Method for in-vitro expansion of treg cells

Abstract

The present disclosure relates to the field of in-vitro cell isolation and expansion, and specifically provides a method for in-vitro expansion of Treg cells. The method comprises the following steps: (1) sorting a CD4+CD25+CD127low / - Treg cell population from PBMCs; and (2) in the presence of rapamycin, ABBV-744 and IL-2, performing in-vitro expansion of the Treg cell population obtained in step (1). The method of the present disclosure achieves a high cell expansion fold while maintaining high purity of Treg cells.

Description

A method for in vitro expansion of Treg cells Technical Field

[0001] This disclosure relates to the field of in vitro cell isolation and expansion, and more particularly to a method for in vitro expansion of Treg cells. Background Technology

[0002] Treg cells are a type of CD4+ T cells with immunosuppressive function and are an important component of the human immune system, playing a crucial role in maintaining autoimmune tolerance and immune homeostasis. This type of CD4+ T cell population develops in the thymus and peripheral tissues of the immune system by expressing an epigenetically stable transcription factor, FOXP3. They constitute a very low proportion of peripheral blood lymphocytes, approximately 4% of CD4+ T cells. Treg cells mediate immunosuppression through various mechanisms, including the production of inhibitory cytokines, regulation of antigen-presenting cell maturation or function, competition for IL-2 and interference with effector cell metabolism, and direct cytolysis of effector cells (VIGNALID A, COLLISON LW, WORKMAN C J. How regulatory T cells work[J]. Nat Rev Immunol, 2008, 8(7):523-32.). In recent years, researchers have been exploring the use of Treg cells to treat autoimmune diseases, transplant rejection, and graft-versus-host disease. Studies have shown that Treg cells can not only suppress harmful immune responses but also promote the reconstitution of immune tolerance. The therapeutic potential of Treg cells has been validated in various preclinical models of graft-versus-host disease (GVHD), solid organ transplantation, type 1 diabetes (T1D), systemic lupus erythematosus (SLE), inflammatory bowel disease, and multiple sclerosis (MS) (RAFFIN C, VO LT, BLUESTONE JA. Treg cell-based therapies: challenges and perspectives[J]. Nat Rev Immunol, 2020, 20(3):158-72.). More than 50 active and completed clinical trials are currently testing the safety and efficacy of Treg cell therapy, including in kidney transplantation, liver transplantation, SLE, inflammatory bowel disease, autoimmune hepatitis, allergies and asthma, GVHD, and T1D (FERREIRA LMR, MULLER YD, BLUESTONE JA, et al. Next-generation regulatory T cell therapy[J]. Nat Rev Drug Discov, 2019, 18(10):749-69.). Although the isolation and expansion protocols for Treg cells vary, early clinical trial results indicate that in vitro expanded Treg cells have immunosuppressive effects in vivo and are extremely safe.

[0003] Because Treg cells are scarce in peripheral blood and lack simple and effective cell surface markers, purification through simple positive or negative selection is difficult. Furthermore, under typical in vitro culture conditions, Treg cells are highly susceptible to contamination by effector cells; even a small amount of effector cells can lead to severe contamination with increasing culture time. These factors make the isolation and in vitro expansion of Treg cells challenging. Although successful isolation and expansion protocols for Treg cells exist, such as tandem sorting with CD8 T cell deletion and CD25-positive selection using magnetic beads, followed by expansion with 100 nM rapamycin in vitro, the purity of the isolated Treg cells is often low, frequently containing a certain number of effector cells. Therefore, a higher concentration of rapamycin is needed during in vitro expansion to maintain the Treg cell proportion. However, since Treg cells are also affected by rapamycin, the expansion fold after each activation is low, typically requiring three rounds of activation and approximately 21 days to obtain a sufficient quantity of Treg cells for clinical treatment. Another approach is to use flow cytometry to divide Treg cells using CD4+CD25+CD127low / - markers. This method has been shown to produce sufficient Treg cells after two rounds of activation and 14 days of expansion without the addition of rapamycin in vitro expansion.

