in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0614] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0615] Example

[0616] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0617] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques.

[0618] Example 1

[0619] Clinical plan

[0620] Patient inclusion criteria

[0621] Ages 12-75

[0622] Patients diagnosed with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), or multiple myeloma (MM).

[0623] Patients with aplastic anemia and severe immunodeficiency or non-malignant bone marrow failure states. Patients with thalassemia major requiring regular blood transfusions or with severe clinical features of sickle cell disease (these include any clinically significant sickle genotype, e.g., hemoglobin SS (Hb SS), hemoglobin SC (Hb SC), hemoglobin Sβ thalassemia (Hb Sβ), or hemoglobin S-OArab genotypes) who have at least one of the following manifestations:

[0624] a. Clinically significant neurological event (stroke) or neurological deficit lasting >24 hours;

[0625] b. A history of two or more acute chest syndrome (ACS) episodes in the 2 years prior to enrollment or referral despite adequate supportive care measures (i.e., asthma treatment);

[0626] c. An average of three or more pain crises per year in the year prior to enrollment or referral (requiring venous pain management in an outpatient or inpatient hospital setting);

[0627] d. Administering regular red blood cell (RBC) transfusion therapy, defined as eight or more transfusions per year (within the 12 months prior to enrollment) to prevent vaso-occlusive clinical complications (i.e., pain, stroke, or acute chest syndrome);

[0628] e. Echocardiographic examination revealed tricuspid regurgitation jet velocity ≥ 2.7 m / sec.

[0629] f. High impact 1 chronic pain that persists on most days of the month for ≥6 months, defined as one or more of the following: chronic pain without contributory SCD 2 complications, or mixed pain pattern, where chronic pain occurs in any location (arm, back, chest, or abdominal pain) not associated with contributory SCD 2 complications (e.g., leg ulcers and / or avascular necrosis of the femoral head).

[0630] Patients with hematologic malignancies must have persistent or progressive disease despite initial chemotherapy and must have achieved stable disease or a partial or complete response to their most recent chemotherapy. Patients with low-grade or chronic disease without additional treatment are eligible. Patients with intermediate-risk or high-risk acute myeloid leukemia in first remission according to the European Leukemia Network (ELN) criteria are eligible.

[0631] Availability of haploidentical related donors

[0632] Karnofsky performance status ≥ 70%

[0633] Adequate major organ system function as demonstrated by:

[0634] - Left ventricular ejection fraction of at least 40%

[0635] - Pulmonary function testing (PFT) demonstrating adjusted diffusion capacity for hemoglobin concentration at least 50% of predicted value

[0636] - Serum creatinine ≤1.5 mg / dL.

[0637] - Serum alanine aminotransferase (SGPT) ≤ 200 IU / mL

[0638] -Bilirubin <1.5 mg / dL (unless Gilbert's syndrome).

[0639] - Negative pregnancy test in females of reproductive potential.

[0640] Patient exclusion criteria

[0641] HIV seropositive.

[0642] Uncontrolled infection or serious medical or psychiatric condition that would limit tolerance to regimen treatment.

[0643] Active CNS malignancy.

[0644] • Availability of medically qualified human leukocyte antigen (HLA) matched related stem cell donors.

[0645] Treatment plan

[0646] exist Figure 1 A schematic diagram of the treatment regimen is provided in .

[0647] There are two collections from the donor, one "naive" mononuclear cell (MNC) for the preparation of counter-suppressor cells, and a second collection of mobilized peripheral blood progenitor cells (PBPC) for the preparation of high-dose T cell-depleted stem cell transplants. Both collections follow the standard of care procedure (SOC), and the donor signs the SOC donation consent form.

[0648] For the generation of antiviral central memory CD8 + Collection of antigen-naive (i.e., non-mobilized) peripheral blood mononuclear cells (PBMCs) for counter-suppressor T cells can be performed at any time, but is generally completed approximately 8 days before the planned transplant date (i.e., day 0, D0). Counter-suppressor cells are either used fresh after their manufacture and will be infused on D+7, or they are cryopreserved after their manufacture.

[0649] If clinically indicated, mobilized PBPCs are harvested using G-CSF and plerixafor for high-dose transplantation. Starting on day -6, the donor is treated with 10 mcg / kg of G-CSF daily for 6 consecutive days, and PBPCs are harvested on days -2 and -1. If clinically indicated, the donor can receive plerixafor on days -3 and -2.

[0650] Remove samples containing ≥1 × 10 6 CD34 + PBPC aliquots were prepared at 100 cells / kg and stored frozen for future use. Cells collected on the first day were stored overnight on day -2 and analyzed for CD34 -Selection and infusion on day -1. A second PBPC collection was performed on day -1; cells were stored overnight and depleted of CD3 using a CliniMacs device (Miltenyi). + / CD19 + cells and infused on day 0. The maximum T cell dose in both T cell-depleted grafts did not exceed 2 × 10 5 CD3 + cells / kg ideal recipient body weight.

[0651] Considerations of mobilization failure

[0652] If T cells are depleted of CD34 in the graft + Total number of cells <5×10 6 Mobilization failure was considered if the patient had PBPCs with sufficient T cell reserve and T cell-depleted cells and was infused on day 0. In this case, the patient did receive countersuppressive cells and was analyzed separately.

[0653] Antiviral central memory CD8 + Generation of suppressor T cells (suppressor cells)

[0654] A detailed protocol for generating repressed cells is provided below in Example 2. This section provides a brief description of some embodiments of the invention.

[0655] The program consists of three steps:

[0656] Step 1: Preparation of Stimuli (Days -8 to -5):

[0657] Preparation of donor DCs from monocytes in PBMCs

[0658] Day -8: Approximately 1 × 10 10 Mononuclear cells were isolated and stored overnight.

[0659] Day -7: Cells were treated by Ficol separation, and half of the mononuclear cells were used for monocyte separation using CD14 magnetic beads (Miltenyi magnetic bead separation system). Thereafter, CD14 was stimulated with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4). + Monocytes differentiate into immature DCs.

[0660] In parallel, CD14 - The (i.e. CD14 negative) cells were combined with half of the initial monocytes obtained after Ficol separation and stored overnight at 37°C in 5% O2 / CO2 in the presence of IL-7. These cells were used for CD8 + Enrichment of memory T cells.

[0661] Day -6: DCs were matured by adding a cytokine cocktail containing lipopolysaccharide (LPS), interferon gamma (IFNγ), GM-CSF, and IL-4 for 16 hours of incubation.

[0662] Day -5: Mature DCs (mDCs) were harvested, loaded with viral peptide cocktail and irradiated.

[0663] Step 2: Preparation of responders and co-culture with virus-loaded mDCs (Day -6 to Day -2):

[0664] A) Memory T cells (CD4 - CD56 - CD45 - ) Purification (from donor PBMC-responders)

[0665] Day -6: Make donor CD14 - The cells were combined with half of the monocytes incubated overnight with IL-7 and CD4 + 、CD56 + and CD45RA + The cells were then cultured and stored overnight at 37°C in 5% O2 / CO2 in the presence of IL-7.

[0666] B) Establishment of responder (donor) stimulator (donor DC loaded with viral peptide) co-culture.

[0667] Day -5 to Day -2: Purified memory responder T cells (CD4 - CD56 - CD45RA - ) were incubated with viral peptide-loaded mDCs in the presence of IL-21 for 3 days.

[0668] Step 3: Differentiation and expansion of antiviral counter-suppressive Tcm (days -2 to +7):

[0669] A) Antiviral central memory CD8 + Expansion of counter-suppressor T cells (Tcm)

[0670] Days -5 to -2: Tcm phenotype was initiated with IL-21 alone as described above.

