Enhancement of graft-versus-tumor effect using ropeginterferon

Abstract

The present invention simultaneously achieves, in allogeneic hematopoietic cell transplantation as a treatment for blood cancer, enhancement of a graft-versus-tumor effect (GVT effect) and / or a graft-versus-leucocyte effect (GVL effect) and suppression of graft-versus-host disease (GVHD).　The present invention relates to a pharmaceutical composition which is for enhancement of a GVL and / or GVT effect in a hematopoietic cell transplantation treatment and which comprises ropeginterferon.

Description

Enhancement of graft-versus-tumor effect using LOPEG interferon

[0001] This patent application claims priority and interest under the Paris Convention and priority under Article 41 of the Japanese Patent Act, based on Japanese Patent Application No. 2024-193170 (filed November 1, 2024), and by reference herein the entire contents of the said application are incorporated herein by reference.

[0002] The present invention belongs to the field of hematopoietic stem cell transplantation therapy. More specifically, the present invention relates to a pharmaceutical composition for enhancing the GVL / GVT effect in hematopoietic stem cell transplantation therapy, and more precisely, to a pharmaceutical composition for enhancing the GVL / GVT effect in hematopoietic stem cell transplantation therapy that contains lopeg interferon.

[0003] Allogeneic hematopoietic stem cell transplantation (GVT) is a treatment method for blood cancers such as leukemia. This method relies on the graft-versus-tumor effect (GVT), in which immune cells, mainly lymphocytes such as T cells, contained in donor-derived hematopoietic cells attack tumor cells remaining in the recipient's (patient's) body, leading to the cure or control of the underlying disease. The GVT effect is expected to suppress recurrence and ultimately lead to a complete cure by continuously attacking and eliminating tumor cells through the immune response. On the other hand, graft-versus-host disease (GVHD) is known as an immune response in which transplanted donor-derived lymphocytes recognize and attack normal body parts such as the patient's skin, liver, lungs, and intestines as foreign bodies. In other words, GVT and GVHD are two sides of the same coin, and if GVHD is suppressed too much by immunosuppressive therapy, the GVT effect, which is an important immunotherapeutic effect for achieving a cure, may be negated. Therefore, in treatments that expect the manifestation of the GVT effect, the onset of GVHD as a side effect and recurrence after transplantation are problems. Tumor recurrence after transplantation is the most common cause of death after transplantation, and separating GVHD from GVT effects has become the ultimate goal in the field of transplantation (Non-Patent Literature 7).

[0004] To enhance the graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia (AML), hypomethylating agents and FLT3 inhibitors have been developed as maintenance therapies to prevent immune evasion by leukemia cells or suppress fatigue of donor T cells. However, relapse of leukemia after transplantation remains a major problem (Non-Patent Documents 1, 2, and 3).

[0005] In addition, it is known that endogenous interferon suppresses GVHD and that interferon administration enhances the GVT effect, in relation to the problems of GVHD onset and recurrence after transplantation (Non-Patent Documents 4 and 5). Furthermore, it has been suggested that lopeg interferon α-2b, described in Patent Document 1, shows a higher therapeutic effect than interferon against acute lymphoblastic leukemia (ALL) (Non-Patent Document 6).

[0006] Patent No. 5613050

[0007] Pollyea DA, et al. Nature Med. 2018: 24(12):1859-1866.Wei Y, et al. Cancer sci. 2021: 112(9):3636-3644.Zhang Z, et al. Bone Marrow Transplant. 2022;57(5):775-780.Robb RJ, et al. Blood. 2011: 118(12):3399-409,Fischer JC, et al. Sci Transl Med. 2017: 9(386):eaag2513.Sakatoku K, et al. Cancer Sci. 2022: 113(7):2246-2257.Wigard, RJ, et al. J Clin Oncol. 2011

[0008] In allogeneic hematopoietic stem cell transplantation as a treatment for blood cancers such as leukemia, it is necessary to enhance the GVT effect and suppress GVHD, which is inextricably linked to the GVT effect. However, if GVHD is suppressed excessively, the GVT effect, which is important for achieving a cure, will be negated. Therefore, a major goal in transplant therapy is to simultaneously achieve both enhancement of the GVT effect and suppression of GVHD.

[0009] Therefore, the present inventors confirmed the efficacy of Ropeg IFN, which contains Ropeg interferon α-2b, against acute myeloid leukemia (AML). They found that it enhances the GVL effect and suppresses GVHD after allogeneic hematopoietic stem cell transplantation for AML, thus completing the present invention.