[0004] Of the two main methods for isolating Treg cells—flow cytometry and magnetic bead sorting—flow cytometry is more attractive from an in vitro experimental perspective. However, in clinical treatment, its open tubing makes it difficult to meet GMP production standards. Although some companies, such as Miltenyi and Sony, have launched fully enclosed flow cytometers with replaceable tubing, their slow sorting speed, high equipment price, and high operating costs have limited their widespread adoption and clinical application. Magnetic bead sorting is generally a better choice for clinical research and production, and it is widely used in CAR-T cell production. However, as mentioned earlier, magnetic bead sorting suffers from low Treg cell purity during Treg cell production, necessitating the addition of rapamycin during culture and expansion, which reduces cell yield and prolongs the production cycle. Summary of the Invention

[0005] The inventors, through CRISPR screening, discovered genes that regulate the in vitro differentiation and stability of mouse Treg cells during in vitro induction. Validation revealed that using the BRD2 protein inhibitor ABBV-744 effectively promotes Treg cell differentiation and improves stability after reactivation. In this paper, we propose a method for separating high-purity Treg cells using magnetic bead sorting. By optimizing the concentration of rapamycin and adding ABBV-744, we obtained a high-yield, high-purity, and high-stability Treg cell production protocol after activation and expansion.

[0006] On the one hand, this disclosure provides a method for in vitro expansion of Treg cells, the method comprising the following steps:

[0007] (1) Isolate CD4+CD25+CD127low / - Treg cells from PBMC cells;

[0008] (2) The Treg cell population obtained in step (1) was expanded in vitro in the presence of rapamycin, ABBV-744 and IL-2.

[0009] In some specific implementations, in step (1), the sorting of the Treg cell population is performed using fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

[0010] In some specific embodiments, step (1) includes the sorting step of enriching CD25+ cells and deleting cells that highly express CD127.

[0011] In some specific embodiments, step (1), the magnetically activated cell sorting method includes:

[0012] (1-1) CD4+CD25+ cells were separated using a mixture of microbeads to remove non-CD4+ and CD25+ cells;

[0013] (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 the Treg cell population of CD4+CD25+CD127low / -.

[0014] In some specific embodiments, step (2) of the in vitro amplification further includes an anti-CD3 / CD28 magnetic bead activation step.

[0015] In some specific embodiments, in step (2), the cell-to-magnetic-bead ratio during the anti-CD3 / CD28 magnetic bead activation is between 1:1 and 1:3.

[0016] In some specific embodiments, in step (2), the anti-CD3 / CD28 magnetic bead activation is performed more than twice; preferably, the magnetic bead activation is performed on day 1 and day 9.

[0017] 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.

[0018] In some specific embodiments, in step (2), the concentration of ABBV-744 is 1-200 nM, preferably 1-150 nM, and more preferably 10-100 nM.

[0019] In some specific embodiments, in step (2), the concentration of IL-2 is 100-1000 U / mL, preferably 300-500 U / mL.

[0020] In some specific implementations, the in vitro amplification time in step (2) is 14-21 days.

[0021] In some specific embodiments, the PBMC cells are derived from tissue samples such as peripheral blood, thymus, and umbilical cord blood of the subject.

[0022] In some specific embodiments, the PBMC cells are derived from healthy subjects.

[0023] On the other hand, this disclosure provides a population of Treg cells obtained by in vitro expansion using the method described above.

[0024] On the other hand, this disclosure provides the use of the Treg cell population described above in the preparation of medicaments for treating diseases.

[0025] On the other hand, this disclosure provides a method for treating a disease in a subject, comprising administering Treg cells as described above to the subject.

[0026] In some specific embodiments, the disease is selected from systemic lupus erythematosus, inflammatory bowel disease, autoimmune hepatitis, allergy, asthma, graft-versus-host disease, type 1 diabetes, and multiple sclerosis.

[0027] Compared with the prior art, the beneficial effects of this disclosure are:

[0028] 1. Using magnetic activation sorting to sort Treg cells results in less cell damage, lower cost, and easier GMP production.

[0029] 2. Compared with traditional CD25 positive selection magnetic bead activation sorting, higher purity Treg cells can be obtained.

[0030] 3. It exhibits a high cell expansion rate while maintaining high purity of Treg cells.

