POLYPEPTIDES FOR TREATMENT OF AML

Abstract

The present invention relates to a polypeptide comprising (i) a binding peptide binding to at least one surface marker of an acute myeloid leukemia (AML) cell, and (ii) an immunogenic peptide comprising at least one T-cell epitope; and to means and methods related thereto.

Claims

1-21. (canceled)

22. A polypeptide comprising: (i) a binding peptide binding to at least one surface marker of an acute myeloid leukemia (AML) cell, and (ii) an immunogenic peptide comprising at least one T-cell epitope.

23. The polypeptide of claim 22, wherein the AML cell is (i) a myeloblast, (ii) a promyelocyte, (iii) a myelocyte, or (iv) a progenitor of any one of (i) to (iii).

24. The polypeptide of claim 22, wherein the AML cell expresses major histocompatibility complex II (MHC-II) or is inducible to express MHC-II.

25. The polypeptide of claim 22, wherein the surface marker of an AML cell is a polypeptide, and optionally wherein the polypeptide is selected from group consisting of CD371, PRAME, CD123, CD138, and TIM-3, preferably from CD371, PRAME, and CD123.

26. The polypeptide of claim 22, wherein the binding peptide is an antibody.

27. The polypeptide of claim 22, wherein the binding peptide is a single-chain antibody.

28. The polypeptide of claim 22, wherein the immunogenic peptide comprises at least one T-cell epitope comprised in a protein of an infectious agent, preferably a virus, commonly infecting the subject, or against which the subject has been vaccinated; or of a tumor antigen.

29. The polypeptide of claim 28, wherein the infectious agent is selected from the group consisting of Epstein-Barr virus (EBV), measles virus, rubella virus, mumps virus, varicella virus, influenza virus, polio virus, hepatitis A virus, hepatitis B virus, rotavirus, papillomavirus, Corynebacterium diphtheriae, Clostridium tetanii, Bordetella pertussis, Haemophilus influenzae, Pneumococcus spec., and Meningococcus spec.

30. The polypeptide of claim 22, wherein: (a) the infectious agent is an infectious agent establishing latent infection, which preferably is EBV or papillomavirus, and (b) the T-cell epitope is an epitope of a latent gene product thereof.

31. The polypeptide of claim 22, wherein the immunogenic peptide comprises an MHC-II peptide.

32. The polypeptide of claim 22, wherein the polypeptide: (a) comprises the amino acid sequence of SEQ ID NO:12; (b) is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:13; (c) comprises the amino acid sequence of SEQ ID NO:14; (d) is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:15; (e) comprises the amino acid sequence of SEQ ID NO:20; (f) is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:21; (g) comprises the amino acid sequence of SEQ ID NO:22; or (h) is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:23.

33. A polynucleotide encoding the polypeptide according to claim 22.

34. A method for the stimulation of AML-specific T-cells, comprising (a) contacting AML cells with the polypeptide according to claim 22, (b) contacting the AML cells of (a) with T-cells, and (c) thereby stimulating AML-specific T-cells.

35. The method of claim 34, wherein the AML-specific T-cells are cytotoxic T-cells, and optionally wherein the cytotoxic T-cells are CD4+ cytotoxic T-cells.

36. The method of claim 34, wherein the method is a method of treating acute myeloid leukemia (AML) in a subject known or suspected to be suffering from AML.

37. The method of claim 36 comprising: contacting the subject with a polypeptide for treatment of AML, thereby treating AML, wherein the polypeptide comprises: (i) a binding peptide binding to at least one surface marker of an acute myeloid leukemia (AML) cell, and (ii) an immunogenic peptide comprising at least one T-cell epitope.

Description

FIGURE LEGENDS

[0115] FIG. 1: (A) workflow of the experiments; (B) to (D) show the results of interferon gamma release assays performed with multiple AML cell lines after exposure to the EBV epitopes BZLF1-3H11 (B), EBNA1-3G2 (C), or EBNA3C-3H10 (D). Some cell lines were treated additionally with interferon gamma prior to the T cell assay as indicated in the caption. LCLs are EBV-positive cells that served as positive controls, T cells alone and cells non-exposed to peptides served as negative controls. Interferon secretion after coculture of AML cell lines exposed to various EBV peptides with T cells specific for this antigen are given in the graph of bars.

[0116] FIG. 2: HLA class II expression at the surface of AML cell line KG-1. The various control antibodies are indicated

[0117] FIG. 3: CLL-1 expression at the surface of various AML cell lines was determined by FACS.

[0118] FIG. 4: Different AgAbs directed against CLL-1 (left) and CD123 (right) were able to bind to their targets as well as non-modified antibodies do. The AML cell lines used to test binding were KG-1 (A), MonoMac-6 (B), and Nomo-1 (C); unstained: unstained control; isotype: isotype control, wt: wildtype antibody (i.e. without immunogenic peptide), 3G2:EBNA1-3G2, 3H10: EBNA3C-3H10, 3H11: BZLF1-3H11.

