GM-CSF or GM-CSF receptor ligands for the treatment of hematologic malignancies in patients who have undergone allo-HCT

Abstract

Described herein is the use of non-agonist ligands, in particular antibodies, that specifically bind to GM-CSF or to one of the GM-CSF receptors consisting of CD116, CD131 and CD131, for treating leukemia in patients who have received allo-HCT or for treating other complications arising from hematopoietic cell transplantation from immunologically different donors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to European Patent Application Nos. EP18158169.5 (filed on February 22, 2018); EP18189562.4 (filed on August 17, 2018); EP18194549.4 (filed on September 14, 2018) and US16 / 269,572. The contents of the aforementioned patent applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to non-agonist ligands, in particular antibodies, that specifically bind to GM-CSF (CSF-2) or its receptor complex consisting of CSF2Rβ (CD131) and CSF2Rα (CD116) for use in the treatment of haematologic malignancies in patients who have received allo-HCT. Background Art

[0004] Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative and life-saving intervention for patients with hematologic malignancies; however, 40% to 60% of all patients develop clinically significant acute or chronic graft-versus-host disease (GvHD), with an aggregate mortality rate of approximately 50%. Donor-derived T cells mediate both processes: alloreactive T cells from the graft attack the host's malignant cells, generating a beneficial graft-versus-leukemia (GvL) effect; whereas this alloreactivity can be directed against healthy tissues (typically the skin, gut, and liver), resulting in GvHD. Although removal of T cells from donor material prior to allo-HCT can prevent / mitigate GvHD, this is at the expense of reduced GvL activity and increased relapse rates. Therefore, there is an urgent need to understand how to disentangle the mechanisms of GvL and GvHD at the T cell level and modulate them to achieve clinical benefit.

[0005] Extensive efforts have been made to define the key T cell subsets and cytokines that support GvHD in murine models, but to date, no consensus has been reached. H 1-mediated pathology, but studies in mice have shown that lack of T H 1 Donor T cells expressing the cytokine IFNγ can exacerbate the disease. H2 cells can both suppress experimental GvHD and induce GvHD affecting the liver and skin. Similarly, despite evidence that T cells that normally produce IL-17A are major mediators of tissue damage in inflammatory diseases, their role in GvHD remains controversial because in murine models, IL-17 appears to promote or ameliorate GvHD, depending on the experimental conditions. H Cells are clearly implicated in the development and perpetuation of GvHD, but so far it has not been possible to identify any specific soluble mediators with reproducible and non-redundant functions in the pathogenesis of the disease in both murine models and humans.

[0006] Based on the above prior art, the object of the present invention is to provide means and methods for reducing GvHD while maintaining the beneficial GvL effect. This object is achieved by the subject matter of the independent claims of the present application. Summary of the invention

[0007] A first aspect of the present invention relates to a method of treating a patient suffering from graft-versus-host disease, or a method of preventing, inhibiting the occurrence of graft-versus-host disease, or reducing the severity of the occurrence of graft-versus-host disease in a patient receiving an allogeneic transplant.

[0008] A second aspect of the invention relates to a method of treating a patient suffering from cancer, particularly a hematological malignancy, who has received an allogeneic hematopoietic stem cell transplant (allo-HCT).

[0009] The method according to any aspect of the invention comprises interfering with the signal transduction of the immune cytokine GM-CSF triggering GvHD. This can be achieved by inhibiting GM-CSF with a ligand, in particular an antibody, more particularly a neutralizing antibody. The object of the invention can also be achieved by interfering with the binding of GM-CSF to its natural receptor, or by inhibiting the signal cascade by administering a non-agonist ligand to these receptors, in particular by administering a neutralizing antibody to the GM-CSF receptor. Another possible mechanism is the temporary or long-term inhibition of the expression of GM-CSF or one of its receptors by nucleic acid interference (RNAi, antisense).

[0010] In a particular embodiment, the invention comprises administering to the patient a non-agonist ligand, particularly an antibody, more particularly a neutralizing non-agonist antibody reactive to GM-CSF or to one of the GM-CSF receptors consisting of CD116, CD131 and CD131.

[0011] Alternatively, this aspect may be formulated as providing a reagent selected from:

[0012] - a neutralizing polypeptide ligand, in particular a neutralizing antibody reactive with one of GM-CSF, CD116, CD131 or a GM-CSF receptor consisting of CD116 and CD131, and

[0013] - a nucleic acid agent that inhibits the expression of GM-CSF, CD116 and / or CD131,

[0014] For use in the treatment or prevention of GvHD in patients with hematological malignancies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A-1H Donor T cells secrete GM-CSF and IFNγ during allogeneic responses

[0017] Figure 1A :CD45.1 alone + WT C57BL / 6TCD-BM or with CD45.1 + WT C57BL / 6 splenocytes combined with lethally irradiated CD45.2 + Survival rate of BALB / c mice. Data from 4 independent experiments were collected, n=5 / group in each experiment. For comparison of survival curves, Lox-rank (Mantel-Cox) test was used, ***p<0.001. Figure 1B : CD45.1 in live singlets in spleen, liver, and skin at 3 and 6 days after allo-HCT + The frequency of cells. Data were collected, n = 10 / group. Figure 1C :3 days after allo-HCT, CD45.1 in mouse liver + CD4+ cells producing IFNγ, GM-CSF and IL-17A in the population + and CD8 + Frequency of T cells. Representative graphs are shown from 3 independent experiments. Figure 1D : CD45.1 in liver, spleen and skin at 3 and 6 days after allo-HCT + CD4+ cells producing IFNγ, GM-CSF, and IL-17A + and CD8 + Frequency of T cells. Data from 3 independent experiments were pooled, totaling n=10 / group. Figure 1E : Serum IFNγ and GM-CSF levels in mice 6 days after allo-HCT. Data from 3 independent experiments were collected, n=2-5 / group. For mean comparison, unpaired two-tailed t-test with Welch correction was used, *p<0.05, **p<0.01. Figure 1F: T cells from WT C57BL / 6 mice and syngeneic (C57BL / 6) or allogeneic (BALB / c) spleen CD11c + IFNγ, GM-CSF and IL-17A in the supernatant of DC co-culture. Data from 3 independent experiments were collected, n=3-5 / group. For mean comparison, unpaired two-tailed t-test with Welch correction was used, *p<0.05, **p<0.01, ***p<0.001. Figure 1G : Syngeneic (C57BL / 6) or allogeneic (BALB / c) spleen CD11c + DC co-cultured C57BL / 6wt, Ifng - / - , Csf2 - / - or Il17a - / - Tritiated thymidine incorporation in mouse T cells. Data from 3 independent experiments, n = 2-5 / group. For mean comparisons, one-way ANOVA with Bonferroni post hoc test was used. Data are in Figure 1B Shown are means + / - SD, Figure 1D-1G Shown are mean values ​​+ / - SEM. Figure 1H : CD45.1 in liver and spleen at 3 and 6 days after allo-HCT + CD4+ cells producing IFNγ and GM-CSF in the population + and CD8 + Frequency of T cells. Representative data collected from 1 of 3 experiments are shown, n=3-4 / group. Abbreviations: TCD: T cell depletion, BM: bone marrow, HCT: hematopoietic cell transplantation, WT: wild type, Cpm: counts per minute.

[0018] Figures 2A-2K GM-CSF is critical for acute GvHD after fully MHC-mismatched allo-HCT

[0019] Figure 2A : WT C57BL / 6TCD-BM alone or with C57BL / 6WT Csf2 - / - Ifng - / - or Il17a - / - Survival rate of lethally irradiated BALB / c mice after allo-HCT with spleen cells from mice. Data from 4 independent experiments were collected, n=5 / group in each experiment. For comparison of survival curves (WT vs. other groups), Lox-rank (Mantel-Cox) test was used, *p<0.05, **p<0.01, ***p<0.001. Figure 2B: WT C57BL / 6TCD-BM alone or with C57BL / 6WT or Csf2 - / - Survival of lethally irradiated BALB / c mice after allo-HCT with T cells purified from mouse spleen. Data were collected from 3 independent experiments, n=5 / group in each experiment. - / - ), using Lox-rank (Mantel-Cox) test, ***p<0.001. Figure 2C : Comprehensive histopathological scores of liver, small intestine and skin of BALB / c mice 6 days after allo-HCT. Data were collected from 3 independent experiments, n=5 / group. For mean comparison (WT vs. Csf2 - / - ), using unpaired two-tailed t-test, *p<0.05. Figure 2D : WT C57BL / 6TCD-BM combined with WT or Csf2 - / - Representative images of hematoxylin and eosin-stained sections of mouse skin and small intestine 6 days after allo-HCT of splenocytes from C57BL / 6 mice, n=4-5 / group (scale bar: 100 μm). Figure 2E-2F : WT C57BL / 6TCD-BM was combined with WT or Csf2 - / - Six days after allo-HCT of splenocytes from C57BL / 6 mice, mice ( Figure 2E ) liver and ( Figure 2F ) Representative images of p22phox labeling in small intestine (scale bar: 100 μm), n=4-5 / group (upper panel). Quantification of the average % of the total area positively labeled with p22phox in each field of view. For mean comparison (WT vs. Csf2 - / - ), using unpaired two-tailed t-test, *p<0.01. Figure 2G : Survival of lethally irradiated BALB / c mice after allo-HCT with WT C57BL / 6TCD-BM alone or in combination with spleen cells from C57BL / 6WT mice. During the experiment, mice were treated with PBS, isotype control antibody or anti-GM-CSF antibody 3 times a week starting 2 days before HCT. Data from 2 independent experiments were collected, n=5 / group per experiment. For survival curve comparison (aGM-CSF vs. isotype), Lox-rank (Mantel-Cox) test was used, ***p<0.001. Data are shown as mean + / - SEM. Figure 2H : Colon length (in cm) of BALB / c mice 6 days after allo-HCT, as described in a. Data from 2 independent experiments were pooled. Fig.2I: Representative images and quantification of apoptotic cells (TUNEL staining) in the colon of BALB / c mice 6 days after allo-HCT, as described in a. Data from 3 independent experiments were pooled (scale bar top 50 μm, bottom 20 μm). Figure 2J : Levels of alkaline phosphatase (AP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and albumin in the serum of BALB / c mice 6 days after allo-HCT. Data are representative of 2 independent experiments, n=3-5 / group. Figure 2K : WT C57BL / 6TCDBM alone or with C57BL / 6WT or Csf2 - / - Mice (CD4WTCD8WT, CD4WTCD8Csf2 - / - 、CD4Csf2 - / - CD8WT and CD4Csf2 - / - CD8Csf2 - / - ) Survival of lethally irradiated BALB / c mice after allo-HCT with a combination of spleen-purified CD4 and CD8 T cells. Mice treated with TCD-BM alone were used as controls. Data represent 2 independent experiments, n=5 / group. For comparison of survival curves (CD4WTCD8WT vs. other groups), Log-rank (Mantel-Cox) test was used, ns (not significant), **p<0.01. Abbreviations: TCD: T cell depletion, BM: bone marrow, HCT: hematopoietic cell transplantation, WT: wild type.

