Treg cell therapy combined with interleukin-2 for GVHD

Abstract

This disclosure relates to the field of cell therapy technology, specifically to a method for treating GVHD using Treg cells combined with IL-2. In this method, IL-2 enhances the persistence and lineage stability of Treg cells, thereby improving the efficacy of Treg cell therapy, while simultaneously reducing or even avoiding the potential side effects of IL-2 therapy activating effector T cells and NK cells.

Description

Technical Field

[0001] This disclosure relates to the field of cell therapy technology, specifically to a method for treating or preventing graft-versus-host disease (GVHD) using Treg cells in combination with interleukin-2 (IL-2). Background Technology

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the key methods for treating benign and malignant hematologic diseases. With the widespread use of unrelated donor transplantation, haploidentical transplantation, and peripheral blood grafts, graft-versus-host disease (GVHD) has become one of the most common complications of allo-HSCT. According to the consensus criteria of the National Institutes of Health (NIH), GVHD is divided into acute GVHD (aGVHD) and chronic GVHD (cGVHD) based on clinical characteristics and time of onset after transplantation (Jagasia MH, Greinix HT, Arora M, et al: National institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: The 2014 Diagnosis and Staging Working Group Report. Biol BloodMarrow Transplant. 2015, 21(3):389–401.). Among patients who survive at least 100 days after transplantation, approximately 30%-70% will develop cGVHD. The incidence rate is closely related to factors such as donor and recipient sex, age, HLA matching, conditioning regimen, and source of hematopoietic stem cells (Flowers MED, Inamoto Y, Carpenter PA, et al: Comparative analysis of risk factors for acute graft-versus-host disease and for chronic graft-versus-host disease according to National Institutes of Health consensus criteria. Blood. 2011, 117(11):3214-3219.).

[0003] Unlike aGVHD, the pathogenesis of cGVHD is not fully understood. It is generally believed that cGVHD is a clinicopathological syndrome caused by the failure of donor-derived B cells and T cells to develop tolerance to allogeneic and autoantigens during the reconstruction of the donor's immune system in allo-HSCT recipients (Pidala J, Kurland B, Chai X, et al: Patient-reported quality of life is associated with severity of chronicgraft-versus-host disease as measured by NIH criteria: report on baseline data from the Chronic GVHD Consortium. Blood. 2011, 117(17):4651-4657.). Its pathogenesis begins with tissue damage, leading to the presentation of co-stimulatory molecules by dendritic cells to T cells, which in turn activates allogeneic and autoreactive T cells. These T cells further activate pathological B cells, releasing harmful immunoglobulins (Zeiser R, Blazar BR: Pathophysiology of chronic graft-versus-host disease and therapeutic targets. N Engl J Med. 2017, 377(26):2565-2579.). Simultaneously, lymphocyte activation is accompanied by macrophage activation and the release of profibrotic growth factors.Thymic injury and loss of central tolerance caused by transplant preconditioning and T cell alloresponsiveness, coupled with poor CD4+ regulatory T cell (Treg) remodeling, jointly lead to peripheral tolerance dysfunction (Kawano Y, Kim HT, Matsuoka KI et al: O37-Low telomerase activity in CD4+regulatory T cells in patients with severe chronic GVHD after hematopoietic stem cell transplantation. Blood. 2011, 118(18):5021-5030., Matsuoka Ki, Kim HT, McDonough S, et al: Altered regulatory T cell homeostasis in patients with CD4+lymphopenia following allogeneic hematopoietic stem cell transplantation. J Clin Invest. 2010, 120(5):1479-1493.). The loss of central and peripheral tolerance allows uncontrolled inflammatory responses to drive the fibrotic process. Fibrosis can affect multiple organ systems, with common sites including the skin, hair, nails, oral cavity, liver, eyes, gastrointestinal tract, genitals, joint fascia, or bones and joints (Socié G, Ritz J: Current issues in chronic graft-versus-host disease. Blood. 2014, 124(3):374-384.). These multi-organ injuries make cGVHD a significant cause of non-recurrence mortality and decreased quality of life for patients after allo-HSCT.

[0004] In hematopoietic stem cell transplantation, the prevention of GVHD is often carried out as a whole, and not just targeting cGVDH. Graft source has become one of the important factors affecting the occurrence of GVHD. In recent years, peripheral blood stem cells have become the main source of grafts, but because they contain more mature donor T lymphocytes, while reducing the risk of disease recurrence, they also increase the incidence of GVHD in recipients (Ohue Y, Nishikawa H:Regulatory T(Treg)cells in cancer:Can Tregcells be a new therapeutic target? Cancer Sci. 2019, 110(7):2080-2089.). Unrelated donors and haploidentical donors are more likely to cause GVHD than sibling fully matched donors, while GVHD is less severe in cord blood hematopoietic stem cell transplant recipients (Shimosato Y, Tanoshima R, Tsujimoto SI, et al: Allogeneic bone marrow versus peripheral blood stem cell transplantation for hematological malignancies in children: a systematic review and meta-analysis. Biol Blood Marrow Transplant. 2020, 26(1): 88-93., Zheng C, Zhu X, Tang B, et al: Transplants of unrelated cord blood or sibling allogeneic peripheral blood stem cells / bonemarrow in adolescent and young adults with chronic myeloid leukemia: comparable outcomes but better chronic GVHD-free and relapse-free survival among survivors with cord blood. Oncotarget. 2017, 9(2): 2848-2857.). However, graft selection is limited in many cases.Another risk factor associated with GVHD is the prevention regimen, specifically the choice of immunosuppressant combination. Commonly used regimens include calcineurin inhibitors (CNI) + methotrexate (MTX) + mycophenolate mofetil (MMF) / sirolimus (SRL), anti-thymocyte globulin (ATG), and post-transplant cyclophosphamide (CTX). Even with GVHD prevention treatment, a significant proportion of patients will still develop moderate to severe cGVHD (≥3 organs involved or a single organ involvement score of 2 or higher). Therefore, the treatment of cGVHD should not be neglected.

[0005] Currently, the main treatment for cGVHD is immunosuppressive drugs. Glucocorticoids have a wide range of immunosuppressive effects and are currently the first-line treatment for moderate to severe cGVHD. However, due to the heterogeneity of cGVHD, the efficacy rate of first-line treatment is less than 50% (Group HSCA, Hematology CSo, Association CM, et al: Chinese consensus on the diagnosis and management of chronic graft-versus-host disease (2021). Chin J Hematol. 2021, 42(4): 265-275.); and long-term exposure to glucocorticoids can lead to disease relapse, significant adverse reactions, and an increased risk of infection. In addition, more than half of the patients develop steroid resistance or dependence, namely hormone-refractory chronic graft-versus-host disease (SR-cGVHD) (Wolff D, Fatobene G, Rocha V, et al: Steroid-refractory chronic graft-versus-host disease: treatment options and patient management. Bone Marrow Transplant. 2021, 56(9):2079-2087.).

[0006] SR-cGVHD requires second-line treatment, but there is currently no standard second-line treatment for SR-cGVHD. Although more and more treatment options are available, relevant research data is limited, and no consensus has been reached on the best approach. The choice of second-line treatment drugs should be based on individual circumstances and the characteristics of the affected organs and tissues. The most frequently reported second-line treatment is extracorporeal photopheresis (ECP), which involves extracting circulating cells and then irradiating them with long-wave ultraviolet light outside the body. Its efficacy is approximately 60% (Lucid CE, Savani BN, Engelhardt BG, et al: Extracorporeal photopheresis in patients with refractory bronchiolitis obliterans developing after allo-SCT. Bone Marrow Transplant. 2011, 46(3):426-429., Malik MI, Litzow M, Hogan W, et al: Extracorporeal photopheresis for chronic graft-versus-host disease: a systematic review and meta-analysis. Blood Res. 2014, 49(2):100-106.). Although ECP is generally well-tolerated, it requires frequent hospital visits, has a long onset time, and cannot be used alone; therefore, it is not currently used in China. Other promising candidate drugs include ruxolitinib and ibrutinib. However, both of these types of drugs have limited efficacy and are prone to causing infections. Other second-line treatments include low-dose methotrexate (MTX), serotonin (SRL), rituximab, imatinib, mesenchymal stem cells (MSCs), azathioprine, low-dose interleukin-2 (IL-2), and mycophenolate mofetil (MMF). Overall, current second-line treatments for SR-cGVHD have not shown good efficacy and have corresponding side effects, therefore, there is an urgent need to develop safe and effective new therapies.