[0671] Days -2 to +7: Depending on the expansion observed in the culture, Tcm cultures were expanded by splitting according to glucose level depletion and adding media supplemented with the following cytokines: IL-7, IL-15, and IL-21.

[0672] Day +7: Harvest and infusion of cells.

[0673] Figure 2 An approximate timeline for Tcm generation and DC preparation from prospective PBMC donors is shown in Figure 2. Cells were harvested and infused on day +7 after hematopoietic stem cell transplantation.

[0674] Antiviral central memory CD8 + Aliquots of the counter-suppressor cells were evaluated for release criteria and functional immune studies.

[0675] Treatment Plan-Conditioning Program

[0676] Figure 1 A schematic diagram of the conditioning protocol is provided.

[0677] Days and treatments:

[0678] -15 admissions / intravenous (IV) hydration

[0679] -14ATG (Thymoglobulin) 2mg / kg

[0680] -13ATG (Thymoglobulin) 2mg / kg

[0681] -12ATG (Thymoglobulin) 2mg / kg

[0682] -11 Fludarabine 30 mg / m 2

[0683] -10 Fludarabine 30 mg / m 2

[0684] -9 Fludarabine 30 mg / m 2

[0685] -8 Fludarabine 30 mg / m 2

[0686] -7 Whole bone marrow irradiation 3Gy

[0687] -6 Whole bone marrow irradiation 3Gy

[0688] -5 Whole bone marrow irradiation 3Gy

[0689] -4 Whole bone marrow irradiation 3Gy

[0690] -3 rest

[0691] -2 rest

[0692] -1 Infusion of a large dose of T cell-depleted PBPC (containing CD34-selected cells)

[0693] Infusion of CD3- / CD19-depleted cells

[0694] +3 Cyclophosphamide (CY) 50 mg / kg / day IV

[0695] +4 cyclophosphamide (CY) 50 mg / kg / day IV

[0696] +7 infusions of antiviral counter-suppressive cells at doses as indicated.

[0697] Of note, immediately prior to the first dose of cyclophosphamide, the patient received a dose of mesna 10 mg / kg via intravenous infusion (IVPB). This was repeated every 4 hours for a total of 10 doses. The patient also received ondansetron (or other antiemetic) prior to each dose of cyclophosphamide (Cy).

[0698] Another group of patients also received rituximab 375 mg / m2 on day -15. 2 Without being bound by theory, rituximab is administered to reduce B cell numbers and prevent autoimmunity.

[0699] Total myeloid irradiation (TMI)

[0700] Patients received four fractions of 12 Gy total myeloid irradiation (TMI) targeting the bone marrow and spleen.

[0701] Radiation Therapy Simulation - The patient is immobilized in the supine position using a whole-body Vac-Lok bag (CIVCO Radiotherapy, Orange, IA, USA). The arms are relaxed at the sides, not akimbo, with the fingers gripping the bag. Additional devices can be used as needed for patient comfort to ensure reproducible positioning. Two planning CT scans are obtained—one supine scan with the head first and one supine scan with the feet first. The field of view of the CT scan should include the immobilization device, and the slice thickness should be ≤3 mm.

[0702] Treatment Planning System: Planning CT scans were imported into the Raystation (RaySearch Laboratories, Stockholm, Sweden) treatment planning system. All contouring and radiation therapy planning were performed in Raystation.

[0703] Definition of target volume - The target volume is outlined on the planning CT scan. The high-dose target volume includes the entire skeleton and spleen. The clinical target volume (CTV) of the bone marrow (CTV_bone) can be defined using a threshold function on the patient's body to include all bones. The CTV of the spleen (CTV_spleen) can be generated by manual delineation or automatic segmentation and reviewed by the attending physician. Taking into account the uncertainty of the patient setting, a high-dose planning target volume (PTV high) is generated by applying a 3-10mm extension to the high-dose CTV. The low-dose target volume includes lymph node sites, liver and brain (CTV_LN, CTV liver, CTV brain, respectively). CTV_LN is delineated by the attending physician. The outlines of CTV liver and CTV brain can be generated by manual delineation or automatic segmentation. All outlines generated using automatic segmentation are checked on each slice and edited as needed. The low-dose planning target volume (PTV low) is generated by applying a 3-10mm extension to the low-dose CTV. The target volumes determined by the attending physician are peer reviewed by members of the hematology section in the radiation oncology department.

[0704] Definition of Organs at Risk - Organs at risk (OARs) are delineated on the planning CT scan. Contouring of the lungs and kidneys is required. Other organs at risk may also be contoured for dose assessment. These optional contours may include the bladder, breast, esophagus, eye, abdominal cavity, small intestine, bowel, larynx, lens, optic nerve, oral cavity, uterus, parotid gland, prostate, rectum, stomach, testicles, thyroid, and genitals, among others. Contouring can be generated by manual delineation or automatic segmentation. If any contours are generated using automatic segmentation, the contours must be reviewed on each slice and edited as necessary. All organs at risk are labeled according to the American Association of Physicists in Medicine (AAPM) Task Force 263 (TG-263) nomenclature. For example, for the required organs at risk, the labels would be "Lung_R," "Lung_L," "Lungs," "Kidney_R," "Kidney_L," and "Kidneys."

[0705] Target dose prescription - The high-dose target dose is set at 12 Gy in four 3-Gy fractions. Treatment planning goals include D90% ≥ 12 Gy (90% of the target volume receives a dose greater than or equal to 12 Gy) and D1% < 120% (the hottest 1% of the target volume receives a dose less than 120% of the prescribed dose). The low-dose target volume is evaluated to ensure the average dose is between 3 Gy and 5 Gy.

[0706] Organs at Risk Targets - The target average dose to individual lungs and kidneys should be less than 5 Gy. Although recommended limits for the lungs and kidneys are provided below, the goal for all non-target structures is to limit the dose to the lowest reasonably achievable (ALARA) without compromising target coverage.

[0707] Dose Adherence Criteria - Radiation therapy plans are evaluated based on the target and OAR dosimetry parameters listed in Table 1. Plans that do not meet these criteria are considered protocol deviations and additional treatment planning is recommended.

[0708] Table 1: Treatment plan parameters

[0709]

[0710] Radiation therapy planning priorities - the priorities of radiation therapy planning goals, in order of importance:

[0711] 1. Minimum and maximum dose determination standards for high-dose target areas

[0712] 2. Average lung dose

[0713] 3. Average renal dose

[0714] 4. Average dose in low-dose target area

[0715] 5. Dose to all other OARs

[0716] Radiation Therapy Planning Techniques - TMI plans utilize volumetric modulated arc therapy (VMAT) for the body and 3D conformal radiation therapy (3D-CRT) for the legs. For the VMAT body plan, 6 to 12 overlapping arcs at 3 to 6 different isocenters may be used. Larger patients may require more arcs and isocenters depending on their height and anatomy. The isocenters and field sizes of these arcs are determined based on the patient's anatomy to optimize the travel of the multileaf collimator. Each arc has a ≥2 cm overlap area with the arc above it and a ≥2 cm overlap area with the arc below it. For the 3D leg plan, the multileaf collimator is aligned with the PTV of the beam view. There is a ≥2 cm overlap area between the VMAT body plan and the 3D leg plan.

[0717] Perform dose calculations on the planning CT scan. The calculation volume should completely cover the patient and the immobilization device. The size of the dose grid should be ≤ 3 mm in all directions.

[0718] The VMAT body plan and the 3D leg plan were set up to be interdependent. Once the dose distribution for the 3D leg plan was calculated, this dose distribution was set as the background dose for the VMAT body plan. The VMAT body plan was then inversely optimized to achieve the planning goals described in the previous section.