[0020] Figures 3A-3H GM-CSF mediates GvHD pathology after partially MHC-mismatched allo-HCT

[0021] Figure 3A : WT C57BL / 6TCD-BM alone or with C57BL / 6WT, Csf2 - / - Ifng - / - Survival of lethally irradiated B6D2F1 mice after partial MHC-mismatched allo-HCT with splenocytes from mice. Data from 5 independent experiments were collected, n=5 / group per experiment. For comparison of survival curves (WT vs. other groups), Lox-rank (Mantel-Cox) test was used, *p<0.05, ***p<0.001. Figure 3B : WT C57BL / 6TCD-BM alone or with C57BL / 6WT, Csf2 - / - Ifng - / -Survival of lethally irradiated B6D2F1 mice after allo-HCT with T cells purified from mouse spleen. Data from 2 independent experiments, n=5 / group in each experiment. For comparison of survival curves (WT vs. other groups), Lox-rank (Mantel-Cox) test was used, *p<0.05, ***p<0.001. Figure 3C : Comprehensive histopathological scoring of liver, small intestine, and skin sections of B6D2F1 mice 11 days after allo-HCT. Data from 2 independent experiments were collected, n = 5 / group. For mean comparison (WT, Csf2 - / - Ifng - / - ), One-way ANOVA with Bonferroni post-test, *p<0.05, ***p<0.001. Figure 3D :WT C57BL / 6TCD-BM and WT, Ifng - / - or Csf2 - / - Representative images of hematoxylin and eosin-stained sections of mouse skin 11 days after allo-HCT with splenocytes from C57BL / 6 mice, n=4-5 / group (scale bar: 100 μm). Figure 3E-3F : WT C57BL / 6TCD-BM was used in combination with cells from C57BL / 6WT or Csf2 - / - Nine days after allo-HCT, mice ( Figure 3E ) liver and ( Figure 3F ) Representative images of p22phox (upper panel) and F4 / 80 labeling in small intestine (scale bar: 100 μm), n=4-5 / group. Quantification of the % of the total area positively labeled with p22phox or F4 / 80 in each field of view. For mean comparison (WT vs. Csf2 - / - ), unpaired two-tailed t-test, ***p<0.001, *p<0.01. Figure 3G : WT C57BL / 6TCD-BM and cells from C57BL / 6WT, Ifng - / - or Csf2 - / - Serum IFNγ and GM-CSF levels in mice 9 days after allo-HCT with spleen cells from mice. Data from 2-3 independent experiments were collected, n=4-5 / group. For mean comparison (WT vs. Ifng - / - or WT and Csf2 - / - ), unpaired two-tailed t-test with Welch correction, *p<0.05, **p<0.01. Data are shown as mean + / - SEM. Figure 3H: Comprehensive histopathological scoring of liver sections of B6D2F1 mice 9 or 28 days after allo-HCT, as described in a. Data are representative of 2 independent experiments, n=4-6 / group. Abbreviations: TCD: T cell depletion, BM: bone marrow, HCT: hematopoietic cell transplantation, WT: wild type.

[0022] Figures 4A-4H GM-CSF is not essential for antitumor activity after allo-HCT

[0023] Figures 4A-4D : In the presence of WT C57BL / 6TCD-BM alone or in combination with C57BL / 6WT or Csf2 - / - T cells purified from mouse spleens were combined with MHC-mismatched allo-HCT. Lethally irradiated BALB / c mice were injected intravenously with A20 tumor cells expressing GFP and luciferase. Mice treated with TCD-BM alone were used as controls. Figure 4A )Tumor growth was monitored by in vivo bioluminescence imaging. Images from one representative experiment of two are shown. ( Figure 4B ) monitors signal intensity in a region of interest (ROI) over time. In each treatment group with lethal GvHD (grey), tumors (black), or survivors (white), ( Figure 4C ) survival rate over time and ( Figure 4D ) Causes of death are expressed as percentages of mice. Data from 2 independent experiments were collected, n = 5 / group. For comparison of survival curves (WT vs. other groups), Log-rank (Mantel-Cox) test was used, ns (not significant), **p < 0.01. ( Figure 4E-4H ) Lethally irradiated BALB / c mice were injected intravenously with A20 tumor cells expressing GFP and luciferase concurrently with MHC-mismatched allo-HCT with WT C57BL / 6TCD-BM alone or in combination with T cells purified from the spleen of C57BL / 6WT mice. Mice treated with TCD-BM alone served as controls. During the experiment, mice were treated with isotype control or anti-GM-CSF antibody 3 times per week starting 2 days before HCT. Figure 4E ) Tumor growth was monitored by in vivo bioluminescence imaging. Images from one experiment with n=6-7 / group are shown. ( Figure 4F ) Signal intensity in the region of interest (ROI) was monitored over time. In each treatment group with lethal GvHD (grey), tumors (black), or survivors (white), ( Figure 4G ) survival rate over time and ( Figure 4H ) Causes of death are expressed as percentage of mice. For survival curve comparison (WT vs. csf2- / - ), using Lox-rank (Mantel-Cox) test, *p<0.05. Data are shown as mean + / - SEM. Abbreviations: TCD: T cell depletion, BM: bone marrow, HCT: hematopoietic cell transplantation, WT: wild type, BLI: bioluminescent imaging, ROI: region of interest.

[0024] Figures 5A-5D Patients with severe GvHD express high levels of GM-CSF in intestinal biopsies

[0025] Figure 5A : Relative expression of GM-CSF at the mRNA level in gastrointestinal biopsies of patients with different GvHD grades, see Table 3. For comparison of means, one-way ANOVA with Bonferroni post hoc test was used, **p<0.01. Data are shown as mean + / - SEM. Figure 5B : Images of GM-CSF labeling in control and GvHD stage IV biopsies of small intestine from allo-HCT patients, see Tables 1 and 2. Brown: anti-human GM-CSF, blue: hematoxylin. Scale bar 100 μm (20 μm in magnified images). Representative images of 3 independent control and patient samples are shown. Figure 5C : Immunofluorescence staining of CD3 (pink), CD68 (green) and GM-CSF (red) from gastrointestinal biopsies of patients with grade IV GvHD. Representative images of 3 individual patient samples are shown. Cell nuclei are depicted in blue (DAPI) (scale bar top 50 μm, bottom 20 μm). Figure 5D Figure : Flow cytometric analysis of PBMCs collected from healthy donors (HD) and GvHD patients (GP) (stimulated with PMA / ionomycin for 4 hours). Representative FACS plots of cytokine-expressing T cells are shown. CD4+ T cells producing GM-CSF, IFN, and IL-17 + and CD8 + T cells are shown as individual frequencies. n=10-9 / group, *P<0.05, **P<0.01. Abbreviations: TCD: T cell depletion, BM: bone marrow, HCT: hematopoietic cell transplantation.

[0026] Figures 6A-6D Comparative phenotypic analysis of T cell populations in WT and Csf2- / - mice

[0027] In the unimmunized Spleen in C57BL / 6WT and Csf2- / - mice ( Fig. 6A )T cell population frequency and ( Figure 6B ) Flow cytometric analysis of activation marker expression frequency. Data from 2 independent experiments were collected, with n=3 / group in each experiment. Figure 6C : Flow cytometric analysis of donor T cell infiltration in recipient lymph nodes (LN), spleen, liver, and skin 2 and 5 days after allo-HCT of lethally irradiated BALB / c mice reconstituted with WT C57BL / 6TCD-BM in combination with spleen cells from C57BL / 6WT or Csf2- / - mice. Cells were gated on H2-Db and CD45; CD4 and CD8 T cells were pre-gated on CD3. One experiment of n=4 / group is shown. Data are shown as mean + / - SEM. Fig.6D : Flow cytometric analysis of donor T cell infiltration in recipient LNH and spleen 5 days after allo-HCT of lethally irradiated BALB / c mice reconstituted with WT C57BL / 6TCD-BM in combination with splenocytes from C57BL / 6WT or Csf2- / - mice. Cells were gated on H2-Db and CD45; CD4 and CD8 T cells. One experiment of n=4 / group is shown. Data are shown as mean + / - SEM. Abbreviations: TCD: T cell depletion, BM: bone marrow, HCT: hematopoietic cell transplantation, WT: wild type.

[0028] Fig. 7A and 7B Killing assays were performed using A20 tumor cells and T cells isolated from the spleen and lymph nodes of C57BL / 6WT or Csf2- / - mice, respectively. Fig. 7A : Representative images of flow cytometric analysis of dead cells stained by TO-PRO3 are shown. Figure 7B : Specific lysis of A20 tumor cells by the indicated T cells at target:effector ratios of 1:1, 1:20, and 1:50. Data are representative of two experiments, n=3 mice / group each. Data are shown as mean + / - SEM. Abbreviations: WT: wild type.

[0029] Fig. 8A and 8B Fig. 8A : Survival of lethally irradiated C57BL / 6WT or Csf2rb- / - mice after allo-HCT with WT Balb / c T cell depleted (TCD)-BM alone or in combination with spleen cells from Balb / c WT mice. Data from 2 independent experiments, n=5-7 / group per experiment. Figure 8B : Survival of lethally irradiated Balb / c WT mice after allo-HCT with C57BL / 6WT or Csf2rb- / -TCD-BM alone or in combination with splenocytes from C57BL / 6WT mice. Data from 2 independent experiments, n=5-7 / group in each experiment.

[0030] Figures 9A-9HGM-CSF drives GvHD via donor-derived bone marrow cells

[0031] Fig. 9A : Survival of lethally irradiated WT C57BL / 6 and Csf2rb- / - mice after MHC-mismatched allo-HCT with WT Balb / c TCD-BM alone or in combination with spleen cells from Balb / c WT mice. Data from 2 independent experiments, n=5 / group per experiment. Fig. 9B : Survival of lethally irradiated Balb / c WT mice after MHC-mismatched allo-HCT with C57BL / 6WT alone or with Csf2rb- / - mouse TCD-BM or in combination with splenocytes from C57BL / 6WT mice. Data from 2 independent experiments, n=5 / group per experiment. For comparison of survival curves (WT vs. Csf2rb- / - recipients / donors), Log-rank (Mantel-Cox) test was used in a and b, **p<0.01. Fig. 9C : Annotated t-SNE plots showing 200,000 randomly sampled cells from the bone marrow of WT C57BL / 6 and Csf2rb- / - mice showing STAT5 phosphorylation (black to yellow gradient) after GM-CSF stimulation, analyzed by flow cytometry analysis. Data are representative of two independent experiments, n=3 / group. Fig.9D : The frequency of GM-CSF-induced pSTAT5 upregulation in monocytes and neutrophils from WT C57BL / 6 and Csf2rb- / - mice, as Fig. 9B Overlap shown. Fig.9E : Flow cytometric analysis of different myeloid cell populations (DCs, neutrophils, monocytes, and MDCs) after HCT. Example gating of the liver is shown. Fig.9F : Myeloid cell populations (from Fig.9E ) Quantification. Shown is a representative from 3 independent experiments, n = 4-5 / group. (g) Frequency of pro-IL-1β-producing neutrophils and monocytes within the H2Db+, CD45+ population of mouse spleens 6 days after allo-HCT. Shown are representative graphs of neutrophils, monocytes, and MDCs (gated as in e). Data are representative of two independent experiments, n = 5 / group. Figure 9H Figure : Flow cytometric analysis of ROS (CellROX reagent) in mice 6 days after allo-HCT. Representative histograms of MFI of neutrophils, monocytes, and MDCs (Figure 2A). Fig.9E). Data represent one experiment, n=5-7 / group. For mean comparison, unpaired two-tailed t-test was used in fh, ***p<0.001, **p<0.01, *p<0.05. Data are shown as mean + / - SEM. Abbreviations: TCD: T cell depletion, BM: bone marrow, HCT: hematopoietic cell transplantation, WT: wild type.

[0032] Brief description of the sequence

[0033] The nucleotide sequences provided herein are represented by the standard letter abbreviations of the nucleotide bases defined in 37 CFR 1.822. Each nucleotide sequence only shows one chain, but it should be understood that any reference to the displayed chain includes the complementary chain. The sequence table is submitted as an ASCII text file (about 2KB) named "Seq protocol uz349wo_ST25.txt", which is incorporated herein by reference.

[0034] SEQ ID No: 1 and 2: Forward and reverse PCR amplification primers for CSF2 gene.

[0035] SEQ ID No: 3 and 4: Forward and reverse PCR amplification primers for GAPDH gene. DETAILED DESCRIPTION

[0036] Terms and Definitions

[0037] In the context of this specification, the term GvHD relates to graft versus host disease, a complication arising from the transplantation of immune cells into a genetically different patient. GvHD is commonly associated with stem cell transplantation, especially in the context of hematological malignancies, but may arise in the context of other transplants.

[0038] In the context of this specification, the term allo-HCT relates to allogeneic hematopoietic cell transplantation.

[0039] In the context of the present specification, the term GM-CSF relates to granulocyte-macrophage colony stimulating factor (Uniprot P04141; CAS number 83869-56-1).

[0040] In the context of the present specification, the term CD116 relates to cluster of differentiation 116, also known as the α chain of the GM-CSF receptor (Uniprot P15509). The GM-CSF receptor consists of a GM-CSF-specific α chain (CD116) and a β chain (CD131, which is also present in the IL-3R and IL-5R interleukin receptors).