[0007] Treg cell therapy, as a novel treatment strategy for graft-versus-host disease (GVHD), exerts its therapeutic effect through its unique immunosuppressive function: on the one hand, Treg cells can secrete inhibitory cytokines such as IL-10 and TGF-β, competitively consuming IL-2 and playing a role in cell lysis, directly inhibiting the proliferation of overactivated effector cells; on the other hand, by regulating the maturation and antigen presentation function of dendritic cells (DCs), it reshapes the immune tolerance microenvironment (VIGNALI DA, COLLISON LW, WORKMAN C J. How regulatory T cells work[J].Nat Rev Immunol,2008,8(7):523-32.). Compared to traditional glucocorticoids or calcineurin inhibitors (CNIs), this therapy not only significantly reduces the risk of infection and drug-related organ toxicity, but also targets and restores immune homeostasis while preserving graft-versus-leukemia (GVL) effects, providing a new approach to balancing GVHD prevention and control with anti-tumor efficacy (GUO WW, SU XH, WANG MY, et al. Regulatory TCells in GVHD Therapy[J]. Front Immunol, 2021, 12: 697854.).

[0008] However, Treg cells account for less than 5% of peripheral blood mononuclear cells (PBMCs), and their low natural abundance limits their direct acquisition, while low-dose Treg cell therapy has limited efficacy. Summary of the Invention

[0009] To address this bottleneck, this paper discloses a highly efficient expansion technology: Treg cells are sorted from PBMCs using the CD4+CD25+CD127low marker, and after specific expansion and culture in vitro, they can achieve a proliferation of 500-2000 times, ultimately obtaining the dose required for clinical treatment. It is worth noting that previous clinical studies have suggested that the survival time of expanded Treg cells after simple infusion is relatively short, which may be related to increased apoptosis caused by insufficient cytokine IL-2 after reinfusion (DONG S, HIAM-GALVEZ KJ, MOWERY CT, et al. The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with type 1 diabetes[J]. JCI Insight, 2021, 6(18).).

[0010] Therefore, this disclosure proposes the combined use of Treg cells and low-dose IL-2: subcutaneous injection of low-dose IL-2 sustainably activates the high-affinity IL-2 receptor on the surface of Treg cells, providing them with survival and function maintenance signals, thereby prolonging the in vivo persistence of infused Treg cells. Simultaneously, it reduces the effect of IL-2 in activating pathogenic effector T cells or NK cells, significantly reducing the risk of excessive immune activation in IL-2 monotherapy. In this combined strategy, Treg cells directly suppress pathological immune responses, while low-dose IL-2 enhances Treg function through metabolic support and homeostatic expansion. The two complement each other mechanistically, ultimately achieving a synergistic therapeutic effect.

[0011] On one hand, this disclosure provides the use of Treg cells and interleukin-2 (IL-2) in the preparation of a pharmaceutical combination for the treatment or prevention of graft-versus-host disease. Furthermore, this disclosure provides the use of Treg cells in the preparation of an synergist for the treatment or prevention of graft-versus-host disease using IL-2. Additionally, this disclosure provides the use of IL-2 in the preparation of an synergist for the treatment or prevention of graft-versus-host disease using Treg cells. Furthermore, this disclosure provides a pharmaceutical combination comprising Treg cells and IL-2 for the treatment or prevention of graft-versus-host disease.

[0012] In some specific embodiments, the Treg cells are at a rate of 1 × 10⁻⁶. 5 Treg cells / kg up to 1×10 8 Administered at a dose of Treg cells / kg, preferably 1×10⁻⁶. 6 Treg cells / kg up to 5×10 7 The preferred dosage of Treg is 1 × 10⁻⁶. 6 Treg cells / kg up to 1×10 7 Administer Treg cells per kg.

[0013] In some specific embodiments, the Treg cells are formulated for intravenous infusion.

[0014] In some specific embodiments, the Treg cells are administered once or multiple times, for example, 1-3 times, preferably once.

[0015] In some specific embodiments, the IL-2 is recombinant human interleukin-2 (rhIL-2).

[0016] In some specific embodiments, the IL-2 is delivered at a rate of 500,000 units / m 2 / d to 2 million units / m 2 / d, preferably 800,000 units / m 2 / d to 1.5 million units / m 2 / d, more preferably 1 million units / m 2 Administer a dose of / day.

[0017] In some specific embodiments, the IL-2 is formulated for subcutaneous injection.

[0018] In some specific implementations, the IL-2 is administered subcutaneously once or in multiple doses (e.g., twice) daily.

[0019] In some embodiments, the proportion of viable cells in the Treg cells is >80%, preferably >90%. In some embodiments, the proportion of CD8+ cells in the Treg cells is <5%, preferably <2%. In some embodiments, the proportion of CD4+ cells in the Treg cells is >95%, preferably >98%. In some embodiments, the proportion of FOXP3+ cells in the Treg cells is >60%, preferably >70%, more preferably >80% or >90%. In some embodiments, the proportion of Helios+FOXP3+ cells in the Treg cells is >50%, preferably >60%, more preferably >70% or >80%. In some embodiments, the TSDR demethylation rate of the Treg cells is >60%, preferably >70%, more preferably >80% or >90%.

[0020] In some specific embodiments, the proportion of live cells in the Treg cells is >80%, the proportion of CD8+ cells is <5%, the proportion of CD4+ cells is >95%, the proportion of FOXP3+ cells is >60%, the proportion of Helios+FOXP3+ cells is >50%, and the TSDR demethylation is >60%.

[0021] In some specific embodiments, the proportion of Helios-FOXP3- cells in the Treg cells is ≤10%, preferably ≤9%, ≤8%, or ≤7%, more preferably ≤6%, ≤5%, or ≤4%, and even more preferably ≤3%, ≤2%, or ≤1%.

[0022] In some specific embodiments, the proportion of Helios-FOXP3+ cells in the CD4+CD25+CD127low / - cell population of the PBMCs used to prepare the Treg cells is ≤16%, preferably ≤15%, ≤14%, or ≤13%, more preferably ≤12%, ≤11%, or ≤10%.

[0023] In some specific embodiments, the Treg cells are autologous or allogeneic Treg cells, such as allogeneic Treg cells.

[0024] In some specific embodiments, the graft-versus-host disease is chronic graft-versus-host disease (cGVHD), preferably refractory chronic graft-versus-host disease, such as hormone-refractory chronic graft-versus-host disease (SR-cGVHD).

[0025] In some specific embodiments, the PBMC cells used to prepare the Treg cells are derived from hematopoietic stem cell transplant donors in subjects suffering from graft-versus-host disease.

[0026] On the other hand, this disclosure provides a kit or drug combination comprising Treg cells and IL-2.

[0027] In some specific embodiments, the IL-2 is recombinant human interleukin-2 (rhIL-2).

[0028] In some specific embodiments, the Treg cells are 4 × 10⁻⁶. 6 Treg cells increased to 1×10 10 Treg cells, preferably 4×10 7 Treg cells increased to 1×10 9 A single-dose form of Treg cells.

[0029] In some specific embodiments, the IL-2 is in a single-dose form of 650,000 to 11,000,000 units, preferably 1,300,000 to 2,200,000 units.

[0030] In some embodiments, the proportion of viable cells in the Treg cells is >80%, preferably >90%. In some embodiments, the proportion of CD8+ cells in the Treg cells is <5%, preferably <2%. In some embodiments, the proportion of CD4+ cells in the Treg cells is >95%, preferably >98%. In some embodiments, the proportion of FOXP3+ cells in the Treg cells is >60%, preferably >70%, more preferably >80% or >90%. In some embodiments, the proportion of Helios+FOXP3+ cells in the Treg cells is >50%, preferably >60%, more preferably >70% or >80%. In some embodiments, the TSDR demethylation rate of the Treg cells is >60%, preferably >70%, more preferably >80% or >90%.

[0031] In some specific embodiments, the proportion of live cells in the Treg cells is >80%, the proportion of CD8+ cells is <5%, the proportion of CD4+ cells is >95%, the proportion of FOXP3+ cells is >60%, the proportion of Helios+FOXP3+ cells is >50%, and the TSDR demethylation is >60%.

[0032] In some specific embodiments, the proportion of Helios-FOXP3- cells in the Treg cells is ≤10%, preferably ≤9%, ≤8%, or ≤7%, more preferably ≤6%, ≤5%, or ≤4%, and even more preferably ≤3%, ≤2%, or ≤1%.

[0033] On the other hand, this disclosure also provides a method for treating or preventing graft-versus-host disease, comprising administering an effective amount of Treg cells and IL-2 to a subject. The Treg cells and IL-2, as well as their dosage and administration method, are as described above.

[0034] In some specific implementations, IL-2 administration begins 5-10 days before Treg cell administration and continues until 8-16 weeks after Treg cell infusion. Preferably, after a 4-week pause, the decision to continue IL-2 administration is made based on the treatment response and the patient's wishes.