[0719] Radiation therapy equipment: Radiation therapy was delivered using a TrueBeam (Varian Medical Systems, Palo Alto, California, USA) linear accelerator equipped with a Millennium 120 multileaf collimator, delivering 6 megavolt (MV) photons. The irradiation output was calibrated according to AAPM TG-51 guidelines. Daily, monthly, and annual quality assurance of the linear accelerator was performed in accordance with state and national guidelines. Before each patient received their first radiation treatment, a certified medical physicist performed a patient-specific quality assurance review of all planned treatment areas.

[0720] Radiation Therapy Schedule - Because systemic therapy and radiation therapy are interdependent within a patient's overall treatment plan, all radiation therapy fractions must be administered on the scheduled dates without interruption in treatment.

[0721] Patient Positioning - The patient's position during treatment is kept as close as possible to the simulation setup, which serves as a reference. Daily image guidance is used to compare the daily patient position to the reference position. Image guidance is performed using an onboard imaging system consisting of a kilovoltage (kV) x-ray source and imaging panel, and a megavoltage (MV) treatment beam.

[0722] All image registration is based on bony landmarks. For VMAT body planning, the patient is positioned head-first in the supine position. After initial patient positioning, a kilovoltage cone-beam CT scan of the chest isocenter is obtained. Translational shifts can be performed remotely by moving the treatment table, while rotational shifts must be performed directly by adjusting the patient's body. Orthogonal kV x-ray images are acquired for each isocenter, sequentially from the head to the pelvis. The radiation therapist uses a combination of automatic and / or manual registration to determine the required shifts and adjusts the patient accordingly. After any adjustments are made, a final set of orthogonal kV x-ray images are acquired for each isocenter to confirm the patient's position before treatment and are sent to the treating physician for approval. MV images are also acquired before processing each isocenter to ensure that the correct isocenter shift has been applied.

[0723] For the 3D leg plan, the patient is placed in a supine position with feet first on the ground. Orthogonal kV images are obtained to match the daily skeletal anatomy to the reference position of the lower limb during simulation.

[0724] Antiviral counter-suppressive cell dose level-CD8 + Cell dose escalation

[0725] Premedication for counter-suppressive cells did not include corticosteroids.

[0726] Dose level 1: 2.5 × 10 6 CD8 + cells / kg ideal body weight

[0727] Dose level 2: 5.0 × 106 CD8 + cells / kg ideal body weight

[0728] Dose level 3: 10×10 6 CD8 + cells / kg ideal body weight

[0729] No additional post-transplant immunosuppressive therapy was administered as GVHD prophylaxis.

[0730] Considerations in Cases Where Manufactured Cell Product is Below Planned Dose Level

[0731] Dose level 1 contained 2.5×10 6 cells / kg ideal body weight. It is important to note that lower doses are not generally considered acceptable.

[0732] For dose level 2, the target dose was 5 × 10 6 cells / kg ideal body weight, however, 2.6 to 5 × 10 6 A dose of 10 cells / kg ideal body weight was considered adequate and was considered dose level 2.

[0733] For dose level 3, the target dose was 10 × 10 6 cells / kg ideal body weight, however, 5.1 to 10×10 6 A dose of 10 cells / kg ideal body weight was considered adequate and was considered dose level 3.

[0734] Supportive care

[0735] No post-transplant growth factors

[0736] • No post-transplant immunosuppressive therapy was administered as GVHD prophylaxis.

[0737] Other supportive care was administered as clinically indicated (e.g., by G-CSF support in cases where engraftment was slow, or by GVHD treatment such as methylprednisolone, tacrolimus, or other immunosuppressive medications). Concomitant medications administered to patients during the study were recorded in the patient's primary medical record.

[0738] Donor lymphocyte infusion (DLI) for persistent or progressive disease

[0739] Patients who have achieved engraftment and have >5% myeloid donor cells and have progressive disease at any time or persistent disease >3 months after transplantation can receive donor lymphocyte infusion as standard of care or as an alternative therapy if GVHD is not present.

[0740] Treatment of graft failure-Proceed as described in Example 1 of International Patent Application Publication No. WO2021 / 024264.

[0741] Interventions for post-transplant GVHD -Proceed as described in Example 1 of International Patent Application Publication No. WO2021 / 024264.

[0742] Assessments during the study

[0743] Strive to adhere to the event schedule and all protocol requirements. Changes to the event schedule and other protocol requirements that do not impact the rights and safety of patients are not considered deviations. Such changes may include off-schedule laboratory evaluations and occasional missed required study samples. Missed samples for study-related purposes do not constitute a protocol deviation.

[0744] Assessment before transplantation (baseline): TM

[0745] Standard work-up for transplantation and disease assessment are completed as part of the diagnostic or routine pre-transplantation evaluation prior to study entry. The following tests are standard care tests prior to transplantation and are not protocol-specific. The results are used to determine transplant eligibility and are not repeated before treatment begins. If treatment is delayed for more than 30 days after consent, the PI or designee should determine which, if any, tests need to be repeated as clinically indicated.

[0746] -CBC, differential, and platelets

[0747] -Bilirubin, serum creatinine and creatinine clearance, ALT, albumin, electrolytes, LDH, alkaline phosphatase

[0748] - Infectious disease panel (hepatitis serology (B, C), HIV, HTLV, I / II, CMV, TPHA screening), toxoplasmosis serology, Strongyloides serology (if indicated), tuberculosis, IRGA (if indicated)

[0749] -PT and PTT

[0750] -ABO and Rh typing

[0751] - Bone marrow aspirate with accompanying cytogenetic and molecular studies if clinically indicated

[0752] - Baseline analysis of chimerism

[0753] -Pregnancy testing in females of reproductive potential

[0754] - Pulmonary function test using DLCO

[0755] - Echocardiography to assess left ventricular EF and pulmonary artery pressure

[0756] - Chest X-ray and ECG

[0757] - Serum for donor-specific anti-HLA antibodies (DSA).

[0758] The active treatment period for patients treated in this study was from the start of the conditioning regimen to day +42 after transplantation. After this, patients had follow-up visits as clinically indicated until day +100. Thereafter, according to standard follow-up visits for stem cell transplant recipients, disease relapse, infection, acute and chronic GVHD, and survival data were collected. After one year, patients were removed from the study. Acute GVHD and chronic GVHD were scored according to NIH consensus criteria.

[0759] Post-standard evaluation

[0760] Standard post-transplant assessments follow the SOC for post-allogeneic transplantation. Bone marrow aspirates and peripheral blood—to assess treatment response, engraftment, chimerism, and immune reconstitution—are performed monthly for 3 months and as clinically indicated.

[0761] The development of acute GVHD was evaluated and scored using the NIH consensus criteria. Disease recurrence or progression was evaluated by morphological recurrence of the patient's malignant tumor. Data on bacterial, viral, fungal, and parasitic infections were collected. The development of chronic GVHD was described according to the NIH consensus criteria [Jagasia MH et al. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: 1. The 2014 Diagnosis and Staging Working Group report. Biol Blood Marrow Transplant. (2015) 21: 389-401e381].

[0762] Evaluation Form

[0763] Table 2: Evaluation Form

[0764]

[0765] 1Baseline laboratory tests: CBC with differential and platelets, bilirubin, serum creatinine and creatinine clearance, ALT, albumin, electrolytes, LDH, alkaline phosphatase, infectious disease panel (hepatitis serology (B, C), HIV, HTLV, I / II, CMV, TPHA screening), toxoplasmosis serology, Strongyloides serology (if indicated), tuberculosis, IRGA (if indicated), PT and PTT, ABO and Rh typing, serum for donor-specific anti-HLA antibodies (DSA).

[0766] 2 Pregnancy test (urine) in females of reproductive potential.

[0767] 3 If clinically indicated.