[0041] The ligand of GM-CSF or one of CD116, CD131 and GM-CSF receptor consisting of CD116 and CD131 according to the present invention can eliminate or neutralize the signal transduction initiated when GM-CSF binds to its receptor.

[0042] In the context of the present specification, the term CLL relates to acute chronic lymphocytic or lymphoblastic leukemia.

[0043] In the context of the present specification, the term ALL relates to acute lymphoblastic leukemia.

[0044] In the context of the present specification, the term CML relates to chronic myelogenous or myeloid leukemia.

[0045] In the context of the present specification, the term AML relates to acute myeloid leukemia.

[0046] In the context of the present specification, the term AMoL relates to acute monocytic leukemia.

[0047] In the context of this specification, the term "antibody" is used with its known meaning in the fields of cell biology and immunology; it refers to complete antibodies, including but not limited to immunoglobulin G type (IgG), A type (IgA), D type (IgD), E type (IgE) or M type (IgM), any antigen binding fragment or single chain thereof, and related or derived constructs. A complete antibody is a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains C H 1. C H 2 and C H Each light chain consists of a light chain variable region (referred to herein as V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses so-called nanobodies or single-domain antibodies, antibody fragments consisting of a single monomeric variable antibody domain.

[0048] The term antibody includes camelid antibodies, particularly humanized camelid antibodies.

[0049] In the context of this specification, the term antibody-like molecule refers to a molecule that can specifically bind to another molecule or target with high affinity / Kd≤10E-8mol / l. The binding of an antibody-like molecule to its target is similar to the specific binding of an antibody. The term antibody-like molecule encompasses repeat proteins, such as the designed ankyrin repeat protein (Molecular Partners, Zürich), an engineered antibody mimetic protein, showing highly specific and high-affinity target protein binding (see US2012142611, US2016250341, US2016075767 and US2015368302, all of which are incorporated herein by reference). The term antibody-like molecule further encompasses, but is not limited to, polypeptides derived from armadillo repeat proteins, polypeptides derived from leucine-rich repeat proteins, and polypeptides derived from triangular tetrapeptide repeat proteins (tetratricopeptide repeat proteins).

[0050] The term antibody-like molecule also encompasses polypeptides derived from protein A domains, polypeptides derived from the fibronectin domain FN3, polypeptides derived from a common fibronectin domain, polypeptides derived from lipocalin, polypeptides derived from zinc fingers, polypeptides derived from Src homology domain 2 (SH2), polypeptides derived from Src homology domain 3 (SH3), polypeptides derived from PDZ domains, polypeptides derived from γ-crystallin, polypeptides derived from ubiquitin, polypeptides derived from cysteine ​​knot polypeptides and polypeptides derived from knottin, polypeptides derived from cystatin, polypeptides derived from Sac7d, triple helix coiled coil (also called alphabody), polypeptides derived from the Kunitz domain of a Kunitz-type protease inhibitor and polypeptides derived from the carbohydrate binding module 32-2.

[0051] The term protein A domain-derived polypeptide refers to a molecule that is a derivative of protein A and is capable of specifically binding to the Fc region and the Fab region of an immunoglobulin.

[0052] The term armadillo repeat protein refers to a polypeptide comprising at least one armadillo repeat, wherein the armadillo repeat is characterized by a pair of alpha helices forming a hairpin structure.

[0053] In the context of this specification, the term "humanized antibody" is used with its known meaning in the fields of cell biology and biochemistry; it refers to an antibody originally produced by immune cells of a non-human species, whose protein sequence has been modified to increase its similarity to antibody variants naturally produced by humans.

[0054] In the context of this specification, the term humanized camelid antibody refers to an antibody consisting only of a heavy chain or a heavy chain variable domain (VHH domain), and its amino acid sequence has been modified to increase its similarity to antibodies naturally produced by humans, so that when administered to humans, it shows reduced immunogenicity. The general strategy for humanizing camelid antibodies is shown in Vincke et al. "General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold", J Biol Chem. 2009 Jan 30; 284 (5): 3273-3284 and US2011165621A1.

[0055] In the context of this specification, the term chimeric antibody is used with its known meaning in the field of cell biology and immunology; it refers to an antibody molecule in which the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or changed class, effector function and / or type or to a completely different molecule (e.g., enzyme, cytokine, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody. For example, an antibody can be modified by replacing its constant region with a cytokine. Due to the replacement with a cytokine, a chimeric antibody can retain its specificity for recognizing an antigen while also having the function or a portion thereof of the original cytokine molecule.

[0056] In the context of this specification, the term dissociation constant (K D ) is used in its known sense in the fields of chemistry and physics; it refers to an equilibrium constant that measures the tendency of a larger object to reversibly dissociate into smaller components (when a complex breaks down into its component molecules). K D It is expressed in molar units [M] and corresponds to the concentration of [Ab] when the [Ag] binding sites are half occupied. In other words, the concentration of unbound [Ab] is equal to the concentration of [AbAg] complex. The dissociation constant can be calculated according to the following formula:

[0057]

[0058] [Ab]: antibody concentration; [Ag]: antigen concentration; [AbAg]: antibody-antigen complex concentration

[0059] In the context of this specification, the terms off-rate (Koff; [1 / sec]) and on-rate (Kon; [1 / sec*M]) are used with their known meanings in the chemical and physical arts; they refer to rate constants measuring the dissociation (Koff) or association (Kon) of an antibody with its target antigen. Koff and Kon can be determined experimentally using methods well known in the art. One method for determining the Koff and Kon of an antibody uses surface plasmon resonance. This is a method used in biosensor systems (such as or They can also be used to determine the dissociation constant KD by the following formula:

[0060]

[0061] As used herein, in one embodiment, the terms "treating" or "treatment" of any disease or condition (e.g., cancer or graft-versus-host disease) refer to ameliorating the disease or condition (e.g., slowing or preventing or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to regulating a disease or condition physically (e.g., stabilization of discernible symptoms), or physiologically (e.g., stabilization of a physical parameter), or both. Unless specifically described below, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0062] In the context of this specification, the term aptamer refers to an oligonucleotide or peptide that can specifically bind to another molecule or target with a high affinity of Kd≤10E-8mol / l. The binding of an aptamer to its target is similar to the specific binding of an antibody. The term aptamer covers RNA or DNA molecules, nucleic acid analogs or peptide molecules. Aptamers can also be coupled to self-cleaving RNA molecules, so-called ribozymes. Methods for obtaining aptamers from scratch are known; these methods include the so-called "selex" method and other methods based on evolving specific binders from random selection.

[0063] Using murine models of fully and partially MHC-mismatched HCT, the inventors demonstrated that GM-CSF (Csf-2), a cytokine that plays an emerging role in a variety of inflammatory diseases, is produced in large quantities by donor T cells early after transplantation. When donor T cells lack GM-CSF, GvHD is significantly improved. Importantly and surprisingly, the lack of GM-CSF does not affect the ability of donor T cells to control tumor growth in mice (GvL effect), and this control can be achieved without the appearance of GvHD even in the case of complete MHC mismatch. The inventors also found high levels of GM-CSF in gastrointestinal biopsies from GvHD patients, which is consistent with parallel effects in human symptoms. Therefore, the inventors propose GM-CSF as a new therapeutic target to attenuate GvHD while maintaining GvL in patients receiving allo-HCT.

[0064] Although the use of anti-GMCSF or anti-GMCSFR antibodies has been proposed in the context of GvHD, the data presented here demonstrate for the first time that GvHD can be treated or suppressed without affecting the therapeutic benefit of allogeneic transplantation, and that there is a clinical rationale for the use of the ligands of the invention, which allow the body to elicit a graft-versus-leukemia response while suppressing GvHD (phagocyte-mediated). This will permit the inclusion of patients (who are not currently scheduled for allo-HCT due to risk of GvHD) for allo-HCT, and may further permit limiting the use of immunosuppressants after allo-HCT.

[0065] The first aspect of the present invention relates to a non-agonist ligand that specifically binds to GM-CSF or to one of CD116, CD131, and a GM-CSF receptor consisting of CD116 and CD131, for use in treating leukemia in patients who have received allogeneic hematopoietic stem cell transplantation (allo-HCT). In other words, the ligand is used for the treatment of leukemia after allogeneic hematopoietic stem cell transplantation.

[0066] The non-agonist ligands of the present invention eliminate the biological signal that GM-CSF exerts on its receptor, thereby leading to the downstream effects of GM-CSF interactions. Therefore, it will be understood that the methods described herein can be used to inhibit or reduce the severity of GvHD in the context of any allogeneic transplantation in which GvHD can occur, but will not significantly reduce the beneficial effects of allogeneic transplantation. In particular embodiments, described herein is a method for treating a hematological malignancy by administering the described non-agonist ligand to a patient while or after providing allo-HCT treatment for the malignant hematologic disease, including leukemia, lymphoma, or multiple myeloma. In other embodiments, the method for inhibiting or reducing the severity of GvHD enables the provision of allogeneic transplants in which there is a risk of inducing GvHD by cells that produce or induce sufficient GM-CSF (which are transplanted together with the allogeneic transplant). Liver transplantation is a non-limiting example of such a transplant.

[0067] In certain embodiments, the non-agonist anti-GM-CSF, anti-CD116, anti-CD131 or anti-GM-CSF receptor ligand is an antibody, antibody fragment, antibody-like molecule, aptamer or polypeptide derived from the domain of protein A. Although the use of antibodies (particularly monoclonal antibodies) is commonly used for therapeutic use in human patients, and indeed GM-CSF-specific antibodies and CD116-specific antibodies have been developed for other therapeutic purposes, it is understood by those skilled in the art that other formats (such as DARPINs, aptamers or antibody-derived molecules) can be used to achieve essentially the same purpose.

[0068] In some embodiments, the non-agonist anti-GM-CSF, anti-CD116, anti-CD131 or anti-GM-CSF receptor polypeptide ligand is an immunoglobulin composed of two heavy chains and two light chains. In some embodiments, the non-agonist anti-GM-CSF, anti-CD116, anti-CD131 or anti-GM-CSF receptor polypeptide ligand is a single domain antibody composed of isolated variable domains from a heavy chain or a light chain. In some embodiments, the non-agonist anti-GM-CSF, anti-CD116, anti-CD131 or anti-GM-CSF receptor polypeptide ligand is a heavy chain antibody composed only of a heavy chain, such as an antibody found in camels.

[0069] In certain embodiments, the non-agonist anti-GM-CSF, anti-CD116, anti-CD131 or anti-GM-CSF receptor polypeptide ligand is an antibody fragment. In certain embodiments, the non-agonist anti-GM-CSF, anti-CD116, anti-CD131 or anti-GM-CSF receptor polypeptide ligand is a Fab fragment, i.e., an antigen-binding fragment of an antibody, or a single-chain variable fragment, i.e., a fusion protein of the variable regions of the heavy and light chains of an antibody connected by a peptide linker.

[0070] The effect of treatment with anti-GM-CSF antibody in a mouse model of post-allo-HCT leukemia treatment is shown in FIG4 .

[0071] In certain embodiments, the ligand is a monoclonal antibody. In certain embodiments, the ligand is a human antibody.

[0072] In certain embodiments, the ligand for use in a method of treating a hematological malignancy, such as a leukemia, in a patient who has received an allogeneic hematopoietic stem cell transplant (allo-HCT) is a humanized antibody. In certain embodiments, the ligand is a chimeric antibody.

[0073] In certain embodiments, the ligand for use in the method of treating a hematological malignancy, such as a leukemia, in a patient who has received allo-HCT is Mavrilimumab (CAS No. 1085337-57-0).

[0074] In certain embodiments, the ligand for use in the method of treating a hematological malignancy, such as a leukemia, in a patient who has received allo-HCT is Namilumab (CAS No. 1206681-39-1).

[0075] In certain embodiments, the ligand used in the method for treating a malignant blood disease (such as leukemia) in a patient who has received allo-HCT is Lenzilumab (CAS No. 1229575-09-0). In certain embodiments, the ligand used in the method for treating a malignant blood disease (such as leukemia) in a patient who has received allo-HCT is Otilimab (MOR103 or GSK-3196165; CAS No. 1638332-55-4). In certain embodiments, the ligand used in the method for treating a malignant blood disease (such as leukemia) in a patient who has received allo-HCT is Gimsilumab (MORAb-022; CAS No. 1648796-29-5).