[0035] In this disclosure, the aforementioned Treg cells can be prepared, for example, by the following in vitro expansion method. This method includes the following steps:

[0036] (1) CD4+CD25+CD127low / - Treg cells were sorted from PBMC cells;

[0037] (2) The Treg cells obtained in step (1) were expanded in vitro in the presence of rapamycin and IL-2, or in the presence of rapamycin, ABBV-744 and IL-2.

[0038] In some specific embodiments, the proportion of Helios-FOXP3+ cells in the CD4+CD25+CD127low / - cell population of the PBMC cells is ≤16%, preferably ≤15%, ≤14%, or ≤13%, more preferably ≤12%, ≤11%, or ≤10%. By using such PBMC cells to prepare Treg cells, the expansion fold of Treg cells can be further increased.

[0039] In some specific embodiments, in step (1), Treg cells are sorted, preferably using fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

[0040] In some specific embodiments, step (1) includes the sorting step of enriching CD25+ cells and deleting cells that highly express CD127.

[0041] In some specific embodiments, step (1) of the magnetically activated cell sorting method includes:

[0042] (1-1) CD4+CD25+ cells were separated using a mixture of microbeads to remove non-CD4+ and CD25+ cells;

[0043] (1-2) The CD4+CD25+ cells pre-enriched in step (1-1) are labeled with CD127 magnetic beads to remove cells with high CD127 expression, thereby sorting out CD4+CD25+CD127low / - Treg cells.

[0044] In some specific embodiments, step (2) of the in vitro amplification further includes an anti-CD3 / CD28 magnetic bead activation step. In some specific embodiments, in step (2), the cell-to-magnetic bead activation ratio is between 1:1 and 1:3. In some specific embodiments, in step (2), the anti-CD3 / CD28 magnetic bead activation is performed more than twice; preferably on day 1 and day 9.

[0045] In some specific embodiments, in step (2), the concentration of rapamycin is 10-200 nM, preferably 20-70 nM, and more preferably 40-60 nM.

[0046] In some specific embodiments, when ABBV-744 is used in step (2), the concentration of ABBV-744 is 1-200 nM, preferably 1-150 nM, and more preferably 10-100 nM.

[0047] In some specific embodiments, in step (2), the concentration of IL-2 is 100-1000 units / mL, preferably 300-500 units / mL.

[0048] In some specific embodiments, step (2) is preferably performed in the presence of rapamycin, ABBV-744 and IL-2.

[0049] In some specific implementations, the in vitro amplification time in step (2) is 14-21 days.

[0050] In some specific embodiments, the PBMC cells are derived from tissue samples such as peripheral blood, thymus, and umbilical cord blood.

[0051] In some specific embodiments, the PBMC cells are human PBMCs.

[0052] Beneficial effects

[0053] The Treg cell combined with IL-2 therapy disclosed herein improves the persistence and lineage stability of Treg cells, thereby enhancing the efficacy of Treg cell therapy, while reducing or even avoiding the side effects of IL-2 therapy that may activate effector T cells and NK cells, thus achieving a synergistic effect. Attached Figure Description

[0054] This disclosure can be more fully understood with reference to the following figures.

[0055] Figure 1 The results of flow cytometry analysis of expanded Treg cells in vitro are shown.

[0056] Figure 2 The study shows the TSDR demethylation level of Treg cells after in vitro expansion detected by qPCR.

[0057] Figure 3 The results of Treg cell prevention and treatment of xenogeneic GVHD in NSG mice are shown.

[0058] Figure 4 This study demonstrated that Treg cells inhibit the proliferation of human T cells in the peripheral blood of xenogeneic GVHD mice.

[0059] Figure 5 The duration and lineage stability of IL-2-promoted Treg cells in mice were demonstrated.

[0060] Figure 6 The correlation analysis of the proportion of Helios-FOXP3+ cells in the CD4+CD25+CD127low / - cell population of PBMC cells with the fold expansion in vitro is shown (A), and the correlation analysis of the proportion of Helios-FOXP3- cells in seeded Treg cells with TSDR demethylation is shown (B). Detailed Implementation

[0061] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0062] As used herein, the term "regulatory T cells" or "Treg" refers to a subset of T cells that constitutively express the transcription factor forkhead box protein P3 (Foxp3). This subset regulates the immune system, maintains tolerance to self-antigens, and eliminates autoimmune and inflammatory diseases. These cells typically suppress or downregulate the induction and proliferation of effector T cells and regulate antigen-presenting cell function. Tregs are cells capable of exhibiting inhibitory activity (i.e., suppressing the proliferation of conventional T cells) through cell-cell contact or through the release of immunosuppressive cytokines. To date, two major classes of Tregs have been identified: CD4 and CD8 Tregs. CD4 Tregs consist of two types: "native" Tregs (nTregs) that constitutively express CD25 and FOXP3, and so-called adaptive or inducible Tregs (iTregs). Native Tregs originate from the thymus, and CD4+ cells express high levels of CD25 and the transcription factor FoxP3. nTregs comprise approximately 5%–10% of the total CD4+ T cell population and can first appear in the single-positive stage of T lymphocyte development. These are positively selected thymocytes with a relatively high affinity for self-antigens. The signaling for the development of Treg cells is thought to originate from the interaction between T cell receptors and MHC II complexes with self-peptides expressed on the thymic matrix.

[0063] As used herein, the term "cell" or "cell population" refers to a population of cells in which the majority (i.e., at least 50% (optionally at least 60%, at least 70%, or at least about 80%)) of the total number of cells have the specified characteristics (e.g., functional characteristics and / or markers of interest) of the cells of interest. Thus, "Treg cells" or "Treg cell population" refers to a population of cells in which the majority of cells are Treg cells but some cells may be non-Treg cells (e.g., some cells may be Teff cells).

[0064] As used herein, “culture” refers to the growth of one or more cell types in vitro under defined or controlled conditions. Examples of defined culture conditions include temperature, gas mixture, time, and culture medium formulation.

[0065] As used herein, the terms “culture medium” and “cell culture medium” and “feeding medium” and “fermentation medium” refer to nutrient solutions used to grow and / or maintain cells, particularly mammalian cells. Without limitation, these solutions typically provide at least one component from one or more of the following categories: (1) an energy source, typically in the form of carbohydrates such as glucose; (2) all essential amino acids, typically the basic set of twenty amino acids; (3) low concentrations of vitamins and / or other organic compounds; (4) free fatty acids or lipids, such as linoleic acid; and (5) trace elements, wherein trace elements are defined as inorganic compounds or naturally occurring elements typically required in very low concentrations (typically in the micromolar range). Nutrient solutions may be selectively supplemented with one or more components from any of the following categories: (1) hormones and other growth factors, such as serum, insulin, transferrin, and epidermal growth factor; (2) salts, such as magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; (5) proteins and tissue hydrolysates, such as peptones or peptone mixtures obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamicin; (7) cell protectants, such as Pranicol polyol; and (8) galactose. Commercially available culture media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing host cells. Any other necessary supplements may also be included in the culture medium at appropriate concentrations.

[0066] Rapamycin is an antifungal antibiotic isolated from *Streptomyces hygroscopicus*. It is a macrocyclic triene antibiotic that binds to and inhibits the molecular target of rapamycin (mTOR); it also forms a complex with FKBP12, binding to and inhibiting the molecular target of rapamycin (mTOR). In some embodiments, the concentration of rapamycin in the cell culture medium can be about 10-200 nM, for example, about 10-20 nM, about 20-30 nM, about 30-40 nM, about 40-50 nM, about 50-60 nM, about 60-70 nM, about 70-80 nM, about 80-90 nM, about 90-100 nM, about 100-150 nM, about 150-200 nM, etc. In some embodiments, the concentration of rapamycin in the cell culture medium is about 20-70 nM or about 40-60 nM.