[0010] Accordingly, the present invention includes the following embodiments: <Pharmaceutical Compositions> [1] A pharmaceutical composition containing lopeg interferon for enhancing the GVL and / or GVT effect in hematopoietic stem cell transplantation therapy. [2-1] The pharmaceutical composition according to [1], characterized by reducing hematopoietic stem cells. [2-2] The pharmaceutical composition according to [2-1], characterized by reducing leukemia stem cells. [2-3] The pharmaceutical composition according to any one of [1] to [2-2], characterized by reducing T cell exhaustion. [3] The pharmaceutical composition according to any one of [1] to [2-3], further characterized by having a GVHD inhibitory effect. [4] The pharmaceutical composition according to any one of [1] to [3] for preventing and / or treating the recurrence of leukemia. [5] The pharmaceutical composition according to [4], wherein the leukemia is acute myeloid leukemia. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the hematopoietic cells are bone marrow. [7-1] The pharmaceutical composition according to [6], wherein the hematopoietic cells are lymphocytes. [7-2] The pharmaceutical composition according to [7-1], wherein the hematopoietic cells are T cells.

[0011] <Combination drug> [8] A pharmaceutical composition according to any one of [1] to [7-2], further characterized by containing hematopoietic stem cell grafts.

[0012] <Identifying Lopeg Interferon> [9] Lopeg interferon is given by formula: [In the formula, R １ , R ２ , R ３ , R ４ , and R ５ H and C are independent of each other. ｌ－５ alkyl, C ２－５ Alkenil, C ２－５Alkynyl, aryl, heteroaryl, C ３－８ cycloalkyl, or C ３－８ heterocycloalkyl, where A １ and A ２ are each independently a polymer moiety, G １ , G ２ , and G ３ are each independently a bond or a bonding functional group, G ３ is a bond, P is a protein moiety where the N-terminal amino group is bonded to G ３ , m is an integer of 0 or 1 - 10, and n is an integer of 1 - 10, G １ and G ２ are each of the formula: , O is bonded to A １ or A ２ , NH is bonded to the carbon atom shown in Chemical Formula I], a protein-polymer conjugate, a pharmaceutical composition according to any one of [1] to [8].

[10] In the protein-polymer conjugate, in the formula, A １ and A ２ are each an mPEG moiety having a molecular weight of 10 - 30 kD, the pharmaceutical composition according to [9].

[11] In the protein-polymer conjugate, in the formula, P is a modified interferon moiety further containing 1 - 4 amino acid residues at its N-terminus, the pharmaceutical composition according to [9] or

[10] .

[12] n is 2, the pharmaceutical composition according to any one of [9] to

[11] .

[13] Ropeg interferon is , mPEG has a molecular weight of 20 kD, IFN is interferon α-2b, the pharmaceutical composition according to any one of [9] to

[12] .

[0013] The present invention also includes the following in other embodiments: <Therapeutic methods>

[101] A method for enhancing the GVL and / or GVT effect in hematopoietic stem cell transplantation therapy, comprising administering lopeg interferon to a subject requiring such treatment, etc. [102-1] The method according to

[101] , characterized by reducing hematopoietic stem cells. [102-2] The method according to [102-1], characterized by reducing leukemia stem cells. [102-3] The method according to any one of

[101] to [102-2], characterized by reducing T cell exhaustion.

[103] The method according to any one of

[101] to [102-3], further characterized by having a GVHD inhibitory effect.

[104] The method according to any one of

[101] to

[103] for preventing and / or treating the relapse of leukemia.

[105] The method according to

[104] , wherein the leukemia is acute myeloid leukemia.

[106] The method described in any of

[101] to

[105] , wherein the hematopoietic cells are bone marrow. [107-1] The method described in

[106] , wherein the hematopoietic cells are lymphocytes. [107-2] The method described in [107-1], wherein the hematopoietic cells are T cells.

[0014] <Combination Therapy>

[108] A method according to any one of

[101] to [107-2], characterized by further administering hematopoietic stem cell grafts.

[0015] <Identifying Lopeg Interferon>

[109] Lopeg interferon is, formula: [In the formula, R １ , R ２ , R ３ , R ４ , and R ５ H and C are independent of each other. ｌ－５ alkyl, C ２－５ Alkenil, C ２－５ Alkynyl, aryl, heteroaryl, C ３－８ Cycloalkyl, or C ３－８ It is a heterocycloalkyl, and A １ and A ２ Each of these is independently a polymer part, G １ G２ , and G ３ Each of these is independently a bond or a bonding functional group, G ３ The bond is P, and the N-terminal amino group is G ３ It is a protein site that is bound to, where m is 0 or an integer from 1 to 10, and n is an integer from 1 to 10, G １ and G ２ Each of these is expressed by: And O is A １ Or A ２ A is a protein-polymer conjugate represented by

[101] to

[108] , wherein A is bonded to a carbon atom shown in chemical formula I.

[110] In a protein-polymer conjugate, A １ and A ２ The method according to

[109] , wherein each of the mPEG sites has a molecular weight of 10-30 kD.

[111] The method according to

[109] or

[110] , wherein in the protein-polymer conjugate, P is a modified interferon site having 1-4 amino acid residues further at its N-terminus.

[112] The method according to any one of

[109] to

[111] , wherein n is 2.

[113] LOPEG interferon is The method according to any one of

[109] to

[112] , wherein the mPEG has a molecular weight of 20 kD and the IFN is interferon α-2b.