[0119] FIG. 5: The graphs show the results of interferon gamma release assays performed with the KG-1 AML cell line after exposure to various amounts of AgAbs specific to CLL-1 or CD123 and carrying the indicated EBV epitopes (BZLF1 3H10, EBNA1 3G2) (upper panel). Native antibodies devoid of antigenic moieties served as negative controls (WT). The lower panel shows the results of T cell assays conducted on AML KG-1 cells exposed to the peptide 3G2 or 3H10 only.

[0120] FIG. 6: The graphs show the results of interferon gamma release assays performed with the MonoMac 6 AML cell line after exposure to various amounts of AgAbs specific to CLL-1 or CD123 and carrying the indicated EBV epitopes (BZLF1-3H10, EBNA1-3G2) (upper panel). Native antibodies devoid of antigenic moieties served as negative controls (WT). The lower panel shows the results of T cell assays conducted on AML MonoMac 6 cells exposed to the peptide 3G2 or 3H10 only.

[0121] FIG. 7: A) The graph shows the results of interferon gamma release assays performed with the MV4-11 AML cell line after exposure to various amounts (100 ng-0,1 ng per 5.10.sup.4 target cells) of IgG2a AgAbs specific to CLL-1, CD123 or FR-beta and carrying the indicated EBV epitope (gp350 1D6). Native antibodies devoid of antigenic moieties served as negative controls (Native—100 ng only). Various amounts of epitopes were also used to demonstrate the superiority of AgAbs stimulation. Per 5.10.sup.4 target cells, 10.sup.5 effector CD4.sup.+ T cells were used (E:T ratio=2:1); B) The graph shows the results of granzyme B release assays performed with the MV4-11 AML cell line after exposure to various amounts (100 ng-0,1 ng per 5.10.sup.4 target cells) of IgG2a AgAbs specific to CLL-1, CD123 or FR-beta and carrying the indicated EBV epitope (gp350 1D6). Native antibodies devoid of antigenic moieties served as negative controls (Native—100 ng only). Various amounts of epitopes were also used to demonstrate the superiority of AgAbs stimulation. Per 5.10.sup.4 target cells, 10.sup.5 effector CD4.sup.+ T cells were used (E:T ratio=2:1).

[0122] FIG. 8: A) The graph shows the results of interferon gamma release assays performed with the Mutz-3 AML cell line after exposure to lOng per 5.10.sup.4 target cells of IgG2a AgAbs specific to CLL-1, CD123 or FR-beta and carrying the indicated EBV epitope (EBNA3C 3H10). Native antibodies devoid of antigenic moieties served as negative controls (Native). Per 5.10.sup.4 target cells, 10.sup.5 effector CD4.sup.+ T cells were used (E:T ratio=2:1); B) The graph shows the results of granzyme B release assays performed with the Mutz-3 AML cell line after exposure to lOng per 5.10.sup.4 target cells of IgG2a AgAbs specific to CLL-1, CD123 or FR-beta and carrying the indicated EBV epitope (EBNA3C 3H10). Native antibodies devoid of antigenic moieties served as negative controls (Native). Per 5.10.sup.4 target cells, 10.sup.5 effector CD4.sup.+ T cells were used (E:T ratio=2:1)

[0123] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1

Expression of Surface Markers on AML Cell Lines

[0124] AML cell lines as indicated were stained using antibodies specific for HLA-DR, CD123, CD138, CD371, TIM-3, PRAME and analyzed by FACS. Some cell lines were stimulated with interferon gamma prior to staining with an HLA-DR specific antibody. Isotype controls were used as negative controls to exclude unspecific staining (Table 1).

[0125] Further, AML cell lines were stained using antibodies specific for HLA-DR and analyzed by FACS; an exemple is shown in FIG. 2. Some cell lines were stimulated with interferon gamma prior to staining with an HLA-DR specific antibody. Isotype controls and unstained samples were used as negative controls to exclude unspecific staining.

[0126] Further (FIG. 3), AML, cell lines were stained using antibodies specific for CLL-1 and analyzed by FACS. Isotype controls and unstained samples were used as negative controls to exclude unspecific staining.

[0127] Table 1 shows the expression of the HLA class II molecule HLA-DR and of multiple cellular markers (CD123, CD138, CD371, TIM-3, PRAME) at the surface of 6 cell lines established from patients with acute myeloid leukemias.

TABLE-US-00001 TABLE 1 Surface markers of AML cell lines; + indicates expression, Neg indicates undetectable expression, inducible means that HLA class II expression can be induced by treating the cells with interferon gamma. CLL-1 FR- Cell line HLA-DR CD123 (CD371) beta PRAME MDM2 CD138 TIM-3 NOMO-1 + + + − + + Neg + KG-1 + + + − + + Neg + MonoMac6 + + + − + + Neg Neg OCI- + + + − + + Neg Neg AML2 HL-60 Inducible Neg + − + + + Neg MOLM-14 Inducible + + − + + Neg Neg Mutz-3 + + + + + + Neg + MV4-11 + + + + + + Neg Neg