[0067] The BET family of proteins consists of two bromodomains (BD1 and BD2) at the N-terminus and an ET domain (Extra terminal) at the C-terminus. The main members of this family are BRD2, BRD3, BRD4, and BDRT (FRENCH CA. Small-Molecule Targeting of BET Proteins in Cancer[J]. Adv Cancer Res, 2016, 131:21-58.). BRD2, BRD3, and BRD4 are widely expressed in mammalian cells, while BDRT is expressed only in the testes. The bromodomain is a conserved protein domain found in all eukaryotes, consisting of four α-helices forming a hydrophobic pocket that can recognize lysine residues acetylated by histones or other proteins (DHALLUIN C, CARLSON JE, ZENG L, et al. Structure and ligand of a histone acetyltransferase bromodomain[J]. Nature, 1999, 399(6735):491-6.). There are 46 proteins containing bromodomains, including HATs, methyltransferases, helicases, chromatin remodeling proteins, transcription coactivators, and BET proteins (CHAIDOS A, CAPUTO V, KARADIMITRISA. Inhibition of bromodomain and extra-terminal proteins (BET) as a potential therapeutic approach in haematological malignancies: emerging preclinical and clinical evidence[J]. Ther Adv Hematol, 2015, 6(3): 128-41.). BET family proteins mainly recognize acetylated lysine residues of histones H3 and H4, and have a higher affinity for multiple acetylated lysine residues within 1-5 amino acids (FILIPPAKOPOULOS P, PICAUD S, MANGOS M, et al. Histone recognition and large-scale structural analysis of the human bromodomain family[J]. Cell, 2012, 149(1): 214-31.).Reports indicate that the bromodomain inhibitor JQ1 can suppress Th17 cell differentiation with little effect on the differentiation of other CD4 T cell subsets. This effect may be related to JQ1's inhibition of various Th17-related cytokines (including IL-17, IL-21, and GMCSF) (MELE DA, SALMERON A, GHOSH S, et al. BET bromodomain inhibition suppresses TH17-mediated pathology[J]. J ExpMed, 2013, 210(11):2181-90.). Subsequent studies using ChIP-seq and other techniques have revealed that the functions of BRD2 and BRD4 are not entirely identical, and that BRD2 is involved in the regulation of more genes, most of which are independent of BRD4. Furthermore, this study revealed the regulatory roles of BRD2 and BRD4 in Th17 differentiation. BRD2 binds to the CTCF-cohesin complex and recruits the stat3-Irf4-Bat complex to transcriptional regulatory elements on chromatin. Simultaneously, BRD4 recruits p-TEFb and CDK9 to promote the transcriptional elongation of RNA polymerase II, thereby regulating the expression of Th17 differentiation-related genes such as IL-17, IL-21, and RORγ (CHEUNG KL, ZHANG F, JAGANATHAN A, et al. Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation[J]. MolCell, 2017, 65(6):1068-80e5.). These studies suggest that although BRD2 and BRD4 are both BET family proteins with two bromine domains, they differ significantly in their mechanisms of gene expression regulation. BRD2 functions by interacting with the CTCF-cohesin complex, promoting the interaction between target enhancer elements and regulatory factor complexes. BRD4 forms a complex with p-TEFb and CDK9, which phosphorylates the Ser 2 site of RNA polymerase II, promoting transcriptional elongation.

[0068] The two bromodomains of the BET protein, BD1 and BD2, also have different functions. BD1-specific inhibitors significantly inhibit cell proliferation, induce cell cycle arrest, and induce apoptosis (GILAN O, RIOJA I, KNEZEVIC K, et al. Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation[J]. Science, 2020, 368(6489):387-94.). BD2-specific inhibitors can specifically inhibit the induced expression of downstream IFNγ signaling transcripts without altering the overall transcript expression. BD2 is not essential for maintaining pre-existing transcriptional programs; it plays a more important role in BET protein recruitment to induce gene expression. Furthermore, BD2 inhibitors have a more significant effect on BRD2 and BRD3, a phenomenon also observed in primary human CD4+ T cell activation.

[0069] ABBV-744 (CAS No.:2138861-99-9) is a selective inhibitor of the BDII domain of BET family proteins, with IC50 values ​​of 4-18 nM for BRD2, BRD3, BRD4 and BRDT (FAIVRE EJ, MCDANIEL KF, ALBERT DH, et al. Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer[J]. Nature, 2020, 578(7794):306-10.).

[0070] In some embodiments, the concentration of ABBV-744 in the cell culture medium can be about 1-200 nM, for example, about 1-10 nM, about 10-20 nM, about 20-30 nM, about 30-40 nM, about 40-50 nM, about 50-60 nM, about 60-70 nM, about 70-80 nM, about 80-90 nM, about 90-100 nM, about 100-150 nM, about 150-200 nM, about 50-150 nM, or about 80-120 nM. In some embodiments, the concentration of ABBV-744 in the cell culture medium is about 1-150 nM or about 10-100 nM.

[0071] As used herein, the term "IL-2" refers to the cytokine and T-cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, forms with conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. The term IL-2 encompasses recombinant human forms of IL-2, such as adefovir (PROLEUKIN), as well as forms of recombinant IL-2 and other commercial equivalents from other suppliers. Adefovir (deallanyl-1, serine-125 human IL-2) is a non-glycosylated recombinant human form of IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses pegylated forms of IL-2, including the pegylated IL-2 prodrug NKTR-214, available from Nektar Therapeutics. The human IL-2 gene was identified by NCBI Gene ID 3558. An exemplary nucleotide sequence of the human IL-2 gene is the NCBI reference sequence: NG_016779.1.

[0072] In some embodiments, the concentration of IL-2 in the cell culture medium is about 100-1000 units / mL, for example, about 100-200 units / mL, about 200-300 units / mL, about 400-500 units / mL, about 500-600 units / mL, about 600-700 units / mL, about 700-800 units / mL, about 800-900 units / mL or about 900-1000 units / mL, preferably about 300-500 units / mL.

[0073] In some implementation schemes, the dosage of IL-2 in the combination therapy is approximately 500,000 units / m². 2 / d to approximately 2 million units / m 2 / d, for example, can be approximately 500,000 units / m 2 / d, approximately 600,000 units / m 2 / d, approximately 700,000 units / m 2 / d, approximately 800,000 units / m 2 / d, approximately 900,000 units / m 2 / d, approximately 1 million units / m 2 / d, approximately 1.5 million units / m 2 / d, approximately 2 million units / m 2 / d, of which approximately 1 million units / m are preferred. 2 / d. Furthermore, IL-2 is preferably administered subcutaneously. And, IL-2 is preferably administered subcutaneously once daily or in multiple divided doses (e.g., twice daily).

[0074] In some implementation schemes, the dosage of Treg cells in the combination therapy is approximately 1 × 10⁻⁶. 5 Treg cells / kg to approximately 1×108 Treg cells / kg, for example, could be approximately 1 × 10⁻⁶. 5 Treg cells / kg, approximately 5 × 10⁻⁶ 5 Treg cells / kg, approximately 1×10 6 Treg cells / kg, approximately 5 × 10⁻⁶ 6 Treg cells / kg, approximately 1×10 7 Treg cells / kg, approximately 5 × 10⁻⁶ 7 Treg cells / kg or approximately 1×10 8 Treg cells / kg. Treg cells are preferably administered via intravenous infusion, and can be given once or multiple times, for example, 1-3 times.

[0075] In some embodiments, the Treg cells in the kits or drug combinations disclosed herein are approximately 4 × 10⁻⁶. 6 Treg cells approximately 1 × 10 10 Treg cells, preferably approximately 4 × 10⁻⁶ 7 Treg cells approximately 1 × 10 9 A single-dose form of Treg cells. Specifically, the Treg cells can be approximately 4 × 10⁻⁶. 6 Treg cells, approximately 1 × 10 7 Treg cells, approximately 5 × 10 7 Treg cells, approximately 1 × 10 8 Treg cells, approximately 3 × 10 8 Treg cells, approximately 5 × 10 8 Treg cells, approximately 1 × 10 9 Treg cells, approximately 5 × 10 9 Treg cells or approximately 1 × 10 10 A single-dose form of Treg cells.

[0076] In some embodiments, in the kits or drug combinations disclosed herein, the IL-2 is in a single-dose form of about 650,000 units to about 11,000,000 units, preferably about 1,300,000 units to about 2,200,000 units. Specifically, the IL-2 may be in a single-dose form of about 650,000 units, about 1,000,000 units, about 1,300,000 units, about 1,500,000 units, about 2,000,000 units, about 2,200,000 units, about 3,000,000 units, about 5,000,000 units, about 8,000,000 units, about 10,000,000 units, or about 11,000,000 units.

[0077] In some implementations, preferably, IL-2 administration begins one week before Treg cell administration and continues until 12 weeks after Treg cell administration, with the decision to continue administration based on treatment response and patient preference.

[0078] As used herein, the term "peripheral blood mononuclear cell (PBMC)" refers to cells in peripheral blood that possess a single nucleus, including lymphocytes and monocytes. In this disclosure, PBMC cells may be derived from a subject in need or a healthy donor.

[0079] The terms “inhibition,” “inhibitor,” “binding antagonist,” or “antagonist” refer to a reduction in certain parameters (e.g., activity) of a given molecule. For example, the term includes inhibition of at least 5%, 10%, 20%, 30%, 40%, or more of the activity of a given molecule. Therefore, inhibition need not be 100%.

[0080] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecules, provided that the antibody exhibits the desired biological activity. Antibodies can be any class of immunoglobulin: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ based on the identity of their heavy chain constant domain. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated with other molecules such as toxins, radioisotopes, etc.

[0081] As used herein, the term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that binds to an antigen. An antigen-binding fragment may contain the antigen-determining region (e.g., complementarity-determining region (CDR)) of the complete antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially produced.