[0076] It will be appreciated that in particular embodiments, antibodies such as mavrilimumab, namilumab, lenzilumab, otilimab or gimsilumab may be used in methods of providing an allogeneic transplant in which the development of GvHD is inhibited or its severity is reduced.

[0077] In certain embodiments, the ligand for use in the method of treating leukemia in a patient who has received allo-HCT is characterized by a K D Less than (<) 10 -7 molL -1, especially K D <10 -8 molL -1 , especially K D <10 -9 molL -1 .

[0078] A second aspect of the invention relates to a nucleic acid molecule encoding a ligand as specified in the first aspect of the invention or any specific embodiment thereof, for use in treating leukemia in a patient who has received allo-HCT.

[0079] In certain embodiments, the nucleic acid molecule used to treat a hematological malignancy, such as a leukemia, in a patient who has received allo-HCT is a single-stranded or double-stranded DNA molecule or a single-stranded or double-stranded RNA molecule.

[0080] In certain embodiments, the nucleic acid molecule used to treat leukemia or other malignancy in a patient who has received allo-HCT according to the present invention is a nucleic acid expression construct comprising the above-specified nucleic acid sequence under the control of a promoter operable in mammalian cells.

[0081] In certain embodiments, the nucleic acid molecule used to treat a hematological malignancy, such as a leukemia, in a patient who has received allo-HCT is an expression construct selected from a DNA plasmid, double-stranded linear DNA, single-stranded RNA, and a virus, particularly a lentivirus, herpes virus, adenovirus, or adeno-associated virus.

[0082] Nucleic acid expression constructs are used to introduce specific genes into target cells and can control the protein synthesis machinery of the cell to produce the protein encoded by the gene. Nucleic acid expression constructs are engineered to contain regulatory sequences that act as promoter regions, and optionally also contain sequences that act as enhancer regions and lead to efficient transcription of the genes carried on the expression construct.

[0083] Nucleic acid expression vectors for use in the methods are common in the art. Illustrative vectors for expression of mammalian proteins include those available from Promega Corp (Madison, WI) and Thermo Fisher Scientific, Inc. (Waltham, MA), among others.

[0084] In another embodiment, as an alternative to administering a non-agonist ligand, the described treatment methods include inhibiting the expression of GM-CSF, its receptors (CD116 and CD131), or a single subunit of the GM-CSF receptor (CD116 and / or CD131). In these methods, a nucleic acid capable of inhibiting the expression of GM-CSF or its receptor peptide is provided to a patient in need thereof (e.g., after or during allo-HCT). In a particular embodiment, the targeting nucleic acid can be antisense DNA, siRNA, etc. Methods for determining suitable targets and targeting sequences are known in the art.

[0085] An alternative to the above aspect of the invention relates to a pharmaceutical composition for treating a hematological malignancy (such as leukemia) in a patient who has received allo-HCT. The composition comprises a non-agonist ligand that specifically binds to GM-CSF or to one of the GM-CSF receptors consisting of CD116, CD131 and CD131, or a nucleic acid expression construct encoding the same, and a pharmaceutically acceptable carrier, in particular it is formulated as a parenteral administration form, more particularly an intravenous administration form.

[0086] Antibodies against GM-CSF and against one of CD116, CD131 and GM-CSF receptors consisting of CD116 and CD131 are known in the art. Monoclonal antibodies specific for GM-CSF have been developed and clinically tested for efficacy in rheumatoid arthritis. Non-limiting examples of antibodies for implementing the present invention include CD116 / 131 antibodies (Mavrilimumab) disclosed in US2014079708, US2012141464, US2009130093, US2014079708 and US2015376285 of Cohen et al., which are incorporated herein by reference.

[0087] Another non-limiting example is the GM-CSF antibody (Otilimab) disclosed in US2015246969 to Haertle et al., and the antibodies disclosed in US2011189082 and US2013071923 to Kirchner et al., all of which are incorporated herein by reference.

[0088] Another non-limiting example is the GM-CSF antibodies disclosed in US2009053213, US2011045000, US2017218061 to Steidl et al., all of which are incorporated herein by reference.

[0089] Another anti-GM-CSF antibody is being developed by Takeda for rheumatoid arthritis under the name Namilumab.

[0090] Another anti-GM-CSF antibody, Lenzilumab (CAS No. 1229575-09-0), is in clinical trials in subjects with previously treated chronic myelomonocytic leukemia (CMML).

[0091] Similarly, within the scope of the present invention is a method of treating a hematological malignancy, such as a leukemia, in a patient who has received allo-HCT, comprising administering to the patient a non-agonist ligand, particularly an antibody, as described above, that specifically binds to GM-CSF or to one of the GM-CSF receptors consisting of CD116, CD131 or CD131.

[0092] Similarly, there is provided a dosage form for preventing or treating leukemia in a patient who has received allo-HCT, comprising a ligand or a nucleic acid construct according to one of the above aspects of the invention.

[0093] The dosage form can be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalant or suppository. Alternatively, parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms, can be used. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0094] Wherever alternatives of single separable features are arranged herein as "embodiments", it should be understood that such alternatives may be freely combined to form discrete embodiments of the invention disclosed herein.

[0095] The present invention is further described exemplarily by the following items:

[0096] Item 1: A non-agonist ligand that specifically binds to and neutralizes the physiological function of:

[0097] -GM-CSF or

[0098] - one of CD116, CD131 and GM-CSF receptor consisting of CD116 and CD131,

[0099] It is used to treat GvHD.

[0100] Item 2: The ligand for treating GvHD according to Item 1, wherein the ligand is an antibody, an antibody fragment, an aptamer or an antibody-like molecule.

[0101] Item 3: The ligand for treating GvHD according to Item 1 or 2, wherein the ligand is a human antibody or a humanized antibody.

[0102] Item 4: A ligand for use in treating GvHD according to any of the preceding items, which is selected from Mavrilimumab, Namilumab, Lenzilumab, MOR103 and MORAb-022.

[0103] Item 5: A ligand for use in treating GvHD according to any of the preceding items, wherein the binding of the ligand to GM-CSF or to one of the GM-CSF receptors consisting of CD116, CD131 and CD131 is characterized by a K D Less than (<) 10 -7 , especially KD<10 -8 , more specifically K D <10 -9 .

[0104] Item 6: A nucleic acid molecule encoding the ligand according to any one of the preceding items 1 to 4, for use in treating GvHD.

[0105] Item 7: The nucleic acid molecule for treating GvHD according to Item 6, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0106] Item 8: A nucleic acid expression construct comprising the nucleic acid sequence according to Item 5 or 7, for use in treating GvHD.

[0107] Item 9: A nucleic acid expression construct for treating GvHD according to Item 8, wherein the expression construct is selected from a DNA plasmid, a double-stranded linear DNA, a single-stranded RNA and a virus, in particular a lentivirus, a herpes virus, an adenovirus or an adeno-associated virus.

[0108] Item 10: The ligand according to any one of items 1 to 5 or the nucleic acid molecule according to item 6 or 7, or the nucleic acid expression construct according to item 8 or 9 for use in treating complications resulting from allo-HCT.

[0109] Item 11: A ligand according to any one of items 1 to 5 or a nucleic acid molecule according to item 6 or 7, or a nucleic acid expression construct according to item 8 or 9 for use in treating complications resulting from allo-HCT in patients suffering from or recovering from a hematological malignancy,

[0110] - in particular a hematological malignancy selected from the group consisting of leukemia, lymphoma and multiple myeloma,

[0111] More particularly the hematological malignancy is selected from chronic myeloid leukemia (CML), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) and acute monocytic leukemia (AMoL), Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0112] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived. These examples are intended to illustrate the invention, but not to limit its scope.

[0113] Example

[0114] Materials and methods

[0115] Study Design

[0116] This study was initiated to determine whether certain T cell-derived cytokines could separate GvHD from GvL and therefore might represent promising new therapeutic targets for the treatment of hematological malignancies. To achieve this goal, we used two different experimental models of GvHD, an experimental model of GvL, and human subjects. For animal studies, mice aged 8 to 12 weeks were used. All animal experiments were approved by local authorities (Swiss Cantonal Veterinary Office) and performed under appropriate experimental permits (76 / 2012 and 052 / 2015). Animals were randomly assigned to experimental groups, in-life clinical scores were performed in a blinded fashion, and organ sections were processed for image analysis. Sample size and disease endpoint time points were selected on the basis of previous studies. Flow cytometry, histopathological analysis, mixed lymphocyte reaction (MLR), killing assays, and cytokine analysis were performed to characterize GvHD / GvL target organs. The effects of antibodies specifically blocking GM-CSF on clinical scores were evaluated by investigators who were blinded to the treatment.

[0117] For reliable statistical analysis, at least three independent experiments were performed for each data presented in the manuscript, unless otherwise stated in the figure legends. All human samples were collected with written informed consent and with approval from the ethics committee of the Albert-Ludwigs University (Freiburg, Germany) (protocol number: 267 / 11). Immunohistochemistry and quantitative RT-PCR were performed on intestinal biopsies, and multiparameter flow cytometry was performed on PBMCs.

[0118] Mice and in vivo procedures. Mice were kept in individually ventilated cages under specific pathogen-free conditions. WTC57BL / 6, BALB / c, and B6D2F1 mice were purchased from Janvier Laboratories, France. Congenic C57BL / 6CD45.1 mice were housed in-house. - / - Mice were obtained from Jackson Laboratories; Csf2 - / - Mice were provided by Jeffrey Whitsett and further backcrossed to C57BL / 6 using speedy congenics. - / - Mice were provided by Y. Iwakura. All animal experiments have been approved by local authorities (Swiss Cantonal Veterinary Office) and were performed under appropriate experimental permits (76 / 2012 and 052 / 2015). Female mice aged 7–12 weeks were used throughout the experiments.

[0119] Induction of GvHD: Single cell suspensions of spleen and BM from donor C57BL / 6 or BALB / c mice were prepared as described below (under “Lymphocyte Isolation”). For T cell depletion of BM, cells were plated at 25 x 10 6 / ml in RMPI complete medium (RPMI 1640 with 10% FCS, 1% penicillin / streptomycin, 2 mM L-glutamine and 0.5 mM β-mercaptoethanol) containing αCD90.2-biotin (1:100, eBioscience) at 4°C with agitation for 30 minutes. Subsequently, after a washing step with cold MACS buffer (PBS with 0.5% BSA and 2 mM EDTA), the cells were plated at 100 x 10 6The concentration of cells / ml was resuspended in MACS buffer and incubated with anti-biotin beads (1:5, Miltenyi) or streptavidin beads (1:10, Miltenyi) at 4°C for 20 minutes under stirring. After washing with MACS buffer, cells were separated using AutoMACS Pro, Miltenyi Biotech and the "depletes" program. Negative fractions were collected and the number of cells was determined using a counting chamber. The removal efficiency was analyzed by flow cytometry. The frequency of T cells in BM was reduced from 1-3% to 0.055+ / -0.022%. In order to separate untouched T cells from splenocytes, a full T cell separation kit (II) from Miltenyi was used according to the manufacturer's protocol. Cells were separated using AutoMACS Pro, Miltenyi Biotech and the "deplete" program. Negative fractions were collected and the number of cells was determined using a counting chamber. Enrichment efficiency was analyzed by flow cytometry, and T cell purity was generally 95.2+ / -2.16%. CD4 and CD8 T cells were isolated using MojoSort Mouse CD4 T Cell Isolation and MojoSort Mouse CD8 T Cell Isolation kits according to the manufacturer's instructions (Biolegend).

[0120] Recipient BALB / c or B6D2F1 mice were lethally irradiated in filter-cages under specific pathogen-free conditions with divided doses of 850 (BALB / c), 1200 (B6D2F1), or 1100 (C57BL / 6) rad at least 5 hours apart. Recipients were injected intravenously with 5x 10 6 BM cells and 0.1-10x 10 6 Splenocytes or 6-7.5x 10 6 T cells / C57BL / 6 or B6D2F1 mouse. Recipients were injected intravenously with 7x 10 6 BM cells and 20x 10 6Splenocytes / BALB / c mice. Mice were treated with 0.1% Borgal (Intervet) in drinking water for three weeks to prevent bacterial infection. Mice were also scored for GvHD symptoms every day, adapted from (Cooke, KR et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood 88, 3230–3239 (1996)) and shown in Supplementary Table 4. Investigators were blinded to group assignment when scoring GvHD. Reference body weight was measured on day 0 before injection of transplanted cells. To block GM-CSF, mice were treated 3 times a week with PBS, 300 μg isotype control (2A3), or 300 μg anti-GM-CSF antibody (MP1) (BioXCell) starting 2 days before HCT.