[0031] 4. By combining rapamycin and ABBV-744 in in vitro expansion, the expanded Treg cells simultaneously possess high purity, high in vitro expansion fold, and high stability. Attached Figure Description

[0032] This disclosure can be more fully understood with reference to the following figures.

[0033] Figure 1 shows the results of flow cytometry analysis of CD4+CD25+CD127low / - Treg cells isolated from PBMCs.

[0034] Figure 2 shows the in vitro expansion fold of Treg cells.

[0035] Figure 3 shows the results of flow cytometry analysis of the purity of Treg cells after in vitro expansion.

[0036] Figure 4 shows the in vitro expansion fold of Treg cells under different culture conditions.

[0037] Figure 5 shows the flow cytometry results of Treg cells expanded under different culture conditions.

[0038] Figure 6 shows the results of in vitro inhibition of Treg cells expanded under different culture conditions.

[0039] Figure 7 shows the TSDR methylation detection results of Treg cells expanded under different culture conditions.

[0040] Figure 8 shows the results of in vitro stability assays of Treg cells expanded under different culture conditions.

[0041] Figure 9 shows the expansion fold of Treg cells after 14 days of in vitro expansion under different concentrations of AB and RAPA combinations.

[0042] Figure 10 shows the statistical results of flow cytometry analysis of Treg cells after in vitro expansion under different concentrations of AB and RAPA combinations.

[0043] Figure 11 shows the flow cytometry results of Treg cells after in vitro expansion under different concentrations of AB and RAPA combinations.

[0044] Figure 12 shows the TSDR methylation detection results of Treg cells expanded under different concentrations of AB and RAPA combinations.

[0045] Figure 13 shows the in vitro expansion fold of Treg cells when AB, GSK and JQ1 were used in combination with RAPA.

[0046] Figure 14 shows the flow cytometry results of Treg cells amplified under the conditions of AB and GSK being coupled with RAPA, respectively.

[0047] Figure 15 shows the TSDR methylation detection results of Treg cells expanded under the conditions of AB and GSK combined with RAPA, respectively. Detailed Implementation

[0048] 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.

[0049] 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.

[0050] As used herein, the term "population" or "cell population" refers to a cell population 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, a "Treg cell population" refers to a cell population 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 C A. 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, KARADIMITRIS A. 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 Exp Med, 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]. Mol Cell, 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.

[0055] 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 the recruitment of BET proteins to induce gene expression. Furthermore, BD2 inhibitors have a more significant effect on BRD2 and BRD3, a phenomenon also observed in the activation of primary human CD4+ T cells.

[0056] ABBV-744 (CAS No.:2138861-99-9) is a selective inhibitor of the BD2 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.).

[0057] 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, about 80-120 nM, etc. In some embodiments, the concentration of ABBV-744 in the cell culture medium is about 1-150 nM or about 10-100 nM.

[0058] 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.

[0059] In some embodiments, the concentration of IL-2 in the cell culture medium is about 100-1000 U / mL, for example about 100-200 U / mL, about 200-300 U / mL, about 400-500 U / mL, about 500-600 U / mL, about 600-700 U / mL, about 700-800 U / mL, about 800-900 U / mL or about 900-100 U / mL, preferably about 300-500 U / mL.

[0060] 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 from a healthy subject.

[0061] 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%.

[0062] 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.

[0063] 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 the 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.

[0064] 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.

[0065] As used herein, the term "treatment" refers to the administration of an effective amount of Treg cells, expanded in vitro according to the methods described herein, 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 disease severity, 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 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, the term "treatment" includes prevention. Alternatively, treatment is "effective" in cases where disease progression is reduced or stopped. "Treatment" may also mean an extension of survival compared to 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.

[0066] The terms “therapeuticly acceptable amount” and “therapeuticly effective amount” are used interchangeably to refer to an amount sufficient to achieve the desired outcome. In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. In some embodiments, a therapeutically 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. The terms “preventatively effective dose” and “therapeuticly effective dose” as used herein can respectively prevent the onset of disease symptoms or reduce the severity of disease symptoms, including those related to the condition.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Example

[0072] 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.

[0073] Example 1: Sorting of Treg cells

[0074] Treg cells were sorted using a human CD4+CD127lowCD25+ regulatory T cell separation kit (StemCell Catalog no. 18063).