TABLE-US-00002 TABLE 2 HLA DRB1 haplotypes of AML cell lines; data are from the TRON cell line portal (Mainz, Germany); n.a.: no data available Cell line HLA DRB1 HL-60 11:30′/13:01′ KG-1 .sup. 11:01/03:17′ MOLM-14 n.a. MonoMac-6 .sup. 01:01/11:01 NOMO-1 04:05′/14:103 OCI-AML2 .sup. 01:03/04:01′ Mutz-3 .sup. 10:01/11:01 MV4-11 .sup. 01:01/13:02

EXAMPLE 2

[0128] Determination of epitopes matching the HLA haplotypes of AML cell lines The EBV peptides binding to HLA subtypes were taken from the literature (cf. Yu et al. (2015), Blood 125(10):1601; Adhikary et al (2006), JEM 203(4):995; Mautner et al. (2004), J. Immunol. 34:2500). The HLA subtypes expressed by the AML cell lines were determined either from the literature or from sequencing. This information allowed the matching of the AML cell lines with EBV peptides that they were expected to be able to present.

TABLE-US-00003 TABLE 3 Epitopes matching the HLA haplotypes of AML cell lines; (?): expression unclear. Cell line Haplotype Matching epitopes HL-60 HLA-DRB1*1301 EBNA1-1C3 EBNA3B-B9 BZLF1-3H11 KG-1 HLA-DRB1*1101 EBNA1-3G2 MonoMac6 EBNA3B-3F7 Mutz-3 EBNA3C-1B2/3H10 HL-60(?) HLA-DRB1*11 EBNA1-3E10 KG-1(?) MonoMac6(?) Mutz-3(?) MV4-11 HLA-DRB1*1302 gp350 1D6

EXAMPLE 3

Presentation of Peptides by AML Cells

[0129] Human T cells specific for the EBV peptides previously isolated from human infected with the virus were stimulated several weeks with these peptides together with interleukin 2. The AML cell lines were exposed to increasing concentrations of the peptides (Example 2) for one day, then extensively washed and mixed with pre-activated T cells specific for the peptide they were exposed to. One day later, interferon gamma release in the supernatant of these cultures was quantified by ELISA using specific antibodies (FIG. 1).

EXAMPLE 4

Binding of Antibody-Immunogenic Peptide Fusion Polypeptides (AgAbs)

[0130] AML cell lines were stained by FACS using AgAbs specific for CLL-1 or CD123. Isotype controls were used as negative controls to exclude unspecific staining. The antibodies that were used to generate the AgAbs were used as controls (FIG. 4).

EXAMPLE 5

Activation of T-cells

[0131] Human T cells specific for the EBV peptides previously isolated from human infected with the virus were stimulated several weeks with these peptides together with interleukine 2. AML cell lines were exposed to increasing concentrations of peptides or of AgAbs containing the same peptides for one day, then extensively washed and mixed with pre-activated T cells specific for the peptide contained in the AgAbs or to the peptide alone they were exposed to. One day later, interferon gamma release in the supernatant of these cultures was quantified by ELISA using specific antibodies. Native antibodies devoid of antigenic moieties served as negative controls (WT) (FIG. 5).

[0132] Further (FIG. 6), Human T cells specific for the EBV peptides previously isolated from human infected with the virus were stimulated several weeks with these peptides together with interleukine 2. AML cell lines were exposed to increasing concentrations of peptides or of AgAbs containing the same peptides for one day, then extensively washed and mixed with pre-activated T cells specific for the peptide contained in the AgAbs or to the peptide alone they were exposed to. One day later, interferon gamma release in the supernatant of these cultures was quantified by ELISA using specific antibodies. Native antibodies devoid of antigenic moieties served as negative controls.

[0133] While Examples 3 to 5 were performed with IgG1 subtype constructs, the following Examples 6 and 7 were performed with IgG2a subtypes:

EXAMPLE 6

Activation by AML cell line MV4-11

[0134] Similar to the proceeding of Example 5, MV4-11 AML cells were used in T cell activation assays (TCAs) using constructs as indicated and determining interferon-gamma secretion (FIG. 7A) or Granzyme B production (FIG. 7B) as parameter of T cell activation.

EXAMPLE 7

[0135] Similar to the proceeding of Example 5, Mutz-3 AML cells were used in T cell activation assays (TCAs) using constructs as indicated and determining interferon-gamma secretion (FIG. 8A) or Granzyme B production (FIG. 8B) as parameter of T cell activation.

LITERATURE

[0136] Adhikary et al (2006), JEM 203(4):995 [0137] Bernardeau et al., (2011), J Immunol Methods, 371(1-2):97-105 [0138] Bordner (2010), PLoS ONE 5(12): e14383 [0139] Galfré (1981), Meth. Enzymol. 73, 3, [0140] Kaech et al. (2002), Nature Reviews Immunology 2(4):251-62 [0141] Köhler and Milstein (1975), Nature 256, 495 [0142] Mautner et al. (2004), J. Immunol. 34:2500 [0143] Nielsen et al., (2004), Bioinformatics, 20 (9), 1388-1397 [0144] Pulendran and Ahmed (2006), Cell 124(4):849-63 [0145] Yu et al. (2015), Blood 125(10):1601