[0768] Related research

[0769] Immune reconstitution was assessed by flow cytometry immunophenotyping panel, and antiviral responses were assessed by tetramer analysis. Immune tolerance testing was performed approximately 3 months after transplantation. Missed samples for related studies did not constitute a protocol deviation.

[0770] Biological sample collection

[0771] Whole bone marrow irradiation may alter the immune response of donor lymphocytes and myeloid cells by better preserving the function of lymphoid organs. Circulating cytokines, immune cell phenotype and function, and myeloid and NK cell phenotype and function were analyzed.

[0772] Blood samples were collected 1-2 weeks before ATG, 4 days before TMI, and 2-4 weeks after cyclophosphamide / antiviral therapy for Tcm. Each patient was provided with a unique identifier. Bone marrow aspiration and biopsy samples were collected on days 30, 60, and 90 of standard treatment as clinically indicated (i.e., no separate bone marrow aspiration / biopsy procedure was performed to obtain samples for related research). Bone marrow aspiration and biopsy samples, peripheral blood mononuclear cells, and serum were obtained from patients.

[0773] Peripheral blood mononuclear cell isolation - Collect blood in 7-10cc green-capped tubes and add heparin. Collect half of the plasma and divide equally. Collect the remaining plasma and PBMCs, wash with PBS, and store in freezing medium (10% DMSO and 90% fetal bovine serum). Isolate PBMCs using Sepmate or lymphocyte separation tubes. Cells are plated at 0.25 to 1 × 10 7The cells were resuspended at a concentration of 100 μL / mL and aliquoted into cryovials in volumes of 500-1000 μL. The cryovials were stored in Mr. Frosty containers at -80°C for 24 hours and then transferred to a liquid nitrogen freezer.

[0774] Serum and ctDNA - Blood samples are collected in 7-10cc red-cap tubes (for serum) and 7-10cc Streck cryovials (for ctDNA). The blood in the serum collection tube should be allowed to clot for 30-60 minutes, and then the serum should be separated by centrifugation. Serum should be stored in 2mL cryovials (Nalgene Cat#5000-0020) in 250 to 500μL aliquots. Samples should be labeled with the biospecimen code number and the date and time of collection. Serum and ctDNA samples should be stored at -80°C.

[0775] Sample Labeling - All bone marrow and blood samples, along with clinical information, are coded and labeled before storage. They are assigned a unique PIN (patient identification number) called TMI. This is a four-digit number followed by the sample type (PBMC: PBMC, P: plasma, S: serum), and then a sequence code (1: before ATG, 2: before TMI, 3: and 4: after TMI + cyclophosphamide). For example, TMI-0001-PBMC1 represents the first PBMC sample collected from an enrolled patient before ATG testing. Samples are also dated.

[0776] Assays - Individual assays performed at core facilities depend on instrument availability and the relative cost of the assays. If improved technologies and / or bioinformatics analyses are available to assess genomic, epigenetic, transcriptional, histological, or other biological differences, we incorporate these into our studies. Biological specimens and sample preparation follow the following procedures:

[0777] TCR Sequencing: PBMCs were isolated using Trizol reagent and RNA was extracted. TCR cDNA libraries were generated by 5' RACE and sequenced on the Illumina platform.

[0778] PMBCs were immunophenotyped using a T-cell functional panel (TIGIT, CD3, FOXP3, TIM3, PD-1, OX40, CTLA4, LAG3, CD56, ICOS, Ki67, CD8, CD25, CD45, and CD4) and a myeloid panel (CD141, CD14, CD11c, CD274, CD1c, CD206, HLA-DR, MerTK, CD86, CD11b, CD45, and CD15).

[0779] Serum Cytokines: Serum or plasma cytokines can be assessed using CodePlex panels on the Isolyte System. These panels can evaluate inflammatory chemokines, cytokines, and growth factors for up to 30 cytokines per sample.

[0780] Immune cell function: T cell function was assessed using the Human Adaptive Immune Panel, a single-cell secretome array from the Isolyte system from Isoplexis. In addition, macrophage and NK cell function was assessed using the Human Innate Immunome.

[0781] Further analysis of circulating immune function can be performed using single-cell RNAseq of PBMCs to assess changes in circulating immune cell subsets. Quantitative differences in immune cell subsets can be assessed using fluorescent cytometry.

[0782] Additional correlation analyses can be performed to understand the characteristics of donor leukocytes and antiviral Tcms. Further biomarker assessments of peripheral blood tumor cells, such as gene expression profiling, clonal hematopoiesis, whole-exome sequencing, or single-cell mass cytometry (CyTOF), can be performed to correlate disease characteristics with transplantation outcomes.

[0783] Statistical considerations, design operating characteristics, and implementation -Proceed as described in Example 1 of International Patent Application Publication No. WO2021 / 024264.

[0784] Secondary End Points, Data Analysis, Reporting Requirements, and Adverse Events -Proceed as described in Example 1 of International Patent Application Publication No. WO2021 / 024264.

[0785] Background Drug Information -Proceed as described in Example 1 of International Patent Application Publication No. WO2021 / 024264.

[0786] Example 2

[0787] Antiviral central memory CD8 + Generation of anti-suppressive cells

[0788] Manufacturing and characterization information for cell preparations

[0789] Cell types and derivatization

[0790] This protocol requires the collection of two separate stem cell preparations. One type of stem cell preparation is from mobilized haploidentical donors, a subset of which are CD34 + The cells were selected and the other part was selected using Miltenyi Device depletes CD3 + / CD19 + The second type of preparation is allogeneic non-mobilized peripheral blood mononuclear (PBMC) cells, which are used to generate antiviral central memory CD8 + PBMCs are used to generate dendritic cells and memory responder T cells (CD4 - CD56 - CD45RA - ), which were then co-cultured to stimulate antiviral central memory CD8 + The production of anti-suppressive cells (Tcm). The Tcm cells are then expanded, harvested and infused. The preparation is used in the manufacture and infusion of Tcm cells to + Cell-enriched T-cell depletion followed by high-dose haploidentical donor grafts achieved engraftment without GVHD.

[0791] Manufacturing Laboratory

[0792] The cell preparation of some embodiments of the present invention is processed in the Cell Therapy Laboratory (Cell Therapy Laboratory) (CTL) of the Stem Cell Transplantation and Cellular Therapy Department (StemCell Transplantation and Cellular Therapy) (SCCT) of MDACC. This facility contains a Cell Processing Laboratory (Cell Processing Laboratory) for minimally regulated cell preparations and Classified Suites (ISO 7 grades) for cell preparations exceeding minimally regulated cell preparations. The cell preparation of some embodiments of the present invention is mainly manufactured in ISO 7 grade suites. CTL also includes flow cytometry and quality control laboratories for supporting clinical trials, research and development.

[0793] Related certifications (FACT, CAP, CLIA)

[0794] SCTCT CTL is registered with the FDA (FEI#0001670014) and is accredited by the Foundation of Accreditation of Cellular Therapy (FACT), the College of American Pathologists (CAP), and holds a Clinical Laboratory Improvement Amendments (CLIA) Certificate for Accreditation from the Centers for Medicare and Medicaid Services (CMS).

[0795] raw materials

[0796] PBMC: This protocol requires two separate cell type preparations. One preparation contains allogeneic non-mobilized peripheral blood mononuclear cells for the generation of antiviral central memory CD8 + One is from a subset of counter-suppressor T cells (Tcm), while the other is from a mobilized haploidentical peripheral blood preparation. Both preparations are collected via leukapheresis.

[0797] Non-mobilized (naive) peripheral blood mononuclear cells for the generation of Tcm cells were collected approximately 8 days before the planned transplant date (day 0). The mononuclear cell yield of the target leukapheresis was 1 × 10 10 Tcm cells are CD34 + The enriched preparation was infused on D+7 after the infusion.