[0016] Furthermore, the present invention includes, in other embodiments: <Use 1>

[201] Lopeg interferon for enhancing the GVL / GVT effect in hematopoietic stem cell transplantation therapy. [202-1] Lopeg interferon according to

[201] , characterized by reducing hematopoietic stem cells. [202-2] Lopeg interferon according to [202-1], characterized by reducing leukemia stem cells. [202-3] Lopeg interferon according to any one of

[201] to [202-2], characterized by reducing T cell exhaustion.

[203] Lopeg interferon according to any one of

[201] to [202-3], further characterized by having a GVHD inhibitory effect.

[204] Lopeg interferon according to any one of

[201] to

[203] for preventing and / or treating relapse of leukemia.

[205] Lopeg interferon according to

[204] , wherein the leukemia is acute myeloid leukemia.

[206] Lopeg interferon according to any one of

[201] to

[205] , wherein the hematopoietic cells are bone marrow. [207-1] Lopeg interferon according to

[206] , wherein the hematopoietic cells are lymphocytes. [207-2] Lopeg interferon according to [207-1], wherein the hematopoietic cells are T cells.

[0017] <Combination Uses> [208-1] A combination of lopeg interferon and hematopoietic stem cell graft to enhance the GVL / GVT effect in hematopoietic stem cell transplantation therapy. [208-2] A combination in any of

[201] to [207-2] in which lopeg interferon is a combination of lopeg interferon and hematopoietic stem cell graft.

[0018] <Identifying Lopeg Interferon>

[209] Lopeg interferon is, formula: [In formula 20, R １ , R ２ , R ３ , R ４ , and R ５ H and C are independent of each other. ｌ－５ alkyl, C ２－５ Alkenil, C ２－５Alkynyl, aryl, heteroaryl, C ３－８ Cycloalkyl, or C ３－８ It is a heterocycloalkyl, and A １ and A ２ Each of these is independently a polymer part, G １ G ２ , and G ３ Each of these is independently a bond or a bonding functional group, G ３ The bond is P, and the N-terminal amino group is G ３ It is a protein site that is bound to, where m is 0 or an integer from 1 to 10, and n is an integer from 1 to 10, G １ and G ２ Each of these is expressed by: And O is A １ Or A ２ LOPEG interferon as described in any of

[201] to

[208] , which is a protein-polymer conjugate represented by

[201] to

[208] , where A is bonded to the carbon atom shown in chemical formula I.

[2010] In a protein-polymer conjugate, in formula A １ and A ２ LOPEG interferon according to

[209] , wherein each of the mPEG sites has a molecular weight of 10-30 kD.

[2011] LOPEG interferon according to

[209] or

[210] , wherein in the protein-polymer conjugate, P is a modified interferon site having 1-4 amino acid residues further at its N-terminus.

[2012] LOPEG interferon according to any one of

[209] to

[211] , wherein n is 2.

[213] LOPEG interferon, The ropeg interferon according to any one of

[209] to

[212] , wherein the mPEG has a molecular weight of 20 kD and the IFN is interferon α-2b.

[0019] The present invention also includes, in further different embodiments: <Use 2>

[301] Use of lopeg interferon for manufacturing a pharmacopoeia for enhancing the GVL / GVT effect in hematopoietic stem cell transplantation therapy. [302-1] Use according to

[301] , characterized by reducing hematopoietic stem cells. [302-2] Use according to [302-1], characterized by reducing leukemia stem cells. [302-3] Use according to any one of

[301] to [302-2], characterized by reducing T cell exhaustion.

[303] Use according to any one of

[301] to [302-3], further characterized by having a GVHD inhibitory effect.

[304] Use according to any one of

[301] to

[303] for preventing and / or treating the relapse of leukemia.

[305] Use according to

[304] , wherein the leukemia is acute myeloid leukemia.

[306] Use according to any one of

[301] to

[305] , wherein the hematopoietic cells are bone marrow. [307-1] Use as described in

[306] , where the hematopoietic cells are lymphocytes. [307-2] Use as described in [307-1], where the hematopoietic cells are T cells.

[0020] <Combinations> [308-1] Use of lopeg interferon and hematopoietic cell grafts for manufacturing a pharmaceutical product to enhance the GVL / GVT effect in hematopoietic cell transplantation therapy. [308-2] Use in any of

[301] to [307-2], wherein lopeg interferon is lopeg interferon and hematopoietic cell grafts.

[0021] <Identifying Lopeg Interferon>

[309] Lopeg interferon is, formula: [In formula 30, R １ , R ２ , R ３ , R ４ , and R ５ H and C are independent of each other. ｌ－５ alkyl, C ２－５ Alkenil, C ２－５ Alkynyl, aryl, heteroaryl, C ３－８ Cycloalkyl, or C ３－８ It is a heterocycloalkyl, and A１ and A ２ are each independently a polymer moiety, and G １ , G ２ , and G ３ are each independently a bond or a bonding functional group, G ３ is a bond, and P is a protein moiety in which the amino group at the N-terminus is bonded to G ３ , m is an integer of 0 or 1 - 10, and n is an integer of 1 - 10, G １ and G ２ are each of the formula: where O is bonded to A １ or A ２ and NH is bonded to the carbon atom shown in Chemical Formula I], a protein-polymer conjugate as described in any one of

[301] to

[308] .