[0082] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, rats, mice, amphibians, reptiles, etc. Unless otherwise stated, the terms "patient" and "subject" are used interchangeably. In this disclosure, the preferred subject is a human.

[0083] As used herein, the term "treatment" refers to the administration of an effective amount of a drug to a subject so that the subject experiences a reduction in at least one symptom of the disease or an improvement in the disease, for example, a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, the reduction of one or more symptoms, a decrease in the severity of the disease, stabilization of the disease state (i.e., no worsening), a delay or slowing of disease progression, an improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Treatment may refer to an extension of survival compared to the expected survival without treatment. Therefore, those skilled in the art will recognize that treatment may improve the disease condition but may not be a complete cure. As used herein, treatment is "effective" when the progression of the disease is reduced or stopped. "Treatment" may also mean an extension of survival compared to the expected survival without treatment. Patients requiring treatment include those already diagnosed with a condition to be treated, and those who may develop such a condition due to genetic susceptibility or other factors.

[0084] As used herein, the term “prevention” may refer to complete protection from disease or to prevention of disease progression. For example, disease prevention may not mean the complete loss of any disease-related effects at any level, but may mean preventing disease symptoms from reaching a clinically significant or detectable level. Disease prevention may also mean preventing the disease from progressing to a later stage. As used herein, “prevention” encompasses the treatment of diseases in mammals, particularly humans, including: (a) preventing the onset of disease or symptoms in susceptible individuals who have not yet been diagnosed with the disease; (b) suppressing disease, such as halting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms.

[0085] The terms “therapeutic / preventive acceptable amount” or “therapeutic / preventive effective amount” are used interchangeably to refer to an amount sufficient to achieve the desired outcome. In some embodiments, a therapeutic / preventive acceptable amount does not induce or cause undesirable side effects. In some embodiments, a therapeutic / preventive acceptable amount induces or causes side effects, but only those acceptable to the healthcare provider in relation to the patient’s condition. A therapeutically acceptable amount can be determined by initially administering a low dose and then incrementally increasing that dose until the desired effect is achieved. As used herein, “preventive effective amount” and “therapeutic effective amount” can respectively prevent the onset of disease symptoms or reduce the severity of disease symptoms, including those related to the condition.

[0086] As used in this article, the term "synergist" refers to a preparation that enhances therapeutic efficacy. In this article, a synergistic effect was achieved by combining Treg cells with IL-2.

[0087] The terms “reduction” and “reduction” are used interchangeably in this document and indicate any change less than the original. “Reduction” and “reduction” are relative terms and need to be compared before and after measurement. “Reduction” and “reduction” include complete depletion.

[0088] As used herein, the term "ex vivo" generally refers to manipulation of cells, tissues, and / or organs that have been removed from a living organism. In some embodiments, the cells, tissues, and / or organs may be returned to the living organism or introduced into another organism by certain methods.

[0089] As used herein, the term "in vitro" generally refers to the removal or release of a portion of an organism from the organism. For example, in vitro assays encompass cell-based assays that may use live or dead cells, and may also encompass cell-free assays that do not use intact cells.

[0090] As used in this article, the term "in vivo" generally refers to the body of a subject. For example, in some cases, "in vivo" can refer to a specific location in the tested tissue or organ.

[0091] As used in this article, graft-versus-host disease (GVHD) is tissue damage caused by donor-derived immune cells reconstituted in the patient's body after hematopoietic stem cell transplantation (HSCT) attacking the recipient's organs. It is a complication specific to allogeneic HSCT (allo-HSCT), and severe GVHD is one of the important causes of transplant failure. Based on the time of occurrence after transplantation, GVHD occurring within 100 days is called acute graft-versus-host disease (aGVHD), and occurring after 100 days is called chronic graft-versus-host disease (cGVHD).

[0092] As used herein, the term "about" refers to a variation in a value that may occur, for example, through real-world measurement or processing procedures, through negligence or errors in these procedures, through differences in the production, origin, or purity of the composition or combination of drugs or reagents, etc. Generally, as used herein, the term "about" means greater than or less than 1 / 10 of the stated value or range of values, for example, ±10%.

[0093] Example

[0094] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments. Unless otherwise specified, the reagents and instruments used in the embodiments of the present disclosure are all known and available in the art. Recombinant human interleukin-2 was purchased from Beijing Sihuan Biopharmaceutical Co., Ltd., with the national drug approval number S10970016.

[0095] Example 1: Preparation of Treg cell injection solution

[0096] Sorting of Treg cells

[0097] Treg cells were sorted using a human CD4+CD127lowCD25+ regulatory T cell separation kit (StemCell Catalog no. 18063).

[0098] For 10 7Personal PBMC cells were resuspended in 2 mL of separation buffer and transferred to a 5 mL sorting tube. 100 μL of CD25 Positive Selection Cocktail was added, mixed, and incubated for 5 minutes. 60 μL of Releasable RapidSpheres and 100 μL of CD4+ T Cell Enrichment Cocktail were added, mixed, and incubated for 5 minutes. The sample volume was adjusted to 2.5 mL with separation buffer, mixed, and the sorting tube was inserted into the magnetic pole and incubated for 10 minutes. The supernatant was aspirated into a new sorting tube for sorting Tconv cells (CD4+CD25-). The sorting tube was removed, resuspended in 2.5 mL of separation buffer, and inserted into the magnetic pole again, incubating for 5 minutes. The supernatant was aspirated, the sorting tube was removed, resuspended in 2.5 mL of separation buffer, and inserted into the magnetic pole again, incubating for 5 minutes. The above steps were repeated once. Aspirate the supernatant, resuspend the cells in separation buffer at the initial sorting volume, add 200 μL of release buffer and resuspend again, pipetting at least 5 times. Add 20 μL of CD127 high Depletion Cocktail and mix well, incubate at room temperature for 5 min. Adjust the sample volume to 2.5 mL with separation buffer, mix well, insert the sorting tube into the magnetic pole, and let stand for 5 minutes. Transfer the supernatant to a new centrifuge tube; this is the sorted Treg cells.

[0099] In vitro expansion of Treg cells

[0100] Centrifuge the sorted Treg cells at 300×g for 10 minutes. Discard the supernatant and resuspend the cells in X-VIVO 15 complete medium supplemented with 5%-10% HumanAB serum to a final volume of 2×10⁻⁶. 5 -1×10 6 Add anti-CD3 / CD28 magnetic beads (Invitrogen) at a cell:magnetic bead ratio of 1:3, along with IL-2 (Beijing Sihuan Biopharmaceutical Co., Ltd.) 300 units / mL and rapamycin (MCE) 50 nM (or rapamycin 10 nM + ABBV-744 100 nM). Mix well and add to a suitable well plate or culture flask. Incubate at 37°C in a 5% CO2 incubator.

[0101] On day 2 of culture, add an equal volume of complete culture medium. Thereafter, count the cells every 2-3 days, and adjust the cell density to 0.25-1 × 10⁻⁶ cells / day by adding fresh complete culture medium based on the count results. 6 The cell / mL concentration was increased, and IL-2 was added.

[0102] On day 9 of culture, collect all cells, centrifuge at 300×g for 10 minutes, and discard the supernatant. Resuspend the cells in 10 mL of complete culture medium and remove the magnetic beads using a magnetic pole. Count the cells and adjust the cell density to 2×10⁶ cells / mL with complete culture medium. 5 -1×10 6 Add anti-CD3 / CD28 magnetic beads at a cell:magnetic bead ratio of 1:1, and add IL-2 300 units / mL. Mix well and add to a suitable well plate or culture flask. Incubate at 37°C in a 5% CO2 incubator.

[0103] Cell counts were performed every 2-3 days from day 9 to day 14. Based on the count results, fresh complete culture medium was added to adjust the cell density to 0.25-1×10⁻⁶ cells / day. 6 Cells were collected at 1 cell / mL, and IL-2 was added to 300 units / mL. Cells were harvested on day 14.

[0104] Example 2: Identification of Treg cells

[0105] To assess the safety and performance of Treg cells after in vitro expansion, cell analysis was performed using flow cytometry and real-time quantitative PCR. Samples were collected 14 days after in vitro expansion, and lymphocytes were identified using FSC / SSC gating. Dead cells were excluded from the analysis using a zombie violet staining method. CD4+ and CD8+ cells were identified based on CD4 / CD8 ratio analysis. Further analysis of CD127 and CD25 expression in CD4+ cells was conducted to assess the purity of CD25+CD127low / - cells. Further analysis of CD4 and FOXP3 expression in lymphocytes was conducted to assess the initial frequency of CD4+ cells expressing FOXP3. Further analysis of FOXP3 and Helios expression in CD4+ cells was conducted to assess the proportion of Helios+FOXP3+ cells in CD4+ cells, an indicator reflecting the lineage stability of Treg cells in vivo.