[0121] Induction of GvL: A20-luciferase was produced on a BALB / c background by transducing A20 (ATCC) with lentivirus encoding CMV-GFP-(T2A)-Luc. + GFP + B-cell lymphoma cell line (A20-luc-gfp, kindly provided by Emma Svensson, Department of Immunology, Genetics and Pathology, Uppsala University, Sweden). WEHI-3-luciferase+ cells were donated by R. Zeiser. On the day of HCT, each mouse was injected intravenously with 2.5x10 5 A20 cells, 5x10 5 A20 cells or 1x10 5 WEHI-3 cells, accompanied by 5x10 6 WT C57BL / 6 BM cells or 1x10 from WT or Csf2- / - C57BL / 6 mice 5 Purified spleen T cells / mouse. On the day of HCT, 2.5x10 per mouse were injected intravenously 5 A20 cells, accompanied by 5x10 6 WT C57BL / 6BM cells or cells derived from WT or Csf2 - / - 1x10 for C57BL / 6 mice 5Purified spleen T cells / mouse. Tumor progression was monitored by bioluminescence imaging (BLI): mice were injected intraperitoneally with 150 mg / kg of D-luciferin (Promega) in PBS ten minutes before imaging using the XenogenIVIS 200 preclinical in vivo imaging system (PerkinElmer, Waltham, MA); exposure time 1-120 s, binning 2-8, FOV 15 cm, f / stop 1, no filter. Mice were irradiated with isoflurane (at 0 °C) before and during imaging. 2 The mice were anesthetized with 2% ethanol (vaporized in 5% ethanol). The total photon flux (photons / second) was measured from fixed regions of interest (ROI) throughout the body using Living Image software (PerkinElmer). Pictures were always taken for each cage and were cropped when mice from different groups were mixed in the same cage. Tumor and GvHD mortality were measured by BLI signal intensity (ROI>1*10 for tumor incidence). 7 ), hind limb paralysis (indicative of tumor development), and clinical manifestations of GvHD (at least grade 2 in two separate GvHD criteria). To block GM-CSF, mice were treated three times per week with 300 μg of isotype control (2A3) or 300 μg of anti-GM-CSF antibody (MP1) (BioXCell) starting 2 days before HCT. Starting from week 3, the antibody dose was reduced to 150 μg.

[0122] Lymphocyte isolation. Using CO 2 Mice were euthanized by inhalation and perfused with 40 ml of cold PBS. Unless otherwise stated below, organs were collected, minced into pieces and incubated with collagenase at 37 °C, followed by mechanical disruption by repeated passage through a 20-gauge needle. Red blood cell lysis was performed. The resulting cell suspension was then filtered through a 70 μm cell strainer and used for further manipulations.

[0123] spleen: To isolate bone marrow cells, organ fragments were incubated in 2 ml collagenase D (0.4 mg / ml, Roche) and 0.1 mg / ml DNase I (Sigma) in RPMI at 37° C. for 30 min. For T cell isolation, spleens were homogenized by mechanical disruption and then filtered through a 70 μm cell strainer. BM: The femur, tibia, and pelvis were flushed with PBS to obtain BM stem cells. liver dirty:The organ fragments were incubated in HBSS (containing 10% FCS) with 1.6 mg / ml type IV collagenase (from Clostridium histolyticum, Sigma) at 37°C for about 45 minutes. The cells were resuspended in 10 ml Percoll (continuous gradient, 27%, GE) and centrifuged at 1700 rpm for 30 minutes at room temperature. The fat and supernatant were removed and the pellet was subjected to erythrocyte lysis. skin: Organ fragments were incubated in RPMI with 1 mg / ml collagenase type IV (from Clostridium histolyticum, Sigma) and 0.1 mg / ml DNase I (Sigma) at 37°C for 1.5-2 hours. Small Intestine: Organs were separated from mesenteric fat before mechanical removal of luminal mucus; organs were then incubated in HBSS without calcium and magnesium containing 2% FCS, 1 mM DTT and 1.35 mM EDTA at 37°C for 15 minutes. After further incubation in HBSS supplemented with EDTA at 37°C for 30 minutes, the colon was cut and digested using 0.4 mg / ml collagenase IV (Sigma Aldrich) at 37°C for 45 minutes. The samples were then homogenized using a syringe with an 18-gauge needle and filtered through a 70 μm cell strainer.

[0124] Flow cytometry. Flow cytometric analysis was performed according to standard methods, which are reviewed in (Perfetto, SP, Chattopadhyay, PK & Roederer, M. Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4, 648–655 (2004)). For all fluorochrome-conjugated antibodies, titration experiments were used to determine the optimal concentration. Mouse CD4 (GK1.5), CD8 (53-6.7), CD3 (17A2), CD45 (30F11), CD45.1 (A20), CD45.1 (104), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), MHC class I H2-D d (34-2-12) and H2-D bAntibody clones specific for (KH95), GM-CSF (MP1-22E9), IFNγ (TC11-18H10), IL-17A (XMG1.2), and FoxP3 (FJK-16s) are available from BD, BioLegend, or eBioscience. For surface staining, cells were incubated with the respective antibodies at 4°C for 20-30 minutes. For all experiments, dead cells were excluded from the analysis using Aqua or Near-IR Live / Dead fixable staining reagents (Invitrogen / BioLegend); doublets were excluded by FSC-Area vs. FSC-Height gating. For intracellular cytokine staining, T cells were incubated at 37°C for 4-5 hours in RPMI containing 10% FCS and PMA (50 ng / ml), ionomycin (500 ng / ml), and GolgiPlug (containing Brefeldin A, BD, 1:1000 dilution). After surface staining, Cytofix / Cytoperm (BD) was used according to the manufacturer's instructions, and Perm / Wash buffer (PBS containing 0.5% saponin and 5% BSA) was prepared in-house. For intracellular staining, cells were incubated with the respective antibodies at 4°C for 20-30 minutes. For intranuclear FoxP3 staining, Fixation / Permeabilization buffer (eBioscience) was used after surface staining, followed by Perm / Wash buffer prepared in-house. Typically, cells were gated based on FSC-Area and SSC-Area to exclude debris, and doublets were excluded by FSC-Area vs. FSC-Height gating. Dead cells were excluded from the analysis using Aqua or Near-IR Live / Dead fixable staining reagents (Invitrogen / BioLegend). Where applicable, CD45.1 cells were gated before gating on CD4 or CD8 T cells. All markers (CD4, CD8, CD3, CD44, CD62L, CD69) used for gating demonstrate the obvious separation of negative colonies and positive colonies. For killing test cells, single cells (singlet) are gated as described above, tumor cells are then identified by Cell trace violet, and a histogram showing TOPRO-3 uptake is produced. Flow cytometry analysis is performed using FACSCanto II (BD) or LSR II Fortessa (specially ordered research products, BD, and equipped with 405nm, 488nm, 561nm and 640nm laser lines) with FACS Diva Software.Data analysis was performed using FlowJo 10.0.x (Treestar).

[0125] Flow cytometric analysis of STAT5 phosphorylation

[0126] The single cell suspension surface of bone marrow was stained for 20 minutes, and thereafter the culture medium (20ng / ml) containing GM-CSF was added to the sample. The cells were incubated at 37°C for 30 minutes to induce STAT5 phosphorylation, and then 4% PFA solution (pH7.4) was added to a final concentration of 2%. The cells were fixed for 20 minutes, washed and resuspended in 1ml 4°C methanol. After 40 minutes, the cells were washed twice and resuspended in 45μl FACS buffer. 5μl anti-pSTAT5 APC (BD Biosciences) was added so that the final staining ratio was 1:10. Before the final washing step and collection, the cells were incubated at 4°C for 45 minutes. Flow cytometry analysis was performed using LSR II Fortessa (specially ordered research products, BD, and equipped with 405nm, 488nm, 561nm and 640nm laser lines) with FACSDiva Software. Data analysis was performed using FlowJo10.0.x (Treestar).

[0127] Phenotypic analysis of human PBMCs by flow cytometry

[0128] Cryopreserved PBMCs were stored in liquid nitrogen until thawed in a 37°C water bath. The cells were gently resuspended in 1 ml of prewarmed cell culture medium (CCM; RPMI-1640 [PANbiotech], 10% FCS [Biochrom], 1× l-glutamine, and 1× penicillin / streptomycin [both from Life Technologies]) supplemented with 1:10,000 benzonase (Invitrogen). The cells were then transferred to a 5 ml tube and washed with CCM. The cells were counted and adjusted to 20 × 10 in CCM. 6 To determine cytokine production by flow cytometry, cells were stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 1 ug / ml ionomycin (Sigma-Aldrich) at 37°C for 4 hours in the presence of GolgiPlug (containing BrefeldinA, BD, 1:1000 dilution) and GolgiStop (containing Monensin, BD, 1:1000 dilution). Nonspecific binding was blocked using Human TruStain FcX (Biolegend).

[0129] Mixed lymphocyte reaction (MLR). - / - Ifng - / - or Il17a – / – Responder T cells were co-cultured with DCs from BALB / c (allogeneic) or C57BL / 6 (syngeneic) mice. Alternatively, BALB / c WT responder T cells were co-cultured with DCs from BALB / c (syngeneic) or C57BL / 6WT and Csf2rb- / - DCs (allogeneic). T cells and DCs were purified from splenocytes by positive selection using anti-CD4 and anti-CD8 (for T cells) or anti-CD11c (for DCs) magnetic beads (Miltenyi Biotec) and AutoMACS Pro, respectively. Cells were cultured in triplicate at a 1 / 10 stimulator cell / responder cell ratio (2*10 4 DC, 2*10 5 T cells) were plated in U-bottom 96-well plates and incubated at 37°C and 5% CO 2 Cultivate in RPMI 1640 complete medium for 3 days.Then collect the culture supernatant and replace it with diluted 3H-thymidine (1 / 100, 50 μCi) for 16 hours.Use 96-well collector (PerkinElmer) to collect cells and measure scintillation by beta counter (1450Microbeta Plus, Wallac).

[0130] Killing test. Spleen and lymph node T cells were isolated using the mouse pan T cell isolation kit II (MACS Miltenyi Biotec). TM Violet Cell Proliferation Kit (ThermoFisher Scientific, C34557) for A20-luciferase + GFP + Cells were stained with soluble α-CD3 (1 ug / ml) and α-CD28 (0.5 ug / ml) at effector / target ratios of 1:1, 20:1, and 50:1 in RPMI supplemented with 10% FCS at 37°C, 5% CO 2 After removing the culture medium, Topro (0.8 μM) was added to the cells and the cells were acquired on a LSRII Fortessa flow cytometer (BD). Data were analyzed using FlowJo Version X (Tree Star). The percentage of specific lysis was calculated as follows: [(experimental lysis-spontaneous lysis) / (maximum lysis-spontaneous lysis)]×100%.

[0131] Treg suppression assay

[0132] T cells were isolated from lymph nodes and spleens of C57BL / 6WT and Csf2- / - mice as described previously. TM CD4 T cells were enriched with Mouse CD4 Nanobeads) and then stained with CD45, CD25, CD62L, and CD4.

[0133] CD4+CD25-CD62L+ (Tconv) and CD4+CD25high (Treg) cells were sorted by flow cytometry using a BD FACS ARIA II cell sorter with a purity of 98-99%. APCs were irradiated with >3400 rad (4 min, 12.2 Gy / min, 225 kV, 17.7 mA) and diluted to 4x10 in 10% cloning medium. 6 / mL (or 2x10 6 In a 96-well round-bottom plate, APCs and Tconv cells were co-cultured with autologous Treg cells at different concentrations (4x10 4 Tconv / well). Cells were stimulated with αCD3 at a concentration of 1 ug / ml at 37°C for 2 days and then incubated with 3H-thymidine for 16 hours. Cells were collected using FilterMate (PerkinElmer) and analyzed by beta counter using program Microbeta. The percentage of Treg inhibition among live cells was calculated as [((cpm Tconv-cpm Treg:Tconv) / cpmTconv alone)*100], cpm is counts per minute, calculated as % inhibition of Tconv cells.