[310] In the protein-polymer conjugate, in the formula, A １ and A ２ are each an mPEG moiety having a molecular weight of 10 - 30 kD, the use described in

[309] .

[311] In the protein-polymer conjugate, in the formula, P is a modified interferon moiety further containing 1 - 4 amino acid residues at its N-terminus, the use described in

[309] or

[310] .

[312] The use described in any one of

[309] to

[311] where n is 2.

[313] Ropeg interferon is where mPEG has a molecular weight of 20 kD and IFN is interferon α-2b, the use described in any one of

[309] to

[312] .

[0022] In this invention, we found that lopeg interferon induces apoptosis and differentiation of LSCs into myeloid cells in AML, delaying leukemia-related death. Furthermore, after hematopoietic stem cell transplantation, lopeg interferon proliferates transiently exhausted T cells in the donor, improves GVHD, and enhances the GVL effect. This invention demonstrates that lopeg interferon is a promising agent for separating the GVL effect from GVHD in maintenance therapy after hematopoietic stem cell transplantation in AML patients. Maintenance therapy is a treatment performed to maintain the therapeutic effect (response) obtained after hematopoietic stem cell transplantation or induction therapy, or to obtain a deeper response.

[0023] Figure 1 shows the experimental scheme for investigating the effect of Ropeg interferon α-2b (RopegIFN) administration on the GVL effect. In the figure, "MLL-AF9 cells" refers to AML cells created by introducing the KMT2A::MLLT3 fusion gene, "TCD-BM" refers to T cell-depleted bone marrow, and "AML" refers to acute myeloid leukemia. Figure 2 shows the enhancing effect of RopegIFN on the GVL effect. Figure 2A is a graph showing the changes in the percentage of peripheral blood AML cells for the TCD-BM + Vehicle administration group (no GVL effect), the TCD-BM + RopegIFN administration group (no GVL effect), the TCD-BM + T cell + Vehicle administration group (GVL effect present), and the TCD-BM + T cell + RopegIFN administration group (GVL effect present). Figure 2B is a graph showing the overall survival rate (Overall survival %) for each group. Figure 3 is a graph showing the results of flow cytometry analysis of the effect of RopegIFN on AML cells. rmIFN-α is recombinant mouse IFN-α. Figure 4 shows the effect of RopegIFN and IFNα administration on enhancing MHC class I expression in leukemia cells and leukemia stem cells (LSCs). Figure 4A is a representative flow cytometry histogram of leukemia cells, and Figure 4B shows the change in the intensity of MHC class I expression on leukemia cells and LSCs from the vehicle administration group. Figure 5 shows the effect of RopegIFN and IFNα administration on enhancing MHC class II expression in leukemia cells and LSCs. Figure 5A is a representative flow cytometry histogram of leukemia cells, and Figure 5B shows the change in the intensity of MHC class II expression on leukemia cells and LSCs from the vehicle administration group. Figure 6 shows the effect of RopegIFN and IFNα administration on enhancing the expression of TRAIL-R2 (a T cell target) on leukemia cells and LSCs. Figure 6A shows a representative flow cytometry histogram of leukemia cells, and Figure 6B shows the change in the intensity of TRAIL-R2 expression on leukemia cells and LSCs from the vehicle administration group. Figure 7 is an experimental scheme for investigating the effect of RopegIFN administration on post-transplant donor T cells.Figure 8 shows that RopegIFN administration suppresses terminal exhaustion of donor T cells after transplantation and increases Transitory exhausted T cells (Transitory Tex). Figure 8A shows increased Transitory Tex in each organ of RopegIFN-administered recipients, and Figure 8B shows increased expression of the cytotoxic molecule granzyme B (GZMB). Figure 9 shows that RopegIFN administration suppresses terminal exhaustion of donor T cells after transplantation, thus promoting T cell proliferation. Figure 9A shows a representative flow cytometry histogram of donor CD8-positive T cells, and Figure 9B shows an increased ratio of the proliferation fraction (Ki67-positive fraction) of donor CD8-positive T cells. Figure 9C shows that RopegIFN administration reduces terminal exhausted cells (Terminal Tex). Figure 10 is an experimental scheme for examining the direct anti-leukemia cell effect of RopegIFN administration. Figure 11 shows that RopegIFN administration selectively reduces LSCs among AML cells. Figure 11A shows a decrease in the number of LSCs, and Figure 11B shows a decrease in the proportion of LSCs among leukemia cells. In the figures, "AML" refers to acute myeloid leukemia, and "LSC" refers to leukemia stem cells. Figure 12 shows, using Annexin V staining, that RopegIFN induces apoptosis in AML cells and LSCs. Figure 12A shows a representative flow cytometry dot plot, and Figure 12B is a graph showing the ratio of apoptosis in AML cells and LSCs. Figure 13 is an experimental scheme for analyzing genetic changes in LSCs after RopegIFN administration by RNA sequencing. Figure 14 shows the results of gene set enrichment analysis (GSEA) in RNA sequencing of apoptosis-related genes for LSCs that were administered and those that were not administered RopegIFN. Figure 15 shows the GSEA results for RNA sequencing of gene groups related to myeloid cell differentiation (A) and gene groups expressed in LSCs (B) for LSCs administered with and without RopegIFN.Figure 16 shows the experimental scheme for performing a limiting dilution test (LDA) to demonstrate that RopegIFN administration reduces the number of LSCs. Figure 17 shows that RopegIFN administration reduces LSC function in AML cells. Figure 17A shows the number of leukemia-grafted mice corresponding to each leukemia inoculation number. Figure 17B shows the number of functional LSCs in leukemia cells based on the results of Figure 17A. "LSC frequency" indicates the LSC frequency. Figure 18 is a graph showing the effect of RopegIFN on GVHD. Figure 18A shows that RopegIFN reduces the pathological score of GVHD in the skin, liver, and small intestine (SI), and Figure 18B shows the effect of Ropeg on extending survival time. In this experiment, leukemia cells were not inoculated to evaluate GVHD.