[0798] After mobilization, peripheral blood cells were collected over two days: D-2 and D-1. Cells collected on D-2 underwent CD34 + The cells collected on D-1 were depleted of CD3 + / CD19 + cells and infused into the patient on D-0. Multiple collections can be pooled prior to the enrichment procedure. An unmodified PBMC reserve fraction containing 2 × 10 6 CD34 + cells / kg ideal body weight and cryopreserved.

[0799] Method Description

[0800] MDACC donors are first evaluated for suitability, including eligibility screening and physical examination. Then the donor's consent is obtained and collection is arranged. The target cell number is protocol-specific and may require multiple collections. After the collection procedure is completed, the cells are transported to the CTL laboratory from the collection facility in a plastic cooler by the CTL staff to protect the cells from temperature fluctuations and physical damage. The preparation is then recorded at the CTL.

[0801] Infectious disease testing and prevention of cross contamination (including donor eligibility, medical history, test list, unidentified donor eligibility of donors, release of cell preparations from ineligible donors, infusion of cell preparations with positive IDM results) - by The process was carried out as described in Example 2 of International Patent Application Publication No. WO2021 / 024264.

[0802] Cell processing

[0803] Antiviral central memory CD8 + Generation of anti-suppressive cells (Tcm cells)

[0804] Program Overview

[0805] For the generation of antiviral central memory CD8 + Antigen-naive peripheral blood mononuclear cells (PBMCs) for counter-suppressor T cells were collected 8 days before the planned infusion date (Day 0). Counter-suppressor cells were infused on Day 7 after the high dose of T cell-depleted cells.

[0806] like Figure 2 As shown in:

[0807] Day -8 to Day -7: Collection of unprimed leukapheresis, MNC isolation, and CD14 + Cell selection

[0808] PBMC isolation, Thrombowash:

[0809] The non-mobilized leukapheresis cell preparation of one embodiment is diluted 1:2 with Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium, and the DPBS is supplemented with 0.5% human serum albumin (HSA). Samples for cell count, trypan blue (TB) viability, and sterility are obtained before Ficoll treatment. MNCs are separated by Ficoll density gradient. After Ficoll, the MNC cell preparation of one embodiment is CD14 + Platelets were depleted twice by manual centrifugation (thrombowash) before isolation and resuspended with wash buffer. Samples for cell count and TB viability were removed for QC testing.

[0810] After the Thrombowash after Ficoll, the cells were divided into two equal fractions and diluted to approximately 500 mL each. Half was processed for CD14 + Selected dendritic cells (DCs) were isolated and the other half (fraction I) was kept overnight for CD8 +Memory T cell enrichment process.

[0811] Fraction I was centrifuged and the 30 × 10 6 The cells were resuspended in T cell growth medium (Click medium with advanced RPMI 1640 supplemented with 1:100 Glutamax and 5% human AB serum) at a concentration of 10 cells / ml along with IL-7 (30 IU / mL). A sample was removed for sterility testing. Fraction I was then plated onto a tissue culture flask and incubated overnight at 37°C with 5% CO2.

[0812] DC Isolation-CD14 + Monocyte isolation and adhesion

[0813] The DC fraction was centrifuged and resuspended in 50 ml of magnetic bead buffer (Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium, supplemented with 0.6% ACD-A and 0.5% HSA). Samples were taken for cell count and TB viability.

[0814] The MNC fraction isolated from DCs was incubated with a CD14 reagent (CD14 monoclonal antibody conjugated to superparamagnetic iron dextran particles). The cells were then washed with magnetic bead buffer to remove excess reagent. Samples were taken for cell counting, TB viability, and immunophenotyping. After washing, the cells were separated using an XS separation column in a SuperMACS ELISA kit according to established SOPs. TM II. Magnetic labeled cells (CD14 + ) are retained by the column and CD14 negative cells are removed. + The cells were released from the column and collected. + The fractions were washed and resuspended in DC medium (CellGro / 1% HSA), while the CD14- fraction was washed and resuspended in T cell growth medium. Samples from each fraction were taken for cell counts, TB viability and immunophenotyping.

[0815] One embodiment of CD14 + The enriched cell preparation was 3×10 6 The cells were resuspended in DC culture medium (CellGro / 1% HSA) at a concentration of 10 cells / ml, supplemented with IL-4 (1000 IU / mL) and GM-CSF (2000 IU / mL). A sample was removed for sterility testing. The cell suspension was then seeded in a Cell Factory plate and incubated overnight at 37°C, 5% CO2 for 16-24 hours.

[0816] The concentration of CD14 negative cells was adjusted to 30 × 106 cells / ml, T cell growth medium and IL-7 (30 IU / mL) were added. A sample was taken for sterility testing. The CD14 negative fraction was then plated into a tissue culture flask and incubated overnight at 37°C with 5% CO2. On the next day, these cells were combined with fraction I and used for CD8 + Memory T cell enrichment process.

[0817] Day -6: Isolation of memory cells and induction of DC maturation

[0818] DC maturation induction

[0819] In CD14 + After 24 hours of incubation of the enriched cells, cytokines: IL-4 (1000 IU / mL), GM-CSF (2000 IU / mL), LPS (40 ng / mL) and IFN-γ (200 IU / mL) were added to CellFactories to induce DC maturation. The cells were then incubated with cytokines at 37°C, 5% CO2 for 16 hours (+ / - 2 hours).

[0820] Isolation of memory cells

[0821] After overnight storage, cells in fraction 1 and CD14 negative fraction were harvested and combined. Once combined (T cell isolation TNC fraction), cells were centrifuged and resuspended in / 0.5% HSA buffer to a minimum ratio of 1:2. The cell pellet was resuspended in Platelet depletion (thrombowash) was performed in 0.5% HSA. Samples were removed for cell count and TB viability.

[0822] The platelet-depleted cell preparation of one embodiment was incubated with IVIg for 10-15 minutes. After the initial incubation, anti-CD4, anti-CD56, and anti-CD45RA reagents (anti-CD4, anti-CD56, and anti-CD45RA antibodies conjugated to superparamagnetic particles) were added to the cell preparation and incubated on an orbital rotator for 30 minutes. After the incubation period, the cells were washed by centrifugation and the cell pellet was resuspended in / 0.5% HSA buffer to remove excess reagents. Take samples for cell counting and immunophenotyping. Then use the depletion tubing set and depletion program. CD4 + / CD56 + / CD45RA + Labeled cells. In this case, magnetically labeled cells (CD4 +、CD56 + and CD45RA + ) are retained by the column, and CD4, CD56 and CD45RA negative cells are passed through the column and are counted as CD4 - CD56 - CD45RA - The depleted fraction (Fraction II) was collected. Fraction II was washed and resuspended in T cell growth medium. Samples from each fraction were taken for cell count, TB viability, and immunophenotyping. Once the volume and TNC of each fraction were determined, the positive fraction was discarded and the negative fraction (Fraction II) was further processed.

[0823] The concentration of fraction II cells was adjusted to 2 × 10 6 cells / ml. Take out a sample for sterility test. The cells were inoculated with 100 μL of culture medium and incubated at 37° C., 5% CO 2 for 24 hours (+ / - 2 hours).

[0824] limit:

[0825] The maximum TNC processed with one reagent kit and tubing set is 200 x 10 6 TNC / mL. The maximum loading volume of the instrument is 300 ml.

[0826] Day -5: Mature DC harvest, viral peptide loading and co-culture

[0827] Mature DC (mDC) harvest

[0828] After 16 hours of incubation, the supernatant was removed and CellFactories was washed gently with warm magnetic bead buffer. Wash buffer and any non-adherent cells were taken out and added to the mDC supernatant. Samples taken out from the mDC supernatant and wash buffer were used for cell counting, TB viability, and immunophenotyping.

