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JP-7854993-B2 - Method for freezing and preserving the operation Treg

JP7854993B2JP 7854993 B2JP7854993 B2JP 7854993B2JP-7854993-B2

Inventors

  • マックギル、イアン

Assignees

  • クウェル セラピューティックス リミテッド

Dates

Publication Date
20260507
Application Date
20211109
Priority Date
20201109

Claims (20)

  1. A method for preparing a composition containing a Treg population for immediate administration to a target , (a) Steps to isolate the Treg population from the sample, ( b ) A step of introducing a polynucleotide encoding the FOXP3 polypeptide into the Treg population , ( c ) A step of cryopreserving the Treg group, and (d) A step of melting the group , The method is one which does not include any further culture or growth steps after step (d) .
  2. The method according to claim 1, further comprising the step of growing the Treg population before step (c).
  3. The method according to claim 1 or 2 , wherein the sample comprises whole blood, umbilical cord blood, leukocyte cones, blood cones, peripheral blood mononuclear cells (PBMCs), or one or more leucopacks.
  4. The method according to any one of claims 1 to 3 , wherein step ( c ) includes the following steps: ( c ) The step of suspending the Treg population in a culture medium for cryopreservation; ( c iii) a step of freezing the Treg group from step ( c i); and ( c iii) a step of storing the Treg group from step ( c iii) at a temperature lower than -130°C.
  5. The method according to claim 4 , further comprising the following steps: Prior to step ( c ), a step of pre-cooling the Treg population and/or one or more reagents and devices used in the cryopreservation step; A step of freezing the Treg group at a controlled freezing rate of approximately -1°C/min in step ( c ); and/or a step of storing the Treg group at -80°C for up to 24 hours prior to step ( ciii ).
  6. The method according to any one of claims 1 to 5 , wherein the melting of the Treg group includes raising the temperature of the Treg group from a temperature lower than -130°C to a temperature between approximately 0°C and 10°C.
  7. The method according to any one of claims 1 to 6 , wherein the Treg population is isolated by selecting (i) or (ii) below: (i) CD4 + CD25 + CD127 - cells and/or CD4 + CD25 + CD127 low cells; or (ii) CD4 + CD25 hi CD127 - cells and/or CD4 + CD25 + CD127 low cells.
  8. The method according to any one of claims 1 to 7 , wherein the Treg population is isolated by selecting CD45RA + cells .
  9. The method according to any one of claims 1 to 8 , wherein the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof that is at least 90 % identical to SEQ ID NO: 1.
  10. The method according to any one of claims 1 to 9 , wherein the polynucleotide encoding FOXP3 is present in the expression vector.
  11. The method according to any one of claims 1 to 10 , further comprising introducing a polynucleotide encoding an exogenous T cell receptor (TCR) or a polynucleotide encoding a chimeric antigen receptor (CAR) into the Treg population .
  12. The method according to claim 11 , wherein the polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the exogenous TCR or the CAR are provided by a single expression vector.
  13. The method according to claim 12, wherein the vector comprises a first polynucleotide encoding the FOXP3 polypeptide and a second polynucleotide encoding the exogenous TCR or CAR, the first polynucleotide and the second polynucleotide being operably linked to the same promoter, and the first polynucleotide being upstream of the second polynucleotide.
  14. The method according to any one of claims 11 to 13 , wherein an internal self-cleavage sequence exists between the polynucleotide encoding FOXP3 and the polynucleotide encoding the exogenous TCR or CAR.
  15. A composition comprising a Treg population for use in the prevention and/or treatment of a disease , wherein the Treg population is obtained by the method of any one of claims 1 to 14, and the Treg population is to be administered to a subject immediately after thawing .
  16. Use of a Treg population in the manufacture of a drug for the prevention and/or treatment of a disease , wherein the Treg population is obtained by the method of any one of claims 1 to 14, and the Treg population is intended to be administered to a subject immediately after thawing .
  17. The composition according to claim 15 , wherein the disease is an autoimmune disease or an allergic disease.
  18. The composition according to claim 15 , wherein the disease is transplant rejection or graft-versus-host disease.
  19. The composition according to claim 15 , used for suppressing the immune response.
  20. The composition according to claim 15 , wherein the disease is a neurodegenerative disease.