[0075] Healthy donor PBMC cells were purchased from Selby Biotechnology Co., Ltd. and used for Treg cell sorting after thawing.

[0076] For 10 7 One PBMC cell was resuspended in 2 mL of separation buffer and transferred to a 5 mL sorting tube.

[0077] Add 100 μL of CD25 Positive Selection Cocktail, mix well and incubate for 5 minutes.

[0078] Add 60 μL of Releasable RapidSpheres and 100 μL of CD4+T Cell Enrichment Cocktail, mix well and incubate for 5 minutes.

[0079] Adjust the sample volume to 2.5 mL using separation buffer, mix well, insert the sorting tube into the magnetic pole, and let stand for 10 minutes.

[0080] The supernatant was aspirated into a new sorting tube for sorting Tconv cells (CD4+CD25-).

[0081] Remove the sorting tube, add 2.5 mL of separation buffer and resuspend it, then insert it into the magnetic pole and let it stand for 5 minutes.

[0082] Remove the supernatant, take out the sorting tube, add 2.5 mL of separation buffer and resuspend, then insert it into the magnetic pole and let it stand for 5 minutes.

[0083] Repeat the above steps once.

[0084] Aspirate the supernatant, add separation buffer to the starting volume for sorting and resuspend the cells, add 200 μL of release buffer and resuspend the cells, and pipette at least 5 times.

[0085] Add 20 μL of CD127 high Depletion Cocktail and mix well. Incubate at room temperature for 5 min.

[0086] 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.

[0087] The supernatant was transferred to a new centrifuge tube, which yielded the sorted Treg cells.

[0088] The sorting method for Tconv cells is as follows: Add 90 μL of Dextran RapidSpheres to the supernatant used for sorting Tconv cells aspirated during the Treg cell sorting process, mix well by pipetting, and incubate at room temperature for 5 min. Insert the sorting tube into the magnetic pole and let it stand for 5 min. Transfer the supernatant to a new centrifuge tube, which contains the sorted Tconv cells.

[0089] Flow cytometry was used to analyze CD4+CD25+CD127low / - Treg cells isolated from PBMCs, and the results are shown in Figure 1. Figure 1A shows that before isolation, CD4 T cells accounted for 48.5% of the PBMC cells, with CD25+CD127low / - cells accounting for 4.61% and FOXP3+ cells accounting for 6.01%. After sorting using a CD4+CD25+CD127low / - regulatory T cell division kit, the proportion of CD4 T cells increased to 93.1%, with almost no CD8 T cells. Furthermore, the proportion of CD25+CD127low / - or FOXP3+ cells among the CD4 T cells reached over 96%. Figure 1B shows the results of four independent experiments, including PBMC cells from three different healthy donors. Two of the experiments used PBMC cells from the same donor, demonstrating good reproducibility.

[0090] Example 2: In vitro expansion of Treg cells

[0091] 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% Human AB serum to a final volume of 2×10⁻⁶. 5 -1×10 6Add anti-CD3 / CD28 magnetic beads (Invitrogen) at a cell:magnetic bead ratio of 1:3, along with IL-2 (Novoprotein) 500 IU / mL and rapamycin (MCE) 100 nM. Mix well and add to a suitable well plate or culture flask. Incubate at 37°C in a 5% CO2 incubator.

[0092] 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.

[0093] 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, along with IL-2 500 IU / mL and rapamycin 100 nM. Mix well and add to a suitable well plate or culture flask. Incubate at 37°C in a 5% CO2 incubator.

[0094] 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 1000 cells / mL, and IL-2 was added to 500 IU / mL. Cells were harvested on day 14.

[0095] Figure 2 shows the results of four Treg cell isolation and in vitro expansion experiments using PBMC cells from three healthy donors, with two isolation and in vitro expansion experiments conducted using the same donor. As shown in Figure 2, PBMC cells from different donors could produce a large number of Treg cells. During the 14-day expansion, the fold increase ranged from 680 to 1500 times, with an average of 1158 times.