[0134] Cytokine analysis. Serum was obtained from allo-HCT recipient mice at the indicated times. After 3 days of co-culture, the culture supernatants of MLR were collected. GM-CSF, IFNγ, and IL-17A in serum or cell culture supernatants were determined by cytokine-specific ELISA according to the manufacturer's instructions (BD Biosciences and Biolegend).

[0135] Liver function parameters

[0136] Liver enzymes (alanine aminotransferase (ALT), alkaline phosphatase (AP)), albumin, and blood urea nitrogen (BUN) in mouse serum were measured using Piccolo Liver Panel Plus (Abaxis).

[0137] Histology. For histopathological evaluation, organs were fixed in HOPE (DCS, Hamburg, Germany) and embedded in paraffin. 3-5 μm sections were cut with a microtome (Micro HM 325, Thermo Scientific) and then stained with hematoxylin and eosin. To generate histological GvHD scores, a semiquantitative tissue-specific scoring system was used: Small Intestine Parameters (including villus blunting, crypt regeneration, crypt cell apoptosis, outright crypt destruction, and lamina propria lymphocyte infiltration) were evaluated by the following scores: 0 = normal; 0.5 = focal and minimal; 1 = focal and mild; 2 = diffuse and mild; 3 = diffuse and moderate; 4 = diffuse and severe, adapted from (Hill, GR et al. Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood 90, 3204–3213 (1997)). skin The following parameters were assessed: Estimated number of hair follicles: 0 = normal (relatively densely packed), 0.5 = slightly reduced number (usually focal); Estimated number of sebaceous glands: 0 = normal (relatively large cells, small groups, at the level of the hair neck level), 1.0 = slightly decreased (number and size, usually focal), 2.0 = markedly decreased (only a few cells); apoptosis and degeneration of sebaceous glands: 0 = absent, 0.5 = very few, 1 = a small number of glands involved, 2 = a moderate number of glands involved; apoptosis and degeneration of hair follicles: 0 = absent, 0.5 = very few, 1 = a small number of follicles involved, 2 = a moderate number of follicles involved, 3 = a large number of follicles involved; leukocyte infiltration (extra-adnexal): 0 = low, 1 = slightly increased, 2 = moderately increased; leukocyte infiltration (adnexal): 0 = absent, 1 = mild, 2 = moderate; epidermis (apoptotic cells, leukocyte infiltration): 0 = absent, 0.5 = very few, 1 = a small number, 2 = a moderate number, 3 = a large number; subcutaneous fat: 0 = continuous, 1 = discontinuous, 2 = absent, adapted from (Kaplan, DH et al. Target antigens determine graft-versus-host disease phenotype. J. Immunol. 173, 5467–5475 (2004). liver Parameters (including portal vein dilatation and infiltration, bile duct infiltration, nuclear bile duct multilayering, pyknotic bile duct cells, intraepithelial bile duct cells, vascular endothelial inflammation, hepatocellular pan-lobular necrosis, eosinophilic bodies, microabscesses, mitotic figures, and hepatocellular steatosis) were evaluated by the following scores: 0 = normal; 0.5 = focal and few; 1 = focal and mild; 2 = diffuse and mild; 3 = diffuse and moderate; 4 = diffuse and severe, adapted from (Hill, GR et al. Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Clin. Invest. 102, 115–123 (1998)). All slides were coded and read blindly on an Olympus BX41TF microscope.

[0138] Human subjects. All human samples were collected with approval from the ethics committee of the Albert-Ludwigs University (Freiburg, Germany) (protocol number: 267 / 11) after written informed consent. Intestinal tissue biopsy samples were collected prospectively from GvHD or healthy control individuals between 2007 and 2014. Intestinal GvHD grading was performed by an experienced pathologist according to the published staging system based on histopathology (Lerner, K Getal. Histopathology of graft-vs.-host reaction (GvHR) in human recipients of marrow from HL-A-matched sibling donors. Transplant. Proc. 6, 367–371 (1974)). Control individuals did not show any abnormalities in intestinal biopsies. The characteristics of the patients, including recipient age, sex, underlying diagnosis, type of donor and graft, conditioning regimen, immunosuppression regimen, and GvHD grade, are detailed in Supplementary Tables 1 and 3 for GvHD patients and in Supplementary Table 2 for control subjects.

[0139] Immunohistochemistry. Immunohistochemistry was performed in fixed paraffin-embedded tissue sections using the two-step IHC staining kit EnVision+SystemHRP DAKO (Glostrup, Denmark) according to the manufacturer's instructions. Sections were subjected to heat-mediated antigen retrieval with Dako Target Retrieval Solution (1×) for 10 minutes. Endogenous peroxidase activity was blocked with the DAKO endogenous peroxidase blocking kit for 30 minutes at room temperature. Primary antibodies were diluted in PBS+5% normal goat serum (NGS). Single immunostaining consisted of incubation with unconjugated mAb anti-human GM-CSF antibody (clone 3209.1; R&D Systems) or IgG isotype control for biopsies from human subjects, and with rat α-F4 / 80 (clone CI: A3-1, BioRad) and rabbit α-p22phox (clone FL-195, Santa Cruz) for mouse tissue sections at 4°C overnight. DAB was used as a chromogen. The sections were then counterstained with hematoxylin, mounted with DPX, and analyzed using a light microscope Olympus BX41 connected to a color camera ColorViwerIIIu (Olympus) and Fiji / ImageJ software package (GNU General Public License).

[0140] Immunofluorescence

[0141] The tissue cryosections (10 μm thick) were used for immunohistochemistry using a Hyrax C60 cryostat (Zeiss) and stored at -80°C. The sections were fixed in 4% PFA, washed in PBS, and blocked with PBS supplemented with 0.1% Triton X-100 and 4% normal goat serum. Subsequently, the sections were incubated with the following primary antibodies, rat anti-GMCSF antibody (BD Pharmingen, clone BVD2-21C11, 1:50), rabbit α-CD3 (Novus, clone SP7, 1:200) and mouse anti-CD68 antibody (DAKO, clone EMB11, 1:50) and diluted in blocking solution at 4°C overnight. The sections were then washed in PBS and incubated overnight at 4°C or at room temperature for 1 hour with AF647-labeled goat anti-rat, AF488-labeled goat anti-mouse, and AF555-labeled donkey anti-rabbit secondary antibodies (LifeTechnologies, 1:500). The sections were mounted with SlowFade Gold antifade reagent containing DAPI (Invitrogen). Fluorescence micrographs were captured using a SP5 Leica confocal laser scanning microscope (SP5; Leica, Heerbrug, Switzerland) equipped with argon and helium lasers using a 40x objective (oil immersion, NA1.25). Images were processed and merged by Imaris imaging software (Bitplane, Zurich, Switzerland).

[0142] TUNEL assay

[0143] Sections fixed in 4% PFA were processed for TUNEL analysis to detect fragmented nuclei in the colon. The ApopTag Red In Situ Apoptosis Detection Kit was used according to the manufacturer's instructions. Briefly, slides were washed with H 2 O 2 Pretreatment and incubation with a reaction mixture containing TdT and digoxigenin-conjugated dUTP at 37°C for 1 hour. Labeled DNA was visualized with a secondary antibody conjugated to AF488. Fluorescence micrographs were captured using a SP5 Leica confocal laser scanning microscope (SP5; Leica, Heerbrug, Switzerland) equipped with argon and helium lasers using a 40x objective (oil immersion, NA1.25). Images were processed and merged by Imaris imaging software (Bitplane, Zurich, Switzerland).

[0144] RNA isolation and quantitative rt-PCR. RNA was isolated from fixed paraffin-embedded tissue sections using the RNeasy FFPE kit (Qiagen, deparaffinized using xylene) according to the manufacturer's instructions. RNA concentration was estimated using a NanoDrop1000 spectrophotometer (Thermo Scientific). For first-strand cDNA synthesis, MLV reverse transcriptase (Invitrogen) was used according to the manufacturer's instructions. Gene expression was measured by real-time quantitative PCR analysis using a CFX 384 real-time detection system (Bio-Rad, Hercules, CA, USA) with SYBR Green Supermix (Bio-Rad). The sequences of PCR primers are listed in the table below. Transcript expression was normalized to the GAPDH housekeeping gene and expressed as 2 -ΔΔC T(ΔΔC T =ΔC T -ΔC 对照 ).

[0145]

[0146] Automated group identification in high-dimensional data analysis

[0147] Data acquired on a BD FACSymphonyTM cell analyzer were compensated, exported in FlowJo Version v10.4 (TreeStar), normalized using Cyt MATLAB (version 2017b), and analyzed unbiased as described in (Nowicka et al. F1000 Res 6, 748 (2017); Hartmann et al., J. Exp. Med. 213, 2621–2633 (2016).).

[0148] Statistics. Graphs were prepared using GraphPad Prism (GraphPad Software). Survival curves were plotted by the Kaplan-Meier method, and the Lox-rank (Mantel-Cox) test was used to compare survival curves. Data are presented as individual data points or mean + / - standard deviation (SD) or standard error of the mean (SEM), as shown in the legends in the figures. Mean comparisons were performed using unpaired two-tailed Student's t-test (with Welch correction, if applicable) or one-way and two-way ANOVA with Bonferroni post hoc test, respectively. No statistical methods were used to determine sample size, but our sample size was similar to that commonly used in the art. No randomization method was used.

[0149] Data availability. The datasets generated and / or analyzed during this study are available from the corresponding author on reasonable request.

[0150] Embodiment 1:

[0151] Tissue-infiltrating donor T cells produce GM-CSF and IFNγ after MHC-mismatched HCT

[0152] To understand the contribution of T cell-derived GM-CSF to acute GvHD, we first used an MHC-mismatched HCT model. We used lethally irradiated wild-type (WT) BALB / c CD45.2 + Mice (MHC haplotype H2d) were then injected intravenously with T cell-depleted (TCD) bone marrow (BM) cells from WT CD45.1 C57BL / 6 (B6) mice (MHC haplotype H2b) with or without B6 CD45.1 + Between days 3 and 6 after allo-HCT, recipients of BM plus spleen cells developed lethal GvHD ( Figure 1A ), which is consistent with donor CD45.1 in spleen, liver and skin. + Cell reconstruction and expansion coincide with each other ( Figure 1B ). + In the donor compartment, the T cell population contains high frequencies of GM-CSF and IFNγ-producing cells, first in the liver ( Figure 1C ) and spleen ( Figure 1D ) and then from day 6 onwards in the skin ( Figure 1D ) were observed in these groups. IL-17A was barely detectable in these groups ( Figure 1C , 1D). Therefore, the inventors found that the amount of GM-CSF and IFNγ in the serum of mice receiving BM plus spleen cells was significantly higher than that of mice receiving BM alone ( Figure 1E The inventors demonstrated the production of GM-CSF and IFNγ during in vitro allogeneic reactions using an allogeneic mixed lymphocyte reaction (MLR) ( Figure 1F ). In addition, wt, Csf2 - / - ,Il17a - / - Ifng - / - T cells proliferated equally after coculture with allogeneic DC, which ruled out a priori differences in the proliferative potential of these cells ( Figure 1G ). In conclusion, GM-CSF and IFNγ, but not IL-17A, are produced by T cells infiltrating target organs during GvHD.

[0153] Embodiment 2:

[0154] GM-CSF production by allogeneic T cells is essential for GvHD pathology

[0155] Considering the alloreactive T H As cells produce GM-CSF and IFNγ, the inventors evaluated the relevance of these cytokines in determining the severity of acute GvHD. As described above, the inventors used lethally irradiated WT BALB / c mice and then compared the effects of injection of TCD-BM cells from WT B6 mice with TCD-BM cells from WT B6 mice with or without TCD-BM cells from WT B6 mice, or from mice lacking GM-CSF (Csf2 - / - ), IFNγ(Ifng - / - ) or IL-17 (Il17a - / - ) of B6 mice. - / - Mice treated with BM plus WT splenocytes were protected from lethal GvHD up to 20 days after allo-HCT, whereas all mice treated with BM plus WT splenocytes died by day 15 after HCT ( Figure 2A ). Use BM plus Ifng - / - or Il17a - / - Splenocyte injection into mice also significantly affected the kinetics of lethal GvHD, but still almost 100% of these mice died by day 20 ( Figure 2A ).