[0106] Figure 1 The results of flow cytometry analysis of expanded Treg cells are shown. From Figure 1 It can be seen that the expanded Treg cells maintained very high purity, with CD4+ cells accounting for >95%, CD8+ cells accounting for <2%, FOXP3+ cells accounting for >90%, and Helios+FOXP3+ cells accounting for >70%.

[0107] Collect 1-5×10 6Genomic DNA was extracted from 10 Treg cells using the QIAamp DNA mini kit (QIAGEN, Catalog no. 51304). Subsequently, the extracted DNA was bisulfite-treated using the EpiTech DNA Fast Bisulfite Conversion kit (QIAGEN Catalog no. 59824) to convert cytosine in unmethylated CpGs in the DNA to uracil.

[0108] Figure 2 The results of Treg-specific demethylated region (TSDR) detection in expanded Treg cells are shown. The methylation level of the TSDR region was quantified using fluorescent probe qPCR (refer to WIECZOREK G, ASEMISSENA, MODEL F, et al. Quantitative DNA Methylation Analysis of FOXP3 as a New Method for Counting Regulatory T Cells in Peripheral Blood and Solid Tissue[J]. Cancer Res, 2009, 69(2):599-608.), thereby assessing the stability of Treg cells. From... Figure 2 It can be seen that the expanded Treg cells still maintain a very high level of TSDR demethylation, demonstrating their excellent lineage stability.

[0109] Example 3: Treg cells for the prevention and treatment of xenogeneic GVHD in NSG mice

[0110] To evaluate the therapeutic effect of Treg cells on GVHD, Treg cells were injected into NSG mice with a xenogeneic GVHD model, and the incidence of GVHD in the mice was observed.

[0111] NSG mice are severely immunodeficient mice lacking mature T, B, and NK cells. Injecting human PBMC cells into sublethal irradiated NSG mice can stably and rapidly induce acute GVHD. This model can be used to indicate the immunosuppressive function of Treg cells in vivo.

[0112] Figure 3 and Figure 4 The results of using Treg cells to prevent or treat xenogeneic GVHD in NSG mice are shown. Treg cell therapy was administered concurrently with or 4 days after PBMC cell injection. Figure 3 A). In the experiment of Treg cell prevention of xenogeneic GVHD in NSG mice, PBMC cells were injected simultaneously with 5×10⁶ cells. 6 Treg cells significantly improved survival in mice and alleviated weight loss and associated GVHD symptoms. Figure 3 B). In the Treg cell therapy experiment for xenogeneic GVHD in NSG mice, 5×10⁻⁶ cells were injected 4 days after PBMC cell injection. 6 Treg cells also significantly improved survival in mice and alleviated weight loss and associated GVHD symptoms. Figure 3 C).

[0113] In the Treg cell prophylaxis experiment for GVHD, peripheral blood flow cytometry analysis was performed on days 10 and 20. On day 10, the proportion of human T cells (hCD45+CD3+) in the peripheral blood of mice treated with Treg cells was lower than that in the control group, while the proportion of Treg cells (CD4+CD25+FOXP3+) was significantly higher than that in the control group. Figure 4 A). On day 20, the proportion of human T cells in the peripheral blood of mice was significantly increased, but the proportion of human T cells in the Treg cell treatment group was half that of the control group, while the number of Treg cells was also significantly higher than that in the control group. Figure 4 B). Peripheral blood lymphocytes showed that Treg cell therapy significantly inhibited the activation and proliferation of human T cells in NSG mouse GVHD models. Although Treg cells gradually decreased in mice, they still had some effect at day 20.

[0114] Example 4: IL-2 increases the duration of Treg cells in mice and promotes Treg cell lineage stability.

[0115] In the treatment of chronic GVHD, low-dose IL-2 primarily promotes the proliferation of Treg cells and increases the Treg / Tconv ratio, thereby achieving a therapeutic effect. Therefore, we investigated whether IL-2 therapy could improve the persistence of Treg cells in vivo.

[0116] Figure 5 This shows that injecting 1×10 into NSG mice... 7Following Treg cell administration, mice were simultaneously injected intraperitoneally with 5000 IUIL-2 daily. The proportion of Treg cells and the expression of FOXP3 and CD25 in peripheral blood were detected by flow cytometry on days 1, 5, and 11. The results showed that IL-2 significantly increased the duration of Treg cell presence in mouse peripheral blood. Figure 5 A, B), and effectively maintained the expression of FOXP3 and CD25 in Treg cells, promoting the lineage stability of Treg cells. Figure 5 C, D).

[0117] Example 5

[0118] This example is a clinical trial to determine the optimal dosage of Treg cells combined with IL-2. The donor was a recipient of hematopoietic stem cell transplantation.

[0119] Research Objective

[0120] Main research objective:

[0121] To evaluate the safety and tolerability of Treg cells combined with low-dose IL-2 treatment in patients with refractory cGVHD after allo-HSCT.

[0122] Secondary research objective:

[0123] (1) To evaluate the changes and persistence of Treg cells in the peripheral blood of the subjects after administration of Treg cell injection;

[0124] (2) Evaluate the content and changes of various lymphocyte subsets in peripheral blood after administration of Treg cells in combination with low-dose IL-2 injection (detection includes T cells, B cells, NK cells and their subsets);

[0125] (3) Changes in the quality of life rating scale compared to baseline;

[0126] (4) Changes in the severity of cGVHD;

[0127] (5) Preliminary evaluation of the overall response rate (ORR) (including CR and PR) after Treg cell therapy combined with low-dose IL-2 in refractory cGVHD subjects;

[0128] (6) To evaluate the duration of response (DOR) in subjects with refractory cGVHD treated with Treg cells in combination with low-dose IL-2;

[0129] (7) Evaluate the survival (including OS, FFS, etc.) of subjects with refractory cGVHD treated with Treg cells in combination with low-dose IL-2;

[0130] (8) Evaluate and analyze patient-reported outcomes (PROs) after drug administration;

[0131] (9) Evaluate the proportion of subjects whose steroid requirements have been reduced to <0.25 mg / kg / day.

[0132] Exploratory purpose

[0133] (1) Evaluate the distribution of Treg cells in vivo after administration of Treg cell injection.

[0134] (2) Single-cell sequencing of Treg cells in the peripheral blood of subjects, changes in clonal ratio and time.

[0135] Overall Design

[0136] This study is a single-arm, open-label, dose-escalation clinical trial to explore the safety, tolerability, changes and persistence of peripheral blood Treg cells, and pharmacodynamic characteristics of Treg cell adoptive therapy, and to preliminarily observe the efficacy of the drug in patients with refractory cGVHD.

[0137] In this study, the recommended dose was determined based on changes and duration of peripheral blood in subjects receiving Treg cells, pharmacodynamics, ORR, safety, tolerability, and previous experience with Treg cell adoptive therapy.

[0138] This study employed a rapid titration of the first dose group and a "3+3" rule design to minimize patient exposure to ineffective doses and reduce risks as much as possible. The study consisted of four dose groups: 1×10⁻⁶. 6 Treg cells / kg dose group, 5.0×10 6 Treg cells / kg dose group, 10×10 6 Treg cells / kg dose group and 5×10 7 Treg cells / kg dose group.

[0139] Low-dose rhIL-2 was administered one week prior to Treg cell infusion to facilitate observation of adverse reactions and differentiation from Treg cell infusion responses, as well as to observe its effect on endogenous Treg cells in vivo; the rhIL-2 dose was 1 million units (1 MIU) / m². 2 / day, subcutaneous injection, continued until 12 weeks after Treg cell infusion (i.e., a total of 13 weeks of rhIL-2 injections), followed by a 4-week break, after which a decision is made on whether to continue using rhIL-2 based on the treatment response and the patient's wishes.

[0140] Selection criteria

[0141] (1) The subjects are aged 18-70 years old, regardless of gender.

[0142] (2) The subjects had previously undergone allo-HSCT, and the stem cell sources included bone marrow, peripheral blood and umbilical cord blood transplantation.

[0143] (3) Subjects are diagnosed with moderate / severe cGVHD according to NIH diagnostic criteria (031) and meet the criteria for hormone-resistant cGVHD (SR-cGVHD), i.e., they meet any of the following criteria:

[0144] (a) No improvement in cGVHD in patients who were initially treated with prednisone (or an equivalent dose of other glucocorticoids) for at least 4 weeks;

[0145] (b) Hormone dependence: relapse or progression of cGVHD when prednisone dose >0.25 mg / kg / day or >0.5 mg / kg / qod; after two failed attempts to gradually reduce the hormone dose at least 8 weeks apart, the prednisone dose should be increased to >0.25 mg / kg / day.