[0096] Example 3: Identification of Treg cells

[0097] To assess the purity of isolated Treg cells and the initial frequency of FOXP3+CD4+ cells, cell analysis was performed by flow cytometry. Analysis was conducted on cell samples collected (before isolation) and on cell samples collected (after isolation). Lymphocytes were identified using FSC / SSC gating. Dead cells were excluded from analysis using a zombie violet staining method. CD4+ and CD8+ cells were identified based on CD4 / CD8 expression in lymphocytes. Further analysis of CD127 and CD25 expression in CD4+ cells was performed to assess the purity of CD25+CD127low / - cells in CD4+ cells. Further analysis of CD4 and FOXP3 expression in lymphocytes was performed to assess the initial frequency of CD4+ cells expressing FOXP3 in lymphocytes.

[0098] Figure 3 shows the results of flow cytometry analysis of the expanded Treg cells from Example 2. Figure 3A shows the results of one expansion, and Figures 3B and 3C show the statistical results of four expansions. As can be seen from Figures 3A, B, and C, the sorted Treg cells maintained very high purity after expansion, with over 90% of the cells being CD25+CD127low / - or FOXP3+, including CD4+>95%, CD8+<2%, and FOXP3+>90%.

[0099] Example 4: Optimization of Culture Conditions

[0100] Treg cells were sorted and expanded in vitro according to the methods in Examples 1 and 2, with the only difference being that rapamycin was replaced in the culture as follows: the control group did not contain rapamycin or ABBV-744 (Selleck); the rapamycin (RAPA) group contained 100 nM rapamycin; the ABBV-744 (AB) group contained 100 nM ABBV-744; and the combination therapy (RAPA+AB) group contained 50 nM rapamycin and 100 nM ABBV-744. The expansion results of each group were identified according to the protocol in Example 3.

[0101] The amplification and identification results of each group are shown in Figures 4 and 5. Figures 4 and 5 show that the control group and the AB group had the best in vitro amplification. However, in these two groups, the proportion of Helios-FOXP3low / - cells was high, exceeding 40%, and these cells do not have immunosuppressive function. The proportions of Helios+FOXP3+ and Helios-FOXP3high cells were lower. These two cell groups are generally considered to be nTreg and iTreg cells, respectively, and have immunosuppressive function. Among them, Helios+ Treg cells have higher stability in vivo. In the RAPA group, although the proportions of Helios+FOXP3+ and Helios-FOXP3high cells were the highest, the in vitro amplification fold of Treg cells was the lowest, less than 100-fold, due to the high concentration of RAPA. The RAPA+AB group, on the other hand, ensured a high Treg cell amplification fold, with the total number of Helios+FOXP3+ and Helios-FOXP3high cells reaching over 95%, and a significantly increased proportion of the more stable Helios+FOXP3+ cells.

[0102] Example 5: In vitro inhibition function detection

[0103] The Treg cells obtained from each group in Example 4 were subjected to in vitro inhibitory function testing according to the following method, and the results are shown in Figure 6.

[0104] The sorted Tconv cells (CD4+CD25-) were used as Tresp cells, and CTV (CellTrace) was used to detect them. TM The cells were labeled with Violet (Invitrogen, Catalog no. C34557). Tresp cells were added to 96-well U-shaped plates at a density of 50,000 cells per well. Treg cells were then added to each well at ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. Finally, 5,000 anti-CD3 / CD28 magnetic beads were added to each well and mixed thoroughly. After culturing at 37°C in a CO2 incubator for 4 days, the CTV signal of Tresp cells was assessed by flow cytometry to evaluate proliferation.

[0105] As shown in Figure 6, the RAPA group and the RAPA+AB group had the best in vitro inhibitory function.

[0106] Example 6: TSDR Methylation Detection

[0107] TSDR is an abbreviation for Treg-specific-demethylation-region, which is the CNS2 non-coding region located on the promoter of the foxp3 gene. Methylation of this region can cause instability in foxp3 gene expression. In nTreg cells, it is completely demethylated and can also be used to reflect the stability of Treg cells. The TSDR methylation level of the Treg cells amplified in each group in Example 4 was detected according to the following method, and the results are shown in Figure 7.

[0108] Collect 1-5×10 6 Genomic 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.