[0156] Since donor splenocyte preparations contain a variety of different immune cell types, the inventors then asked whether T cells were indeed a biologically important source of GM-CSF in the GvHD model of the present invention. The inventors injected lethally irradiated BALB / c recipients with TCD-BM cells from WT B6 donors (with or without TCD-BM cells from WT or Csf2 - / - B6 mouse spleens and purified T cells isolated from the spleen); this approach confirmed that Csf2 - / - Mice that received WT T cells were completely protected from lethal GvHD up to 70 days after allo-HCT, in stark contrast to mice that received WT T cells ( Figure 2B There was no evidence that this protective effect was associated with either WT or Csf2 - / - The innate differences between splenic T cells of mice were related to the presence of Fig. 6A ), activation state( Figure 6B ), the ability to infiltrate GvHD target organs after HCT ( Figure 6C ), activated (CD44 high )T cell frequency( Fig.6D), the frequency of regulatory T cells (Treg) and granzyme production (data not shown) or their ability to produce proinflammatory cytokines (such as IFNγ, TNFα or IL-17A). The inventors then examined the relative contribution of GM-CSF from CD4 and CD8 T cells to GvHD pathology. To this end, the inventors used 4 combinations: CD4 WT T cells + CD8 WT T cells, CD4 WT T cells + CD8 Csf2 - / - T cells, CD4 Csf2 - / - T cells +CD8 WT T cells and CD4 Csf2 - / - T cells +CD8 Csf2 - / - T cells, a “criss-cross” experiment was performed. Only the combination lacking GM-CSF in CD4 T cells provided protection against lethal GvHD ( Figure 2K ), which supports the view that alloreactive TH cells are an important source of GM-CSF in GvHD pathology. Therefore, the inventors concluded that T cell-derived GM-CSF is responsible for triggering lethal GvHD after MHC-mismatched HCT.

[0157] The survival advantage conferred by allo-HCT of GM-CSF-deficient T cells was also evident at the level of individual GvHD target tissues. Histopathological analysis 6 days after allo-HCT revealed that Csf2 - / - Transplantation of spleen cells resulted in significantly less tissue damage to the skin and small intestine ( Figure 2C , 2D). In more detail, accepting Csf2 - / - Mouse skin expressing WT cells showed reduced apoptosis and cellular infiltration around and within hair follicles and epidermis, while the small intestine showed reduced complete crypt destruction ( Figure 2D ). The inventors further report herein that Csf2 - / - Colonic pathology was reduced upon cell transplantation, as indicated by increased colon length and decreased apoptotic index ( Figure 2H , 2I). In addition, screening for subclinical signs of liver dysfunction revealed that Csf2 - / - Reduced serum levels of alanine aminotransferase (ALT), alkaline phosphatase (AP), and blood urea nitrogen (BUN) in splenocyte-transplanted mice ( Figure 2G ). Furthermore, in the absence of GM-CSF, systemic TNFα serum levels were reduced, whereas the amount of IL-6 did not change (data not shown).

[0158] Since reactive oxygen species (ROS) have been implicated in GvHD tissue damage, the inventors asked whether this reduced tissue damage was associated with lower levels of ROS production. The inventors found that slices from Csf2 mice receiving WT splenocytes were significantly higher than those from slices from mice receiving Csf2 mice. - / - Sections of liver and small intestine from mice on day 6 after splenocyte transplantation contained significantly fewer cells expressing p22phox, a key component of the phagocyte ROS-generating machinery ( Figure 2E , 2F).

[0159] To further confirm the functional role of GM-CSF in GvHD, the inventors treated recipient mice with anti-GM-CSF antibodies before MHC-mismatched HCT of BALB / c mice with WT B6 TCD-BM alone or with BM and WT splenocytes. Systemic blockade of GM-CSF successfully replicated the effects of Csf2 - / - The survival advantage of spleen cells transplanted into these mice ( Figure 2G ).

[0160] Although mouse models of fully MHC-mismatched HCT have been informative, we next asked whether the role of GM-CSF in GvHD pathology is preserved in the more clinically relevant setting of a haplo-mismatched model of acute GvHD. - / - Ifng - / - splenocytes and transplanted into lethally irradiated B6D2F1 mice, which are a cross between B6 and DBA mice and thus have the MHC haplotype H2b / d. Here again, Csf2 - / - Transplantation of splenocytes conferred a significant survival advantage, protecting mice from lethal GvHD to a level nearly comparable to that of TCD-BM alone ( Figure 3A ), which in purified Csf2 - / - Replication of spleen T cells after transplantation ( Figure 3B In the spleen cell transplantation experiment and T cell transplantation experiment, the inventors also noticed that the mortality rate increased significantly after Ifng- / - cell transplantation ( Figure 3A , 3B), which is consistent with the known protective immunomodulatory function of this cytokine in GvHD. At day 9 after HCT, the skin was the most affected organ with the highest histological score; here, the inventors found that compared with WT splenocytes, in Csf2 - / - There was less tissue damage in mice with - / - More tissue damage in recipients of splenocytes ( Figure 3C, 3D). This is consistent with changes in the number, organization, and cellular infiltration of sebaceous glands around and within hair follicles and epidermis ( Figure 3D At a later stage after allo-HCT (28 days), Csf2 - / - Transplantation of splenocytes also resulted in a reduction in liver pathology ( Figure 3H ), characterized by portal vein infiltration and bile ductal changes. In this partially mismatched model, the skin is the most affected organ with the highest GvHD scores; here, the inventors found that compared with WT splenocytes, in Csf2 - / - Tissue damage was significantly less in mice with Ifng - / - Tissue damage was significantly greater in recipients of splenocytes ( Figure 3C , 3D). At the cellular level, the inventors also observed that Csf2 - / - Signs of GvHD were reduced in the liver and small intestine of mice expressing p22phox + Significantly fewer cells, with F4 / 80 + Reduced infiltration of bone marrow cells ( Figure 3E , 3F). Given the GvHD protective effect of IFNγ observed in these mice, the inventors then asked whether the absence of GM-CSF was associated with the levels of this cytokine: 10 days after haplo-mismatched HCT, the inventors found that compared with WT splenocytes, IFNγ in mice receiving csf2 - / - IFNγ levels were significantly higher in mice with Figure 3G ). Interestingly, the inventors also found that accepting Ifng - / - Evidence for increased GM-CSF in mouse serum from splenocytes ( Figure 3G ), suggesting that GM-CSF may also mediate enhanced GvHD due to the absence of IFNγ.

[0161] In summary, we conclude that in both murine models of allo-HCT, T cell-derived GM-CSF has a crucial role in the development of GvHD immunopathology.

[0162] Embodiment 3:

[0163] GM-CSF mediates GvHD lethality via donor-derived bone marrow cells

[0164] Following lethal conditions, radiosensitive host antigen presenting cells (APCs) are lost and replaced by donor-derived APCs within the first few days after HCT. Although host APCs are required for the initiation phase of GvHD, donor APCs play a minor role in disease induction but may be involved in permanent tissue damage. To characterize whether the GM-CSF-responsive cell types associated with pathology are of donor or host origin, the inventors used Csf2rb, a GM-CSFR lacking the β subunit. - / - Mice were used as donors or hosts for HCT. GM-CSFR deficiency in the recipient compartment did not affect survival in GvHD ( Fig. 9A ). In contrast, from Csf2rb - / - Transplantation of BM in mice resulted in a significant delay in mortality ( Fig. 9B ), phenocopying Csf2 - / - Transplantation of spleen cells ( Figure 2A ). The inventors used unsupervised nonlinear dimensionality reduction (t-SNE) to identify and visualize GM-CSF-responsive cells in donor grafts. Using this approach, the inventors found that monocytes and neutrophils were particularly sensitive to GM-CSF, as assessed by single-cell analysis of phosphorylated STAT-5, a transcription factor downstream of the GM-CSF receptor complex. - / - Mice were used as a source of GM-CSF unresponsive cells ( Fig. 9C , 9D). Having demonstrated that cells that respond to GM-CSF are restricted to the bone marrow compartment, the inventors comprehensively characterized the bone marrow cell subsets found in inflamed target organs 5 days after allo-HCT. - / - Transplantation of splenocytes results in a decrease in the frequencies of several myeloid subsets, including dendritic cells (DCs), neutrophils, monocytes, and monocyte-derived cells (MDCs). Fig.9E , 9F). Neutrophils and monocytes / MDCs were also found to be reduced in target organs after anti-GM-CSF treatment, while the systemic numbers of these bone marrow subsets remained unchanged. The inventors observed that in the absence of GM-CSF, donor monocytes / MDCs produced less IL-1β ( Figure 9G ). Importantly, IL-1β is a hallmark cytokine of the pathogenic program initiated by GM-CSF and has been shown to play a key role in the development of GvHD. - / - Given the low levels of Csf2- / - T cells in tissue sections of mice and their reported role in driving tissue damage, the inventors also investigated their expression in donor bone marrow cells after allo-HCT. The inventors found that transplantation of Csf2- / - splenocytes resulted in reduced ROS production by both monocytes / MDCs and neutrophil subsets ( Figure 9H). Together, these findings suggest that GM-CSF-permissive donor-derived bone marrow cells are critical for GvHD pathology.

[0165] Example 4

[0166] Preserved effective GvL in the absence of GM-CSF

[0167] The immune responses that lead to tissue damage in GvHD and tumor elimination in GvL are widely believed to be mediated by the same mechanisms. Since donor T cell-derived GM-CSF is essential for lethal GvHD, the inventors investigated whether it also mediates GvL in the MHC-mismatched HCT model. To test this, the inventors injected A20 B cell lymphoma cells (BALB / c-derived) co-expressing GFP and luciferase (A20-GFP-Luc) into lethally irradiated BALB / c recipients intravenously, together with WT B6 TCD-BM cells alone or with purified B6 WT or Csf2 - / - When A20 cells were injected alone with TCD-BM, all recipients died from growing tumors within 35 days ( Figures 4A-4C ), which is consistent with the key role of T cells in the GvL effect. - / - Mice that received T cells showed a significant improvement in survival ( Figure 4C ), but in BM transplantation, WT or Csf2 - / - T cells lead to effective control of tumor growth ( Figures 4A-4C ). Lymphoma-bearing mice that received TCD-BM plus WT T cells died mainly from severe GVHD, whereas mice that received TCD-BM plus Csf2 - / - Mice that lacked T cells showed an unabated GvL effect but were protected from GvHD ( Figure 4C In another GvL model using the monocytic myeloid cell line WEHI-3, Csf2 - / - Similarly, when the inventors increased the number of A20 lymphoma cells inoculated, Csf2 - / - T cells even more efficiently executed GvL than WT T cells, resulting in a significant increase in overall survival. or Csf2 isolated from mice undergoing allo-HCT - / - Similar to WT T cells, WT T cells were able to kill A20 lymphoma and WEHI-3 cells ex vivo, expanded similarly in spleen and LN, and showed similar activation (CD44 high), proliferation (Ki67+), and frequency of granzyme-producing B cells. To test the translatability of these findings to the clinical setting, the inventors used a neutralizing mAb against GM-CSF. Strikingly, leukemic mice receiving anti-GM-CSF treatment with TCD-BM plus T cells showed a potent GvL effect and had significantly better survival than isotype-treated control mice ( Figure 4E-4H ). Better survival was associated with a reduced incidence of GvHD ( Figure 4H ). Notably, neutralization of GM-CSF had no effect on tumor growth in mice that did not receive HCT.