[0829] Adherent mDCs were detached from the CellFactories and harvested by adding ice-cold magnetic bead buffer and letting it stand on a frozen gel pack for 30 minutes. After 30 minutes, the mDCs were harvested and the CellFactories were washed once with ice-cold magnetic bead buffer. The Cell Factories were examined microscopically to determine whether all mDCs were removed. If it was observed that not all mDCs were removed, the washing process was repeated. The harvested (adherent) mDC suspension was then centrifuged, washed, and resuspended in magnetic bead buffer. Samples from the adherent mDCs were taken for cell counting and TB. After determining the mDC volume and TNC, the cell concentration was adjusted to 1×10 7 Cells / ml were used for peptide loading. In-process samples were removed for immunophenotyping. Once the volume and TNC of each fraction were determined, the mDC supernatant fraction was discarded and the mDC cell preparation of one embodiment was further processed.

[0830] Viral peptide loading

[0831] After harvesting mDCs, the calculated peptides (AdV5 hexon, HCMV pp65, EBV selection, and BKV LT) were added to the cells and incubated for 1 hour at 37°C, 5% CO2. Following incubation, the viral peptide-loaded mDCs were washed with magnetic bead buffer and centrifuged. The viral peptide-loaded mDCs were then resuspended in T cell growth medium and irradiated with 30-25 Gy via an X-ray source.

[0832] Once irradiation was complete, viral peptide-loaded mDCs were washed, centrifuged, and resuspended in T cell growth medium. Samples were removed for cell counts, TB viability, immunophenotyping, and sterility.

[0833] Day -5 to Day -2: Co-culture preparation and starvation phase

[0834] In fraction II (CD4 - CD56 - CD45RA - After 24 hours of incubation, cells were removed from the incubator, centrifuged and resuspended in T cell growth medium. Samples were taken for cell count, TB viability, immunophenotyping and sterility. - CD56 - CD45RA - ) cells were washed and 1×10 6 cells / ml(100ml / 100) were inoculated into the rats together with mDC loaded with viral peptide at a ratio of 5:1 (Fraction II cells: dendritic cells (DC)). The co-cultured cells were incubated at 37° C. with 5% CO 2 for 3 days for the starvation period.

[0835] Day -2 to Day +6: Differentiation and expansion of antiviral suppressor cells (Tcm)

[0836] After the 72-hour starvation period, carefully remove the A sample of the supernatant was taken at 100 μl without disrupting the cell layer to determine the pH and glucose level of the culture. The pH should be in the physiological range (pH 7.2-7.6) and the glucose should be at least 50 mg / dl. Fresh T cell growth medium supplemented with IL-7 (30 IU / mL), IL-15 (125 IU / mL) and IL-21 (100 IU / mL) was added to each culture at 50% of the culture volume. 100. The cells were then incubated for another 48 hours at 37°C with 5% CO2. This process was repeated every 48 hours until the end of the culture on day +7.

[0837] Cells are cultured for a maximum of 12 days. Depending on pH and glucose levels, fresh medium containing cytokines or cytokines alone is added every 48 hours. If glucose levels are between:

[0838] Glucose levels of 170 mg / dL to 130 mg / dL: Only cytokines IL-7, IL-15, and IL-21 were added to the culture.

[0839] 129 mg / dL to 100 mg / dL glucose level: Add 100 ml ( Fresh T cell growth medium (25% of 100 volume) plus cytokines IL-7, IL-15, and IL-21 were added to the culture.

[0840] 99 mg / dL to 50 mg / dL glucose level: Add 200 ml ( 100% of the volume of fresh T cell growth medium + cytokines IL-7, IL-15, IL-21 were added to the culture.

[0841] It is worth noting that: The maximum volume level of 100 is 400ml. If reached If the maximum volume is 100, then the volume of the culture is replenished.

[0842] Day +4:

[0843] On day +4, a sterility sample was obtained for quality control release testing.

[0844] Day +6: Assessment of culture cell counts

[0845] On day +6, samples were removed for cell count, TB viability, and immunophenotyping for TNC determination.

[0846] Day +7: Final preparation and infusion of antiviral CD8 central memory cells (Tcm)

[0847] On day +7, Tcm cells were inspected for macroscopic contamination, harvested, and washed with cell suspension medium (Plamalyte-A / 0.5% of 25% HSA). Samples were removed for non-release testing: cell count, TB viability, sterility, and for release testing: endotoxin and mycoplasma before cell washing.

[0848] After washing and resuspending the cell preparation of one embodiment, a release test sample is taken for cell count, viability, and immunophenotyping. After cell count and TNC determination, the cell preparation of one embodiment is resuspended in approximately 50-100 ml of cell suspension medium to a Tcm cell dose according to the dose specified in the clinical protocol. A sample for release testing is obtained for Gram staining and sterility as a non-release test. Before the cell preparation is released, the final Tcm cell preparation meets the release criteria.

[0849] The final Tcm cells are transported from the CTL to the patient floor by CTL staff in plastic coolers to protect the cell preparation from temperature fluctuations and physical damage. CTL staff deliver and distribute the cell preparation to the infusionist.

[0850] CD34 + Generation of enriched cells

[0851] Program Overview

[0852] Mobilized hematopoietic progenitor cells (also called HPC-A) were collected within two days (D-2 and D-1). 6 CD34 + A spare fraction of unmodified PBMCs at 100 cells / kg ideal body weight was set aside and stored frozen. The remaining D-2 pool was stored overnight to deplete CD3 + / CD19 + cells and infused at D0. The maximum T cell dose for transplantation of whole T cell-depleted stem cells collected from all fractions was 2 × 10 5 CD3 + cells / kg ideal body weight.

[0853] Both procedures were performed in the CTL Core Laboratory using the Miltenyi CTL has a well-established SOP for the use of this device and has used it to manufacture cell preparations for multiple clinical trials. Depending on the cell number and timeline, individual HPC-A preparations can be split or pooled.

[0854] CD34 + Enrichment process

[0855] set up

[0856] Prepare a 0.5% human serum albumin (HSA) solution before processing according to a well-established laboratory SOP. PBS / EDTA buffer bag (1000ml).Determine the weight of HPC-A preparation.Take out the sample from day-collection (Day-collection) and be used for following test: cell count, viability, sterility, immunophenotyping.Each buffer and cell preparation bag are marked with patient's name, MDACC numbering and preparation date.

[0857] CD34 + Separation

[0858] The cells collected in D-2 of one embodiment are incubated with a CD34 reagent (CD34 antibody conjugated to superparamagnetic particles). The cells are washed to remove excess reagent. A sample is taken for cell counting and immunophenotyping. The sample is then used Piping set and CD34 + Select the program, In this case, magnetically labeled cells (CD34 + ) are retained by the column and CD34 negative cells are removed. CD34 + The cells were then released from the column and collected. Samples from each fraction were taken for cell count, viability, and immunophenotyping.

[0859] CD34 + The enriched cell preparation can be cryopreserved for infusion or can be used as fresh cells.

[0860] limit:

[0861] The maximum TNC processed with one reagent kit and tubing set is 6 × 10 10 .about The maximum loading volume of the instrument is 300 ml.

[0862] CD3 + / CD19 + exhaustion process

[0863] set up

[0864] Prepare a 0.5% human serum albumin (HSA) solution before processing according to a well-established laboratory SOP. PBS / EDTA buffer bags (1000 ml). Determine the weight of the HPC-A cell preparation. Remove samples from HPC-A for the following testing: cell count, viability, sterility, and immunophenotyping. Each buffer and cell preparation bag is labeled with the patient's name, medical record number (MRN), and preparation date.