[0109] The stability of Treg cells was assessed by using fluorescent probe qPCR to quantify the methylation level of the TSDR region using fluorescent probes targeting methylated (5'-ATGGCGGTCGGATGCGTC-3', SEQ ID NO: 1) and unmethylated (5'-ATGGTGGTTGGATGTGTTGGGTT-3', SEQ ID NO: 2) sites at the CNS2 site (refer to WIECZOREK G, ASEMISSEN A, 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.).

[0110] As shown in Figure 7, the RAPA group and the RAPA+AB group had the highest demethylation levels, indicating that the Treg cells expanded in vitro under these two conditions had the highest stability.

[0111] Example 7: In vitro stability of Treg cells

[0112] The Treg cells obtained from each group in Example 4 were used to detect the expression of Helios and FOXP3 using the following method. The results are shown in Figure 8.

[0113] Treg cells cultured for 7 days were activated and expanded for another 7 days under conditions additionally containing 10 ng / mL IL-1β, 10 ng / mL IL-6, and 10 ng / mL IL-23, inducing the loss of foxp3 gene expression in Treg cells. After three rounds of activation and expansion under these conditions, some Treg cells transformed into non-immunosuppressive Tconv cells. The expression of Helios and FOXP3 was detected by flow cytometry to determine the proportions of nTreg (Helios+FOXP3+), iTreg (Helios-FOXP3high), and non-Treg cells (Helios-FOXP3low / -).

[0114] As shown in Figure 8, most cells in the control group had transformed into Helios-FOXP3low / - non-Treg cells. The AB group still contained approximately 17.5% nTreg cells and approximately 5.8% iTreg cells, while the RAPA+AB group contained approximately 41.1% nTreg cells and approximately 7.7% iTreg cells. This indicates that Treg cells expanded in vitro under RAPA+AB conditions exhibited higher stability under certain inflammatory cytokine conditions.

[0115] Example 8: Effects of different concentrations of ABBV-744 combined with rapamycin on Treg cell expansion and stability

[0116] Treg cells were sorted and expanded in vitro according to the methods described in Examples 1 and 2, except that rapamycin in the original culture system was replaced with the following treatment combination: the control group (DMSO) did not add any inhibitors; the experimental group was added to the culture medium with ABBV-744 (Selleck) and rapamycin (RAPA) in the following concentration combinations: ABBV-744 (1nM, 10nM, 100nM, 200nM) was used in combination with rapamycin (10nM, 50nM, 100nM, 200nM).

[0117] The amplification results of Treg cells in each group are shown in Figure 9. The DMSO group showed the best amplification effect, while the amplification fold of ABBV-744 and rapamycin at the lowest concentration combination (1 nM ABBV-744 + 10 nM RAPA) was closest to that of the DMSO group. The amplification levels of Treg cells in the other groups were lower than those of these two groups. Furthermore, as the concentrations of ABBV-744 and rapamycin increased, the amplification fold of Treg cells showed a decreasing trend, with the effect of rapamycin concentration being more significant.

[0118] As shown in Figures 10 and 11, in the expanded Treg cells, all experimental groups that received rapamycin and ABBV-744 showed increased Helios levels. + FOXP3 + The proportion of Helios cells in the DMSO group was significantly higher than that in the DMSO group; while in the DMSO group, the proportion of Helios cells was significantly higher. - FOXP3low / - The proportion of cells is relatively high.

[0119] The results in Figure 12 show that the TSDR region demethylation level was higher in all combination drug groups than in the DMSO group.

[0120] In summary, the combined use of ABBV-744 and rapamycin within a certain concentration range can effectively maintain the functional epigenetic stability of Treg cells while maintaining their ability to expand in vitro.

[0121] Example 9: Comparison of the effects of ABBV-744, GSK046, and JQ1 in Treg cell expansion

[0122] GSK046 and ABBV-744 are both small-molecule inhibitors that selectively target the second bromodomain (BD2) of the BET protein family, specifically regulating the expression of downstream genes. JQ1, on the other hand, is a broad-spectrum BET inhibitor that cannot distinguish between the BD1 and BD2 domains. It competitively binds to acetylated lysine recognition sites, blocking the interaction between BET protein and chromatin, thereby inhibiting the expression of multiple oncogenes, including c-Myc.