[0168] The inventors compared the cause of death (tumor or GvHD) between different groups (lymphoma-bearing mice that did not receive HCT, reached withdrawal criteria and required euthanasia). Lymphoma-bearing mice that received TCD-BM plus WT T cells showed delayed tumor progression but died of severe GVHD, while those that received TCD-BM plus Csf2 - / - Mice that were negative for Csf2 T cells showed a strong GvL effect and were protected from GvHD (Fig. 4d). - / - and WT T cells have the same ability to kill A20 lymphoma cells in vitro ( Fig. 7A , 7B). To test the translatability of these findings to the clinical setting, the inventors used neutralizing mAbs against GM-CSF. Strikingly, leukemic mice treated with anti-GM-CSF TCD-BM plus T cells showed a potent GvL effect and had significantly better survival than isotype-treated control mice ( Figure 4E-4H ). Better survival was associated with a reduced incidence of GvHD ( Figure 4H ). Notably, neutralization of GM-CSF had no effect on tumor growth in mice that did not receive HCT.

[0169] Notably, compared with WT donors, recipients of Csfrb2 - / - Mice receiving allo-HCT from donors showed a significant improvement in survival ( Fig. 8A and 8B ), indicating that neutralization of the GM-CSF receptor is effective in alleviating GvHD.

[0170] Thus, in the absence of GM-CSF, donor T cells are potent mediators of therapeutic GvL responses in mice without inducing lethal GvHD, even in the highly immunogenic context of MHC-mismatched allo-HCT.

[0171] Embodiment 5:

[0172] GM-CSF is elevated in human GvHD biopsies

[0173] The inventors then asked whether there was evidence that GM-CSF plays a parallel role in GvHD in human HCT recipients. The inventors evaluated GM-CSF gene expression and protein abundance in gastrointestinal biopsies of patients with different grades of clinically documented GvHD after HCT under a range of different conditions (Tables 1-3). At the transcriptional level, the inventors detected significantly higher expression of GM-CSF in gastrointestinal biopsies of patients with grade IV GvHD than in those with grade I GvHD ( Figure 5A ). In addition, in samples from patients with grade IV GvHD, the inventors observed strong immunoreactivity for GM-CSF in the interstitial compartment; whereas the inventors did not detect GM-CSF in biopsies from patients without GvHD (Figure 5b). Taken together, these results suggest a correlation between GM-CSF and the presence of severe GvHD after allo-HCT in human patients.

[0174] discuss:

[0175] Although there is a large literature discussing different T H Although T cell subsets and their signature cytokines have the potential to contribute to GvHD, the underlying mechanisms of inflammation and subsequent tissue destruction are not fully understood. H Plasticity in lineage commitment and in vitro manipulation of transplanted polarized T cells also limits the interpretation of these results. Here, we reveal that GM-CSF plays a previously unknown role in driving lethal GvHD in mice and is similarly elevated in human allo-HCT patients with severe GvHD. Furthermore, GM-CSF appears dispensable for therapeutic GvL effects and may therefore represent a promising novel therapeutic target in GvHD prevention and treatment. Our findings also exclude a role for T cell-derived IL-17A in acute GvHD but demonstrate a protective effect of IFNγ.

[0176] GM-CSF has been implicated in a variety of pathological inflammatory diseases and has potent proinflammatory effects on myeloid cells. Here, the inventors used two different acute GvHD murine models to show that donor T cell-derived GM-CSF directly mediates severe GvHD, which is associated with massive myeloid cell infiltration and increased tissue staining for components of the ROS production machinery, which is normally used by phagocytes for host defense against pathogens. The same mechanism is clearly deleterious in the immunopathology of GvHD, where tissue damage and leukocyte infiltration go hand in hand. - / -This notion is supported by the reduction in bone marrow-driven oxidative stress in GvHD-prone organs observed in animals with T cells expressing GM-CSF. The key role of GM-CSF in chronic intestinal inflammation and the finding of large amounts of GM-CSF in gastrointestinal biopsies of GvHD patients suggest that modulating the amount of this cytokine in this specific organ could be highly beneficial in the prevention and / or treatment of GvHD.

[0177] The inventors also found evidence for potential reciprocal regulation of GM-CSF and IFNγ during GvHD in mice. IFNγ produced by activated donor T cells has previously been shown to both promote and protect against GvHD; here, mice that received partially MHC-mismatched HCT with splenocytes lacking IFNγ were found to be more susceptible to lethal GvHD than mice that received WT spleens. Interestingly, these mice exhibited elevated serum GM-CSF levels, whereas mice that received splenocytes lacking GM-CSF exhibited elevated serum IFNγ. IFNγ has previously been reported to be involved in the regulation of effector T cells. H However, GM-CSF does not directly affect T cells that do not express the receptor complex, so the mechanism by which GM-CSF affects IFN production requires further investigation.

[0178] A long-standing assumption is that donor T cell-driven GvHD and therapeutic GvL effects are mediated by the same mechanism: data presented herein challenge this view. Treatment of mice with an antibody blocking GM-CSF prior to allo-HCT significantly protected them from the development of lethal acute GvHD; whereas tumor-bearing mice treated with anti-GM-CSF benefited from effective control of tumor growth and protection from GvHD. Thus, GM-CSF cannot control T cell-mediated killing of malignant lymphohematopoietic cells; rather, the inventors propose, without wishing to be bound by theory, that in GvHD, GM-CSF acts as a conduit of communication between pathogenic T cells and myeloid cells, allowing the latter to cause tissue destruction. Although GvL may rely on direct cytotoxicity of T cells, GM-CSF-driven myeloid cell responses may be more relevant to GvHD pathology, thus representing a potential mechanism to uncouple GvL effects from GvHD.

[0179] The pathological role of GM-CSF in autoimmune and inflammatory diseases has led to the development of monoclonal antibodies targeting the GM-CSF pathway in human disease. Given the beneficial effects of neutralizing GM-CSF on experimental GvHD shown here, it is clear that blocking GM-CSF in allo-HCT patients will improve clinical outcomes. The marked increase in levels of this cytokine observed in affected tissues of patients with severe GvHD further supports this notion. However, as GM-CSF is essential for surfactant homeostasis and pulmonary host defense, close monitoring of lung disease parameters when blocking GM-CSF in the clinical setting will be important. In mice, there is evidence that GM-CSF plays a key role in IL-23-mediated immune responses, and anti-IL-23p19 therapy improves syngeneic GvHD-associated colitis. Therefore, direct blockade of GM-CSF may provide a valuable addition to any clinical trials targeting IL-23. The fact that depletion of GM-CSF can terminate the development of GvHD without impairing GvL responses lends itself to testing this therapeutic strategy for the prevention and treatment of GvHD after allo-HCT in clinical trials. Furthermore, the fact that GM-CSF was elevated in a sample of patients with the most severe manifestations of GvHD means that this treatment strategy holds particular promise in the patient population with the worst outcomes and highest risk of death.

[0180] Table 1. Clinicopathological characteristics of GvHD patients group I

[0181]

[0182] CVID: common variable immunodeficiency; sAML: secondary acute myeloid leukemia; T-ALL: T-cell-acute lymphoblastic leukemia; MDS: myelodysplastic syndrome; MRD: matched related donor; MMURD: mismatched unrelated donor; MURD: matched unrelated donor; PBSC: peripheral blood stem cells; BM: bone marrow; FLU: fludarabine; BCNU: bischloroethyl nitrosourea; MEL: melphalane; TBI: total body irradiation; VP16: etoposide; TT: thiotepa; ATG: antithymocyte globulin; CyA: cyclosporine A; MMF: mycophenolate mofetil; MTX: methotrexate.

[0183] Table 2. Characteristics of control subjects

[0184] Patient number gender age Biopsy 1 male 59 No pathological findings 2 male 60 No pathological findings 3 male 46 No pathological findings

[0185] Table 3. Clinicopathological characteristics of GvHD patient group II

[0186]

[0187] AML: acute myeloid leukemia; B-ALL: B-cell acute lymphoblastic leukemia; CVID: common variable immunodeficiency; MDS: myelodysplastic syndrome; RAEB: refractory anemia of immature excess; T-NHL: T-cell non-Hodgkin lymphoma; sAML: secondary acute myeloid leukemia; MPN: myeloproliferative neoplasms; MM: multiple myeloma; MMURD: mismatched unrelated donor; MRD: matched related donor =MURD: matched unrelated donor; PBSC: peripheral blood stem cell; FLU: fludarabine; BCNU: bischloroethyl nitrosourea; MEL: melphalan; TREOS: treosulphan; TBI: total body irradiation; VP16: etoposide; TT: thiotepa; BU: busulfan; CY: cyclophosphamide; CyA: cyclosporine A; MTX: methotrexate; MMF: mycophenolate mofetil; ATG: antithymocyte globulin.

[0188] Table 4. GvHD scoring system for allo-HCT mice

[0189] Sequence Listing <110> University of Zurich <120> GM-CSF or GM-CSF receptor ligands for the treatment of hematologic malignancies in patients who have undergone allo-HCT <130> uz349wo <160> 4 <170> PatentIn version 3.5 <210> 1 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 1 cactgctgct gagatgaatg aaa 23 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 gtctgtaggc aggtcggctc 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 gaaggtgaag gtcggagtca ac 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 tgattttgga gggatctcgc tc 22

Claims

1. Use of a non-agonist ligand that specifically binds to granulocyte macrophage colony stimulating factor in the preparation of a pharmaceutical composition for treating a patient with a malignant blood disease who has received an allogeneic hematopoietic stem cell transplant, wherein the malignant blood disease is leukemia, lymphoma or multiple myeloma, wherein the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia and acute monocytic leukemia, wherein the non-agonist ligand that specifically binds to granulocyte macrophage colony stimulating factor is a neutralizing humanized anti-granulocyte macrophage colony stimulating factor antibody lenzilumab.

2. The use according to claim 1, wherein the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

3. The use according to any one of claims 1 to 2, wherein the non-agonist ligand lenzilumab is a protein encoded by a nucleic acid.

4. Use of a non-agonist ligand that specifically binds to granulocyte macrophage colony stimulating factor in the preparation of a pharmaceutical composition for treating a patient with a hematological malignancy, thereby inhibiting the development of graft-versus-host disease or reducing the severity of graft-versus-host disease, wherein the patient has received an allogeneic hematopoietic stem cell transplant, wherein the hematological malignancy is leukemia, lymphoma or multiple myeloma, wherein the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia and acute monocytic leukemia, wherein the non-agonist ligand that specifically binds to granulocyte macrophage colony stimulating factor is a neutralizing humanized anti-granulocyte macrophage colony stimulating factor antibody lenzilumab.

5. The use according to claim 4, wherein the allogeneic hematopoietic stem cell transplant is provided to the patient for the treatment of hematological malignancies.

6. The use according to claim 4 or 5, wherein said lenzilumab is provided to said patient simultaneously with or after said allogeneic transplantation.

7. A use of a nucleic acid molecule in the preparation of a pharmaceutical composition for treating a hematological malignancy in a patient who has received an allogeneic hematopoietic stem cell transplant, wherein the nucleic acid molecule encodes a non-agonist protein ligand that specifically binds to granulocyte macrophage colony stimulating factor, and the non-agonist protein ligand that specifically binds to granulocyte macrophage colony stimulating factor is the neutralizing humanized anti-granulocyte macrophage colony stimulating factor antibody lenzilumab, wherein the hematological malignancy is a leukemia, a lymphoma or multiple myeloma, and the leukemia is selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia and acute monocytic leukemia.

8. The use according to claim 7, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule.

9. A use of a nucleic acid expression construct comprising a nucleic acid molecule in the preparation of a pharmaceutical composition for treating a malignant blood disease in a patient who has received an allogeneic hematopoietic stem cell transplant, wherein the nucleic acid molecule encodes a non-agonist protein ligand that specifically binds to granulocyte macrophage colony stimulating factor, and the non-agonist protein ligand that specifically binds to granulocyte macrophage colony stimulating factor is a neutralizing humanized anti-granulocyte macrophage colony stimulating factor antibody lenzilumab; wherein the nucleic acid molecule is a DNA molecule or an RNA molecule, wherein the malignant blood disease is a leukemia, a lymphoma or multiple myeloma, and wherein the leukemia is selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia and acute monocytic leukemia.

10. Use according to claim 9, wherein the expression construct is a DNA plasmid.

11. The use according to claim 9, wherein the expression construct is a double-stranded linear DNA.

12. The use according to claim 9, wherein the expression construct is a single-stranded RNA and a virus.

13. The use according to claim 9, wherein the expression construct is a virus.

14. The use according to claim 9, wherein the expression construct is a lentivirus, a herpes virus, an adenovirus or an adeno-associated virus.

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