[0024] <Pharmaceutical Composition> In one embodiment, the present invention relates to a pharmaceutical composition containing lopeg interferon for enhancing the GVL and / or GVT effect in hematopoietic stem cell transplantation therapy. In the present invention, "GVL effect" refers to the effect in allogeneic hematopoietic stem cell transplantation, a treatment method for leukemia, in which immune cells, mainly lymphocytes such as T cells, contained in donor-derived hematopoietic stem cells attack tumor cells remaining in the recipient's (patient's) body, resulting in the cure or control of the original disease, i.e., the graft-versus-leukemia effect. The present invention enhances this GVL effect while minimizing graft-versus-host disease (GVHD), a side effect that is inextricably linked to the GVL effect. The GVT effect similarly refers to the graft-versus-tumor effect.

[0025] In this invention, "hematopoietic stem cell transplantation therapy" refers to therapy using hematopoietic stem cell grafts. Transplant therapy for malignant tumors is a powerful immunotherapy that utilizes donor immune cells, mainly lymphocytes such as T cells, to exert GVT or GVL. "Hematopoietic stem cell grafts" are donor-derived cells that are infused during transplantation, and specifically include umbilical cord blood, bone marrow cells, and peripheral blood stem cells.

[0026] In the present invention, pegylated interferon means the protein-polymer conjugate described in Patent JPB5613050. Specifically, pegylated interferon has the formula: [wherein, R １ , R ２ , R ３ , R ４ , and R ５ are each independently H, C ｌ－５ alkyl, C ２－５ alkenyl, C ２－５ alkynyl, aryl, heteroaryl, C ３－８ cycloalkyl, or C ３－８ heterocycloalkyl, A １ and A ２ are each independently a polymer moiety, G １ , G ２ , and G ３ are each independently a bond or a bonding functional group, G ３ is a bond, P is a protein moiety in which the N-terminal amino group is bonded to G ３ , m is an integer of 0 or 1-10, and n is an integer of 1-10, G １ and G ２ are each of the formula: where O is bonded to A １ or A ２ and NH is bonded to the carbon atom shown in Chemical Formula I].

[0027] Preferably, pegylated interferon is a protein-polymer conjugate in which, in the formula, A １ and A ２ are each an mPEG moiety having a molecular weight of 10-30 kDa. More preferably, pegylated interferon is a protein-polymer conjugate in which, in the formula, P is a modified interferon moiety further containing 1-4 amino acid residues at its N-terminus. Even more preferably, pegylated interferon is a protein-polymer conjugate in which, in the formula, n is 2.

[0028] Even more preferably, pegylated interferon has the formula: As shown, the mPEG has a molecular weight of 20 kD, and the IFN is interferon α-2b. In this invention, "Ropeg interferon α-2b" is a novel long-acting interferon α (IFNα), and may be referred to as RopegIFN in this specification. Ropeg interferon α-2b has a longer half-life compared to conventional pegylated IFNα, and site-selective pegylation technology prevents the production of isomers that cause adverse events (Hsu S, et al. J Formos Med Assoc. 2021; Edahiro Y, et al. Int J Hematol. 2022). Ropeg interferon α-2b is marketed under the trade name "Bethremi®" and its usefulness and safety in polycythemia vera have been confirmed and it is currently on the market.

[0029] The pharmaceutical composition of the present invention is a pharmaceutical composition containing lopeg interferon for enhancing the GVL and / or GVT effect in hematopoietic stem cell transplantation therapy, and includes embodiments in which hematopoietic stem cell grafts and lopeg interferon are used in combination. In the pharmaceutical of the present invention, hematopoietic stem cell grafts and lopeg interferon may be formulated separately and administered simultaneously or separately with a time difference, via the same route or different routes, to the same subject. Alternatively, in the pharmaceutical of the present invention, hematopoietic stem cell grafts and lopeg interferon α-2b may be formulated simultaneously and contained in the same formulation, and administered to the subject. That is, the pharmaceutical of the present invention includes a pharmaceutical in which a pharmaceutical composition containing hematopoietic stem cell grafts and a pharmaceutical composition containing lopeg interferon are formulated separately and used in combination, and a pharmaceutical in which hematopoietic stem cell grafts and lopeg interferon α-2b are contained in a single formulation.