[0123] Treg cells were sorted and expanded according to the methods in Examples 1 and 2, with the difference that rapamycin was replaced with the following treatments: AB+RAPA group was treated with 100 nM ABBV-744 and 50 nM rapamycin; GSK+RAPA group was treated with 1 μM GSK046 (Selleck) and 50 nM rapamycin; JQ1+RAPA group was treated with 1 μM JQ1 (MCE) and 50 nM rapamycin. The expanded Treg cells were analyzed according to the methods in Examples 5-7.

[0124] Figure 13 shows that the fold increase of Treg cells in the AB+RAPA group and the GSK+RAPA group was similar, but on day 14 of culture, the fold increase in the AB+RAPA group was slightly higher than that in the GSK+RAPA group. Cell proliferation in the JQ1+RAPA group was significantly inhibited, making effective proliferation difficult.

[0125] Figure 14 shows that the flow cytometry analysis results indicate that Helios cells in the expanded Treg cells of the AB+RAPA group and the GSK+RAPA group... + FOXP3 +Helios - FOXP3high and Helios - FOXP3low / - The proportions of cell subsets were basically the same. Figure 15 further shows that there was no significant difference in TSDR demethylation levels between the two groups.

[0126] In summary, when ABBV-744 and GSK046 are used in combination with rapamycin, ABBV-744 is slightly more effective than GSK046 in the in vitro expansion of Treg cells, and ABBV-744 can achieve similar or even better expansion effects at lower concentrations. In contrast, JQ1, due to its lack of BD2 selectivity, has a significant inhibitory effect on the expansion of Treg cells and is not suitable for the in vitro expansion system of this type of cell.

[0127] By incorporating references

[0128] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0129] Equivalence

[0130] 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. A method for in vitro expansion of Treg cells, characterized in that, The method includes the following steps: (1) Isolate CD4+CD25+CD127low / - Treg cells from PBMC cells; (2) The Treg cell population obtained in step (1) was expanded in vitro in the presence of rapamycin, ABBV-744 and IL-2.

2. The method according to claim 1, wherein, In step (1), Treg cell populations are sorted using fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

3. The method according to claim 2, wherein, Step (1), the steps of the magnetically activated cell sorting method include: (1-1) CD4+CD25+ cells were separated using a mixture of microbeads to remove non-CD4+ and CD25+ cells; (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 the Treg cell population of CD4+CD25+CD127low / -.

4. The method according to any one of claims 1-3, wherein, In step (2), the in vitro amplification also includes anti-CD3 / CD28 magnetic bead activation.

5. The method according to claim 4, wherein, In step (2), during the anti-CD3 / CD28 magnetic bead activation, the cell-to-magnetic-bead ratio is between 1:1 and 1:3; and / or The anti-CD3 / CD28 magnetic bead activation is performed more than twice; preferably, the anti-CD3 / CD28 magnetic bead activation is performed on day 1 and day 9.

6. The method according to any one of claims 1-5, wherein, In step (2), the concentration of rapamycin is 10-200 nM, preferably 20-70 nM, more preferably 40-60 nM; and / or The concentration of ABBV-744 is 1-200 nM, preferably 1-150 nM, more preferably 10-100 nM; and / or The concentration of IL-2 is 100-1000 U / mL, preferably 300-500 U / mL.

7. The method according to any one of claims 1-6, wherein, In step (2), the in vitro amplification time is 14-21 days.

8. The method according to any one of claims 1-7, wherein, The PBMC cells were derived from peripheral blood, thymus, or umbilical cord blood samples from the subjects.

9. The Treg cell population obtained by in vitro expansion using the method of any one of claims 1-8.

10. Use of the Treg cell population of claim 9 in the preparation of a medicament for treating a disease selected from systemic lupus erythematosus, inflammatory bowel disease, autoimmune hepatitis, allergy, asthma, graft-versus-host disease, type 1 diabetes, and multiple sclerosis.

11. A method of treating a disease in a subject, comprising administering the Treg cells of claim 9 to the subject, wherein the disease is selected from systemic lupus erythematosus, inflammatory bowel disease, autoimmune hepatitis, allergy, asthma, graft-versus-host disease, type 1 diabetes, and multiple sclerosis.

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