[0865] Platelet depletion

[0866] In one embodiment, the HPC-A cell preparation has CD3 + / CD19 + Platelet depletion was performed prior to depletion using a COBE 2991. Once the cells were removed from the COBE 2991, a sample was taken for cell counting. The cell preparation was then incubated with a CD3 reagent and a CD19 reagent.

[0867] CD3 + / CD19 + Exhaustion

[0868] The platelet-depleted HPC-A cell preparation of one embodiment was incubated with IVIg for 10-15 minutes. + and CD19 + Reagents (CD3 and CD19 antibodies conjugated to superparamagnetic particles) were added to the cell preparation and incubated on an orbital rotator for 30 minutes. After the incubation period, the cells were washed using a COBE 2991 to remove excess reagents. Samples were taken for cell counting and immunophenotyping. The depletion tubing set and depletion program were then used to deplete the cells. Processing CD3 + / CD19 + Labeled cells. In this case, magnetically labeled cells (CD3 + / CD19 + ) are retained by the column, and CD3 and CD19 negative cells are passed through the column and are expressed as CD3 + / CD19 + The depleted fractions were collected and samples were taken from each fraction for cell count, viability, and immunophenotyping.

[0869] CD3 according to one embodiment + / CD19 + The depleted cell preparation is cryopreserved for infusion (as discussed below) or can be used as fresh cells.

[0870] Limitations: The maximum TNCs processed with one reagent kit and tubing set is 8 × 10 unless a higher number is approved by the Laboratory Director or designee. 10 .

[0871] Second CD3 + Depletion (not a necessary step, to be used as needed)

[0872] If there are more than 2×10 5 If the CD3+ / CD19+ depleted preparation has a CD3 / kg ideal body weight, a second cycle of CD3 depletion can be performed on the CD3+ / CD19+ depleted preparation to further reduce the CD3 population in the cell preparation. Samples for cell count and immunophenotyping, viability, and sterility are obtained from each fraction.

[0873] Alternatively, utilize only a portion of the T cell-depleted fraction for infusion in order to avoid infusion of more than 2 × 10 5 CD3 cells / kg ideal body weight.

[0874] CD34 + Enrichment and CD3 + / CD19 + Optional cryopreservation of depleted cell preparations:

[0875] After cell selection or depletion and sampling, the cell preparation of one embodiment is concentrated and cryopreserved according to the procedure verified in CTL. Before cryopreservation of the cell preparation, a final cell count, viability, immunophenotyping, Gram staining and sterility sample are obtained. After cell count and TNC determination, the cell preparation of one embodiment is cryopreserved in freezing medium (50% Plasma-Lyte A, 7.5% DMSO and 35% HSA 25%) according to SOP. Containing CD34 + Enriched cells or CD3 + / CD19 + Bags of depleted cells were frozen in a controlled rate freezer and stored in the vapor phase in a liquid nitrogen freezer.

[0876] It is important to note that the final cell preparation is the pre-cryopreservation preparation, as thawing of the cells occurs at the bedside without further manipulation.

[0877] Cell preparation release and infusion

[0878] On the day of infusion, CD34 + Enriched cells or CD3 + / CD19 +The spent cell preparation is delivered to the patient floor by CTL staff and distributed to the infusion staff. The final cell preparation meets batch release criteria before the cell preparation is released. These cells are then infused into the patient according to the protocol-specific dose. Of note, when cryopreserved, the cells are thawed on the floor and distributed to the infusion staff according to standard operating procedures. In addition, when cryopreserved, the final cell preparation is transported from the CTL to the floor by CTL staff in an approved polystyrene foam LN2 transport container with a seamless metal insert filled with approximately 2 inches of liquid nitrogen.

[0879] All cell preparation testing was protocol-specific and performed according to SOPs.

[0880] Reagents -As described in Example 2 of International Patent Application Publication No. WO2021 / 024264.

[0881] Testing of cell preparations- For details, see Example 2 of International Patent Application Publication No. WO2021 / 024264.

[0882] Cell preparation release standard testing and other tests

[0883] Table 3: Batch release testing and specifications for Tcm formulations

[0884]

[0885] Table 4: CD34 + Enriched formulation batch release testing and specifications

[0886]

[0887]

[0888] Table 5: Additional in-process testing (non-release)

[0889] test: Specification Sterility No growth at 14 days Immunophenotype For reference only TNC For reference only Mycoplasma (PCR) Negative

[0890] Example 3

[0891] Whole bone marrow irradiation combined with antiviral central memory CD8 + Anti-suppressive cells in haploidentical stem cell transplantation for the treatment of AML and MDS cancers

[0892] To generate countersuppressive cells, donor memory CD45RO+CD8 T cells were isolated and co-cultured with donor dendritic cells pulsed with peptides from four viruses (EBV, CMV, BKV, and adenovirus). Cultures were performed under cytokine-deprived conditions for 3 days to allow the death of anti-host clones by neglect, and then IL15, IL21, and IL7 were added to allow the expansion of antiviral central memory CD8 T cells. In 11 runs, a single leukapheresis yielded >1×10 10 CD45RO+CD3+CD8+CD62L+T cells. At the end of 12 days of culture, the proportion of CD3+CD8+T cells was 96.4±2.5%, of which 80.1±10.6% (range 71.5 to 92.8) presented the CD62L+CD45RO+CM phenotype. Limiting dilution analysis revealed that alloreactive T cell clones were depleted by >3 log levels. Antiviral activity was detected by intracellular expression of TNF-α and INF-g, and the results showed that after 6 hours of stimulation with the viral peptide mixture, the average positive cell rate was 38.8±19.6%.

[0893] Three patients (two with AML and one with MDS, see Table 6 below) received 2 mg / kg anti-thymocyte globulin daily on days -8 to -6 and 30 mg / m daily on days -5 to -2. 2 Fludarabine treatment, and 12Gy whole bone marrow irradiation and spleen irradiation for 4 consecutive days (see Examples 1 and Figure 1 On days 0 and 1, two collections of donor G-CSF-mobilized peripheral blood progenitor cells were performed. The first infusion was of CD34-selected cells, and the second was of CD3+ / CD19+-depleted cells. The infusion consisted of 1-2 × 10 CD3+ T cells / kg. Cyclophosphamide (CY) at 50 mg / kg was administered on days 3 and 4. Countersuppressive cells (5 × 10 / kg) were infused on day 7.

[0894] All three patients successfully engrafted (see Table 6 below). One of the AML patients developed a steroid-responsive rash, which likely represented overall grade 2 GVHD of the skin, although a skin biopsy suggested an allergic reaction to the drug. GVHD was not detected in the second AML patient or the MDS patient. All patients had significant donor myeloid and T cell chimerism at all time points tested (see Table 7 below).

[0895] In conclusion, our data suggest that haploidentical TCDHSCT combined with antiviral CM countersuppressive CD8 T cells after well-tolerated reduced-intensity conditioning including TMI results in reliable engraftment and shows a low incidence of GVHD on day 4 in the absence of immunosuppression other than CY treatment.

[0896] Table 6: Date of engraftment (>0.5 ANC), GVHD, discharge, and relapse following transplantation of T cell-depleted high-dose HSCT with 5 million / kg antiviral central memory suppressor cells

[0897]

[0898]

[0899] *Recovered from autoimmune neutropenia and awaiting recovery from thrombocytopenia

[0900] Table 7: Myeloid T cell chimerism

[0901]

[0902] Although the present invention has been described in conjunction with its specific embodiments, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0903] All publications, patents and patent applications mentioned in this specification are incorporated herein in their entirety, as if each individual publication, patent or patent application were clearly and individually indicated to be incorporated herein by reference when cited. In addition, the citation or identification of any reference in this application should not be construed as admitting that such reference can be used as prior art of the present invention. With regard to the use of section headings, they should not be construed as necessary limitations. In addition, any priority document of the present application is incorporated herein by reference in its entirety.