Description

This invention relates to a method for cryopreserving regulatory T cells (Tregs). In particular, it relates to a method for increasing FOXP3 expression in Tregs before cryopreservation in order to preserve the inhibitory function of Tregs after cryopreservation. The invention also relates to cryopreserved manipulated Tregs, pharmaceutical compositions comprising cryopreserved manipulated Tregs, and their therapeutic uses. In recent years, there has been growing interest in using regulatory T cells (Tregs) in clinical settings for adoptive cell therapy (ACT) to treat various different pathological conditions. Due to their immunosuppressive function, Tregs have been proposed for use in controlling undesirable immune responses. For example, Tregs have been used in the treatment of autoimmune or allergic diseases, immunomodulation in transplantation, and improvement and/or prevention of immune-mediated organ damage in inflammatory diseases. In addition, genetic engineering of Tregs to express novel T cell receptors (TCRs) or chimeric antigen receptors (CARs) has been performed, resulting in the advantage of targeted immunosuppression. Various Phase I trials have demonstrated the safety and efficacy of Treg therapy, and several Phase II trials are currently underway. The primary sources of Treg for therapeutic use are the patient's own blood (collected directly from a blood vessel or as a leukocyte apheresis product) or umbilical cord blood. While the number of Treg obtained from these sources is small, a significant amount is needed to effectively suppress the immune system. Therefore, ex vivo expansion is necessary to obtain a sufficient number of Treg for patient injection. A typical isolation and expansion protocol following Good Manufacturing Practice (GMP) guidelines takes approximately 9 to 21 days. Processing and freezing these cells in sufficient quantities, making them readily available for patient use when needed, would be highly advantageous, as it would avoid the prolonged processing that can negatively impact Treg quality. This approach would allow for greater flexibility in therapy planning and injection timing, and would enable cell harvesting before subsequent Treg injections and pharmacological therapy. Therefore, the ability to cryopreserve Treg is crucial. The effects of freeze-thaw cycles on Treg cell populations are not clearly defined. Several research groups have reported that cryopreservation may have detrimental effects on Treg cells, potentially reducing their viability, causing abnormal cytokine secretion, and altering the expression of surface markers essential for proper Treg suppression and migration. In particular, Florek et al. (PLoS One, 2015, DOI: 10.1371 /journal.pone.0145763) revealed that freeze-thawing of Treg leads to loss of CD62L (L-selectin) expression, impairing its protective ability against graft-versus-host disease (GvHD). They found that thawed Treg exhibits reduced binding ability to MADCAM1, the binding partner of CD62L, and impaired homing to secondary lymphoid organs. Furthermore, other studies have shown that the expression of CD62L (L-selectin) and CCR5 on T cells is lost after cryopreservation (De Boer et al, Bone Marrow Transfer, 1998, 22:1103-10 and Hattori et al, Exp Hematol, 2001, 29:114-22). Treg cells lacking CD62L expression showed reduced transport capacity and localization, resulting in decreased compartmentalization of the Treg-regulated immune response (Sakaguchi et al, Cell, 2008, 133:775-87). Loss of CD62L expression after thawing can be restored by culturing overnight (De Boer et al., 1998 and Hattori et al., 2001). However, resting cells in this way may not be practical in clinical applications because thawing is usually performed at the bedside, making further culturing impossible. Golab et al. (Oncotarget, 2018, vol. 9, (No. 11), pp: 9728-9740) evaluated two cell banking strategies for Treg therapy: cryopreservation of CD4+ cells for subsequent Treg isolation/proliferation, and cryopreservation of ex vivo proliferated Treg (CD4 + CD25 hi CD127 lo/- cells). Ex vivo proliferated Treg was found to be more sensitive to the cryopreservation process than CD4+ cells, with significantly reduced cell viability after thawing and decreased Treg marker expression (i.e., a reduced frequency of cells with the phenotypes CD4 + CD25 hi CD127- and CD4 + FoxP3 + ). The low Treg viability and phenotypic impairment after thawing were overcome by restimulating Treg during a subsequent 13-day ex vivo proliferation. Similarly, Peters et al. (PLoS One, 2008, 3:e3161) showed that the inhibitory ability of Treg after thawing can be restored by 10 days of stimulation and proliferation. However, as mentioned above, these additional culture and proliferation steps are not feasible in clinical settings, and are time-consuming and carry additional risks, thus negating the advantages of using cryopreserved Treg cells compared to newly isolated cells. Therefore, an improved