[0146] (4) The prednisone dose of the subject before enrollment is >0.25mg / kg / d and lasts for more than 4 weeks; the subject must maintain a stable prednisone dose for 4 weeks before the first Treg cell infusion and not increase or discontinue other immunosuppressants (including cyclosporine, tacrolimus and sirolimus).

[0147] (5) The subjects’ ECOG score was 0-2.

[0148] (6) The subject’s expected survival time is more than 3 months.

[0149] (7) Liver, kidney, heart, and lung functions meet the following requirements (excluding liver and kidney insufficiency caused by cGVHD):

[0150] (a) Creatinine clearance (calculated using the Cockcroft-Gault formula) ≥ 60 mL / min;

[0151] (b) Cardiac ejection fraction greater than 50%, with no clinically significant electrocardiographic changes;

[0152] (c) Baseline forced expiratory volume in one second (FEV1) ≥ 50%; FEV1 decrease caused by cGVHD is eligible for enrollment;

[0153] (d) Total bilirubin ≤ 2 ULN; ALT and AST ≤ 3 ULN; Elevated ALT and AST due to cGVHD are eligible for enrollment;

[0154] (8) Hematopoietic function: Neutrophils > 1 × 10 9 / L, platelets >25×10 9 / L; except for supportive treatment such as platelet transfusion and other cytokines.

[0155] (9) The subject or the subject's guardian understands this trial and has signed the informed consent form.

[0156] (10) Donors must be between 14 and 70 years old, regardless of gender.

[0157] (11) The donor’s ECOG score is 0-1.

[0158] (12) The donor must be the hematopoietic stem cell donor from the subject’s original allo-HSCT.

[0159] (13) Female donors of childbearing age must have a negative serum or urine β-human chorionic gonadotropin (HCG) test within 3 weeks of blood collection.

[0160] (14) The donor can establish the necessary venous access for collection and has no contraindications for leukocyte collection; if peripheral venous leukocyte collection is insufficient, the donor is willing to have a central catheter inserted.

[0161] (15) The donor agrees to make the donation and signs a consent form.

[0162] Exclusion criteria

[0163] Individuals with any of the following conditions are ineligible to be selected as participants:

[0164] (1) The subject had a relapse of primary hematologic disease before receiving Treg treatment;

[0165] (2) The subject has persistent, recurrent or delayed acute GVHD;

[0166] (3) Continuous use of prednisone >1 mg / kg / day is required;

[0167] (4) The severity of cGVHD in the subject cannot be assessed by physical examination or laboratory tests;

[0168] (5) Overlap syndrome;

[0169] (6) Subjects with non-cGVHD-related major organ (cardiovascular, cerebrovascular, pulmonary) dysfunction, a history of active gastrointestinal bleeding within 3 months; uncontrolled hypertension or a history of hypertensive crisis or hypertensive encephalopathy, or a history or evidence of significant cardiovascular or cerebrovascular risk, including any of the following: congestive heart failure, unstable angina, clinically significant arrhythmia (such as ventricular fibrillation, ventricular tachycardia, etc.); a history of arterial thrombosis within the past 3 months (such as stroke, transient ischemic attack); a history of symptomatic deep vein thrombosis or pulmonary embolism within the past 6 months, or a history of coronary angioplasty, defibrillation, or any clinically relevant complications or diseases that may pose a risk to the safety of the subject or interfere with the study assessment, procedures, or completion;

[0170] (7) The subjects were hemodialysis patients;

[0171] (8) The subject or donor has an active bacterial, viral or fungal infection that requires treatment and is not under control (infection progression is defined as hemodynamic instability caused by sepsis, new symptoms or signs, worsening of physical signs or imaging findings caused by infection); HBsAg or HBcAb is positive and peripheral blood HBV DNA is ≥ detection limit; hepatitis C virus antibody is positive and peripheral blood HCV RNA is positive; syphilis TRUST test is positive; human immunodeficiency virus HIV antibody is positive;

[0172] (9) The recipient is a pregnant or lactating woman; and a subject who plans to become pregnant during the study after infusion, or within one year after completion or withdrawal from the study;

[0173] (10) Subjects who received IL-2 treatment within 4 weeks prior to enrollment, or subjects treated with drugs targeting IL-2 (such as basiliximab, denileukin, diftitox, etc.);

[0174] (11) Received lymphocyte infusion (DLI) therapy within 100 days prior to enrollment;

[0175] (12) Received CAR-T or similar engineered cell therapy within 100 days prior to enrollment;

[0176] (13) Had received new cGVHD treatments (including imatinib, BTK inhibitors, rituximab and other immunosuppressants) within 4 weeks prior to enrollment after transplantation.

[0177] (14) Individuals with a history of microvascular diseases such as thrombotic microangiopathy (TMA), hemolytic uremic syndrome, or thrombotic thrombocytopenic purpura;

[0178] (15) Subjects with poor adherence to IL-2 treatment;

[0179] (16) Subjects who participated in other cGVHD-related clinical studies within 4 weeks prior to enrollment;

[0180] (17) Subjects known to be allergic to any component of Treg cell injection;

[0181] (18) Any situation that the researcher believes would compromise the safety of the subject or interfere with the research objectives, or any situation that the researcher believes is unsuitable for the subject to participate in this trial;

[0182] (19) Those who are ill and unable to sign a written informed consent form or comply with the research procedures; those who are unwilling or unable to comply with the research requirements;

[0183] (20) The donor is a pregnant woman.

[0184] Definition of dose-limiting toxicity (DLT)

[0185] The severity of adverse events is classified according to the NCI-CTCAE version 5.0, and the investigator determines whether the subject has DLT.

[0186] Considering the specific characteristics of the subjects, DLT is defined as: any of the following drug-related conditions that occur within 28 days after the subject receives Treg cell infusion, despite other treatment measures:

[0187] 1. Grade 4 or 5 cytokine release syndrome caused by Treg cell injection therapy;

[0188] 2. Grade 3 cytokine release syndrome caused by Treg cell injection therapy and which does not improve to grade 2 or below within 7 days;

[0189] 3. Grade 3 or higher neurotoxicity occurs;

[0190] Grade 4 hematologic toxicity: lasting longer than 28 days (≥28) and not caused by the primary disease (excluding lymphopenia, neutropenia and thrombocytopenia caused by chemotherapy pretreatment);

[0191] 5. Any unexpected toxicity that necessitates termination of treatment based on the investigator's judgment.

[0192] Definition of Maximum Tolerable Dose (MTD)

[0193] MTD is defined as the highest dose at which no more than 2 out of 6 subjects experience DLT.

[0194] Key evaluation indicators

[0195] DLT and incidence of adverse reactions exceeding CTCAE grade ≥3

[0196] Secondary evaluation indicators

[0197] (1) Changes in the expansion of Treg cells in the peripheral blood of the subjects after administration of Treg cell injection and the relationship between duration and time.

[0198] (2) The content and changes of various lymphocyte subsets in peripheral blood after administration of Treg cells in combination with low-dose IL-2 injection (the detection included T cells, B cells, NK cells and their subsets).

[0199] (3) Changes in the quality of life rating scale relative to the baseline.

[0200] (4) Changes in the severity of cGVHD were assessed according to the Chronic Graft-versus-Host Disease Grading and Scoring System.

[0201] (5) Evaluate the efficacy of Treg cells combined with low-dose IL-2 in refractory cGVHD subjects after administration, including ORR (CR+PR) at 12 and 24 weeks.

[0202] a) Complete remission (CR): The symptoms and signs of chronic GVHD completely disappear.

[0203] b) Partial remission (PR): Symptoms improve in at least one organ or site, with no progression in any other organ or site.

[0204] (6) Patient-reported outcomes (PROs) after drug administration: These are reports directly from patients regarding their health status, functional status, and treatment experience, excluding interpretations by healthcare professionals or any other personnel. A Lee cGVHD symptom scale score ≥7 is associated with improved quality of life.

[0205] (7) The proportion of subjects who were able to reduce their steroid requirement to <0.25 mg / kg / day.

[0206] (8) Duration of response after administration (DOR): defined as the time from the first remission to disease progression, new cGvHD systemic treatment or all-cause death (whichever occurs first).

[0207] (9) Failure-free survival (FFS) after administration: the time from the start of cell infusion to the first disease progression, relapse after remission, or death from any cause.

[0208] (10) Overall survival (OS) after administration: the time from the start of cell reinfusion to death from any cause. For subjects who detached before death, the time of death was the last follow-up date; if the subject received another new therapy, the time of death was the start date of the new therapy; if the study ended, the time of death was the end date.

[0209] Exploratory assessment endpoint

[0210] (1) Distribution of Treg cells in the body, and distribution of Treg cells in extramedullary lesions, pleural effusion, ascites, bone marrow, cerebrospinal fluid, etc.

[0211] (2) Single-cell sequencing of Treg cells in the peripheral blood of subjects, changes in clonal ratio and time.