[0030] The pharmaceutical composition of the present invention can be safely administered intravenously, for example, by mixing hematopoietic cell grafts and / or lopeg interferon with a pharmacologically acceptable carrier according to known methods, as a pharmaceutical composition, such as a liquid or injectable preparation. The lopeg interferon injectable preparation can be administered intravenously, intramuscularly, subcutaneously, or intra-organally.

[0031] The present invention relates, in another embodiment, to a pharmaceutical composition containing lopeg interferon for enhancing the GVL and / or GVT effect in hematopoietic stem cell transplantation therapy, and to a pharmaceutical composition for reducing hematopoietic stem cells. In the present invention, "hematopoietic stem cells" include, but are not limited to, leukemia stem cells (LSCs). In the present invention, "reducing leukemia stem cells (LSCs)" means selectively reducing leukemia stem cells among leukemia cells by causing apoptosis or promotion of differentiation of leukemia stem cells. Furthermore, it also includes increasing immunogenicity by promoting the expression of MHC and TRAIL-R2 in leukemia stem cells, making them more susceptible to the GVL effect after allogeneic hematopoietic stem cell transplantation, and thereby reducing LSCs.

[0032] In this embodiment, specifically, hematopoietic cells are cells contained in the graft (umbilical cord blood, bone marrow cells, or peripheral blood). TCD-BM, the graft used in this study and clinical practice, is bone marrow cells from which T cells have been removed. Since it does not exhibit the GVT effect, it is used for benign diseases in clinical transplantation, and in this study, it was used to perform transplantation without causing the GVL effect. When a graft with T cells added to TCD-BM is used, the GVT effect and GVL effect are exhibited.

[0033] This invention relates, in one embodiment, to a pharmaceutical composition for enhancing the GVL and / or GVT effects in hematopoietic stem cell transplantation therapy, characterized by further having a GVHD inhibitory effect. Simultaneously achieving enhancement of the GVT effect and suppression of GVHD is a major goal in transplant therapy. This invention has been found to make this possible.

[0034] As described above, it is known that endogenous interferon suppresses GVHD and interferon administration enhances the GVT effect (Non-Patent Literature 4 and 5), and that lopeg interferon α-2b shows a higher therapeutic effect than interferon against acute lymphoblastic leukemia (ALL) (Non-Patent Literature 6). As shown in Example 6 below, lopeg interferon showed an unexpected effect: while the GVT effect was higher with lopeg interferon than with interferon, the GVHD incidence was lower with lopeg interferon than with interferon.

[0035] This invention relates, in one embodiment, to a pharmaceutical composition for preventing and / or treating leukemia relapse. In this invention, leukemia relapse refers to the proliferation of leukemia cells after transplantation, which are detected in peripheral blood, bone marrow, organs, etc.

[0036] In the present invention, “treatment” in the prevention and / or treatment of leukemia relapse means a method or process aimed at (1) delaying the relapse of leukemia; (2) slowing or stopping the progression, exacerbation, or worsening of the relapse of leukemia or the symptoms of leukemia; (3) bringing about remission of the relapse of leukemia or the symptoms of leukemia; or (4) curing the relapse of leukemia or the leukemia condition. Treatment may be administered as a preventive measure before the onset of the disease or condition, or treatment may be administered after the onset of the disease.

[0037] In this invention, "prevention" means preventing the recurrence or onset of leukemia.

[0038] In this embodiment, the present invention relates to a pharmaceutical product of the present invention, wherein the leukemia is acute myeloid leukemia. Acute myeloid leukemia is a fatal disease in which undifferentiated bone marrow cells proliferate uncontrollably due to genetic abnormalities in hematopoietic cells or progenitor cells, causing impaired normal hematopoiesis and organ damage. In acute myeloid leukemia (AML), it is known that silencing of MHC molecules and increased expression of immunosuppressive molecules (e.g., PD-L1) lead to immune evasion (Christopher MJ: NEJM. 2018, Vago L, et al. Cancer Discov. 2022), and exhaustion of donor T cells lead to AML recurrence after transplantation (Toffalori C: Nat Med. 2019).

[0039] The present invention will be described in detail below with reference to examples and embodiments, but it should be noted that these are merely illustrative and not intended to limit the scope of the present invention.

[0040] Example 1: Investigation of the effect of RopegIFN administration on the graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation. Bone marrow (BM) cells of B6 mice (wild-type mice treated with 5-fluorouracil) or BDF1® mice (acute myeloid leukemia (AML) mice) were infected with a retroviral vector to express the KMT2A::MLLT3 fusion gene and eGFP (enhanced green fluorescent protein). Subsequently, after in vivo passage, eGFP-expressing cells were used as AML cells in the following experiment. The experimental scheme is shown in Figure 1.