Claims

1. A method of treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject, the method comprising: (a) conditioning the subject under a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI); and subsequently (b) transplanting a therapeutically effective amount of T cell-depleted immature hematopoietic cells into the subject; and subsequently (c) administering to the subject a therapeutically effective amount of cyclophosphamide; And then (d) administering to the subject a therapeutically effective amount of cells that suppress non-graft-versus-host disease (GVHD) inducing cells, thereby treating the disease in the subject.

2. A combination of a pre-transplant conditioning regimen comprising a therapeutically effective amount of total bone marrow irradiation (TMI), a therapeutically effective amount of T cell-depleted immature hematopoietic cells, a therapeutically effective amount of cyclophosphamide, and a therapeutically effective amount of anti-graft versus host disease (GVHD) inducing cells for use in treating a disease in a subject in need thereof, wherein the disease comprises pathological cells present in the bone marrow of the subject.

3. The method according to claim 1 or the combination for use according to claim 2, wherein the disease is cancer.

4. The method or combination for use according to claim 3, wherein the cancer comprises a myeloid malignancy or multiple myeloma.

5. The method or combination for use according to claim 4, wherein the myeloid malignancy is selected from the group consisting of AML, CML and MDS.

6. The method or combination for use according to claim 3, wherein the cancer comprises AML or MDS.

7. The method of claim 1 or the use of the combination of claim 2, wherein the disease is non-cancerous.

8. The method or combination for use according to claim 7, wherein the disease is selected from the group consisting of sickle cell anemia, aplastic anemia, thalassemia and metabolic genetic diseases.

9. The method or combination for use according to any one of claims 1 to 8, wherein the counter-suppression non-GVHD-inducing cells and the T cell-depleted immature hematopoietic cells are obtained from the same donor.

10. The method or combination for use according to any one of claims 1 to 9, wherein the conditioning further comprises a therapeutically effective amount of spleen irradiation.

11. The method or combination for use according to any one of claims 1 to 10, wherein the TMI is administered on day -7 to -1 before transplantation of the T cell-depleted immature hematopoietic cells.

12. The method or combination for use according to any one of claims 1 to 11, wherein the therapeutically effective amount of the TMI comprises 8-18 Gy in total.

13. The method or combination for use according to any one of claims 1 to 12, wherein the therapeutically effective amount of the TMI is administered in at least 2 doses administered on consecutive days.

14. The method or combination for use according to any one of claims 1 to 13, wherein the therapeutically effective amount of the TMI is administered consecutively in 4 doses.

15. The method or combination for use according to any one of claims 1 to 14, wherein the T cell-depleted immature hematopoietic cells are from a donor that is not syngeneic with the subject.

16. The method or combination for use according to claim 15, wherein the non-syngeneic is allogeneic.

17. The method or combination for use according to claim 16, wherein the allogeneic donor is an HLA-matched sibling, an HLA-matched unrelated donor, an HLA haploidentical related donor, or a donor displaying one or more different HLA determinants.

18. The method or combination for use according to any one of claims 1 to 17, wherein the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises less than 5×10 5 CD3 + T cells / kg ideal body weight of the subject.

19. The method or combination for use according to any one of claims 1 to 17, wherein the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises less than 2×10 5 CD3 + T cells / kg ideal body weight of the subject.

20. The method or combination for use according to any one of claims 1 to 19, wherein the therapeutically effective amount of the T cell-depleted immature hematopoietic cells comprises at least 5×10 6 CD34 + cells / kg of the subject's ideal body weight.

21. The method or combination for use according to any one of claims 1 to 20, wherein the therapeutically effective amount of the T cell-depleted immature hematopoietic cells is depleted of CD3 + and / or CD19 + expressing cells.

22. The method or combination for use according to any one of claims 1 to 21, wherein the therapeutically effective amount of the cyclophosphamide comprises 25-200 mg cyclophosphamide per kilogram of the subject's ideal body weight.

23. The method or combination for use according to any one of claims 1 to 22, wherein the therapeutically effective amount of the cyclophosphamide is administered to the subject in two doses between 2 and 5 days after the transplantation of the T cell-depleted immature hematopoietic cells.

24. The method or combination for use according to any one of claims 1 to 22, wherein the therapeutically effective amount of the cyclophosphamide is administered to the subject in two doses 3 and 4 days after the transplantation of the T cell-depleted immature hematopoietic cells.

25. The method or combination for use according to any one of claims 1 to 24, wherein the counter-suppression non-GVHD inducing cells comprise a central memory T lymphocyte (Tcm) phenotype.

26. The method or combination for use according to any one of claims 1 to 25, wherein the counter-suppressed non-GVHD inducing cells have antiviral activity.

27. The method or combination for use according to any one of claims 1 to 26, wherein the counter-suppression non-GVHD inducing cells can be obtained by: (i) a first population of peripheral blood mononuclear cells (PBMC) from a donor is subjected to a cytokine receptor capable of binding to CD14 + The cells expressing the antibody were contacted and CD14 that could mature into antigen presenting cells were selected. + expressing cells; (ii) loading the antigen-presenting cells with viral peptides; (iii) using a drug that can deplete CD4 + 、CD56 + and CD45RA + A second population of PBMCs from the same donor as the first population of PBMCs is treated with one or more agents expressing cells to obtain cells enriched for CD45RA expression. - CD8 + Phenotypic memory T cell populations; (iv) contacting the cell population comprising the T cells enriched in the memory T cells with the antigen-presenting cells loaded with the viral peptides of step (ii) in the presence of IL-21, so as to allow enrichment of virus-reactive memory T cells; and (v) culturing the cells obtained in step (iv) in the presence of IL-21, IL-15 and / or IL-7 to allow proliferation of cells comprising the Tcm phenotype.

28. The method or combination for use according to any one of claims 1 to 27, wherein the therapeutically effective amount of the counter-suppressive non-GVHD inducing cells is administered on day 6-9 after the transplantation of the T cell-depleted immature hematopoietic cells.

29. The method or combination for use according to any one of claims 1 to 28, wherein the therapeutically effective amount of the counter-suppression non-GVHD inducing cells comprises at least 2.5×10 6 CD8 + cells / kg of the subject's ideal body weight.

30. The method or combination for use according to any one of claims 1 to 29, wherein the subject is not treated long-term with GVHD prophylaxis after the transplant.

31. The method or combination for use according to any one of claims 1 to 30, wherein the conditioning further comprises anti-B cell therapy.

32. The method or combination for use according to claim 31 , wherein the anti-B cell therapy comprises an anti-B cell antibody.

33. The method or combination for use according to any one of claims 31 to 32, wherein the anti-B cell therapy comprises rituximab.

34. The method or combination for use according to any one of claims 1 to 33, wherein the conditioning further comprises T cell depletion.

35. The method or combination for use according to claim 34, wherein the T cell depletion is achieved by antibodies, and optionally wherein the antibodies comprise at least one of an anti-thymocyte globulin (ATG) antibody, an anti-CD52 antibody, or an anti-CD3 antibody.

36. The method or combination for use according to claim 34, wherein the T cell depletion is achieved by anti-thymocyte globulin (ATG) antibodies.

37. The method or combination for use according to any one of claims 1 to 36, wherein the conditioning further comprises a chemotherapeutic agent.

38. The method or combination for use according to claim 37, wherein the chemotherapeutic agent comprises at least one of fludarabine, busulfan, melphalan, thiotepa and cyclophosphamide.

39. The method or combination for use according to claim 37, wherein the chemotherapeutic agent comprises fludarabine.

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