[0212] Statistical methods

[0213] General principles:

[0214] Statistical analysis will be performed using SAS statistical analysis software version 9.2 or later. For continuous variables, descriptive statistics will include the mean, median, standard deviation, maximum, and minimum values. For categorical variables, descriptive statistics will include the number of subjects and the percentage.

[0215] The main statistical analysis set is as follows:

[0216] All Subjects Enrolled Set: All subjects who signed informed consent, regardless of whether they were successfully screened or received Treg cell injection treatment;

[0217] Full Analysis Set (FAS): This is the dataset of subjects who underwent Treg cell infusion according to the trial protocol within the selected subject dataset. The FAS set is the primary dataset for analyzing changes in the number of Treg cells in vivo.

[0218] Safety Set: This is the set of subjects who received Treg cell injections and had subsequent safety visits within the screening subject dataset. The Safety Set is an analytical dataset that summarizes safety data.

[0219] Dose escalation analysis set: includes subjects who experienced DLT during the dose escalation phase and subjects who did not experience DLT and completed cell reinfusion.

[0220] Efficacy Set: This set comprises all subjects in the selected subject dataset who received Treg cell injections and underwent subsequent efficacy evaluations. The efficacy set is an analytical dataset that summarizes efficacy data.

[0221] Test results

[0222] This study employed a dose-escalation design, with the first dose group undergoing rapid titration. Following a "3+3" design principle, a total of four dose groups were established, each consisting of 1×10⁻⁶ doses. 6 Treg cells / kg, 5.0 × 106 Treg cells / kg, 10×10 6 Treg cells / kg and 5×10 7 Treg cells / kg. To date, four subjects have been enrolled. The first subject received 1×102 6 Treatment with Treg cells / kg dose, combined with IL-2 (1 million units / m²) 2 (Daily) subcutaneous injection, continued for 13 weeks; the second to fourth subjects received 5.0 × 10 6 For the Treg cell / kg dose group, the IL-2 administration regimen was the same as before.

[0223] The first patient was followed up for more than 24 weeks after completing Treg cell infusion. No dose-limiting toxic events occurred during treatment, and the patient's overall condition remained stable. Dermatitis symptoms appeared at week 5, which were relieved after IL-2 administration was discontinued. No similar adverse reactions occurred after IL-2 administration was resumed at week 9.

[0224] The second subject had completed Treg cell infusion and was followed up for more than 24 weeks. No dose-limiting toxic events were observed during treatment, clinical symptoms improved, and the cGVHD score decreased.

[0225] The third subject also completed Treg cell infusion and was followed up for more than 24 weeks. No dose-limiting toxic events occurred during the treatment, clinical symptoms improved, and the cGVHD score decreased.

[0226] In the fourth patient, the cell expansion rate was too low during Treg cell production, resulting in a substandard TSDR test result for the final product. Therefore, Treg cell reinfusion was not performed. Retrospective analysis and independent parallel control experiments confirmed that this problem was unrelated to the production process and was mainly attributed to the cell characteristics of the donor PBMCs.

[0227] Table 1 summarizes the data related to Treg cell production from four subjects and five batches in the preclinical study, including the proportions of Helios+FOXP3-, Helios+FOXP3+, Helios-FOXP3-, and Helios-FOXP3+ cells in the CD4+CD25+CD127low / - cell population of the donor PBMC, the proportions of each subpopulation in the CD4+CD25+CD127low / - cell population obtained after sorting by the method described in Example 1, the fold increase of Treg cells after 9 days of in vitro expansion, and the TSDR demethylation level of the Treg cell injection solution.

[0228] Figure 6This demonstrates the correlation between the proportion of Helios-FOXP3+ cells and the fold increase in Treg cells within the CD4+CD25+CD127low / - cell population of donor PBMCs. Figure 6 A), and the correlation between the proportion of Helios-FOXP3- cells inoculated with Treg cells and the level of TSDR demethylation after expansion ( Figure 6 B).

[0229] Analysis of data from four enrolled subjects and five batches of Treg cell production from preclinical studies showed that the proportion of Helios-FOXP3+ cells in the CD4+CD25+CD127low / - cell population of donor PBMCs was significantly negatively correlated with the fold expansion of Treg cells after inoculation (see Table 1). Figure 6 A). Therefore, during the subject screening phase, the proportion of Helios-FOXP3+ cells in the donor's peripheral blood CD4+CD25+CD127low / - cell population can be used as one of the screening indicators for enrollment. It is recommended to prefer donors with a Helios-FOXP3+ proportion of less than 16%.

[0230] Furthermore, the proportion of Helios-FOXP3- cells inoculated with Treg cells was negatively correlated with the level of TSDR demethylation after expansion (see Table 1). Figure 6 (B) Therefore, the proportion of Helios-FOXP3- cells can be detected in the sorting process of Treg cell production as an intermediate quality control indicator to assess the quality of the final product early in production. If the proportion of Helios-FOXP3- cells in the inoculated Treg cells exceeds 10%, the subsequent TSDR test results of the product are likely to be unqualified, and production should be rescheduled.

[0231] Table 1

[0232]

[0233] By incorporating references

[0234] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0235] Equivalence

[0236] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. Use of Treg cells and interleukin-2 (IL-2) in the preparation of a combination of drugs for the treatment or prevention of graft-versus-host disease (GVHD) in subjects.

2. The use according to claim 1, wherein, The Treg cells were at a rate of 1 × 10 5 Treg cells / kg up to 1×10 8 Administered at a dose of Treg cells / kg, preferably 1×10⁻⁶. 6 Treg cells / kg up to 5×10 7 The preferred dosage of Treg is 1 × 10⁻⁶. 6 Treg cells / kg up to 1×10 7 Administer Treg cells per kg.

3. The use according to claim 1 or 2, wherein, The Treg cells were formulated for intravenous infusion.

4. The use according to any one of claims 1-3, wherein, The Treg cells are administered 1-3 times, preferably once.

5. The use according to any one of claims 1-4, wherein, The IL-2 mentioned is recombinant human interleukin-2 (rhIL-2).

6. The use according to any one of claims 1-5, wherein, The IL-2 is at a concentration of 500,000 units / m 2 / d to 2 million units / m 2 / d, preferably 800,000 units / m 2 / d to 1.5 million units / m 2 / d, more preferably 1 million units / m 2 Administer a dose of / day.

7. The use according to any one of claims 1-6, wherein, The IL-2 is formulated for subcutaneous injection.

8. The use according to any one of claims 1-7, wherein, The IL-2 is administered subcutaneously once or in multiple daily doses.

9. The use according to any one of claims 1-8, wherein, The proportion of live cells in the Treg cells is >80%, the proportion of CD8+ cells is <5%, the proportion of CD4+ cells is >95%, the proportion of FOXP3+ cells is >60%, the proportion of Helios+FOXP3+ cells is >50%, and the proportion of TSDR demethylation is >60%. Preferably, the proportion of Helios-FOXP3- cells in the Treg cells is ≤10%, more preferably ≤9%, ≤8%, or ≤7%, more preferably ≤6%, ≤5%, or ≤4%, and even more preferably ≤3%, ≤2%, or ≤1%.

10. The use according to any one of claims 1-9, wherein, The proportion of Helios-FOXP3+ cells in the CD4+CD25+CD127low / - cell population of the subject's PBMCs is ≤16%, preferably ≤15%, ≤14%, or ≤13%, more preferably ≤12%, ≤11%, or ≤10%.

11. The use according to any one of claims 1-10, wherein, The graft-versus-host disease is a chronic graft-versus-host disease, preferably a refractory chronic graft-versus-host disease.

12. A kit comprising Treg cells and IL-2.

13. The kit according to claim 12, wherein, The IL-2 mentioned is recombinant human interleukin-2 (rhIL-2).

14. The kit according to claim 12 or 13, wherein, The number of Treg cells was 4 × 10 6 Treg cells increased to 1×10 10 Treg cells, preferably 4×10 7 Treg cells increased to 1×10 9 A single-dose form of Treg cells.

15. The kit according to any one of claims 12-14, wherein, The IL-2 is in a single-dose form of 650,000 to 11,000,000 units, preferably 1,300,000 to 2,200,000 units.

16. The kit according to any one of claims 12-15, wherein, The proportion of live cells in the Treg cells is >80%, the proportion of CD8+ cells is <5%, the proportion of CD4+ cells is >95%, the proportion of FOXP3+ cells is >60%, the proportion of Helios+FOXP3+ cells is >50%, and the TSDR demethylation is >60%. Preferably, the proportion of Helios-FOXP3- cells in the Treg cells is ≤10%, more preferably ≤9%, ≤8%, or ≤7%, more preferably ≤6%, ≤5%, or ≤4%, and even more preferably ≤3%, ≤2%, or ≤1%.

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