[0041] Lethally irradiated (10 Gy) BDF1® mice were transplanted with T-cell-depleted bone marrow (TCD-BM) cells, either with or without purified T cells from an allogeneic B6 donor. TCD-BM cell transplantation is a transplant that does not have a GVL effect. To evaluate the GVL effect, 1 × 10⁶ cells were transplanted on the day of allogeneic hematopoietic stem cell transplantation (allo-HCT). 4Individual AML cells were intravenously administered to recipient mice. After allo-HCT, eGFP+ AML cells in peripheral blood (PB) were quantified weekly. Ropeg interferon α-2b (RopegIFN) was provided by PharmaEssentia. Specifically, the peripheral blood AML cell ratio and overall survival rate (Overall survival %) were examined for the TCD-BM + Vehicle administration group, the TCD-BM + RopegIFN administration group, the TCD-BM + T cell + Vehicle administration group, and the TCD-BM + T cell + RopegIFN administration group.

[0042] The results obtained are shown in Figure 2. Figure 2 shows that, in the group treated with RopegIFN after transplantation with TCD-BM alone (TCD-BM + RopegIFN group), AML cell proliferation was mildly suppressed and survival was slightly extended, but long-term remission was not achieved, indicating no GVL effect. Furthermore, Figure 2 shows that when T cells were transplanted and RopegIFN was administered (TCD-BM + T cells + RopegIFN group), AML cells were completely eliminated and long-term survival was achieved, indicating a GVL effect. This effect was a stronger antitumor effect compared to the GVL effect when T cells were transplanted but RopegIFN was not administered (TCD-BM + T cells + Vehicle group). In other words, it was found that RopegIFN enhances the GVL effect after allogeneic hematopoietic stem cell transplantation.

[0043] Example 1 (2): Exploring the mechanism of the direct anti-AML effect of RopegIFN To explore the mechanism of the direct anti-AML effect of RopegIFN, AML cells were inoculated into allogeneic B6 mice, and then 6 μg of RopegIFN was subcutaneously injected on days 5, 12, and 19. Flow cytometry (FCM) was used to examine the proliferative effects of RopegIFN and recombinant mouse IFN-α (rmIFN-α) on AML cells. The results are shown in Figure 3. RopegIFN, and to a lesser extent rmIFN-α, significantly delayed the proliferation of AML cells in PB.

[0044] Example 2: Investigation of the effect of RopegIFN administration on enhancing the GVL effect It is known that immune evasion in leukemia (such as decreased HLA expression) weakens GVL. Here, to investigate the effect of RopegIFN on the GVL effect, we tested its effect on the expression of MHC and other T cell targets (such as TRAIL-R) in leukemia cells. In detail, lethally irradiated B6 mice were transplanted with bone marrow cells derived from syngeneic B6 donors. AML cells were inoculated at the time of transplantation, and RopegIFN and vehicle were administered. After transplantation, the expression of MHC class I (MHCI) and MHC class II (MHCII) on eGFP-positive cells (AML cells) and leukemia stem cells (LSCs) in the bone marrow was examined.

[0045] The results showed that RopegIFN promotes the expression of MHC and other T cell targets (such as TRAIL-R) on leukemia cells, enhancing the immunogenicity of AML cells and suppressing immune evasion. These effects were not observed with the administration of conventional IFN-α. Specific results are shown in Figures 4-6. Figure 4 shows that RopegIFN enhances the expression of MHC class I. Figure 5 shows that RopegIFN enhances the expression of MHC class II. Figure 6 shows that RopegIFN enhances the expression of TRAIL-R2.

[0046] Example 3: Examination of the effects of RopegIFN administration on post-transplant donor T cells. It is known that GVL is attenuated by T cell exhaustion. Here, to investigate the effect of RopegIFN on the GVL effect, the effect of RopegIFN on terminal exhaustion of T cells after allogeneic hematopoietic stem cell transplantation was tested. Specifically, BDF1® mice were subjected to whole-body irradiation, and then grafts containing purified T cells from allogeneic donor mice (TCD-BM cells) were infused. To examine cytotoxic T cells against recipient alloantigens (allogeneic antigens), 2C TCR transgenic (2CTg) CD8+ T cells that recognize antigens presented on recipient-derived MHC were used, and other T cells were collected from CD45.1-positive B6 mice, mixed, and infused. RopegIFN was administered the day before transplantation and two weeks later. The experimental scheme is shown in Figure 7.

[0047] As a result, RopegIFN suppressed terminal exhaustion of T cells after allogeneic hematopoietic stem cell transplantation and increased the number of donor T cells. Furthermore, RopegIFN induced transient exhausted T cells (transient Tex) with potent antitumor effects. Specific results are shown in Figures 8 and 9. Figure 8 shows that RopegIFN administration increased transient exhausted T cells and increased donor CD8 T cells expressing GZMB, a molecule with tumor-killing effects. Figure 9 shows that RopegIFN administration increased Ki67 expression in donor CD8-positive T cells and promoted donor T cell proliferation. Previous studies have shown that terminal exhaustion of T cells after transplantation weakens the GVL effect and leads to leukemia relapse. These results indicate that RopegIFN suppresses terminal exhaustion and increases transient exhausted T cells that can exert the GVL effect.

[0048] Example 4: Investigation of the direct effects of RopegIFN administration on leukemia cells. It is known that the persistence of leukemia stem cells attenuates GVL. Here, in order to investigate the direct effects of RopegIFN on leukemia cells, we used a syngeneic transplantation model in which the GVL effect is not exerted to test the effects on apoptosis and differentiation induction of leukemia cells and leukemia stem cells (LSCs). Specifically, B6 mice were irradiated, then bone marrow cells and AML cells from syngeneic B6 mice were infused, and RopegIFN was administered after transplantation. AML cells and LSCs in the bone marrow were analyzed 3 weeks after transplantation. The experimental scheme is shown in Figure 10.

[0049] The results obtained are shown in Figures 11 and 12. Figure 11A shows that RopegIFN reduces the number of leukemia stem cells (LSCs) in the bone marrow. Figure 11B shows a decrease in the proportion of LSCs in AML cells, indicating that RopegIFN reduces AML cells in general, but particularly selectively reduces LSCs. Figure 12 shows that RopegIFN enhances apoptosis in LSCs. These results indicate that RopegIFN reduces leukemia stem cells by inducing apoptosis.

[0050] Example 4(2): Comprehensive analysis of LSC transcriptional changes after RopegIFN administration. LSCs were purified after RopegIFN administration, RNA sequencing was performed, and the transcriptional state of each gene was comprehensively examined. The experimental scheme is shown in Figure 13. In detail, B6 mice were irradiated, then bone marrow cells and AML cells from syngeneic B6 mice were infused, and RopegIFN was administered after transplantation. LSCs in the bone marrow three weeks after transplantation were purified and RNA sequencing was performed.

[0051] The results obtained are shown in Figures 14 and 15. Figure 14 shows the results of gene set enrichment analysis (GSEA) performed on RNA sequencing. It was shown that the expression of apoptosis-related genes was upregulated in LSCs of mice administered with RopegIFN. Figure 15A shows the results of GSEA on RNA sequencing, indicating that the expression of genes expressed by myeloid (myeloid) cells, which differentiate from leukemia cells, was increased in LSCs of mice administered with RopegIFN. On the other hand, Figure 15B shows that the expression of genes that were highly expressed in LSCs was decreased. From these results, it was found that RopegIFN induces apoptosis in LSCs while promoting differentiation into myeloid cells.

[0052] Example 5: Examination of the effect of RopegIFN administration on LSC function. Although it was shown that the number of LSCs decreased with RopegIFN administration, limiting dilution assays (LDA) were performed to prove that LSC function also deteriorates (Li Y, et al. Blood. 2022, Li Y, et al. Cell stem cell. 2023, Zhang, et al. Cell Death Dis. 2023). The experimental scheme is shown in Figure 16. RopegIFN or vehicle was administered to B6 mice with AML, and then AML cells were purified from the bone marrow of the mice. These purified AML cells were then infused into irradiated B6 mice mixed with bone marrow cells taken from healthy B6 mice. Three different numbers of AML cells were used for infusion: 20, 100, and 500.

[0053] The results obtained are shown in Figure 17. Figure 17 shows that RopegIFN administration significantly reduces LSC function per AML cell. Specifically, treatment with RopegIFN reduced the estimated LSC frequency from 1 per 149 leukemia cells (CI: 67.3-331) to 1 per 501 cells (CI: 205.4-1225). This demonstrates that RopegIFN administration reduces the number of functional LSCs.

[0054] Example 6: Effect of RopegIFN on GVHD The results obtained are shown in Figure 18. Figure 18 shows that RopegIFN significantly improved pathological acute GVHD in the liver and intestines and extended survival time after allogeneic hematopoietic stem cell transplantation. In recipients of T cell-depleted bone marrow (TCD-BM) cells alone, RopegIFN only slightly slowed the progression of AML and extended survival time.

Claims

A pharmaceutical composition containing lopeg interferon for enhancing the effects of GVL and / or GVT in hematopoietic stem cell transplantation therapy. 　 The pharmaceutical composition according to claim 1, characterized by reducing hematopoietic stem cells. 　 The pharmaceutical composition according to claim 1, further characterized by having a GVHD inhibitory effect. 　 A pharmaceutical composition according to claim 1 for preventing and / or treating the recurrence of leukemia. 　 The pharmaceutical composition according to claim 4, wherein the leukemia is acute myeloid leukemia. 　 The pharmaceutical composition according to claim 1, wherein the hematopoietic cells are bone marrow. 　 The pharmaceutical composition according to claim 1, wherein the hematopoietic cells are T cells. 　 The pharmaceutical composition according to claim 1, further characterized by containing hematopoietic stem cell grafts. 　 The pharmaceutical composition according to claim 1 or 8, wherein lopeg interferon is lopeg interferon α-2b.

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